VI. NATURAL IMMUNITY AGAINST PATHOGENIC MICRO-ORGANISMS…

VI. NATURAL IMMUNITY AGAINST PATHOGENIC MICRO-ORGANISMS

Natural immunity and the composition of the body fluids.—Cultivation of the bacteria of influenza and pleuro-pneumonia in the fluids of refractory animals.—Resistance of Daphniae to the Blastomycetes.—Examples of natural immunity in Insects and Mollusca.—Immunity of Fishes against the anthrax bacillus.—Immunity of frogs against anthrax, Ernst’s bacillus, the bacillus of mouse septicaemia and the cholera vibrio.—Natural immunity in the cayman.—Immunity of the fowl and pigeon against anthrax and human tuberculosis.—Immunity of the dog and rat against the anthrax bacillus.—Immunity of Mammals against anthrax vaccines.—Immunity of the guinea-pig against spirilla, vibrios, and streptococci.—Natural immunity against anaerobic bacilli.—Fate of Blastomycetes and Trypanosomae in the refractory organism.

[Sidenote: [136]]

In the third chapter reference has been made to the frequency of cases of natural immunity against infective diseases. Examples of this immunity occur in the lower animals—the Invertebrata—and are widely met with among the Vertebrata. We have already mentioned that this natural immunity can be attributed neither to insusceptibility to microbial toxins nor to the elimination of the micro-organisms by the excretory channels. Nevertheless the pathogenic agents which have penetrated into the tissues of the refractory organism disappear, without being eliminated. To facilitate the study of their disappearance it has been necessary to pass in review the phenomena that follow the introduction of foreign bodies into the organism and to present a brief analysis of the process of resorption of cell elements in its relations to digestion. We have tried to demonstrate that resorption is nothing more than a process of digestion which, instead of going on in the intestinal canal, takes place in the tissues; that it is, indeed, an intracellular digestion exactly comparable to that which serves for the nutrition of certain of the lower animals.

[Sidenote: [137]]

A knowledge of all these facts is necessary before we can deal with the subject to which the present chapter must be devoted—the innate natural immunity of animals and man against pathogenic micro-organisms. As, under natural conditions, it is the micro-organism and not its toxic products which invades the organism, it is clear that we must give the first place to the study of immunity against the micro-organism. The more so because this form of immunity is much more frequently met with than is an insusceptibility to toxins.

Since the animal organism has a very variable composition it might be concluded that the micro-organisms find in the refractory species simply a chemical medium in which they cannot live. We cannot go far in the discussion of this supposition without seeing that it may be rejected. Among the pathogenic micro-organisms some are distinguished by a great fastidiousness and sensitiveness as regards the medium in which they are placed. Such, for example, are the parasites of malaria and their allies. They live inside the red blood corpuscles of Vertebrata and appear to be extremely discriminating in regard to their requirements. All animals, even monkeys, are refractory to human malarial fevers. It might be concluded from this that here at least the immunity may be due to the fact that the chemical composition of the contents of the red corpuscles in the immune animals is different from that of the red corpuscles of man. But when we see, as was first demonstrated by Ross[175], that the malaria parasite of Laveran, having made its way into the digestive canal of certain mosquitos (Anopheles), there develops abundantly, it is difficult to maintain this thesis.

Among other micro-organisms of animal origin we have the Trypanosoma, the parasite of the terrible disease propagated by the Tsetse fly which commits such ravages amongst mammals. Man alone escapes it, exhibiting a natural immunity that nothing apparently can overcome. Are we to affirm that it is the difference in the chemical composition of the human body which assures to man his immunity against a parasite that attacks indifferently an herbivorous animal, such as the ox or rabbit, or a carnivorous animal, such as the dog? In these examples I have chosen merely those micro-organisms which it has never been possible to cultivate on any artificial nutrient medium and which are kept alive with very great difficulty outside the living organism.

[Sidenote: [138]]

What is to be said then of the vegetable micro-organisms which, in this respect, are much less exacting? The most important of these and the most numerous of all pathogenic micro-organisms, the Bacteria, can as a rule be cultivated without difficulty not only in the blood and fluids of animals that are susceptible or refractory to their morbific action, but also on all kinds of vegetables and artificial media: broths, fluids composed of mineral salts and of certain organic substances. It is really not possible to attribute the natural immunity of the dog and the fowl against the anthrax bacillus—so fatal to a great number of mammals, man included,—to its incapacity to feed on the fluids of these animals, when we see that this same bacillus is capable of killing lower animals, such as the cricket, and can thrive on carrots, potatoes and other vegetables.

Even when, among the bacteria, we take those that are most exacting in the choice of their food, we still find it impossible to explain natural immunity as being due to the want of power on the part of these organisms to obtain their nutriment from the juices of refractory species. The bacillus discovered by R. Pfeiffer[176] in influenza does not develop on media that are ordinarily employed in bacteriology in the cultivation of a great number of micro-organisms. It needs a special food, which is prepared for it by spreading a drop of fresh blood on the surface of agar. Pfeiffer has established the fact—confirmed by many observers—that the best species of blood to use for this purpose is that of the pigeon. We should have to believe, then, did the immunity really depend on the composition of the fluids, that the pigeon is the least refractory of all animals. Experiment has demonstrated the erroneousness of such a supposition: the pigeon is quite as refractory to Pfeiffer’s bacillus as are most other species of animals.

[Sidenote: [139]]

As a second example the bacterium of bovine pleuro-pneumonia may be cited. It is the smallest of all known bacteria. The difficulties surrounding the discovery and identification of this organism were very great, and the ingenuity of Nocard and Roux[177] was required for the demonstration of its existence. Very exacting in its choice of nutritive material, it was first cultivated in the fluids of the rabbit, a species endowed with an absolute immunity against bovine pleuropneumonia. It is unnecessary to multiply examples to obtain a general proof that natural immunity against micro-organisms cannot be explained by the incapacity of these pathogenic agents to live in the fluids of the refractory organism.

We must, however, ascertain what takes place in resistant animals inoculated with micro-organisms. Here, again, it is preferable to begin with the lower animals of simple organisation. We have already seen that examples of natural immunity are not rare in the Invertebrata. When engaged in the study of the disease found in Daphniae, small crustacea so common in fresh water, I was able to show that the special Blastomycetes which cause it meet with a vigorous resistance on the part of the organism. As the Daphniae are small, transparent, and consequently easily observed under the microscope, I was able without difficulty to establish the main phenomena observable in these organisms. I can be the more brief in describing these phenomena of resistance as, in addition to devoting a special memoir to the Daphnia disease[178], I have, in my Lectures on Inflammation (pp. 97–103)[179], described at some length the reaction of their organism to the Monospora. It is nevertheless necessary that I should recall, very briefly, the mechanism by which these small crustaceans secure immunity.

The spores of the parasite—very delicate and rigid needles—are swallowed with the food. By means of their sharp points they perforate the intestine and penetrate into the body cavity, full of blood, where they find themselves exposed to the attacks of leucocytes. These leucocytes, guided by their tactile sense, gather around the foreign body, ingest it completely and destroy it. It is remarkable that the spore, which is furnished with a very resistant membrane, once in the interior of the mass of leucocytes, undergoes modifications which afford evidence of the presence in these cells of an extraordinary digestive power. The surface of the spore, from being smooth and regular, becomes pitted and sinuous, the spore breaks up into fragments and is reduced to a mass of débris which, in the form of brown granules, remains indefinitely in the contents of the leucocytes. From this it is evident that these phagocytes must produce a ferment which is capable of digesting the cellulose or analogous substance which forms the membrane of the spore. Unfortunately, the small size of the Daphniae, so useful for the direct observation of the phenomena of immunity, presents an insurmountable obstacle to the study of its leucocyte ferments, especially in vitro.

[Sidenote: [140]]

The destruction of the spores of the parasite by the leucocytes secures to the Daphnia a real immunity. Of a hundred Daphniae taken in my aquarium and carefully examined under the microscope, fourteen only were found to be infected by the budding conidia of the parasite, whilst fifty-nine of the others contained the remains of spores that had been destroyed by the phagocytes. When transferred to pure water containing no new source of contagion, these Daphniae flourished and lived a normal life, giving birth to a numerous progeny.

The immunity of the Daphnia, due to the intervention of phagocytes, is an example of natural, individual immunity. It is not the specific or racial possession of these crustacea, for when the leucocytes do not seize the spore, at once, on its penetration into the body cavity, it commences to germinate and gives rise to a whole generation of budding cells. These cells, then, secrete a poison which not only repels the leucocytes, but kills and completely dissolves them. Under these conditions the Daphnia is disarmed; the parasites grow in the organism, deprived of its arm of defence, as in a culture tube, and the animal rapidly succumbs.

Since I first observed this struggle between the Daphnia and its parasite, some eighteen years ago, no other example has been found that is so easily observed and so demonstrative of the protective action of phagocytes in an animal that can be kept under observation, alive, under the microscope. Cases, however, are not wanting in the Invertebrata in which the different phases of this struggle may be observed with sufficient accuracy to warrant the conclusion that in these cases also the phenomena are analogous to those observed in the case of the Daphniae.

[Sidenote: [141]]

It has already been stated in Chapter III. that the larvae of the rhinoceros beetle (Oryctes nasicornis), although very sensitive to the cholera vibrio, are very refractory to anthrax and diphtheria. In order that we may obtain some idea of the mechanism of this immunity let us inject into the body cavity of these large white grubs a trace of anthrax culture. In the blood, drawn off the following morning, the injected bacilli are found, not in the plasma, but inside many of the leucocytes. Here there has occurred, as in the Daphnia, an ingestion of the parasites which have then been destroyed by the intracellular digestion of phagocytes. The process is the same, then, as that by which the resorption of the red corpuscles of the goose takes place when they are injected into the blood of cockchafer larvae. In both cases the foreign bodies are ingested and destroyed by the leucocytes of the blood; this act of resorption, however, taking a very long time.

Although the leucocytes of the larvae of the rhinoceros beetle exhibit a positive chemiotaxis for the bacillus, these same cells behave in a very different fashion in presence of the cholera vibrio. Very small quantities of this vibrio, when injected into the blood of the larvae, give them a fatal disease: the vibrios excite in the leucocytes a negative chemiotaxis and flourish without hindrance in the blood plasma. The larva is soon transformed into a culture vessel and the numerous vibrios that develop in it cause the death of the animal.

The difference in action of the two bacteria cannot be explained by any corresponding difference in their mode of life in the blood. Removed from the organism the blood plasma of the white larvae of the rhinoceros beetle is a culture medium just as favourable to the growth of the anthrax bacillus as to that of the cholera vibrio. Moreover, the former of these micro-organisms is quite capable of setting up a fatal disease in other representatives of the class of Insects. Kovalevsky[180] has discovered in the house cricket four phagocytic organs, with a great appetite for all kinds of foreign particles that may penetrate into its body. The blood of mammals, when injected below the skin of the cricket, is rapidly absorbed by the cells of the four “spleens” (for so Kovalevsky designates these phagocytic organs). The resorption of the red blood corpuscles goes on within these phagocytes owing to their power of intracellular digestion. When Kovalevsky kept crickets at a temperature of 22°–23° C. and injected them with anthrax bacilli he noted that these bacilli also were ingested by the cells of the spleens. There was, thus, no manifestation of negative chemiotaxis of these elements towards the bacillus. The ingestion of the bacilli by the phagocytes was not sufficient, however, to protect the animal. The bacilli reproduced themselves rapidly in the blood fluid; the intracellular lacunae of the spleens were full of them and the crickets quickly succumbed to the infection.

[Sidenote: [142]]

Nevertheless these crickets are quite capable of resisting certain other bacteria. Balbiani[181] has shown that they are refractory to a great number of bacilli belonging to the group of Bacillus subtilis. He observed that when injected into the body of the cricket these bacilli are devoured and destroyed by the leucocytes of the blood and by the large cells of the pericardial tissue corresponding to the elements of the spleens of Kovalevsky. Whilst the crickets and other Orthoptera, which are rich in phagocytes, exhibit a real immunity against these bacilli, insects which have very few leucocytes such as butterflies, flies and Hymenoptera are found to be much more susceptible to infection by the same bacilli. In this case the direct relation between immunity and phagocytosis is very marked.

[Sidenote: [143]]

The Mollusca also furnish some interesting examples of natural immunity. Karlinsky[182] has observed that anthrax bacilli, when injected into the blood of slugs and snails, soon disappear from their bodies; these pulmonate Gasteropods are absolutely unaffected by this bacillus so formidable for many species of animals. From the rapidity of this disappearance of the bacilli it has even been concluded that it was impossible for this bacillus to live in the fluids of Mollusca. Kovalevsky (l.c. p. 443) has studied this question with the carefulness that characterises all his work. He confirms the fact that snails (Helix pomatia) resist the introduction of a large quantity of anthrax bacilli into their bodies; he notes also that these bacteria disappear from the blood. But he finds them again in the tissues of the foot, and especially in the cells which surround the pulmonary vessels. “The greater number of the bacteria are found in the cells of that part of the pulmonary region in Helix which adjoins the heart and kidney. All the bacteria were ingested by the cells and I easily succeeded in demonstrating this not only in sections but also in bulk” (p. 444). The snails remained in good health in spite of the presence in their phagocytes of numerous bacteria which maintained themselves there for some time. At the end of ten or twelve days and more these bacteria still presented their usual aspect; this accords well with the slowness with which intracellular digestion goes on in the majority of the Invertebrata. These bacteria were, however, no longer living, although still undigested. Morsels of the pulmonary tissue of the snails that were injected with anthrax bacilli still gave cultures 48 hours after injection and contained bacilli capable of giving fatal anthrax to mice. Later, media seeded with similar particles remained sterile, and mice inoculated therewith continued to live. From these experiments it may be accepted that bacteria, living in the blood plasma, become the prey of phagocytes which render them inoffensive and kill them. This example demonstrates once again that the organism gets rid of bacteria by the same mechanism as that which serves for the resorption of any of the formed elements. The snail reacts to bacteria as it does to the red corpuscles of the goose.

It is unnecessary to insist further on the natural immunity of the Invertebrata, and it is useless to multiply examples which always point in the same direction: to the importance of phagocytic reaction and of intracellular digestion in resorption and immunity. We must pass on to the examination of the reaction phenomena of the vertebrate organism towards pathogenic micro-organisms, following, as hitherto, the comparative method. We will commence with the study of the natural immunity of fishes as lower representatives of the great group of the Vertebrata.

[Sidenote: [144]]

It is well known that fishes are liable to infective diseases and pisciculture has often to deplore considerable losses brought about sometimes by certain of the lower Fungi (e.g. Saprolegniae), sometimes by Bacteria. The pathogenic microbes which produce epidemics in fishes are still little understood; but among the bacteria which kill many of the higher animals are some which cause fatal maladies in certain fishes. Thus the anthrax bacillus so virulent for many mammals is capable also, as we have seen, of producing an infection in the cricket, and may cause the death of small marine osseous fishes, the Hippocampi. Sabrazès and Colombot[183], who have studied this question, have demonstrated that the anthrax bacillus, which is virulent for the rabbit, when inoculated into these fishes first produces swellings at the seat of inoculation and ultimately becomes generalised throughout the body, producing a fatal septicaemia. As these experiments have given this result at a temperature of 14°–16° C., it is quite evident that the bacillus, in order to manifest its pathogenic effect, in no way needs the high temperature of the mammalian body for its action.

Now among fishes there are not wanting species which resist the anthrax bacillus. Mesnil[184] has, in our laboratory, thoroughly studied the mechanism of this immunity. He has shown that several fresh-water fishes, e.g. the perch (Perca fluviatilis), the gudgeon (Gobio fluviatilis), and the gold-fish (Carassius auratus), will resist an injection of a considerable number of bacilli into the abdomen. When kept at temperatures of 15°–20° C. or even 23° C., a temperature at which the bacilli are able to develop very abundantly, these fishes destroy a large number of the bacteria in their bodies. Soon after the introduction of the bacilli into the peritoneal cavity, the numerous leucocytes accumulate around them and ingest them by the same mechanism that is observed in the Invertebrata or in the same fishes when absorbing the red blood corpuscles of alien species. In the gudgeon, at as early as six and a half hours, a very marked, nay, an almost complete phagocytosis is set up.

It is impossible to doubt the fundamental fact that the bacilli, at the moment of their ingestion, are in a perfect condition of vitality and virulence. The fluid of the peritoneal exudation, when withdrawn from the animal, is of itself incapable of preventing the development of the anthrax bacilli. The peritoneal lymph of the above-mentioned fishes is, in vitro, even a good culture medium for these bacilli.

[Sidenote: [145]]

When, long after the completion of the phagocytosis by the leucocytes of the peritoneal exudation, a drop of the exudation is withdrawn and kept outside the organism under suitable conditions of temperature and moisture, a number of the ingested bacilli begin to multiply and give an abundant culture. This experiment proves, indisputably, that the bacilli are devoured in the living state. If a little of the peritoneal exudation, withdrawn several (up to nine) days after the injection of the bacilli, be injected below the skin of guinea-pigs these animals die from generalised anthrax, a fact which demonstrates that the bacilli, which have been ingested alive, have retained their virulence a long time after they have been devoured by the leucocytes. But, if the peritoneal exudations that have been withdrawn at still longer periods after injection be examined, it is found that they no longer contain bacilli capable of developing in culture media or of setting up the disease in the most susceptible animal. Hence it follows that in the organism of the refractory fish, the bacteria are not destroyed by the fluids but by the phagocytes, which take a long time to bring about the complete intracellular digestion of ingested micro-organisms.

The phagocytes which assure immunity to the osseous fishes that were studied by Mesnil belong principally to the group of haemomacrophages. These are leucocytes with abundant protoplasm which stain readily by basic aniline dyes, mononuclear cells whose nucleus, however, is sometimes divided into lobes. It is to be noted that in the perch these are the sole representatives of the motile phagocytes, and that in this fish not only the eosinophile but every other variety of granular leucocyte is completely wanting. In the gudgeon, in addition to haemomacrophages, some microphages whose protoplasm stains faintly with acid aniline colours are met with. These facts will be useful to us when we come to study the part played by phagocytes in immunity from a general point of view.

Another class of cold-blooded animal, the Amphibia, has been much more frequently studied from the point of view of infection and immunity. The frog, an animal so convenient for many physiological and pathological researches, has been much employed for the study of immunity against pathogenic micro-organisms. Quite a literature, which has been excellently summarised in the memoir of Mesnil already cited, and to which we shall have occasion to return more than once, has been accumulated on the subject.

[Sidenote: [146]]

The immunity of frogs against the anthrax bacillus was early demonstrated and studied in Robert Koch’s celebrated memoir[185] on anthrax. This observer, after injecting an emulsion of anthrax spleen into the lymph sac of the frog, recovered the bacilli from the interior of round cells which burst readily when transported into water. Koch, accepting the view then generally held, thought that the bacilli found a favourable culture medium in the contents of certain cells, but that, in spite of this, the frog was capable of manifesting a real immunity against anthrax. Gibier[186] made the interesting discovery that frogs when subjected to the influence of high temperature (about 37° C.) lose their natural immunity and readily contract fatal anthrax.

Since that time the mechanism by which the organism of the frog secures immunity against the anthrax bacillus has repeatedly been studied. In a memoir which appeared in 1884[187] I insisted that the principal part played in this immunity belonged to the phagocytes which devour the injected bacteria and subject them to intracellular digestion. The round cells described by Koch are nothing but the leucocytes of the lymph sac which have seized upon the anthrax bacilli. These bacilli instead of thriving in the cell contents find there a very unfavourable medium, and perish at the end of a longer or shorter period. When the activity of the phagocytes is impeded by unfavourable influences, e.g. high temperature, they exhibit a very feeble reaction, incapable of assuring to the frog that immunity which, under normal conditions, it possesses. The conclusions I have just summarised have raised very lively opposition from a large number of observers. Baumgarten[188], with his pupils Petruschky[189] and Fahrenholtz[190], have endeavoured to demonstrate that phagocytosis plays no part in immunity and that the frogs resist anthrax simply because the bacilli are incapable of maintaining themselves alive in the fluids of this Batrachian. Nuttall[191], of Flügge’s school, also maintained that frogs resist anthrax owing to the bactericidal power of their fluids. This view has been defended by several other observers and appeared for some time to become quite dominant.

[Sidenote: [147]]

Nevertheless, it is possible to demonstrate that the plasmas of the frog not only are not inimical to the life of the bacillus, but serve as a good culture medium for it[192]. All that is necessary for the demonstration of this fact is to introduce below the skin of frogs anthrax spores enclosed in a sac of reed pith, or simply enveloped in a small piece of filter paper. The plasma of the lymph sac at once permeates the spores and allows them to germinate and produce quite a generation of bacilli. But, as soon as the leucocytes pass through the paper, they seize upon the young bacilli, digest them in their substance and prevent their pathogenic action. The germination of the spores may take place even where they have been introduced below the frog’s skin without being protected in any way whatever. But, under these conditions, only a certain number of the spores germinate, the majority not having time to do so before the arrival of the leucocytes. The small, very short bacilli which proceed from the germinated spores, are, along with the spores that have not germinated, soon ingested by the phagocytes. But, whilst the rods are in the end digested within these cells, the ingested spores remain intact for a very long time: they do not germinate, but they are not destroyed and retain their vitality indefinitely, in spite of the influence of the phagocytes. It is sufficient to withdraw from a frog, that has been inoculated with anthrax spores some time before and kept at a moderate temperature (15°–25° C.), a little lymph and sow it in any nutrient medium (of those employed in the culture of bacteria), in order to see the spores germinate and produce a whole generation of absolutely normal filamentous bacilli. All these phenomena have been carefully studied by Trapeznikoff[193] in a work executed in my laboratory. It is obvious from his experiments that the phagocytes of the frog are quite capable of protecting the organism against the anthrax bacillus by ingesting and digesting the bacilli in the vegetative state and by preventing the germination of the ingested spores. This phagocytic action is very important in presence of the fact that the plasmas of the frog allow the spores to germinate and the bacilli to develop and produce abundant cultures.

[Sidenote: [148]]

The immunity of frogs against the anthrax bacillus that we have just described and which is guaranteed by the activity of the phagocytes, is constant under the conditions of temperature above mentioned (15°–25° C.), conditions which are sufficient, however, to ensure the death of susceptible cold-blooded animals, such as the cricket or Hippocampus, from anthrax. The edible frog, a species that readily accommodates itself to a temperature of 35° C., resists, even under these conditions, infection by the bacillus, as pointed out by Mesnil in a work already cited when treating of the immunity of fishes. The green frog (Rana esculenta) when kept for a long time at this high temperature, so suitable for the development of the anthrax bacillus, reacts by the same phagocytic mechanism. The leucocytes of the lymph and blood, the cells of the splenic pulp and Kupffer’s stellate cells of the liver, seize the introduced bacilli and digest them as in any other case of phagocytosis. The brown frog (Rana temporaria) adapts itself but slightly and with great difficulty to the high temperature and dies whether it has been inoculated with anthrax or not. Under these conditions the bacteria develop in the body of the dead or dying frogs, but Mesnil insists on the fact that a true anthrax infection is not produced, as has been maintained by Gibier as the outcome of his researches.

[Sidenote: [149]]

Dieudonné[194], however, has found a method of removing the natural immunity of the frog against the anthrax bacillus, by inoculating it with an artificial bacterial race which he had adapted to develop fairly luxuriantly at the low temperature of 12° C. Under these conditions all the inoculated frogs, even those which had resisted the inoculation with ordinary bacteria (grown at 37°·5 C.), died within a period of 48 to 56 hours, containing many bacilli in the blood and organs. Dieudonné has not studied the essential mechanism that accompanies this loss of immunity; but it is very probable that, for one thing, we have here to do with a reinforcement, special for the frog, of the bacillus that has become accustomed to develop at a low temperature. This bacillus must multiply, in frogs that have been maintained at a low temperature, much more rapidly and profusely than would the ordinary bacillus. On the other hand, the susceptibility of Dieudonné’s frogs must depend on a less resistance of the organism under the conditions of his experiments. Unfortunately, we cannot find in his memoir sufficient data on these points; he does not even state the temperature at which the frogs that had been inoculated with bacteria adapted to cold lived. Dieudonné invokes the analogy of his results with those obtained in the case of the immunity and susceptibility of frogs as regards a septicaemic bacillus.

This bacillus (Bacillus ranicida) has been made the subject of an interesting study by Ernst[195]. It is a small, very slender bacillus, which, in frogs, produces a fatal malady epidemic in spring, but ceasing completely during summer. Taking this fact as a basis, Ernst has succeeded in conferring immunity upon frogs in autumn by placing them in an incubator at 25° C. In spite of the injection of a considerable dose of the small bacillus, the frogs living at this temperature remained in good health, whilst control animals exposed to a low temperature died of septicaemia. The counter-test was made in summer. Inoculated frogs that were kept in the laboratory were unaffected, whilst those that had been kept in a refrigerating apparatus at 6°–10° C. invariably died. It may be asked, Is this evident influence of temperature on immunity and receptivity exercised on the organism of the frog or upon the pathogenic bacillus? In the case where a bacillus can only develop at low temperatures its harmlessness at the higher temperature may be readily understood. The experiments of Ernst have demonstrated, however, that this small bacillus develops much better at 22° C., and even at 30° C., than at lower temperatures. It must be concluded, therefore, that the high temperature which confers immunity acts not by weakening the bacillus, but rather by reinforcing the resisting power of the organism. The low temperatures (6°–10° C.) that are favourable to a fatal infection have a different action; that is to say, they weaken the reaction of the inoculated frogs.

[Sidenote: [150]]

Although Ernst has not studied the mechanism of this resistance fully, it is evident, from the data he has supplied, that it consists in a phagocytic reaction. He was able to demonstrate the ingestion of the bacilli by the phagocytes in the susceptible refrigerated frogs, as well as in the refractory frogs, kept at a higher temperature; but in the former case the phagocytosis was so feeble that 24 hours after inoculation a considerable number of free bacilli were still found in the lymph of the dorsal sac, whilst in the refractory frogs the much more active phagocytosis brought about the disappearance of the free bacilli during the first day. If, as is very probable, the analogy of this septicaemia with anthrax in frogs, upon which Ernst insists, really exists, it must be concluded that the susceptibility of these Batrachians to the modified race of the bacillus depends on their weak phagocytic resistance.

Since, in these two examples of natural immunity in the frog, we have seen that the phagocytic activity exhibits itself in an active form against bacteria which readily develop in the fluids of the same animal, we might conclude that the reaction of the phagocytes constitutes a general mode of defence in cold-blooded animals. But Lubarsch[196], a very cautious observer, has expressed an opposite view, based on his studies on the bacillus of mouse septicaemia. He convinced himself that frogs will resist injections of even considerable quantities of this bacillus, without any co-operation on the part of the phagocytes. As we have, here, to do with a matter of fact, Mesnil (l.c.) set himself to verify these observations, with the object of establishing whether it was a case of a real exception or of a simple misunderstanding. He was able to demonstrate, by irrefutable observations and experiments, that the bacilli of mouse septicaemia when inoculated into frogs, set up a very pronounced positive chemiotaxis on the part of the phagocytes, which seized and digested the bacilli just as they do the anthrax bacillus. This apparent exception, therefore, becomes transformed into an additional argument in favour of phagocytic reaction being a general factor in immunity. In support of this hypothesis I may adduce a further example, already mentioned in a preceding chapter when discussing another question. The frog is very refractory against the cholera vibrio. When these vibrios are inoculated into the dorsal lymphatic sac or into any other part of the body the animal retains its health unimpaired. An examination of the exudation at the point of inoculation demonstrates that the vibrios meet with a vigorous opposition on the part of the phagocytes, which ingest and completely digest them. This is of special interest from the fact that the frog is very sensitive to the toxin of the cholera vibrio. When injected in a weak dose it kills the frog very quickly. Two small frogs died in less than an hour from the effect of 0·5 c.c. of cholera toxin.

[Sidenote: [151]]

The natural immunity of the frog against the cholera vibrio affords, then, an example in which the organism, destroying the vibrio by phagocytosis, prevents the production of the poison, which, otherwise, would infallibly kill it.

Having demonstrated that phagocytic reaction manifests itself in the frog in all cases of natural immunity that have been sufficiently studied, we must dwell for an instant on the question of the condition of the bacteria at the moment of their ingestion by the phagocytes. It is very evident that this phagocytic defence is only efficient on condition that it is exercised against bacteria which, in its absence, might injure the organism by their multiplication and their virulence. For this reason the question as to whether the micro-organisms, before being ingested, were living and capable of producing their pathogenic action has been widely discussed. It has even been suggested that the phagocytes are only capable of ingesting the dead bodies of micro-organisms that have been killed by other agents. Frogs are very suitable for a study of this question. When a drop of the exudation is removed some time after inoculation with a motile organism, such as the Bacillus pyocyaneus or the cholera vibrio, the organism was often found moving rapidly within the vacuoles inside leucocytes. The experiment will succeed even more completely if a drop of frog’s lymph be mixed, on a slide, with a trace of a culture of these motile micro-organisms, the latter being soon found in the clear vacuoles included in leucocytes and executing extremely rapid movements.

Besides this direct proof we can assure ourselves of the living condition of the micro-organisms in another way. Withdraw a drop of the exudation at an advanced stage of the process when there are no longer any free micro-organisms; inside the phagocytes a few scattered bacteria, more or less well preserved, can still be seen. It is sufficient to keep a hanging drop of such an exudation at a temperature of about 30° C., care being taken to keep it from drying, but without adding to it any nutrient medium. Under these conditions the leucocytes die more or less rapidly, but the bacteria regain vigour: they begin to multiply, and at the end of a short time produce a generation of bacteria within the dead leucocyte. The multiplication of the bacteria goes on progressively and the hanging drop is transformed into a real pure culture. Mesnil was able to confirm these data with the exudations of frogs that had been inoculated with either the bacilli of anthrax or of mouse septicaemia.

[Sidenote: [152]]

The bacteria, ingested in the living state by phagocytes, retain their original virulence. Some authors think, and I was formerly of this opinion, that at the end of a more or less prolonged sojourn within the leucocytes, anthrax bacilli undergo an attenuation in their virulence. Later, numerous researches have, however, demonstrated that this opinion is incorrect, and that the virulence is maintained in the bacteria included in the phagocytes of frogs the whole time that these bacteria remain alive. Dieudonné has insisted on this fact as regards the anthrax bacillus. Mesnil has confirmed it for this same species and for the bacillus of mouse septicaemia. It is impossible, therefore, to doubt this general result, that frogs which are refractory against certain bacteria resist because of the phagocytosis which is exercised against living and virulent micro-organisms.

We have insisted sufficiently on the analysis of the natural immunity of the frog, and need not tarry over the facts relating to other amphibia which, moreover, have been much less studied. The reptiles, those higher representatives of the Vertebrata called cold-blooded, often present examples of really remarkable immunity. Thus alligators will resist enormous doses of various bacteria, such as the anthrax bacillus, that of human tuberculosis or the cocco-bacillus of typhoid fever. When, some time after an injection is made, the exudation at the point of inoculation is withdrawn there is found a large number of leucocytes, amongst which may be recognised many eosinophile microphages, though the majority are macrophages with one, two or more nuclei. Really giant cells are found in the exudation. It is the macrophages which specially manifest phagocytosis and they are often found crammed with the injected bacteria, as I was able to assure myself after injections of typhoid cocco-bacilli. The natural immunity of alligators (Alligator mississipiensis) persists not only at the temperature of the incubator (37° C.), but also at room temperature (20°–22° C.).

[Sidenote: [153]]

Passing in review the animal kingdom we must pause for a moment to consider the natural immunity of birds or lower warm-blooded Vertebrates. The classic example of this immunity is that of the fowl against anthrax. It has long been known that birds resist inoculation with anthrax or only exhibit a feeble receptivity; though smaller birds are for the most part susceptible to anthrax, the pigeon is much less so and the fowl presents a case of the most pronounced immunity. It was believed to be absolutely refractory until the experiments of Pasteur and Joubert[197], who found a sure method of suppressing this immunity. Fowls that had been inoculated with the bacillus were immersed up to the thighs in cold water in order to bring down their temperature. It was found that, under these conditions, the anthrax bacillus developed at the seat of inoculation and later became generalised in the blood, and invariably caused death. It was concluded from this that the natural immunity of the fowl was dependent on its very high normal temperature (41°–42°) which interfered with the pathogenic functions of the anthrax bacillus.

Hess[198] studied the mechanism of this immunity of the fowl and pointed out the important part that phagocytosis plays in the destruction of the inoculated bacteria.

These researches were resumed in my laboratory by Wagner[199]. Having established that the anthrax bacillus develops readily in the blood and the blood serum of fowls, outside the organism, at high temperatures (42°–43° C.), he came to the conclusion that the lowering of the temperature of the body of the fowls by immersing them in water produced, not a reinforcement of the bacillus, but a weakening of the resisting power of the animal. He was able to convince himself that this resistance exhibits itself in the activity of the phagocytes which ingest and destroy the anthrax bacillus in its vegetative state. In the normal fowl the phagocytosis is rapid and very pronounced, whilst in a fowl that has been refrigerated this reaction is very slight or absent. To corroborate this general conclusion, Wagner, instead of lowering the temperature by means of cold water, made use of antipyrin and chloral. The application of this treatment likewise caused enfeeblement of the natural defence of the organism and suppressed the immunity of the fowl against anthrax.

[Sidenote: [154]]

Trapeznikoff[200] has studied carefully the fate of anthrax spores when injected into fowls. He observed that most of them are devoured by the leucocytes. Some of the spores were first transformed into small rods, sometimes growing into real bacilli, but finally they all became the prey of phagocytes and perished in their interior. Those in the vegetative condition are soon digested, the spores, however, persist for some time inside the phagocytes, but ultimately disappear. The phagocytosis in fowls inoculated with spores is very marked, and preparations, stained by Ziehl’s method, demonstrate most clearly the reality of this reaction phenomenon. These preparations have for long been used in the course in bacteriology at the Pasteur Institute for the demonstration of phagocytosis.

[Sidenote: [155]]

In the face of these facts, well established and confirmed many times, it is impossible to accept Thiltges’[201] denial of the ingestion of these bacteria by the phagocytes of the fowl. Some fault of technique, which I am not at the moment in a position to indicate exactly, has evidently slipped into this author’s work. The positive data, however, on phagocytosis in the fowl, obtained by Hess, Wagner, and Trapeznikoff, data confirmed by myself, render unnecessary any fresh researches for the purpose of explaining the negative results obtained by Thiltges. As regards his experiments on the bactericidal action of defibrinated blood and of blood serum of fowls against the bacillus and its spores, experiments whose results are opposed by those of Wagner, the contradiction may be explained pretty easily, at least in part. Thiltges mentions several times that the bacilli, when sown in the blood serum of the fowl, were aggregated in clumps. Nevertheless, he has failed to guard against this source of error and has attributed the diminution in number of the colonies on plates to the destruction and not to the agglutination of the bacilli. Thiltges gives so few particulars of the conditions under which his experiments were performed that we do not even know at what temperature he kept his tubes containing blood and serum sown with bacilli. As Wagner kept his at 42°–43° C., a temperature which corresponds to that of the body of the fowl, I asked M. Gengou to make a series of experiments on the bactericidal power of the plasma and blood serum of fowls on the anthrax bacillus, keeping his tubes at 37° C. The result of his experiments was in complete accord with those of Wagner. Under the conditions that I have just stated the fluids of the fowl are no more bactericidal than they are under the conditions maintained in Wagner’s experiments.

In summing up these data on the natural immunity of fowls against anthrax, we are certainly justified in concluding that it is due to the phagocytosis and not to any bactericidal property of the “humours.”

The pigeon is more susceptible than the fowl to the action of the anthrax bacillus, still it manifests a certain degree of resistance against the microbe. After what we have said on the subject of the fowl we need make but few remarks on the pigeon, in spite of the very animated discussions that have taken place on the mechanism of its immunity. When Baumgarten was offering a systematic opposition to the part played by phagocytic reaction in immunity, he set his pupil Czaplewski[202] to investigate the resistance of pigeons against anthrax. The results of this investigation were absolutely negative as regards phagocytosis. The latter was said to have no importance in the defence of the organism, which resisted simply because it was impossible for the bacillus to live in the body of the pigeon. I then set myself to study this question[203], and I was able to demonstrate that the anthrax bacillus is quite capable of keeping alive in the pigeon, that it can develop in its fluids, but that it is unable to defend itself against the aggression of the phagocytes which ingest it and completely digest it. By isolating the phagocytes that had ingested the bacilli injected into the body of the pigeon, I was able to prove that a number of these bacilli were still alive. The enfeeblement and death of the phagocytes when outside the body allowed the anthrax bacilli again to get the upper hand in this struggle, to develop and to give virulent cultures. The part played by phagocytes in this example of natural immunity was thus placed beyond doubt.

[Sidenote: [156]]

Later, Czaplewski[204] himself became convinced that his previous negative results would not stand criticism, and Thiltges, in his work already mentioned, when discussing the fowl, was able to confirm the importance of phagocytosis in the defence of the organism of the pigeon against anthrax. He was struck by the difference between these two species of birds. In the pigeon it was easy for him to prove that in the individuals that succumb to anthrax the phagocytic reaction is very feeble, whilst in those which ultimately resist the bacillus it is very pronounced. Thiltges likewise observed that the blood and blood serum of pigeons when sown in vitro with the anthrax bacillus, manifest only an insignificant bactericidal power, a fact that further warrants him in attributing great importance to phagocytosis in the maintenance of the natural immunity of the pigeon. It is remarkable that, in presence of these facts, it did not occur to the author to ask whether this fundamental difference in the mechanism of the resistance, which he thought possible in two birds so closely allied as are the pigeon and the fowl, really did exist in nature. I infer that his experiments on the fowl were made before those on the pigeon, and that the difference in his results depended specially on the fact that he had acquired greater skill in executing his later experiments.

Having observed that frogs die readily when inoculated with an anthrax bacillus that was adapted to develop at a low temperature, Dieudonné (l.c.) endeavoured to suppress the immunity of the pigeon by using bacilli adapted to a high temperature. But the inoculation of a second generation of the anthrax bacillus, cultivated at 42° C., was borne by five pigeons without inconvenience. Even bacilli that were rendered capable, by cultivation through sixteen generations, of developing at this temperature were not in a condition to kill more than five pigeons out of thirteen inoculated. These attempts to explain immunity as due to the properties of the bacilli rather than to those of the organism of the pigeon, have therefore led to a result very different from that anticipated by Dieudonné.

[Sidenote: [157]]

The pigeon is further of interest to us because of its natural immunity against the bacillus of human tuberculosis. It resists considerable doses of this bacillus, so virulent for man and for the majority of mammals, and even for some birds (canaries and parrots). Dembinski[205], studying the mechanism of this immunity, was able to prove that the bacilli of human tuberculosis encounter in the organism of the pigeon a very great resistance from the phagocytes, especially from the macrophages. These cells fuse together around masses of bacilli and imprison them within real giant cells or polynucleated macrophages (Fig. 21). The microphages in this struggle play only a secondary part, but the resistance offered by the macrophages is a most effective one. Incapable of completely destroying the bacilli, these phagocytes exercise over them an unfavourable influence and prevent them from multiplying and exhibiting their noxious action. The importance of the defence by the macrophages comes out still more clearly when compared with what takes place if, instead of the bacillus of human tuberculosis, we inoculate into pigeons the bacillus of avian tuberculosis. In the latter case the microphages certainly promptly seize the bacilli, but being powerless against them they perish, whilst the macrophages only intervene later on and in small numbers. The result is that in the pigeon the avian bacillus becomes generalised in the organism and sets up a fatal tuberculosis.

It must be admitted, then, that the immunity of the pigeon against the bacillus of human tuberculosis is due to the defence by the macrophages. This conclusion is corroborated by the fact that in the fowl—equally refractory against the same bacillus—there is also observed a very strong macrophagic reaction.

[Sidenote: [158]]

Nocard[206], who for several years has been carrying on studies on the relations between the bacilli of human and avian tuberculosis, conceived the idea of adapting the former to the organism of the fowl. With this object he enclosed a culture of the bacillus of human tuberculosis in a sac of collodion which he then introduced into the peritoneal cavity of fowls. Under these conditions the bacillus, protected against the aggression of phagocytes, continued to live inside the sac through whose walls the fluid part of the peritoneal lymph could diffuse. After several passages from sac to sac the human bacillus becomes acclimatised to the body of the fowl and is transformed into a variety quite comparable to the bacillus of avian tuberculosis. This experiment has definitely settled the question so long under discussion of the specific difference between the two tubercle bacilli. It has resolved it in the sense of affirming their unity; the avian bacillus is only a modified race of the same bacillus which sets up tuberculosis in man and other mammals.

In spite of the great difference between the anthrax bacillus and that of human tuberculosis, the immunity against these two bacteria, which is shown in birds, depends in every case upon the reaction of the phagocytic system.

Having rapidly glanced at natural immunity as we ascend the scale of the animal series we now come to it as it presents itself in the highest class, Mammals, a question on which it is necessary to dwell at greater length because of its great importance, and also because of the fuller study that has been given to it.

[Sidenote: [159]]

As the immunity of the Invertebrata and of the lower Vertebrata against the anthrax bacillus has furnished us with several important indications we will first endeavour to throw light on the mechanism of the resistance offered to anthrax by certain mammals. The representatives of this class being, however, for the most part extremely susceptible to this disease, examples of true natural immunity are very rare. The first place among resistant mammals is occupied by the dog. Although young dogs, as demonstrated by Strauss[207], readily take fatal anthrax, the canine species may nevertheless be regarded as possessing a real immunity, as adult dogs withstand, without inconvenience, the inoculation of large quantities of bacilli. When introduced beneath the skin these bacilli excite a local inflammation, accompanied by a very marked diapedesis of white corpuscles which at once begin to devour the bacilli. This phagocytosis has already been observed by Hess[208], Malm[209], myself, and several other investigators, so that its existence cannot be doubted. Recently, Martel[210] has demonstrated a very distinct phagocytic reaction in all those cases where he has had to deal with dogs that were refractory or not very susceptible. This reaction is shown by the ingestion of the bacteria and by the large accumulation of leucocytes at the seat of inoculation. His researches are of special interest by reason of the counter-test that he was able to make upon dogs that were susceptible to anthrax. It was demonstrated some years ago that the natural immunity of the dog against the bacillus, although very real, is, nevertheless, relative and limited. Thus Bardach[211] established the fact that dogs from whom the spleen, an organ full of phagocytes, had been removed, became susceptible to anthrax. Even dogs into whose veins he injected fine wood-charcoal powder suspended in water, with the purpose of “diverting” the phagocytosis, readily succumbed to anthrax.

Martel endeavoured to suspend the natural immunity of dogs by injecting into them phloridzin or pyrogallic acid. But he obtained much more constant results by inoculating the bacillus into rabid dogs. The organism, weakened by this terrible disease, became very susceptible to anthrax, and the rabid animal succumbed to anthrax before the rabies had completed its evolution. By its passage through the rabid dog the anthrax virus is so augmented in virulence that it becomes fatal for normal dogs. Martel succeeded also in reinforcing the bacillus isolated from a cow affected with anthrax. In all these cases where the reinforced bacilli set up a severe and rapidly fatal infection, Martel could demonstrate only a feeble phagocytic reaction.

Researches on the phagocytosis of dogs, inoculated with the anthrax bacillus, have always demonstrated a regular and constant relation between this reaction and the resistance of the organism. On the other hand, experiments undertaken for the purpose of establishing the part played by the body fluids in this immunity, have always given negative results.

[Sidenote: [160]]

As the dog, of all mammals, exhibits the greatest natural immunity from anthrax, it is very natural that in the bactericidal property of its blood the key to the enigma has been sought. Thus Nuttall[212] concludes from his experiments that the anthrax bacillus is readily destroyed by defibrinated dog’s blood. But, as this result was not in accord with my observations[213] that the bacillus is easily cultivated in dog’s blood, and as several observers, especially Lubarsch[214], had arrived at conclusions opposed to those of Nuttall, systematic researches were made for the purpose of solving this complicated problem. Denys and Kaisin[215] sought to remove the objections formulated against the explanation of the immunity of the dog as due to the bactericidal property of its blood by affirming that this power, which is absent in the inoculated dog, develops whilst the animal is under the influence of the bacillus. Immunity is reduced, then, in this case to the establishment of a new property in the fluids during the course of the struggle of the organism against the inoculated bacillus. None of the observers, however, who have repeated these experiments, e.g. Lubarsch[216] and Bail[217] were able to confirm the results of the Belgian observers. Denys himself, indeed, having resumed this study with Havet[218], had to reject the conclusions of his former work executed in collaboration with Kaisin. He is persuaded that their error was due to the fact that in their experiments in vitro, the living leucocytes ingested the bacilli and prevented their development. As the result of these new researches Denys and Havet have come to the conclusion “that the main, the predominating part of the bactericidal power of the dog’s blood must be ascribed to the leucocytes acting as phagocytic elements” (loc. cit. p. 15).

[Sidenote: [161]]

As a result of the investigations I have summarised the conclusion is forced upon us that the natural immunity of the dog from anthrax is a function of the phagocytes. In presence of this uniformity of the experimental results it becomes very important to make a more profound study of the phenomena that manifest themselves during the destruction of the bacilli by the phagocytes of the dog. What are the phagocytic elements which play the principal part in this struggle, and by what means do they attain this result? Gengou[219] undertook a detailed investigation in my laboratory to answer these questions. He was able to convince himself, in agreement with the statements of his predecessors, that not only was the serum of dog’s blood not bactericidal for the anthrax bacillus, but that the plasma of the blood is no more so. The fluid of the aseptic pleural exudation obtained after injection of gluten-casein, was likewise incapable of killing the anthrax bacillus. When Gengou, by means of centrifugalisation, isolated the leucocytes from these exudations, washed them in physiological salt solution, froze them, and then macerated them in broth, he obtained suspensions of white corpuscles, to which he added bacilli. He was able to demonstrate that when the exudations contained macrophages principally, as is observed in exudations taken at the end of two or three days, the bactericidal power of the suspensions was nil or insignificant. When, on the other hand, the leucocytes came from exudations only twenty-four hours old and were composed almost exclusively of microphages, the destructive action on the bacilli of the extract of the microphages in broth was most marked. Now it is fully demonstrated that in the exudation set up in the refractory dog by the injection of anthrax bacilli, it is especially the microphages which exhibit the phagocytic reaction against this bacillus.

This is how the question of the immunity of the dog from anthrax stands at present. The natural immunity of this species, which although not unlimited, is very real, depends on the activity of phagocytes. These elements, under the stimulus of the bacillus and its products, exhibit a positive chemiotaxis of the most marked character, they approach the bacilli, ingest them by a physiological act, and destroy them by means of a substance which is not found in either the plasma or the blood serum, but which can be demonstrated in an extract of the microphages.

[Sidenote: [162]]

In spite of the uniformity and precision of these data, it is impossible to rest satisfied with describing, as an example of natural immunity from anthrax, the single case of the dog. If the resistance of the rat against this disease was merely of historical interest because of the large number of works devoted to this question, we might relegate it to the chapter reserved for the history of our knowledge on immunity. But it is not so. The anthrax of rats is a subject full of very valuable instruction, and von Behring was quite justified in saying that whoever wished to get a true conception of natural immunity from a virus should pay special attention to this example.

As a matter of fact, it may be stated that the grey rat (Mus decumanus), the black rat (Mus rattus), and white rats are far from enjoying a true immunity from anthrax. They, nevertheless, exhibit a more or less marked resistance against this disease and are always less susceptible than are the other laboratory rodents: mice, guinea-pigs and rabbits. Rats resist attenuated bacilli (anthrax vaccines) better than do these three species, and in order to induce in them fatal anthrax it is necessary to inoculate a much larger number of virulent bacilli. On the other hand, rats are distinguished by a great irregularity in the resistance they offer to the bacillus. At times they resist very virulent bacilli; at others they contract a fatal disease after an injection of very attenuated bacilli (Pasteur’s first vaccine).

[Sidenote: [163]]

In my first memoir on anthrax[220] I noted the fact that in rats the phagocytosis against the bacillus when injected subcutaneously was more marked than after the same inoculation into the rabbit and guinea-pig. Later, this fact was disputed by several observers, who refused to accept the extent and importance of the phagocytic reaction in the rat. This opposition was strengthened by a very interesting discovery made by von Behring[221], namely, that the blood serum of the rat possessed a remarkably destructive power for the anthrax bacillus. When this observer added a certain quantity of anthrax bacilli to some blood serum of the rat, instead of elongating into filaments and dividing they underwent a change, lost their normal refraction and took on staining reagents very imperfectly. The membranes alone remained of the bacilli thus treated. Von Behring considered that this bactericidal action of the serum depends on the presence of an organic base dissolved in the blood fluid. He had merely to neutralise the serum by means of an acid, and there was at once a very abundant development of the bacillus. From these researches von Behring came to the conclusion that the natural immunity of the rat from anthrax can be reduced to terms of the chemical action of the blood on the bacillus.

In one of his most recent publications this author[222] returns to the question of anthrax in rats and sums up his present point of view as follows. He regards the immunity of these rodents as being relative, not absolute. “The anthrax bacilli”—he says—“die in rat’s serum in vitro; and in the cases where the inoculation of these animals with the anthrax virus is not fatal, it is at least reasonable to assume that the blood fluid likewise produces this protection in the organism of the living rat. Now, an immunity that manifests itself without the aid of any activity of the cell must undoubtedly be regarded as being of a humoral character” (loc. cit. p. 202).

[Sidenote: [164]]

Let us begin by analysing the facts as presented in rats into whose subcutaneous tissue we have injected anthrax virus. A certain number of them resist, without exhibiting any lesion other than a certain exudative inflammation at the seat of inoculation. The exudation is, in this case, very rich in leucocytes which quickly exert their phagocytic function and destroy the ingested bacilli. In this reaction it is the microphages that play the chief part, the macrophages intervening later and in a much less pronounced fashion. Usually, however, the inoculated rats exhibit a more serious illness: the bacilli multiply at the point of inoculation and excite the formation of an extensive oedema, rich in serous fluid, transparent, and very poor in leucocytes. It is only later that these cells intervene in any considerable number. The exudation becomes thicker and turbid, the numerous white corpuscles devour the bacilli and cause their disappearance. Under the influence of this marked reaction the animals in most cases recover, as has already been established by Frank[223]. But even in those individuals which succumb to anthrax death occurs more or less tardily, an examination of the internal organs then revealing a considerable phagocytic reaction. The spleen, often of enormous size, contains numerous macrophages which are filled with normal or more or less altered bacilli. In the liver these macrophages, which have devoured several microphages and some bacteria, are also found (Figs. 22 and 23).

When instead of bacteria in the condition of rods, anthrax spores are inoculated subcutaneously or into the anterior chamber of the eye, we can observe their germination. There is developed a whole generation of bacilli which behave like those we have already described, that is to say, they excite an exudation and are ultimately digested within the phagocytes (Figs. 24 and 25). All these phenomena of phagocytosis I described in detail more than ten years ago in my memoir on the anthrax of rats[224]. Since then not a single fact has been brought forward to invalidate the results there set forth.

[Sidenote: [165]]

How is this paradoxical fact to be explained, that anthrax which grows in the body of the rat, there setting up a disease more or less grave and sometimes fatal, is so readily destroyed by the serum and blood when removed from the organism? From numerous experiments, carried out by Hankin[225] and by Roux and myself[226], it has been demonstrated that the bactericidal power of the fluids of the rat cannot be invoked as the cause of the animal’s resistance to anthrax. Those rats which show themselves very susceptible to this disease and die from anthrax infection, furnish, nevertheless, a serum that will prevent anthrax in other rats, and which will protect even mice into which the bacilli have been injected. Rats into which we inoculate on one side of the body a little anthrax culture, and on the other side the same quantity of bacilli mixed with blood serum from the same animal, manifest oedema at the former place only. It is from this latter point that the general infection takes place, the side where the anthrax bacilli mixed with serum was introduced remaining unaffected. Sawtchenko[227], who has investigated the immunity of the rat in my laboratory, has to the facts just mentioned added the observation that when the injection of bacilli causes haemorrhage the rat survives. When, on the contrary, the injection is made with a fine needle and without effusion of blood, the rat contracts a fatal anthrax.

[Sidenote: [166]]

[Sidenote: [167]]

It follows from these facts that the blood, immediately it has escaped from the vessels, undergoes a change in its composition and becomes bactericidal for the anthrax bacillus, whilst, when it is circulating in the organism, it exhibits no such power. Sawtchenko has studied the substance in the serum which kills the bacilli and has demonstrated that it will resist heating to 56° C.; even when heated to 61° C. the serum still exercises a certain amount of bactericidal power for very attenuated bacilli (Pasteur’s first vaccine). Researches on the distribution of this bactericidal power in the living rat have convinced Sawtchenko that none of it passes into the fluid of the passive oedema set up by the slowing of the circulation, nor into that of the active oedema developed as the result of the inoculation of anthrax bacilli. He observed that even the bacillus of Pasteur’s first vaccine grows abundantly in the oedematous fluid produced by the injection of virulent bacilli. The peritoneal lymph, however, exerts a very marked bactericidal action on the bacilli. Having demonstrated this fact Sawtchenko put to himself the question: May not the great difference between the action of these fluids depend on the fact that the lymph is rich in leucocytes, whilst in the fluid of the oedema they are almost absent? Pursuing this question, Sawtchenko made a comparative study of the bactericidal power of the serum, prepared outside the body, and of the blood plasma obtained by means of an extract of the heads of leeches, and he concluded from his researches that the bactericidal substance circulates in the plasma of the living rat and that it is not derived from the microphages, but must be looked upon rather as a secretion of the macrophages in the blood and of endothelial cells. This result was not confirmed by Gengou[228], who also took up the study of this important question in my laboratory. Instead of preparing the plasma by means of the addition of an extract of leeches he made use of a method much more perfect and free from sources of error. He introduced no foreign substance capable of affecting the results of his experiments. Collecting the rat’s blood in paraffined tubes, and centrifugalising it in similar tubes, he obtained a fluid which approaches much more closely the plasma of circulating blood than does serum. This fluid, however, will coagulate at the end of a fairly long interval, which proves that it cannot be looked upon as blood plasma. Gengou examined the bactericidal power of the fluid portion of the “plasma,” obtained by the process just described, on the anthrax bacillus, and also that of serum prepared in tubes in the ordinary way. The difference between the two fluids is very marked; whilst the serum destroys the bacilli sown in it very rapidly and dissolves their contents, the fluid of the “plasma” has no similar action. These results, confirmed several times, demonstrate very definitely that the plasma of the circulating blood does not contain any bactericidal substance. This, during the life of the animal, is found inside leucocytes and only escapes from them when the cells burst or undergo profound lesions, this taking place when the clot is formed and when the serum is prepared outside the organism, or in the effused and coagulated blood, or again in the peritoneal lymph during phagolysis. This phagolysis is inevitably produced as a result of rapid injection of foreign fluids into the peritoneal cavity, e.g. of broth or of physiological salt solution, containing bacteria in suspension.

[Sidenote: [168]]

The facts we have brought together on the subject of anthrax in rats form a whole whose several parts are in complete harmony. The phagocytes of this species of rodent contain a bactericidal ferment, a kind of cytase, which resists temperatures approaching 60° C. This cytase is very active against the bacilli, but in the living animal it can only act within the phagocytes, or, in a transitory and incomplete fashion, outside these cells, when phagolysis is taking place in the peritoneal cavity. The resistance offered by the rat to anthrax depends, then, on this phagocytic activity. For its manifestation it is necessary, first of all, that the phagocytes should manifest a positive chemiotaxis for the bacilli, and then that they should seize and ingest these organisms. These are the vital acts that decide the result of the struggle. When the phagocytes show themselves inactive the bacilli multiply in the oedematous fluid which contains no bactericidal cytase, and pass into the plasmas of the lymph and of the blood, which also are incapable of killing these bacteria. The animal may, then, die of anthrax, in spite of the presence in its body of a large quantity of bactericidal cytase which is to be found in situations to which the bacilli have not penetrated. In those cases, on the other hand, where the phagocytes accomplish their function, where they rush up to the menaced point and devour the inoculated bacteria, these bacilli are placed in contact with the intracellular cytase and undergo complete digestion. The organism in this case gets rid of its enemies and victoriously resists infection.

[Sidenote: [169]]

Anthrax in rats, then, presents one of the most instructive examples of natural immunity. But the detailed analysis of the mechanism of this resistance demonstrates very clearly the great part played by the phagocytes in this process. In this respect the organism of the rat presents, in a general fashion, a great analogy to the natural immunity of the dog, of birds, and of other representatives of the animal kingdom that we have examined. Under these conditions it is useless to insist at any length on other examples of resistance against anthrax which, moreover, have relation much more often to a natural immunity against attenuated bacilli than to one against true anthrax virus. Rabbits and guinea-pigs, so sensitive to this virus, often resist the inoculation of Pasteur’s vaccines. The rabbit is, in general, refractory to the first anthrax vaccine; it may even resist the second vaccine. The guinea-pig, a more sensitive animal, does not exhibit any natural immunity except against the first vaccine. In all these cases the mechanism is similar to that which the rat and the dog oppose to virulent anthrax. The bacilli, into whatever part of the body they are injected, set up an exudative inflammation which brings up a large number of leucocytes to the point menaced. These cells readily exert their phagocytic function and rid the organism of the introduced bacteria. In order to obtain a complete grasp of the part played by this reaction it will be found useful to inject beneath the skin of one ear of a rabbit a little anthrax vaccine and beneath the skin of the other the same quantity of virulent bacilli. The difference between the reaction in the two cases is very striking. The ear inoculated with vaccine soon becomes the seat of a circumscribed inflammation with a purulent exudation, all the bacilli in which have been devoured by the leucocytes. The other ear, on the contrary, presents, around the injected virus, only a serous or blood-tinged exudation containing no, or few, leucocytes; the bacilli are found free in the liquid and multiply without let or hindrance. Meeting with no opposition the virus becomes generalised throughout the organism and brings on death by anthrax septicaemia. Rabbits, into which anthrax vaccines only are introduced, oppose to the invasion of the bacilli a leucocytic barrier which arrests their extension. The natural immunity of the sheep, rabbit and guinea-pig is also a phagocytic immunity, but it is only capable of being exercised against bacilli previously attenuated in virulence. The researches of Mme Metchnikoff[229] on the reaction of the phagocytes of these animals to the bacilli of Pasteur’s two anthrax vaccines have demonstrated the importance of the destruction of these bacilli by the leucocytes. All the other examples of natural immunity against anthrax are also merely relative. The fowl that resists an anthrax virus strong enough to kill an ox or a horse, succumbs to a special variety of anthrax cultivated by Levin[230]. The dog, as we have seen, in spite of its pronounced natural immunity against anthrax, is killed by the special anthrax bacillus prepared by Martel.

In this immunity against anthrax we have to deal with a bacillus capable of living and reproducing itself in extremely varied media. Hence the reason, it may be said, that the bactericidal influence of the fluids is so little pronounced in this case. To bring it into relief we must, therefore, choose a bacterium less capable of adapting itself to the chemical composition of various culture media. In this matter we cannot do better than select pathogenic spirilla of extremely delicate nature and analyse the mechanism of the natural immunity of certain species of animals with respect to them. It must not be forgotten, however, that here we are making use of representatives of an infinitely small minority of pathogenic bacteria, the majority resembling the anthrax bacillus in the facility with which they can be cultivated in all sorts of nutritive media.

[Sidenote: [170]]

[Sidenote: [171]]

The spirillum of recurrent fever of man (Spirochaete obermeyeri) was the first pathogenic microbe found in an infective disease distinctly human. Discovered a third of a century ago, it has passed through the hands of the most skilful bacteriologists, who have tried all possible methods of cultivating it outside the body. Koch himself tried to solve the problem, but, in spite of his incomparable skill, did not succeed. Later, Sakharoff[231], at Tiflis, discovered a spirillum very similar in appearance which produced a fatal septicaemia in the goose. He, also, tried to cultivate it, but in vain. His successors have not been more fortunate in this respect. Here, then, are two micro-organisms, against which natural immunity should be easily obtainable and in a fashion quite other than that against anthrax. Nothing, indeed, is more frequent than examples of very stable natural immunity against the spirilla of Obermeyer and of Sakharoff. As I wished to obtain a clear idea of the mechanism by which the guinea-pig resists injections of the spirillum of goose septicaemia (Spirochaete anserina) I made injections of goose’s blood, containing a quantity of these organisms, into the peritoneal cavity of guinea-pigs. This injection, as usual, causes the disappearance of most of the leucocytes, as the result of a very marked phagolysis. We know that, under these conditions, the damaged leucocytes allow a certain quantity of the bactericidal cytase to escape. In spite of this the spirilla remain intact and exhibit very active movements in the peritoneal exudation. This exudation, after a period of phagolysis, which lasts for two or three hours, begins to be stocked again with leucocytes which come up in increasing numbers, a fact that does not prevent the spirilla moving about with great rapidity. Even seven hours after the injection of goose’s blood we still find many extremely active spirilla among a large number of recently migrated leucocytes, some of which even at this stage contain red corpuscles of the blood of the goose. It is not until later that the ingestion of these spirilla by the leucocytes commences, the leucocytes at last damaging and completely destroying them. This act of phagocytosis may be readily observed in hanging drops of the peritoneal exudation of inoculated guinea-pigs. The attention of the observer is drawn to certain macrophage leucocytes which throw out one or two conical-looking processes (Figs. 26–28). These pseudopodia attach themselves to spirilla which exhibit very violent movements as though wishing to extricate themselves from the grasp of the leucocyte. Sometimes the spirillum succeeds in escaping, but usually it becomes surrounded by the protoplasm and sinks more and more deeply into the substance of the leucocyte. Even when almost surrounded the free part of the spirillum still continues to move (Figs. 29–31). These movements cease only after the complete ingestion of the spirillum. Once inside the phagocyte the spirillum is digested and soon becomes unrecognisable.

[Sidenote: [172]]

Recently, Sawtchenko[232] took advantage of an epidemic of recurrent fever at Kazan to make similar investigations on the natural immunity of the guinea-pig against Obermeyer’s spirillum. He observed that these organisms, when injected into the peritoneal cavity, remained there, alive, for 24 and even 30 hours, whilst these same spirilla, when kept at 37° C. outside the organism in their natural medium, died at the end of some (4–7) hours. The injection of human serum containing spirilla into the peritoneal cavity of guinea-pigs set up a phagolysis succeeded by a considerable afflux of leucocytes. In spite, however, of the arrival of quite an army of these cells, the spirilla continued to move rapidly; for a long time they evaded the phagocytes which, however, in the end always ingested them. But it is only the macrophages which fulfil their phagocytic function (Figs. 32 and 33); the microphages obstinately exhibit an absolutely negative chemiotaxis. Now, as the macrophages do not make their way into the peritoneal cavity until after the microphages have appeared, it is easy to understand that phagocytosis can only take place at a late period. Sawtchenko came to the conclusion that “in the peritoneal cavity of animals naturally refractory, the spirochaetes perish as the result of a slow phagocytosis and not from the action of the bactericidal substances of the fluids.” In conformity with this result this observer has often noted the ingestion of living spirilla by the macrophages, in hanging drops of the peritoneal exudation of inoculated guinea-pigs. The phenomenon corresponds exactly to that described in connection with the spirillum of the goose.

In spite of the great difference between the spirillum and the anthrax bacillus from the point of view of their adaptation to surrounding media, the general result is the same with both these microbes: animals endowed with natural immunity get rid of them through the agency of their phagocytes.

[Sidenote: [173]]

[Sidenote: [174]]

It would be impossible and even useless here to pass in review all the cases of natural immunity against infective micro-organisms. We must consequently limit ourselves to several examples which may have an interesting bearing on the study of the problem as a whole. The spirilla, whose history we have just recorded, remain in the peritoneal fluid, without change of form, up to the moment when they are captured by the macrophages. Let us see by what mechanism the natural immunity against micro-organisms, characterised by a very special sensitiveness to external influences and by a considerable change of shape, is produced. The cholera vibrio and its allies best satisfy this postulate. When they find themselves placed under unfavourable conditions, these vibrios immediately become transformed into small spherical bodies which are much more like cocci than vibrios. The cholera vibrio is pathogenic for the laboratory rodents, especially for the guinea-pig, when a fairly large quantity of a culture is injected into the peritoneal cavity. Against smaller doses, however, the natural immunity is a most marked one. If we take a race of the cholera vibrio of medium virulence, and inject into the peritoneal cavity of guinea-pigs a sublethal dose of a culture, the following phenomena may be observed[233]. The inoculated vibrios move actively in the peritoneal fluid, from which almost all the leucocytes have disappeared. There remain only a few lymphocytes which appear to be indifferent to the influences that set up a real phagolysis. But, little by little, fresh leucocytes come into the exudation and engage in a struggle with the vibrios which, so long as they are free, retain their curved form and complete motility. The microphages, especially, swarm into the peritoneal cavity. Some of them begin to ingest vibrios, but this phagocytosis is at first slight. Later it becomes much more active. The microphages and macrophages seize vibrios that are evidently living and uninjured, which, sometimes, may be observed inside the vacuoles of the leucocytic contents exhibiting very lively movements. Once ingested, however, many of the vibrios become transformed into round granules. This change of shape is constant when inside microphages, but is completely absent when inside macrophages (Figs. 34 and 35). Finally, the phagocytosis becomes complete, and the organism gets rid of the vibrios solely by means of this reaction. Even seven hours after injection of the vibrios, when the peritoneal fluid, crammed with leucocytes, has become thick and turbid, there still remain a few scattered vibrios which always retain their shape and their normal activity. A drop of this exudation, maintained at 38° C. outside the organism, gives, in a few hours, an abundant culture of very active vibrios. It must, therefore, be concluded that the fluid part of the exudation was powerless to destroy the vibrios or even to render them motionless, whilst the living leucocytes have shown themselves capable of ingesting and digesting them. The peritoneal exudation, withdrawn at a period when it no longer contains any free vibrios, still gives cultures of the organism for some time. Soon, however, there comes a period when the inoculated exudation remains sterile, this proving that the vibrios, ingested in a living state by the phagocytes, have at length been killed by the microphages and macrophages.

[Sidenote: [175]]

When, instead of cholera vibrios of medium virulence, we take a variety completely deprived of pathogenic activity, it is sometimes observed that certain of these organisms, when injected into the peritoneal cavity of the normal guinea-pig, become transformed into spherical granules in the fluid of the exudation without any direct co-operation of the phagocytes. This transformation into granules was first studied by R. Pfeiffer[234] and hence has been termed Pfeiffer’s phenomenon. It is of limited occurrence in natural immunity and is produced, as I have been able to demonstrate, only under certain well defined conditions. Pfeiffer’s phenomenon is observed in the peritoneal fluid. It commences soon after the injection of the vibrios and takes place during the period of phagolysis. In other parts of the body of the guinea-pig, notably in the subcutaneous tissue and in the anterior chamber of the eye, Pfeiffer’s phenomenon does not manifest itself; the animal, none the less, resists the inoculation of the vibrios. Even in the peritoneal cavity, moreover, it is easy to check the granular transformation of the vibrios by means which prevent the production of phagolysis. When we inject into the peritoneal cavity of a guinea-pig a foreign fluid, capable of exciting the phagocytic action, e.g. veal broth, physiological salt solution, urine, etc., we first excite a transitory phagolysis. To this stage succeeds another in which the leucocytes become very numerous and much more resistant than before. If we take advantage of this period of leucocytic stimulation to inject vibrios which have been attenuated as much as possible, we shall observe that they soon become the prey of the peritoneal phagocytes, without manifesting any sign whatever of Pfeiffer’s phenomenon.

It is evident, then, that this extracellular destruction of the vibrios, sometimes observed in the peritoneal cavity, is really the work of the microcytase that has escaped from the phagocytes during their period of transient injury.

[Sidenote: [176]]

[Sidenote: [177]]

Having analysed the mechanism of natural immunity against certain bacilli, spirilla and vibrios, it will be interesting to determine whether the same rules are to be applied in the case of the cocci. Choice is not difficult since we may equally well fix upon the staphylococci, the pneumococci, streptococci or gonococci. Should we decide upon the streptococcus it is solely because the natural immunity against this micro-organism has attracted the special attention of several observers. A second advantage of the streptococcus, however, is the high degree of natural immunity manifested against it by a laboratory animal so convenient as the guinea-pig. Dr Jules Bordet[235] studied this subject in my laboratory. He observed that the injection of streptococci into the peritoneal cavity sets up a marked leucocytosis which ends in a complete destruction of the micro-organisms. The leucocytes rapidly ingest the great majority of the streptococci and destroy them; there remain only a few isolated and free individuals which are protected by a clear zone (aureola) which develops around them, but in the end they also become the victims of the voracity of the phagocytes. When we increase the dose of streptococci injected, phagocytosis still goes on, but some of the streptococci succeed in escaping, and we see a new generation produced which is distinguished by the thickness of the protective aureola. In spite of the afflux of a large number of leucocytes, they no longer ingest the streptococci and generalisation of the infection results, followed by the death of the animal. Natural immunity, then, can be suppressed under certain definite conditions. Dr Jules Bordet[236] wished to satisfy himself whether the leucocytes failed to fulfil their phagocytic function because of the paralysis of their movements, or as the result of some other weakness. With this object he injected into the peritoneal cavity of guinea-pigs, at the moment when the streptococci begin to get the upper hand of the leucocytes, a definite quantity of a culture of Proteus vulgaris. These small bacilli in a short time become the prey of phagocytes which, however, still refuse to ingest streptococci (fig. 36). There is thus in the peritoneal cavity a kind of selective process as regards the ingestion of these microbes. The Proteus disappears as the result of phagocytosis, whilst the streptococci thrive in the fluid of the exudation and continue to multiply. This experiment, which readily succeeds, demonstrates very clearly the difference between the positive susceptibility of the leucocytes (with respect to the Proteus) and the negative (with respect to the streptococcus). Bordet, in accordance with the view now generally accepted, regards this sensitiveness as a chemiotaxis, that is to say a perception of the chemical composition of the surrounding medium. It must be admitted that the substance which excites the chemiotaxis of the leucocytes does not readily diffuse and may not, therefore, be found in a state of solution in the plasma of the peritoneal exudation. Otherwise the leucocytes would refuse to ingest, not only the streptococci, but also the small Proteus bacilli, bathed in the same repellent fluid. It is more probable that the substance which excites the negative chemiotaxis is contained in the aureola that surrounds the streptococci, from which it only escapes with difficulty and for a short distance.

[Sidenote: [178]]

Marchand[237] continued the investigation of the same subject in Denys’ laboratory at Louvain. He studied the natural resistance of the guinea-pig, rabbit and dog against the streptococcus. He, also, came to the conclusion that phagocytosis constitutes the principal means of defence of these mammals in their struggle against one of the most formidable of the pathogenic micro-organisms. Starting from a single colony, Marchand obtained two distinct races, one very virulent for the rabbit, the other encountering a most effective natural resistance. This resistance is due to the activity of the phagocytes which destroy the streptococci in the ordinary fashion. He states as the general result of his investigation that “an attenuated streptococcus is a streptococcus readily devoured by phagocytes” whilst “a very virulent streptococcus is a microbe that is not attacked by the leucocytes,” and he adds that “a streptococcus is virulent because it is not devoured by phagocytes” (l.c. p. 270). Up to this point the views of Marchand are in accord with those of Bordet; but here they diverge, in fact as soon as it becomes a question of the explanation of the origin of the difference in the behaviour of the leucocytes. Marchand refuses to apply the theory of chemiotaxis and asserts “that the phagocytosis depends on some physical property of the streptococcus and is consequently dependent on the tactile functions of the leucocytes” (p. 292). The experiments upon which he founds his conclusion cannot, however, be regarded as absolutely demonstrative. Thus, Marchand observed that the attenuated streptococci, when conveyed in the culture-fluid of the virulent variety, are as readily devoured by the phagocytes as when they were injected alone. According to him, therefore, there was in the culture-fluid of the virulent streptococcus no soluble substance capable of exciting the negative chemiotaxis of the leucocytes. But is it quite proved that this substance must necessarily pass into the filtrate of a virulent culture? If it adheres closely to the glairy aureola, as we have suggested, may it not remain behind with the bodies of the streptococci, without passing through the filter in any appreciable amount? The question cannot be regarded as definitely settled, but probability appears to be on the side of the theory of chemiotaxis.

Marchand also investigated whether the immunity against the attenuated streptococcus might not be explained by the bactericidal activity of the fluids of refractory animals. His results were unvarying and definite. The blood serum of his animals never exhibited any bactericidal power against the streptococcus, and the attenuated race, like the virulent one, grew well in the serums of the rabbit, dog and guinea-pig.

More recently, Wallgren[238] has taken up the study of the immunity and susceptibility of rabbits with respect to the streptococcus. His conclusions are, on the whole, in accord with those of his predecessors. He found that if the injected streptococci were not very virulent phagocytosis began immediately after the injection into the peritoneal cavity and continued as long as there were any streptococci to be attacked. In those cases, on the other hand, where the streptococcus was endowed with a greater virulence, a transitory phagocytosis took place at the beginning of the infection; but the streptococci soon succeeded in adapting themselves to the struggle with the leucocytes and kept them at a distance. The multiplication of the streptococci could then go on without restraint and the animal soon succumbed to a generalised infection. Wallgren considers that, in the defence of the organism against the streptococcus, the products of the destroyed leucocytes may, sometimes, play a part.

[Sidenote: [179]]

As the mechanism of natural immunity against the groups of bacteria—bacilli, spirilla (and vibrios) and cocci—presents a very great analogy in all three, it might be considered superfluous to continue our analysis of this phenomenon. Our review, however, would be incomplete if we omitted to take note of the natural immunity of the animal organism against micro-organisms which are distinguished by an exceptional toxicity. The first place in this group must undoubtedly be assigned to the bacillus of tetanus.

[Sidenote: [180]]

It may appear very inconsequent to be told that animals very susceptible to tetanus, such as the guinea-pig and rabbit, are endowed with a natural immunity against the tetanus bacillus. And yet this fact, paradoxical as it may seem, has been demonstrated beyond doubt by Vaillard and his collaborators Vincent and Rouget[239]. When a small quantity of a culture of the tetanus bacillus was injected into one of the animals just mentioned, tetanus was not long in declaring itself. After a period of incubation, certain muscles became stiff and a tetanus, local at first, soon became general and had a fatal issue. Now, when much larger quantities of bacilli are inoculated, but care is taken to rid them of the tetanus poison elaborated in the culture-fluid, the animals resist without exhibiting any trace of tetanus. This experiment, repeated many times, always with the same result, demonstrates that the tetanus bacillus, when deprived of the co-operation of the toxin, encounters, in these animals so susceptible to the latter, a most effective opposition. Why is this? It was supposed that, in diseases like tetanus so markedly toxic in character, the resistance was in no way dependent on the phagocytic function. Thus Vaillard and Vincent were quite prepared to attribute no share to the phagocytes in the example of natural immunity which they had discovered. A detailed analysis of the facts convinced them, however, that in this they were in error. Guinea-pigs and rabbits do not contract tetanus, after the inoculation of a quantity of spores and bacilli of tetanus deprived of their toxin, solely because of the occurrence of very pronounced phagocytosis. Such an injection is soon followed by a very marked invasion of leucocytes which cram themselves with spores and bacilli without being in any way inconvenienced thereby (Fig. 37). Once the phagocytes have devoured all these organisms, the latter become incapable of producing their morbific effect. The spores cannot germinate within the phagocytes, but there undergo a marked degeneration and finally, after a longer or shorter interval, disappear.

When, on the other hand, the tetanus bacilli or their spores are accompanied by the pre-formed toxin, the latter, according to Vaillard, excites a negative chemiotaxis of the leucocytes which keep away from the organisms and which are thus allowed to multiply and to secrete fresh quantities of toxin. The natural immunity of the animal’s organism against the tetanus bacillus can be suppressed whenever the phagocytic defence is hampered in any way. Under natural conditions it is usually the adjuvant micro-organisms that aid the tetanus infection by hindering the phagocytes from seizing the spores with sufficient rapidity to prevent their germination. This fundamental result, established by Vaillard and Vincent, has often been gainsaid on the evidence of insufficient experiments (Sanchez-Toledo, Klipstein, Roncali), but, ultimately, its accuracy has been completely confirmed. Cases have been cited in which the tetanus spores, deprived of their toxin, still set up a fatal tetanus. When a small fragment of an agar culture of tetanus, previously heated to 85° C. for the purpose of destroying the toxin, is inoculated, we produce tetanus. Vaillard and Rouget have demonstrated that, under these conditions, the leucocytes penetrate merely into the superficial layer of the agar, the spores germinating and the bacilli multiplying in the deeper part. We can also set up a fatal tetanus in animals by inoculating, along with sterilised earth, spores deprived of their toxin by means of heat. The particles of soil protect the spores against the aggression of the phagocytes, allow them to germinate and then to poison the organism. Lactic acid produces an analogous effect, by destroying or weakening the phagocytes. Micro-organisms, most often inoffensive in themselves, also prevent the phagocytosis of the tetanus spores and thus aid the intoxication.

[Sidenote: [181]]

The facts above summarised have been demonstrated to be the rule for several species of anaerobic pathogenic bacteria. Thus, Besson[240] showed that the septic vibrio is, by itself, incapable of setting up septicaemia; in order to do this it needs the co-operation of other micro-organisms. Leclainche and Vallée[241] have extended the same rule to the bacillus of symptomatic anthrax (Bacillus chauvaei), so important as being the cause of an epizootic disease of the Bovidae. The spores of this bacillus when heated to 80°–85° C. lose the preformed toxin and at once become incapable of producing infection.

In this case also, these spores soon after injection become the prey of phagocytes, which seize them, prevent their germination and check their pathogenic action. If to these heated spores, however, we add a small quantity of toxin, they are enabled to germinate in the tissues and set up a typical infection. If heated spores are mixed with sterile sand, and the mixture introduced into guinea-pigs, these animals almost invariably acquire a fatal symptomatic anthrax. The spores in the superficial part of the sandy mass are readily devoured by the phagocytes; but those which are included within the central part of the mass, being protected for some time against these cells, germinate as soon as they become permeated with the fluids of the animal organism. If we envelope the sand in a paper sac the protection against the phagocytes is still more complete and allows almost all the spores to germinate and in every case to set up a fatal infection. Leclainche and Vallée conclude from their experiments “that we only require to protect the spore mechanically in order to see an infection produced; here we cannot allege an increase of its virulence, as when we associate a chemical substance with the virus, and the exclusive part played by the phagocytosis in the protective process stands out clearly” (p. 221).

The history of these three anaerobic organisms clearly proves that the natural immunity against them cannot be made dependent on either the bactericidal power of the fluids, or on any antitoxic property, or on the incapacity of the micro-organism to secrete its toxin in the fluids of the refractory animal. The cause of this immunity resolves itself into the reaction of the phagocytes which prevent the micro-organisms from producing their poisons.

[Sidenote: [182]]

All that has been said on the subject of the natural immunity of the Vertebrates has had reference to cases of resistance against Bacteria. But may not the immunity against micro-organisms belonging to other groups depend on other factors with which the reader has not yet been made sufficiently acquainted? Amongst the lower plants there are Blastomycetes (Torulae and Yeasts) which are capable of producing infections, e.g. the disease amongst the Daphniae.

[Sidenote: [183]]

Some observers, no doubt, have come to the conclusion that the various Blastomycetes, when introduced into a refractory organism, undergo complete destruction within a few hours without any intervention of phagocytosis. Thus Jona[242] explains the disappearance of yeast-cells injected into the veins or peritoneal cavity of the rabbit as due to the sole influence of the microbicidal property of the bloodfluid. Gilkinet[243] looks at it from the same point of view. He injected beer yeast (Saccharomyces cerevisiae) into a rabbit and observed that it had disappeared shortly afterwards. The destruction of the yeast-cells, according to this observer, “is effected by means of plasmatic juices” and “is due to a specific property of the organic fluids” whose nature is “quite unknown as regards its essential principle.” Phagocytosis is said to play no part in this phenomenon. Let us hasten to say that before the publication of the two works just cited, a memoir by Schattenfroh[244] had appeared on the same subject. This observer, who carried out his experiments in Buchner’s laboratory at Munich, accurately observed and described the destruction of injected yeasts by phagocytes, whilst his experiments on the microbicidal power of the blood and serum failed. This testimony is the more important that it emanates from a school by whom the microbicidal power of the “humours” is regarded as the principal factor in the defence of the animal organism. The facts described by Schattenfroh are perfectly accurate and have been confirmed in my laboratory by Skchiwan[245], who did not restrict himself to injecting ordinary yeasts (pink yeast, Saccharomyces pastorianus) but inoculated guinea-pigs with pathogenic yeast-cells, isolated by Curtis[246] from a case of myxomatous tumour in man. The guinea-pig is refractory to small doses of this yeast but succumbs to injections of larger quantities: Skchiwan convinced himself that the ingestion of the non-pathogenic yeast-cells takes place with great rapidity. Thus the Saccharomyces pastorianus, in the peritoneal cavity of the guinea-pig, is ingested almost exclusively by microphages at the end of two hours. Some (3–4) hours after injection, “sowings” of the peritoneal exudation no longer yield growths. On the other hand Curtis’ pathogenic yeast-cells resist the action of the phagocytes for a much longer time. After a period of phagolysis in the peritoneal cavity, the leucocytes that have just arrived in large numbers begin to seize the yeast-cells. Usually several macrophages fuse around the same yeast globule forming a very characteristic kind of rosette. Sometimes the macrophages run together to produce a giant cell, whose centre contains the yeast-cell. This latter defends itself against phagocytosis by secreting a fairly thick membrane. The struggle between the two living elements is a fairly prolonged one; 24 to 48 hours after inoculation all the yeasts are surrounded by phagocytes, amongst which microphages are exceptional. But the parasites remain alive for 4–6 days after their injection into the peritoneal cavity, as proved by the cultures that are obtained from the exudation when the fluid is “seeded” out. It must be concluded, therefore, that the yeast-cells were surrounded by the phagocytes whilst still presenting all the signs of life. Skchiwan was no more successful than Schattenfroh in demonstrating any kind of microbicidal action of the fluids on the Blastomycetes.

There is, consequently, no doubt whatever that the resistance of the animal organism against yeasts follows the same rules that hold in the defence against bacteria.

[Sidenote: [184]]

The animal micro-organisms are much rarer in infective diseases than are the microphytes; moreover the impossibility of obtaining cultures of them renders their investigation much more difficult. Yet there exist facts that are capable of affording us information as to the means made use of by the refractory organism against certain parasitic Protozoa. Amongst these latter the Trypanosomae play a most important part. One species of this genus (T. lewisi) produces an infective disease in rats, especially in the grey rat (Mus decumanus), the blood of these rodents often containing a very large number of them, whilst the small flagellated organisms flourish well in the serum prepared from the blood of affected animals. Laveran and Mesnil[247], in their studies on the Trypanosomae, injected defibrinated blood containing numerous Trypanosomae into the peritoneal cavity of guinea-pigs, which exhibit a natural immunity against this parasite. The parasites remained alive for some days and then disappeared completely. Here again it is the phagocytes of the peritoneal exudation which rid the animal of the Trypanosomae by ingesting them. Laveran and Mesnil were able, by the examination of hanging drops of the peritoneal exudation of their guinea-pigs, to detect leucocytes in the act of devouring Trypanosomae which showed, by their active movements, that they were still alive. Once the parasites were completely enclosed within the macrophages, their final disappearance took place with extraordinary rapidity.

In this chapter we have attempted to place before the reader a complete series of the phenomena observed in natural immunity in animals. We have passed in review the resistance of the animal organism against the principal groups of bacteria, and we have dwelt on certain of them which are most capable of adapting themselves to various media, and on others which present examples of microorganisms more exacting and more delicate. We have examined the immunity against Blastomycetes and parasitic animalcules. Above all, in the lower animals, just as in the Vertebrata of all classes, we have always observed this general phenomenon: phagocytic resistance as the principal and constant factor in natural immunity.

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VII. THE MECHANISM OF NATURAL IMMUNITY AGAINST MICRO-ORGANISMS

The destruction of micro-organisms in natural immunity is an act of resorption.—Part played by inflammation in natural immunity.—Importance of microphages in immunity against micro-organisms.—Chemiotaxis of leucocytes and ingestion of micro-organisms.—Phagocytes are capable of ingesting living and virulent micro-organisms.—The digestion of micro-organisms in phagocytes is most often effected in a feebly acid medium.—Bactericidal property of serums.—Phagocytic origin of the bactericidal substance.—Theory of the secretion of the bactericidal substance by leucocytes.—Comparison of the bactericidal power of serums and of blood plasmas.—The bactericidal substance of blood serums must not be considered a secretion-product of leucocytes; it remains within the phagocytes, so long as they are intact.—The cytases.—Two kinds of cytases: macrocytase and microcytase.—Cytases are endo-enzymes, allied to trypsins.—Changes in the staining properties and in the form of micro-organisms in the phagocytes.—Absence or rarity of fixatives in the serums of animals endowed with natural immunity.—The agglutination of micro-organisms does not play any important part in the mechanism of natural immunity.—Absence of antitoxic property of the body fluids in natural immunity.—The phagocytes destroy the micro-organisms without their ingestion being preceded by neutralisation of the toxins.

[Sidenote: [185]]

The facts we have set forth in the preceding chapter clearly justify us in concluding that the destruction of the micro-organisms in natural immunity is reduced to their resorption by the phagocytes.

We have now, therefore, returned to the point arrived at and already studied in Chapter IV, where we attempted to establish certain fundamental laws. It remains to be seen up to what point these laws apply to the phenomena of natural immunity against infective micro-organisms.

[Sidenote: [186]]

The introduction into the animal organism of foreign blood, of spermatozoa belonging to the same or a different species, or of any other cells, as in the case of the penetration of micro-organisms into the tissues or cavities of the body of a refractory animal, determines, primarily, a localised inflammation, associated with which is a diapedesis of many white corpuscles. Instead of aseptic inflammation, as in the case of the resorption of cells, there is produced, in antimicrobial immunity, a septic inflammation at the point of invasion of the micro-organisms. In this inflammation the redness and heat are slight, the fluid part of the exudation is insignificant, but what is especially characteristic is the large number of leucocytes which come up towards the point menaced. This constancy of the inflammatory reaction in natural immunity is one of the best proofs of the accuracy of the view that inflammation is a phenomenon useful to the animal organism, especially in its struggle against microbial invasion. As we have devoted a whole volume to the discussion of the comparative pathology of inflammation it is here unnecessary to discuss it further. Since the publication of this book numerous articles on inflammation have appeared, but none of them have undermined, in the least degree, the fundamental bases of the phagocytic theory of inflammation. The view that this phenomenon really constitutes a healing reaction of the organism is at present accepted by many investigators in all countries. It is therefore needless to re-defend it.

Although there still remain a certain number of points that are not sufficiently cleared up in the essential mechanism of inflammation, it is now proved beyond doubt that the sensitiveness of the cell elements which here play a part, is one of the essential factors in the process. The nerve cells which govern the vascular dilatation, the endothelial cells which allow of the passage of leucocytes, and the leucocytes themselves which escape from the vessels in order to reach the point of entrance of the micro-organisms, all must be influenced in a special fashion. In natural immunity the phagocytes exhibit a positive chemiotaxis and this form of sensitiveness is a condition indispensable to a state of immunity and to the disappearance of the micro-organisms.

In my eighth lecture on inflammation I have already set forth the fundamental facts upon which rests the doctrine of the chemiotaxis of leucocytes. During the last ten years numerous data corroborating these results, obtained first by Leber, Massart, and Charles Bordet, and since confirmed by numerous other observers, have been accumulated.

[Sidenote: [187]]

In the resorption of blood corpuscles and of animal cells in general, it is especially the macrophages which intervene, but in natural immunity against micro-organisms positive chemiotaxis is exhibited by the microphages more than by the macrophages. When we examine an inflammatory exudation and find a preponderance of microphages we are satisfied that there has been an intervention of micro-organisms. Even in the examples where it is, at first, principally the macrophages which destroy the micro-organisms (as in the case of the resistance of the animal organism against the tubercle bacillus), there is also a great afflux of microphages. The sensitiveness of the two chief categories of phagocytes often exhibits a marked difference. We need merely recall to the reader the example of the spirilla, ingested and destroyed exclusively by the macrophages of the guinea-pig, which alone exhibit the necessary positive chemiotaxis. In many other examples of natural immunity the part played by the macrophages is masked by that of the microphages.

In natural immunity the motile phagocytes, having come up to the invaders, perform a second physiological function; they ingest the micro-organisms. Sometimes the leucocytes devour at one swoop whole masses of these organisms, and carry out their work in a very short time. In other cases, especially when actively motile micro-organisms, such as the spirilla of Obermeyer or of Sacharoff, have to be dealt with, the ingestion takes place with more difficulty and requires special conditions. Thus, in order to ingest a spirillum, the macrophages of the guinea-pig throw out long conical processes. Never in the ingestion of micro-organisms have I observed methods comparable to that by which the macrophages seize upon the red corpuscles of birds or upon other animal cells.

[Sidenote: [188]]

Some observers have expressed the opinion that micro-organisms make their way into the cells spontaneously and do not need to be drawn in by means of protoplasmic processes thrown out by the phagocytes. It is of course indisputable that certain micro-organisms may pass into the interior of the cell independently of any act of phagocytosis. Such is the case with the malaria parasite and allied species which make their way into the red blood corpuscles. But here we are dealing with amoeboid organisms, quite capable of perforating the wall of the red blood corpuscle by means of their own pseudopodia. Bacteria, which do not possess amoeboid movements, are deprived of this power of invasion. There are, however, very rare cases in which such penetration does take place. For example, Bizzozero[248] has described spirilla in the stomach of the dog; these may be found inside epithelial cells. But here these actively motile bacteria make their way into the interior of vacuoles which open on the free surface. Attracted, probably, by the epithelial secretions the spirilla first draw near to the cells and then take advantage of small openings through which they pass into the secretory vacuole. In almost all cases, however, living and even actively motile bacteria are incapable of penetrating into cells. Thus, when we observe the spirilla of recurrent fever or of goose septicaemia in the neighbourhood of leucocytes, we often see them exhibit very brisk corkscrew movements on the surface of these cells without ever being able to invade them. On the other hand, when the leucocyte sends out a process towards the spirillum ingestion rapidly takes place. In anthrax exudations, or in the spleen of animals that have succumbed to anthrax, large numbers of bacilli may often be observed in the immediate neighbourhood of the leucocytes or of the cells of the splenic pulp, without a single bacillus being found within these cells. Nor do we ever see any bacteria (which develop abundantly in a drop of exudation withdrawn from the organism) invade the dead leucocytes, lying alongside them. Whilst on the other hand we see the micro-organisms swarming outside the neighbouring leucocytes and occupying the free spaces between these cells.

[Sidenote: [189]]

Almquist[249] has recently described a method by means of which micro-organisms can be taken into the substance of dead leucocytes. He collects leucocytes from mammalian blood, mixes them with bacteria, and centrifugalises the mixture for some time. He convinced himself that after a not very prolonged contact the bacteria are found within leucocytes. Here Almquist excluded phagocytosis, properly so-called, that is to say, the ingestion of the bacteria by the active movements of the leucocytes; but he does not give sufficient proof that the cells, in his experiments, were actually dead. He thinks that the relatively low temperature (below 15° C.) excluded the possibility of amoeboid movement in the leucocytes of warm-blooded animals. This argument, however, does not accord with actual fact, for it is indisputable—and we have often convinced ourselves of this—that the leucocytes of man and warm-blooded vertebrates maintained at even a lower temperature than 15° C. are quite capable of motion and of ingesting foreign bodies. In all cases, the data as a whole, some of which we have cited above, leave no doubt that the ingestion of micro-organisms unprovided with amoeboid powers takes place by means of active movements of the living protoplasm of the leucocytes. To dissipate any remaining doubt on the part of the reader I need only recall Bordet’s investigations, cited in the preceding chapter, of the behaviour of leucocytes in the peritoneal cavity of guinea-pigs inoculated with streptococci and Proteus bacilli. The leucocytes of the peritoneal cavity allow the virulent streptococci to develop freely, not ingesting a single one, whilst the Proteus bacilli, injected later, are quickly devoured and at the end of a very short time are all found in the substance of these same phagocytes. This example, so demonstrative, of the chemiotaxis (positive as regards Bacillus proteus and negative as regards the streptococcus), is at the same time the best proof of the fact that the ingestion of the micro-organisms is a vital, physiological act and not merely a simple phenomenon of mechanical penetration of micro-organisms into the soft protoplasm of the leucocytes.

It was formerly thought that leucocytes, charged with micro-organisms, provide the latter with a good culture medium and serve also as vehicles of transport for them from one place to another in the living organism. This view has often been affirmed without any proof whatever being given of it. It has now been demonstrated to be erroneous. The micro-organisms, with some rare exceptions, find within the leucocytes a very unfavourable medium. Usually they perish there, or, in the case of very resistant micro-organisms, such as the tubercle bacilli in refractory animals or the endospores of certain bacteria, without being actually destroyed, they are prevented from germinating and multiplying.

[Sidenote: [190]]

Later, another view has been advanced that phagocytes are capable of ingesting only those micro-organisms that have been previously killed by some substance which is found outside the defensive cells. This view is quite as erroneous as the one we have just analysed. The phagocytes are perfectly capable of seizing and devouring living micro-organisms. We have only to recall on this point the facts cited in the preceding chapter on the subject of living bacteria ingested by the leucocytes of various animals, or the history of the very active spirilla which retain their motility up to the moment when they become completely enclosed by the protoplasmic processes of the leucocytes of the guinea-pig. Observations in vitro have, as already described in the same chapter, afforded a demonstration of the ingestion of living flagellated Infusoria by the leucocytes of refractory animals.

These facts, fairly numerous in themselves, are not, however, the only ones that might be cited in favour of the fundamental thesis that phagocytes possess all the means for incorporating living micro-organisms. In my first works on phagocytosis I cited the example of amoeboid cells, in the Invertebrata, containing motile bacteria[250], and that of leucocytes of the frog charged with motile bacilli[251] of an artificial septicaemia. Since then the number of similar cases has increased considerably. Nothing is easier than to observe the phagocytosis of living micro-organisms in vitro. Take a drop of frog’s lymph and add to it a few of the Bacilli pyocyanei, we soon observe the struggle between the leucocytes and the very motile bacteria, and inside the digestive vacuoles bacilli executing very pronounced and active movements.

[Sidenote: [191]]

The same result may be obtained by another method, by which at the same time we gather information as to the virulence of the micro-organisms ingested by the phagocytes. The view has often been expressed that phagocytes seize only those bacteria that have been deprived of their virulence by a previous action of the fluids of the animal organism; consequently search has been made for some attenuating property of these fluids. We have already answered this objection in the previous chapter by the citation of cases in which the exudations of refractory animals, containing only micro-organisms ingested by the phagocytes, were, nevertheless, very virulent for susceptible animals. This question has been especially discussed in relation to the anthrax of frogs, on which subject several investigations have been carried out, the result of which is completely convincing. Bacilli ingested by the leucocytes of these Batrachians retain their full virulence for a long time. Exudations which contain only intraphagocytic bacilli, the majority of which have already lost their normal staining by aniline dyes, produce fatal anthrax in susceptible animals, such as the mouse and the guinea-pig. Mesnil has demonstrated the same fact by using the exudations of fresh-water fishes that are refractory to anthrax. The same rule applies equally to the exudations of dogs and fowls that have been inoculated with the bacillus.

Long before these experiments on anthrax were made, Pasteur[252] had shown that the virus of fowl cholera, which in the guinea-pig sets up a mild affection and gives rise to the formation of abscesses, retains its virulence for a considerable time in the pus of these abscesses. When he injected rabbits with a small quantity of guinea-pig’s pus developed at the point of inoculation of the cocco-bacillus of fowl cholera, the animals succumbed to a generalised and rapid infection. The conviction has since been arrived at that, in the guinea-pig, these micro-organisms readily become the prey of the leucocytes that are present in the exudations.

The rule, therefore, is general that in animals endowed with natural immunity the phagocytes seize and ingest even living micro-organisms that have retained their initial virulence.

[Sidenote: [192]]

[Sidenote: [193]]

Once within the phagocytes, the micro-organisms are surrounded by a clear fluid, which accumulates in vacuoles, or they are lodged directly in the protoplasm. In both cases the micro-organisms are subjected to a digestive action which usually dissolves them completely. It is not always easy to form an idea of the conditions under which the intracellular digestion takes place. At first[253] I used a weak solution of vesuvin for the purpose of gaining some idea as to the condition of the micro-organisms that have been ingested by the leucocytes and demonstrated that the living bacteria remain unstained in this solution, whilst the dead bacteria take on a somewhat deep brown stain. Thanks to this reaction I was able to furnish one of the proofs of the fact that in immunised animals ingested bacteria are killed inside the phagocytes. The use of Ehrlich’s neutral red (Neutralroth) gives us further valuable indications. This colour, quite innocuous for living elements, is an excellent indicator of acid or alkaline reaction. Plato[254], in Breslau, has carried out numerous researches on the staining of micro-organisms by a weak aqueous solution (1%) of this substance. He has shown that “free” micro-organisms remain alive in this solution without taking on any tinge of colour. On the other hand, the same micro-organisms, when ingested by the phagocytes, are stained brownish-red. Most of these stained organisms no longer exhibit any sign of vitality; but amongst those within the phagocytes are some which, in spite of being deeply stained, are certainly alive. Plato insists on the fact that ingested micro-organisms remain stained as long as the phagocytes are alive, for, shortly after the death of these cells, decoloration of the micro-organisms and of the intracellular granules takes place. When neutral red is added to an exudation in which the leucocytes are dead, the staining of the ingested micro-organisms—dead or living—does not take place. I have myself verified these observations, and Himmel[255], who has carried out an elaborate investigation on this subject in my laboratory, has confirmed them in numerous cases. In the third and fourth chapters of this work I have already brought forward arguments in favour of the view that the staining of the ingested elements indicates a feebly acid reaction inside the phagocytes. Sometimes this reaction manifests itself in the digestive vacuoles; in other cases it is exhibited only in the micro-organisms directly lodged in the protoplasm (Fig. 38). Whilst the phagocyte is still living the acid juice which fills the vacuoles or permeates the ingested organisms does not mix with the protoplasm which is always alkaline. But shortly after the death of the phagocytes this mixture is effected without difficulty, and the alkalinity of the protoplasm is then amply sufficient to neutralise or even render alkaline the feebly acid juices. This interpretation of the facts is in complete harmony with all the data, collected up to the present, on the staining by neutral red of phagocytised micro-organisms.

All ingested bacteria do not, however, stain in the way we have indicated. Tubercle bacilli, even in cases of natural immunity, remain unstained inside the phagocytes or take on only a very slight straw-yellow tint. Himmel made this observation on the bacilli of avian tuberculosis that had been ingested by the peritoneal leucocytes of the guinea-pig, a species resistant to this micro-organism. It might be thought that such a resistant membrane as that of the tubercle bacillus, with its waxy layer, would prevent the penetration of the acid leucocytic juice; but several bacilli which resist decoloration by acids, as do the tubercle bacilli, notably the bacilli of Moeller and their allies, are stained a bright red by neutral red as soon as they are ingested by the phagocytes. It is, therefore, more probable that, in the case of true tubercle bacilli, the reaction in the cells is no longer acid, but alkaline. This conclusion is confirmed by what is observed in the giant cells of the Algerian gerbil (Meriones shawii), a species of rodent which exhibits a great natural resistance against the bacillus of human tuberculosis[256]. The bacilli, ingested by these phagocytes, secrete a series of concentric membranes which become impregnated with phosphate of lime (Fig. 5). The process causes the death of the bacilli, of which there remain only the calcified membranes. The precipitation of the lime salt around bacillary membranes itself indicates the alkaline reaction of the medium. The use of certain staining substances fully confirms this conclusion. Thus, with alizarin sulpho-acid the giant cells stain deep violet, this affords clear proof of a very distinct alkaline reaction.

[Sidenote: [194]]

We arrive then at the general conclusion that phagocytic digestion usually takes place in a medium weakly acid, but that it can also go on in an alkaline medium. It is impossible, in the present state of our knowledge, to define the nature of the acid secreted by the phagocytes. H. Kossel[257] has expressed the view that the intracellular digestion of micro-organisms is effected by the nucleic acid, secreted by the cell nucleus and accumulated in the vacuoles of the contents of the phagocytes. He has brought forward in support of this view the fact that nucleic acid is distinctly bactericidal, killing certain pathogenic micro-organisms, and giving a precipitate composed of albumen and nucleic acid. Later H. Kossel pointed out the presence in these formed elements of albuminoid substances which have an alkaline reaction but which also destroy bacteria. Thus he has isolated from the spermatic fluid of the sturgeon a protamine, “Sturin,” which, even in very weak solutions, exhibits a strong bactericidal action on the typhoid bacillus, staphylococcus, etc. It is possible that these substances play a part in intracellular digestion. On the other hand, however, we must regard it as well established that in phagocytes there is a soluble ferment which kills and digests micro-organisms. We have already seen, in connection with the resorption of animal cells, that it is the ferment alexine, or cytase, which plays the principal part in the digestive function. We must now ask ourselves whether the same substance acts also on micro-organisms.

For more than fifteen years a study of the bactericidal power of the blood and other fluids drawn from the animal organism has been carried on. Based on the not very definite results of Traube and Gscheidlen[258], Fodor[259] drew attention to the property of the defibrinated blood of the rabbit to destroy the bacteria sown in it. Under the inspiration of Flügge[260], Nuttall[261] carried out a whole series of experiments on this bactericidal property of defibrinated rabbit’s blood, of the aqueous humour, and of some other fluids. After confirming Fodor’s general result, Nuttall went further and showed that the bactericidal power of the fluids is due to a substance of undetermined nature which is destroyed by heating to 55° C. for one hour. This discovery was confirmed by a large number of observers, and soon became an accepted fact.

[Sidenote: [195]]

Flügge now considered that he could base a theory of immunity on the presence of the bactericidal substance of the body fluids. Bouchard[262] and his school adopted and developed this view, especially with reference to researches on the microbicidal power of blood serum. Buchner[263] soon came forward as the chief advocate of this theory, and enriched it by numerous investigations carried out by himself or along with collaborators in his school at Munich. It is to him that we owe the suggestion of the term alexine (protective substance) to designate the bactericidal substance of blood serum and other fluids of the animal organism which are capable of killing micro-organisms. Buchner determined the conditions under which alexine acts best as a bacterial poison and developed the humoral theory of natural immunity, according to which the latter is reduced to the bactericidal property of the body fluids.

[Sidenote: [196]]

As the postulates of this theory are often not in accord with the real facts, as Lubarsch[264], especially, has demonstrated in many of his papers, we[265] expressed the opinion that a portion at least of the bactericidal power might come from substances that had escaped from the leucocytes during the preparation of the defibrinated blood and of the blood serum. This hypothesis remained for several years unnoticed, but later several observers have, quite independently, arrived at the conclusion that alexine is nothing but a leucocytic product. Denys and Havet[266] were the first to show that exudations rich in white corpuscles exhibited a bactericidal power much higher than that of the corresponding blood serums. Shortly afterwards H. Buchner[267] showed the same thing on comparing the bactericidal power of exudations rich in leucocytes with the blood serum of the same animals. As this property disappeared from both fluids after they had been heated to 55° C., Buchner concluded that the bactericidal substance of the exudations must be identical with the alexine of the blood serum. Several other observers, amongst whom Bail, Schattenfroh, Jacob and Löwit, may be cited, obtained results more or less in accord with the above, though obtained by different methods, so that it has now for some time come to be recognised that the leucocytic origin of the alexines is generally accepted, especially since Jules Bordet[268], in an investigation carried out in my laboratory, arrived at the same result from various very demonstrative experiments.

Nevertheless several authoritative voices have been raised against this interpretation of the facts. R. Pfeiffer especially, with his school, has pronounced against the leucocytic origin of the bactericidal substance found in the blood serum. Pfeiffer and Marx[269] and Moxter[270] have insisted on the fact that the fluids of exudations rich in leucocytes are often much less bactericidal than is the serum of the blood of the same animals.

For some years, struck by the marked difference between the phagocytic function of the macrophages and that of the microphages, I have thought that the contradictory results of the observers cited might be explained by some difference in the nature of the leucocytes of the various exudations and of the blood which served for the preparation of the serums. I therefore asked Gengou to devote his attention to this particular point and to compare the bactericidal power of exudations, rich in microphages, with that of others containing many macrophages and also with the blood serum of the same animals. Gengou[271] has carried out his experiments with remarkable exactness and care, and as I have followed them closely I am in a position to speak as to their extreme accuracy.

[Sidenote: [197]]

In order to obtain exudations very rich in microphages Gengou injected gluten-casein by Buchner’s method into the pleural cavity of dogs and rabbits. Usually at the end of 24 hours he was able to collect a large quantity of fluid containing numerous leucocytes, almost exclusively microphages. To obtain macrophagic exudations Gengou injected washed red blood corpuscles of the guinea-pig into the pleural cavity of his animals; two days afterwards he withdrew from the pleural cavity a very viscid fluid, containing, as regards formed elements, macrophages almost exclusively. After isolation of the leucocytes by centrifugalisation of the exudations, Gengou washed the cells with physiological salt solution and then added to them an equal volume of broth. This mixture was frozen by Buchner’s method, and was then submitted to a temperature of 37° C. Under these conditions the leucocytes, killed by cold, gave up to the fluid their bactericidal substance.

Studied in this way, the bactericidal power of the extract of microphages showed itself always superior to that of the corresponding blood serum. The greatest difference was observed in the dog, where, as already mentioned in the preceding chapter, the serum of the blood has no bactericidal property as regards the anthrax bacillus, whilst the extract of microphages manifests this property very strongly. The microphagic extract of the exudations of rabbits was more active in the destruction of the bacilli of anthrax and typhoid, Bacillus coli and the cholera vibrio, than was the blood serum.

The result of these experiments leaves no room for doubt. The microphages, collected in the aseptic exudations of the dog and rabbit, contain more bactericidal substance than does the blood serum of the same animals. Nor can there be a doubt that this bactericidal substance is the same whether it appears in the microphages or in the blood serum: in both cases it is destroyed by heating to 55° C. and, in all other respects, it behaves in the same manner.

The experiments of Gengou with the extracts of macrophages have demonstrated, on the other hand, that this fluid exerts no bactericidal power. Let it be understood at the outset that this fact is in no way an indication of the absence of the bactericidal ferment in the macrophages. Direct examination of the phenomena which are manifested inside these cells demonstrates most clearly that the macrophages kill and digest micro-organisms. But this process usually goes on much more slowly in the macrophages than in the microphages, owing probably in the former to the presence of a smaller quantity of the bactericidal substance. Under these conditions we can readily understand that this substance does not pass, or passes only in small amount, into the extracts. There is nothing remarkable in the fact that, with so imperfect a method of preparing the extracts, the greater part of the bactericidal substance should remain in the bodies of the cells.

The facts just set forth afford a sufficient explanation of the marked difference in the results obtained by various observers as to the bactericidal power of the exudations. When the latter are rich in microphages, the bactericidal property is very marked: when, on the other hand, the exudations contain a large number of macrophages, the bactericidal power may be very weak or even nil.

[Sidenote: [198]]

The experiments above summarised confirm the conclusion that the microphages must be regarded as the source of the bactericidal substance of the body fluids. But here arises the question: Do the microphages secrete the substance during life, giving it up to the blood plasma, or does this substance escape only after the death of the leucocytes and the damaging of the cells, due to various external causes? We here touch on a problem which has been the subject of much discussion and one of very great importance in connection with the question of Immunity in general.

After the discovery of the bactericidal power of serums, several investigators set to work in search of the source of the bactericidal substance. Hankin[272], and shortly afterwards Kanthack and Hardy[273], expressed the view that this substance is the secretion-product of the eosinophile leucocytes which would thus appear to be a kind of motile unicellular glands. This theory could not be supported by solid arguments and must be regarded as generally abandoned, because it is now completely out of accord with well-established facts. Thus, various osseous fishes, in spite of the total absence of eosinophile or pseudo-eosinophile granules are none the less capable, thanks to their leucocytes, of destroying a large number of pathogenic micro-organisms (Mesnil, l. c.).

[Sidenote: [199]]

A similar theory was enunciated by H. Buchner[274], though he holds that it is not the eosinophile leucocytes only that secrete the bactericidal substance, but the leucocytes in general. Being attracted to the point menaced by the micro-organisms, these cells secrete their bactericidal product, which diffuses into and along with the plasma of the exudations and of the blood. In these fluids the micro-organisms undergo a more or less complete destruction, or at least severe injury which renders them more susceptible to the attack of the phagocytes. At the International Congress of Hygiene, held at Budapest in 1894, Buchner proclaimed the thesis that “the leucocytes fulfil an important function in the natural defence of the organism ... by means of soluble substances which they secrete.” Later, his pupils, Hahn[275] and Schattenfroh[276], endeavoured to support this theory by exact experiments, but they found it impossible to do this at all satisfactorily. Later, another of Buchner’s pupils, Laschtschenko[277], published a paper in which he maintains that he has found a convincing argument. It is as follows. A blood serum, by itself void of bactericidal property, some minutes after white corpuscles from another species of mammal have been added to it acquires this property. Thus the rabbit’s leucocytes added to dog’s serum immediately give to it the bactericidal power, so long as a large number of cells remain alive and motile. But when the leucocytes of the same species are added to rabbits’ serum the fluid becomes no more bactericidal than before. The same result may be obtained by mixing rabbits’ leucocytes with the blood serum of the horse, pig and other species. Laschtschenko concludes from these observations that the vital secretion of the bactericidal substance by the leucocytes of the rabbit takes place when they are irritated by the serum of a different species. As an analogous effect has been observed with mixtures of rabbits’ leucocytes with the serum of a different species heated to 60° C., Laschtschenko believes himself safe from the objection that the giving up of the bactericidal substance results from the death or injury of the white corpuscles. According to him this injurious effect on the white corpuscles can only be produced by an unstable substance which is destroyed by heating to 60° C. Laschtschenko forgets that the leucocytes are in general delicate cells, capable of being affected even by fluids which do not actually kill them. Now we know that serums, when heated to 60° C., still retain their power of agglutinating the leucocytes, a power which must hamper these cells in their normal function.

[Sidenote: [200]]

Trommsdorff[278], in an investigation carried out in Buchner’s laboratory, endeavoured to supplement Laschtschenko’s results and to support them by new and more convincing experiments. But he only succeeded in a few cases in obtaining a bactericidal serum after adding rabbits’ leucocytes to the blood serum of other animals. “In a great number of my experiments,” says Trommsdorff, “I very often did not succeed in extracting the alexines from the rabbit’s leucocytes by the use of Laschtschenko’s method” (p. 385). On the other hand, Trommsdorff, wishing to establish the living condition of the leucocytes mixed with a foreign serum, arrived at the following result: “In the majority of the cases, as in fresh exudations, the number of living leucocytes after their treatment with active horse’s serum, as well as with inactive serum (heated to 60° C.) of dog, ox and horse, varied between 60 and 80%” (p. 391). In spite of these verifications, Trommsdorff comes to the conclusion that the presence of alexine in those serums to which leucocytes had been added, must “in all probability” be attributed to its secretion by the living leucocytes. We regard it as much more probable that the alexine, in those cases where it passed into the serum, was due to the breaking up of the dead leucocytes, whose numbers rose to 40 %, that is to say, almost half their total number. Our conclusion is, in any case, much more in accord with the more constant and more exact results obtained by other methods.

[Sidenote: [201]]

In spite of the insufficiency of proofs in favour of the theory of bactericidal secretions by the leucocytes it has been very favourably received by many investigators. As, however, it came into collision with the general fact that, in the refractory animal, the microorganisms remain alive in the plasmas of the exudations and are, in this condition, ingested by the phagocytes, it was therefore very important that this fundamental contradiction should be settled by decisive experiments. The attempt has often been made to obtain blood plasma and to compare its bactericidal action with that of serum from the same animal. In the preceding chapter we have already mentioned an attempt in this direction made by Sawtchenko. Hahn[279] had previously attempted to prepare plasma by adding histon to blood. As this “plasma” was found to be just as bactericidal as the blood serum Hahn concluded that the bactericidal substance, secreted by the living leucocytes, circulates in the living blood. In all the experiments carried out by this method it was impossible to avoid certain sources of error, and in my laboratory Gengou[280] undertook a new series of researches, endeavouring to obtain from blood a fluid resembling normal plasma as closely as possible. The method he employed has been described in detail in a memoir, on an anticoagulating serum, which he published along with Bordet[281]. The blood was drawn into paraffined tubes and centrifugalised at once in other tubes whose walls were likewise covered with a layer of paraffin. The fluid thus prepared is certainly more allied to circulating plasma than is the blood serum obtained after the coagulation of the blood. Nevertheless, it is still far from being identical with true normal plasma; it still coagulates, though tardily. Gengou compared, in their bactericidal action, the blood serum and the serum, decanted after the tardy coagulation of the fluid analogous to plasma. He carried out a great number of experiments with the two fluids, obtained from dogs, rabbits and rats, making a comparative study of their bactericidal power as regards the anthrax bacillus, the typhoid bacillus, and the cholera vibrio. I have closely followed all these experiments and can confirm the results described by Gengou, namely, that the fluid, in this plasma serum, possesses an insignificant bactericidal power or none at all, whilst the blood serum almost always exhibits this property to a marked degree.

As a result of the researches just summarised it is no longer possible to maintain the theory of bactericidal secretions by leucocytes or by any other category of cells. The bactericidal substance does not circulate in the blood plasma nor in that of the exudations, and this is a sufficient reason for refusing to it the title of a secretion-product. Its presence in the blood serum is due, like that of the fibrin-ferment, to the destruction or more or less grave injury of the phagocytes.

[Sidenote: [202]]

This fact, upon which we must insist most strongly, is in flat contradiction to the view recently formulated by Wassermann[282]. In a work devoted to natural immunity against micro-organisms, this author describes how he submits his animals (guinea-pigs) to the action of an anticytase (or anti-alexine) serum whose preparation, described in the fifth chapter of this work, offers no difficulties. Under the influence of this serum, the guinea-pigs, into the peritoneal cavity of which a strong dose of typhoid cocco-bacilli is inoculated, die from infection, whilst the control animals, inoculated in a similar manner, but which have received in addition some normal rabbit’s serum, heated to 60° C., entirely resist the infection. Wassermann concludes that the first series of guinea-pigs succumbed because of the impossibility of fighting against the typhoid bacillus by means of the free cytase, this being neutralised by the anticytase serum. The fact pointed out by Wassermann is perfectly accurately stated and has been confirmed by Besredka[283], in an investigation carried out in my laboratory. Nevertheless, it is impossible to accept Wassermann’s view as to the part played by anticytase in his experiment. As clearly demonstrated by Besredka, the anticytase serum acts not merely by neutralising the bactericidal ferment, but also by its other properties, especially by one which prevents the stimulation of the phagocytes.

In the struggle of the guinea-pig’s organism against a strong dose of typhoid cocco-bacilli (in Wassermann’s experiments 40 times the lethal dose), the free cytase plays a part so infinitely small that even the injection into a guinea-pig of a large quantity of serum (3 c.c.) from a normal guinea-pig (containing much cytase) does not prevent the death of the animal. It is only the blood serum of other species (rabbit or ox) that is capable of protecting a guinea-pig against such a large quantity of typhoid bacilli.

Wassermann was in error in supposing that his experiment was a case of natural immunity. It comes entirely within the range of the phenomena of acquired immunity. In fact, the natural immunity of the guinea-pig is only exhibited against a dose 40 times less than that employed by Wassermann. Consequently the control guinea-pigs which received such a huge quantity of the typhoid cocco-bacilli, going beyond 40 times the limit of their natural immunity, require to be preserved from death by the injection of a large quantity of blood serum heated to 60° C. from the normal rabbit. This serum, deprived of its cytase, retains its other properties, by which the organism of the guinea-pig profits, especially exercising a stimulating action on the phagocytes of the guinea-pig. The immunity of Wassermann’s control animals was, then, really an acquired immunity, the result of the introduction into their organism of the stimulating serum of the rabbit. For this reason an analysis of the work of this observer must be postponed until we treat of the phenomena of acquired immunity under the influence of normal serums.

We must, then, persist in the opinion that the plasmas of the normal animal, containing no cytases, cannot play a bactericidal part in natural immunity, a part which devolves upon the cytase contained within the phagocytes.

[Sidenote: [203]]

This result accords well, also, with the whole of the facts bearing on the destruction of micro-organisms in the animal body. The transformation into granules of the attenuated cholera vibrios that is sometimes observed in the peritoneal cavity during the period of phagolysis, and the absence of this transformation under conditions where the peritoneal leucocytes are protected against this injury, is clearly explained. In the first case, Pfeiffer’s phenomenon is set up by the bactericidal substance which has escaped from the leucocytes that have been altered by the foreign substances injected into the peritoneal cavity; in the second case, this phenomenon is not produced because the leucocytes remain intact. The absence of this granular transformation in the anterior chamber of the eye and in the subcutaneous tissue is also readily explained by the fact that the bactericidal substance, not being present in the blood plasma, cannot pass into the exudations of the eye and subcutaneous tissue[284].

The bactericidal substance, then, is essentially some substance which remains inside the uninjured phagocytes in the living animal but which escapes from these cells when they are injured, either in the body of the animal or outside in the blood withdrawn from the organism. Buchner has given to this substance the name of alexine and we must now determine whether this substance is the same cytase which digests the formed elements on their resorption.

[Sidenote: [204]]

Since his first researches on the power of one normal blood serum to dissolve the red corpuscles of another species, Buchner[285] has maintained the identity of the haemolytic substance with the bactericidal substance of the same serum. In both cases we have to do, according to him, with one and the same substance of an albuminoid nature, with the same “alexine.” In his later work, Buchner attempted to confirm and develop this thesis. Bordet[286] has, on several occasions, brought forward arguments in favour of the same view; but against this Ehrlich and Morgenroth[287] have declared themselves. According to these observers a single serum may contain several alexines or “complements.” The same serum may even contain two complements, one of which is destroyed by heating to 55° C., whilst the other, much more stable as to the action of heat, resists this temperature. In one of their most recent memoirs, Ehrlich and Morgenroth lay special stress on the importance of an experiment which has enabled them, by means of filtration, to separate two complements from the normal serum of the goat, one of them attacking the red corpuscles of the guinea-pig, the other those of the rabbit.

Max Neisser[288] has adopted this view of the plurality of alexines. According to Ehrlich and Morgenroth, the same serum may possess several complements which attack the red blood corpuscles of various species and other complements which attack micro-organisms. In favour of this thesis Neisser gives a summary of his experiments on the absorption of complements which, in his opinion, prove the plurality of alexines. By centrifugalising rabbit’s blood serum to which he had previously added a certain number of anthrax bacilli, he obtained a fluid which no longer destroyed this bacillus but which still dissolved the red corpuscles of goat and sheep. There are then, according to Neisser, in the normal serum of the rabbit, at least two different complements; one for the bacilli and one for the red corpuscles.

[Sidenote: [205]]

With the object of explaining the discrepancy between these results and those of his previous experiments, Bordet[289] undertook a new series of researches on the absorption of cytases. He first made it clear that the normal red corpuscles, when plunged into a normal haemolytic serum, are incapable of fixing all the cytase. When such a serum is centrifugalised, after a prolonged contact with red corpuscles of a different species, the fluid no longer dissolves normal red corpuscles. But if these latter be sensibilised by means of a specific fixative, the red corpuscles are dissolved in large numbers. It must be admitted that in this experiment we have to do with a single cytase because, before centrifugalisation, as after it, the red corpuscles of the same species are added. In the first case, however, these corpuscles were normal, whilst in the second they were sensibilised by the fixative.

When, after the first part of this experiment, that is to say, after the fixation of a certain quantity of cytase by the red corpuscles, we centrifugalise the mixture and add, not the sensibilised red corpuscles of the same species but the normal red corpuscles of a different species, we find that the latter still dissolve and fix a certain quantity of cytase. As the first experiment (with sensibilised red corpuscles) has shown that the whole of the cytase has not been absorbed by the red corpuscles, we readily understand that the portion remaining in the fluid will act on the normal red corpuscles of another species.

[Sidenote: [206]]

But when we fix the cytase to the sensibilised red corpuscles the absorption becomes complete and the addition of other species of red corpuscles no longer produces any solution. It is easy, therefore, by means of sensibilised red corpuscles, to take out the whole of the cytase from a serum. When to such a serum, thus deprived of the whole of its haemolytic cytase, we add bacteria, these latter show no sign of disintegration; whilst previously, that is before the absorption of the cytase by the sensibilised red corpuscles, the same serum was highly bactericidal. Let us take a concrete example so that the reader may form some definite idea of the phenomena observed. Take a normal rat’s serum which, in a very short time, transforms cholera vibrios into granules or deforms and dissolves anthrax bacilli. The same serum dissolves the red corpuscles of a different species. We will first leave this serum in contact with these red corpuscles sensibilised by the specific fixative. After the solution of a quantity of these red corpuscles, let us add to the serum a few cholera vibrios or anthrax bacilli. The vibrios, in this serum, are no longer transformed into granules and the anthrax bacilli undergo no change at all; they stain in the normal fashion by basic aniline dyes, they present neither deformations nor solution of their contents. In other words, no bactericidal action takes place in a serum that is deprived of its cytase by sensibilised red corpuscles.

Is it necessary to conclude from this and other analogous experiments that the cytase, fixed by the sensibilised formed elements (red blood corpuscles or micro-organisms), is always one and the same cytase? May it not be that these elements, impregnated with specific fixatives, become so greedy for cytases that it is easy for them to absorb not only one variety but several species of cytases?

The facts we have summarised in Chapter IV concerning the cytases, indicate that very probably there exist two kinds of cytases, connected with the two great groups of phagocytes. Extracts of the mesenteric glands, of the omentum and of the exudations, which are composed for the most part of microphages, do not dissolve the red corpuscles, but are, on the other hand, specially bactericidal. Sarassewitch has carried out numerous experiments on this point in my laboratory and has brought forward a large number of data in favour of this theory of two phagocytic cytases. He found that, even when specific fixative is added to the extract of microphagic exudations (of rabbit), the sensibilised red corpuscles are not dissolved. It must then be accepted that microcytase, so active against bacteria, is entirely powerless against animal cells.

As the microphages seize, though rarely, and digest red blood corpuscles, spermatozoa and other cells of animal origin, it must be admitted that they also contain a small quantity of macrocytase, or that the microcytase, given time, is capable of dissolving these elements. On the other hand, the macrophages, in spite of their marked predilection for animal cells, also ingest and digest certain bacteria. This is due perhaps to the presence of a little microcytase or to the power that the macrocytase has of attacking micro-organisms. These questions are too subtle to be definitely resolved at present.

[Sidenote: [207]]

The duality of the cytases does not clash with the experiments of Bordet summarised above. We have only to admit that the formed elements, once they are impregnated with specific fixatives, become capable of absorbing not only the cytase which digests them, but also another which, without dissolving them, is simply fixed to them. Here we should have a phenomenon analogous to the fixation by fibrin of diastases, other than trypsin and pepsin, or to the fixation by silk threads of all kinds of soluble ferments.

It may be accepted, then, that the phagocytes elaborate two cytases: macrocytase, active for animal cells, and microcytase, which digests bacteria. This result up to a certain point has been anticipated by Schattenfroh’s[290] experiments and foreseen by Max Neisser (l.c.).

It has already been noted that the reaction inside the phagocytes is usually feebly or very feebly acid, and only rarely distinctly alkaline. On the other hand, it is well known that cytases, in serums, act in an alkaline medium. It is certain therefore that these soluble ferments can carry on the process of digestion under varied conditions. Hegeler[291], working in Buchner’s laboratory, has studied the influence of the alkalinity and acidity of the medium on the bactericidal action of serum. He comes to the conclusion that the destruction of micro-organisms can take place in a serum to which has been added small quantities of alkali (carbonate of soda) and also in a weakly acid serum (from the addition of small quantities of sulphuric acid). Once the serum becomes distinctly acid the bactericidal power disappears at once.

Our knowledge of the cytases, as a whole, leads us to approximate these diastases to the group of trypsins, papain, amoebodiastase and actinodiastase. The cytases are elaborated by the phagocytes, but are not secreted into the plasmas and they remain inside the cells so long as these cells remain uninjured.

[Sidenote: [208]]

In this respect the cytases must be placed in the group of the “Endo-enzymes,” according to the nomenclature of Hahn and Geret[292]. These observers have carefully studied the proteolytic diastase of the yeast of beer which likewise acts inside the cells without ever being excreted. This diastase, to which they give the name of “yeast endotrypsin” (Hefeendotrypsin), presents in general an undeniable relationship with the phagocytic cytases, from which it is distinguished however by a greater sensitiveness to alkalis. Kutscher[293] in his researches on autodigestion in yeast has established analogous facts.

The cytases and endotrypsin are consequently endo-enzymes, as are also amoebodiastase, actinodiastase, plasmase (fibrin ferment) and the zymase of E. Buchner. All remain confined within the cells which have manufactured them and are not secreted or excreted, as are the sucrase and invertin produced by yeasts or Mucedinae.

Our present knowledge on the cytases is as yet far from perfect, which is not astonishing, seeing how recently the question has been brought forward. The cytases found in the serum of the same animal are the same, for we have seen that the macrocytase which dissolves red blood corpuscles is the same which digests spermatozoa; whilst the same microcytase digests bacilli, spirilla, and cocci. But in the serums of different species, the cytases differ. Thus the cytases of the dog are not the same as are those found in the serums of the rabbit or horse. Whilst the majority of the cytases are very sensitive to heat and are destroyed at a temperature of 55°–56° C., some, e.g. the microcytase of rat’s serum, resist this temperature and are only destroyed at 65° C., presenting, consequently, an example of cytase stable to heat similar to that discovered by Ehrlich and Morgenroth.

[Sidenote: [209]]

It is as yet very difficult to establish whether, besides the cytases, there exist other endo-enzymes within phagocytes, that is to say, soluble ferments which do not pass into the serums on the destruction of the phagocytes, but continue within these cells. Our present methods of investigation do not enable us to come to any conclusion on this point. We know only that the digestion of the formed elements is more complete inside the phagocytes than in the serums. Thus, as we have seen in Chapter IV, the best spermotoxic and haemolytic serums never digest either spermatozoa or the nuclei of the red corpuscles of birds. And yet these elements are completely dissolved in the phagocytic contents. Does this difference depend on the fact that, in the serums, we get only a very small part of the macrocytase, or upon the injurious influence of the alkalinity of the serums on the macrocytase which acts better in weakly acid media, or on the presence in the phagocytes of other endo-enzymes still unknown? These are questions to which at present no definite answer can be given.

Just as animal cells, when ingested by phagocytes during resorption (see Chap. IV), immediately become permeable to stains, so in natural immunity do micro-organisms taken into phagocytes acquire the same property. Very often, under the influence of the phagocytic action, the ingested micro-organisms become stainable by eosin (fig. 36). This eosinophile transformation has been observed in the cholera vibrio, the anthrax bacillus and in Proteus vulgaris. It is probably widely diffused among the phagocytised bacteria. This fact demonstrates clearly that at least some of the eosinophile granules are derived from foreign bodies ingested by the phagocytes. Others of these granules are probably the result of the transformation of soluble substances absorbed by the phagocytes. In fact, during inflammation, many microphages which contain no foreign solid body, may often be seen charged with a quantity of small pseudo-eosinophile granules.

Certain vibrios and bacilli, when ingested by microphages, become transformed, almost immediately, into spherical granules. The cholera vibrio undergoes the same transformation in the peritoneal exudation at the moment of phagolysis, as also in blood serum. The Bacillus coli, the typhoid bacillus, and certain other cocco-bacilli do not change in the least, or change very slightly in serum, but exhibit the transformation into granules when inside microphages. The macrophages, on the other hand, digest the same bacteria (vibrios and cocco-bacilli) without these bacteria presenting any signs of this change of form. The bacterial membrane resists the influence of the phagocytic digestion longer than do the contents, but is in the long run also completely digested. After the ingestion and destruction of micro-organisms by the phagocytes, débris of indeterminate form may, for long, be found in the cells, but I have never been able to demonstrate any solid excreta from them. We must suppose, then, that the undigested portions are not thrown out from the phagocytes.

[Sidenote: [210]]

When describing the solution of red blood corpuscles by normal serums, we have mentioned Ehrlich and Morgenroth’s view that the cytases are incapable of fixing themselves to these cells without the help of fixatives. They cite in support of their opinion several examples of fixatives (intermediary substances or “Zwischenkörper”) discovered by them in the serums of various species of mammals. Is this so with microcytase in respect to micro-organisms? If this soluble ferment is incapable alone of fixing itself upon the bodies of these parasites, the help of fixatives would be indispensable to it. The bactericidal property of the microcytase, then, would depend on the existence of another body (fixative) which, perhaps, might not owe its origin to phagocytes. The problem, then, has a wide general range.

In one of his memoirs, Bordet[294] had already raised the question of the existence of this sensibilising (or fixative) property in normal serums. By mixing two normal serums coming from different species, he was sometimes able to demonstrate the existence of such fixatives. Thus the cholera vibrios, which do not undergo granular transformation in either the normal serum of the horse (which is capable only of arresting their movements and agglutinating them into a mass) or in that of the normal guinea-pig, readily become transformed into granules when placed in contact with a mixture of the two serums. Bordet, however, refrains from any hasty generalisation on this observation and proposes to make fresh researches on this subject. Independently, Moxter[295] has attempted to demonstrate the presence of fixative in the normal serum of the guinea-pig. When deprived of cytases by heat, this serum is incapable of transforming the cholera vibrios into granules; but when fluid from the peritoneal exudation of the same guinea-pig is added, the transformation takes place very rapidly. Nevertheless, as this exudation was already, by itself, capable of producing Pfeiffer’s phenomenon, Moxter’s conclusions on the presence of the fixative in the normal guinea-pig’s serum cannot be accepted without a fuller analysis of the facts, and this demands fresh researches.

[Sidenote: [211]]

A recent investigation, carried out by Bordet[296] in collaboration with Gengou, devoted to the study of the absorption of cytases by micro-organisms that have been sensibilised by means of fixatives, also gives us information on the question which now occupies us. It was easy to demonstrate the presence of fixative in the serums in the case of the cholera vibrio and its allies, by reason of their transformation into granules, appreciable on microscopical examination. When a serum, which of itself is incapable of setting up this transformation, produces it directly we add another serum heated to 55° C., we must conclude that the latter fluid contains the cholera fixative, whilst the former contains only cytases. But, as the majority of bacteria do not undergo any analogous transformation in serums, we are, in these cases, without any criterion as to the presence of fixative. Bordet and Gengou have eliminated this inconvenience in determining the fixation of alexine by bacteria which undergo neither granular transformation nor any other visible change. A normal unheated serum, which always contains a sufficient quantity of cytases, is mixed with any micro-organism, e.g. with the anthrax bacillus or the cocco-bacillus of plague. The serum, decanted after a prolonged contact with these bacteria, remains quite as capable of dissolving the red corpuscles of a determined foreign species as it was originally. This proves that cytases remain in the serum and that they have not been absorbed by the bacteria. Repeat the same experiment with this difference, that instead of normal anthrax bacilli or plague cocco-bacilli we introduce into the unheated normal serum these bacteria after they have been sensibilised by the corresponding fixatives (that is to say, previously submitted to the influence of specific serums heated to 55° C.). After contact for a certain length of time with these bacteria the serum is no longer capable of dissolving the red corpuscles of a determined foreign species, thus demonstrating that the cytases have, thanks to the help of the fixatives, been linked to the bacteria. We see, therefore, that it is easy to determine whether a serum, whose properties are unknown, contains fixatives or not. It is heated to 55° C. and mixed with normal unheated serum to which bacteria are added. If, after contact with these latter the normal serum has lost the power of dissolving the red corpuscles (which it was capable of dissolving previously), it is because its cytases, thanks to the fixative which must be present in the heated serum, have been absorbed by the bacteria. In the other case, we conclude the non-existence of the fixative.

[Sidenote: [212]]

In their researches, Bordet and Gengou often employed normal unheated serums to which they added several species of bacteria. They demonstrated that in these mixtures the cytases remained intact or nearly so. These soluble ferments were scarcely, if at all, absorbed by the bacteria, which proves that in the normal serums there are no fixatives in any appreciable quantity. Of all their experiments the one that interests us most was carried out with Proteus vulgaris. This organism placed in prolonged contact with normal guinea-pig’s serum showed itself incapable of absorbing or fixing anything beyond the most minute quantities of the cytases. There is consequently no fixative for Proteus in normal guinea-pig’s serum, or, if any exists, it is only in negligible quantity. And yet this same Proteus vulgaris, when injected into guinea-pigs, was in a short time ingested and destroyed by the phagocytes which assure to the animal a natural immunity of the most stable character. The facility with which the leucocytes of the guinea-pig devour the Proteus follows, among others, from an experiment by Bordet[297] carried out with quite another object. A guinea-pig, very ill as the result of the injection into its peritoneal cavity of a very virulent streptococcus, contained in the peritoneal exudation a quantity of empty microphages incapable of ingesting these streptococci. At this critical moment there was injected into the same position a mass of Proteus vulgaris. “At the end of a very short time, it is seen that the leucocytes which energetically refuse to ingest streptococci greedily seize upon the new organism offered to them; and at the end of half-an-hour the whole of these organisms are found inside phagocytes.”

Here, then, we have an actual proof of the fact that the phagocytes, in order to rid the animal organism of a microbe and assure to it a natural immunity, have no need of any previous help from an extraphagocytic fixative. The phagocytes act, so to speak, motu proprio, and themselves bring about the resorption of the intruders. The question of fixatives in normal serums, then, loses its importance for us and their origin no longer presents any essential interest for the problem with which we are at present occupied.

[Sidenote: [213]]

Can we conclude, from the data just summarised, that the cytases, which in several respects approximate to the trypsins, have this further feature in common with them that they can act without the help of any fixative? It is known, as mentioned in Chapter III, that trypsin can digest alone, or in collaboration with enterokynase, that ferment of the intestinal juice which acts as such a powerful adjuvant to the pancreatic ferments. Is this also the case with the cytases? The fact that when Proteus vulgaris is placed in contact with normal unheated guinea-pig’s serum, it is incapable of absorbing cytases, although it is so readily digested by phagocytes, indicates rather that, for the fixation of cytases, the help of the fixative is indispensable. But, as this fixative is absent from the serum, and since, nevertheless, it must exist for the needs of digestion, it must clearly be concluded that it is found inside the phagocytes. Its quantity is perhaps so small that when it has passed into the serum its action is entirely lost or nearly so. Fresh researches are necessary to elucidate this delicate point.

But perhaps the phagocytes which, as we have just seen, can engage in a struggle with and ingest the micro-organisms without the latter being previously modified by the fixative, may be incapable of fulfilling their functions without the help of some other substance circulating in the blood plasma? Amongst these substances is one which manifestly acts upon the micro-organisms by rendering them motionless and agglomerating them into masses. This agglutinative property is met with in the normal fluids of many species of animals and is exercised upon many bacteria. It may be demonstrated not only in the blood serum, but also in the fluids of transudations and exudations and in certain secretions such as milk, tears, and urine. Little is known as yet of the mechanism of this agglutinative action, and we can the more readily refrain from entering into details concerning it as it is of no great importance from the point of view of natural immunity.

In the preceding chapter we have already spoken of the ingestion of cholera vibrios in the peritoneal cavity of guinea-pigs. In those cases in which the animals exhibit an effective resistance, the phagocytes devour the vibrios whilst they still exhibit very active movements. Even when a large majority are already seized by the leucocytes and only a few isolated free vibrios remain, these latter still continue to exhibit normal movements. These facts, repeatedly observed, clearly demonstrate that phagocytosis may take place without any previous agglutinative action; this does not, however, prevent the micro-organisms, when united into motionless masses, from being ingested by the leucocytes with greater ease.

[Sidenote: [214]]

In the case of the typhoid bacillus, one of the most active of bacteria, the same facts may be observed as in the case of the cholera vibrio. In animals that remain unaffected we often see the last free bacilli moving about actively between the leucocytes filled with microbes. In many other examples of natural immunity we constantly meet with phagocytes containing but a single or a small number of micro-organisms (streptococci, yeasts, etc.).

The presence of motile micro-organisms inside phagocytes proves also that it is possible for these cells to do without the help of agglutinative substance in carrying on their protective work. The most carefully studied case of the relations between natural immunity and agglutination is that met with in the anthrax bacillus. We owe it to Gengou[298], who at the Liège Bacteriological Institute carried out a very detailed investigation on this question. He showed that the bacillus of Pasteur’s first anthrax vaccine is agglutinated by the blood serum of a great number of animals. But he also showed that the serums which have the greatest agglutinative action on this bacillus do not come from the most refractory species. Human serum agglutinates most strongly the bacillus of the first vaccine (in the proportion of one part of serum to 500 parts of culture) but man is far from being exempt from anthrax. Pigeon’s serum, on the other hand, is completely without any agglutinative power, although this species resists not only the first vaccine but very often even virulent anthrax. The serum of the ox, a species susceptible to anthrax, is more agglutinative (1 : 120) than that of the refractory dog (1 : 100). There are, however, exceptional cases in which the agglutinative property corresponds to the degree of susceptibility. Thus the serum of the mouse has not the slightest agglutinative action on the bacillus of the first vaccine. But alongside this example is that of the rat, a species of moderate susceptibility to anthrax, whose serum possesses the least agglutinating power of all, acting only in the proportion of 1 : 10. All these facts fully justify the conclusion formulated by Gengou that “we cannot establish any relation between the agglutinating power and the refractory state of the animals to anthrax” (p. 319). This conclusion may be extended to the phenomena of the agglutination of micro-organisms and to those of natural immunity in general.

[Sidenote: [215]]

Amongst the properties of humours, there exists one which might play a part in natural immunity against micro-organisms. I mean the power possessed by the blood and certain other fluids of the animal body to neutralise the action of microbial poisons. Perhaps, it may be suggested, the phagocytes are not capable of commencing to do their work except after a previous action of antitoxins. After the neutralisation of the principal means possessed by the micro-organisms to injure the organism, these parasites, having been rendered innocuous, might be readily destroyed by the phagocytic cells. We have already had occasion to treat this fundamental question. Thus, we have insisted in the preceding chapters on the absence of any parallelism between immunity against micro-organisms and that against their toxins, taking as our examples anaerobic bacteria (tetanus bacillus, septic vibrio, bacillus of symptomatic anthrax) in connection with which phagocytosis takes place without any help from the antitoxic function.

We must now pass directly to the examination of the question of antitoxins in the fluids of animals naturally refractory to the micro-organisms and of the ultimate part played by them in this immunity.

[Sidenote: [216]]

Examples of the presence of antitoxic serum in normal animals are very rare. It might be supposed that animals endowed with natural immunity against micro-organisms and at the same time against their toxins, present an appreciable natural antitoxic power. Let us examine some of the more typical examples. The fowl enjoys a very marked immunity against the tetanus bacillus and its toxin; its blood and its serum, however, as demonstrated by Vaillard[299], exhibit no antitoxic power; this observation has been confirmed by several other workers. The rat is very refractory to diphtheria; it resists subcutaneous inoculation of a large quantity of diphtheria bacilli and vigorously withstands diphtheria toxin when injected anywhere but into the brain. It has been demonstrated by Kuprianow[300], in an investigation carried out under Loeffler’s direction, that the blood serum and the emulsion of the organs of the grey rat (Mus decumanus) possess no antitoxic property. This fact has been confirmed by other observers. Von Behring[301], in a review of the phenomena of immunity in general, sums up this question as follows: “we find no antitoxin in the blood of individuals that are naturally refractory.” There are, however, certain exceptions, perhaps only apparent, to this rule. Thus Wassermann[302] has shown that blood serum from healthy human beings is sometimes antitoxic to the diphtheria poison. The individuals who furnished this antitoxin maintained that they had never suffered from diphtheria. We know, however, that this disease is sometimes present in so benign a form that it may pass unnoticed. More conclusive appears the example of normal horses whose blood serum, as demonstrated by Meade Bolton[303] and Cobbett[304], is very often antitoxic for the diphtheria toxin. This property, however, is not found in every horse; in certain individuals it is entirely absent. This last fact affords an indication that the antitoxic property in the blood of horses has been acquired as the result of some affection produced by a bacillus allied to the diphtheria bacillus. This view has not yet been sufficiently examined and consequently cannot claim to be accepted as settled. Recently, Max Neisser and Wechsberg[305] have discovered an antitoxin in human blood which is capable of preventing the solution of the red corpuscles by the toxin of staphylococci. This antitoxic power varies considerably in different individuals and is probably to be accounted for by the fact that the staphylococcus is one of the most widely diffused organisms among the bacterial flora of the human body. The small lesions produced by these micro-organisms (acne, boils, etc.) are so frequent in man that they may readily bring about the production of an antitoxin. In this case, however, we have again an example of acquired antitoxic power.

The examples just summarised can in no way affect the general thesis that the phagocytes, in order to fulfil their microbicidal function in an animal endowed with natural immunity, have no need of any previous action of the body fluids to neutralise the corresponding toxins.

[Sidenote: [217]]

The facts and views analysed in these two chapters afford us a general picture of the phenomena exhibited in natural immunity against micro-organisms. The dominant feature is represented by the phagocytic reaction that is observed throughout the animal series and that is exercised against parasites belonging to all the microbial groups. Phagocytosis is exhibited not only by the macrophages but also, in a high degree, by the microphages which stand out as the defensive cells par excellence against micro-organisms. Their action is divided into a series of vital physiological acts, such as sensitiveness to the micro-organisms and their products, amoeboid movements which serve to ingest the micro-organisms, and into chemical and physico-chemical processes, such as the destruction and digestion of the devoured organisms.

The phagocytes enter into a struggle against the micro-organisms and rid the animal organism of them without requiring any previous help on the part of the body fluids. Phagocytosis, exercised against living and virulent micro-organisms, is sufficient to ensure natural immunity. The bactericidal power of the serum, which for a long time served as the basis for a humoral theory of immunity, represents merely an artificial property, developed in consequence of the setting free of the microcytase of the leucocytes that have become disintegrated after the blood has been drawn. The agglutinative power of the normal fluids of the body plays no important part in natural immunity.

The phagocytes, in order to fulfil their function, can attack micro-organisms that are capable of producing toxins. Any antitoxic action against these bacterial poisons is in no way necessary to allow of phagocytosis coming into action.

Taken as a whole, the data collected on natural immunity against micro-organisms clearly demonstrate that the destruction of these parasites in the refractory animal organism represents merely a special phase of the resorption of formed elements.

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