IV. RESORPTION OF THE FORMED ELEMENTS
Digestion in the tissues.—Resorption of cells in the Invertebrata.—Resorption of red corpuscles by the phagocytes of the Vertebrata.—Phagocytes.—Various categories of these cells.—Macrophages and microphages.—Part played by macrophages in the resorption of the formed elements.—Digestive property of the macrophagic organs.—Solution of the red blood corpuscles by the blood serums.—The two substances which operate in haemolysis.—Macrocytase and fixative.—Analogy of the latter with enterokynase.—Escape of the macrocytase during phagolysis.—Suppression of phagolysis.—Resorption of the spermatozoa.—Presence of fixatives in plasmas.—Origin of fixatives.
[Sidenote: [72]]
It is usually understood that nutritive substances must necessarily be subjected to the influence of the digestive juices in the gastrointestinal canal before they can be utilised for the nutrition of the organism. This is a very old idea. It was based on a well-known experiment by Schiff who injected several animals intravenously with solutions of cane sugar and egg albumen and others with the same substances after they had been artificially digested. In the first case the food substances passed into the urine, in the second they only appeared there when injected in large quantities.
[Sidenote: [73]]
At the recent International Congress of Medicine held in Paris in 1900, the question of extra-buccal nutrition was much discussed[90]. It has been accepted that fats, when injected into the subcutaneous tissues, are, at least in part, absorbed by the organism, but that carbo-hydrates and albuminoids are never absorbed. This is perhaps true from the point of view of clinical medicine. But, in principle, it must be admitted that food substances of very diverse natures, when introduced into the organism by channels other than the gastrointestinal canal, still undergo profound changes.
When we inject milk, blood serum, or white of egg, that is to say, materials very rich in albuminoid substances, under the skin or into the peritoneal cavity of laboratory animals, we find that after a time they disappear. At the same time they give rise to modifications of the organism which indicate that these injected substances have there undergone profound changes.
After injecting eel’s serum into rabbits, Th. Tchistovitch[91] found a substance in the blood of the injected animals which gave a precipitate with eel’s serum. Shortly afterwards Bordet[92] observed that the blood of animals into which he had injected cow’s milk acquired a new property: it gave a precipitate with this milk, a condition never observed in the serum of untreated animals.
The injection of white of egg into rabbits, carried out by Myers[93] and Uhlenhuth[94], brought about the same changes in the blood serum. The researches of the latter of these two observers have for our present purpose a special interest. He demonstrated first that the injection of white of egg into the peritoneal cavity of rabbits was followed by the appearance in the blood serum of these animals of a substance which precipitates egg albumen in vitro. Uhlenhuth then obtained this same acquired property of the blood in rabbits which had been made to swallow a considerable quantity of the white of hens’ eggs. Twenty-four days after the commencement of this regimen the serum of the rabbits precipitated white of egg in the test-tube. This example affords a marked analogy between the results of digestion in the alimentary canal and those of resorption into the tissues. Uhlenhuth points out, indeed, that his rabbits which received the injections of white of egg into the peritoneal cavity flourished under this treatment.
[Sidenote: [74]]
A certain number of similar examples are now recognised. They all indicate that various nutritive substances, when introduced into the peritoneal cavity or under the skin of animals, are retained there for a longer or shorter time and are subjected to certain modifying influences on the part of the organism. The proof that these substances are not eliminated intact by the kidneys has been furnished by a large number of experiments. Recently Lindemann[95] and Néfédieff[96], working in my laboratory, have established the fact that normal blood serum, when injected under the skin of animals, does not provoke albuminuria at all, or at least produces it in a very insignificant and transitory degree.
The mechanism by which the organism modifies these nutritive substances, introduced by a channel other than the digestive canal, is not as yet sufficiently known; and is therefore not easy to define. But we know, very definitely, that each injection of serum, whether of white of egg, milk or fatty matter, is followed by a rather considerable aseptic inflammation at the point at which these substances are introduced. We might conclude from this that the organism digests the food substances outside the gastro-intestinal canal, by means of an inflammatory reaction. In order to determine more exactly the phenomena that appear under these conditions, it may be useful to consider first, not the fluid substances but the solid elements that are introduced into the tissues and cavities.
Let us begin with the lower animals in which the anatomical organisation and all the functions are of a much more simple character than they are in the Vertebrata. In my Comparative Pathology of Inflammation (Lecture IV) I have directed some attention to the digestion of the Sponges.
The nutritive substances—small organisms—whether they may have entered by the small openings, so numerous on the surface of Sponges, or have been introduced through a rent in the body wall, undergo the same fate. They are seized by vibratile or amoeboid cells which ingest the food and digest it by an intracellular digestion. These two kinds of cells, which come under the category of Phagocytes, have a great resemblance to one another, and we may say that digestion and resorption are two very closely related phenomena.
[Sidenote: [75]]
When we examine somewhat higher Invertebrata, such as the Medusae or certain other Coelenterates, we can still trace a close analogy between the true digestion of the food that goes on within the epithelial cells of the entoderm and the resorption of certain foreign bodies which make their way by an extra-buccal channel into the intermediary tissue. Here these bodies are surrounded by amoeboid cells which fulfil their function as phagocytes by ingesting and digesting the substances that have come from outside.
It is, here, unnecessary to go over the whole gamut of the perfecting of the organisation of the Invertebrata, in its relation to the resorption of foreign bodies, especially as it has already been treated in my Lectures on Inflammation. Let us choose merely some of the more common and better-known representatives of the Invertebrata and dwell for a few moments on the phenomena manifested in their organism, into the midst of which have been introduced a few nucleated red blood corpuscles[97].
If a small drop of defibrinated blood from a goose be injected beneath the skin of a snail and another under the skin of a cockchafer larva, the red corpuscles are disseminated in the blood fluid which, of itself, is incapable of modifying them, but at the end of a few hours the leucocytes of the two invertebrates that we have chosen for the experiment will have ingested a certain number of the injected red blood corpuscles. The next day red blood corpuscles are still to be found intact in the blood plasma, but the great majority have been devoured by the leucocytes (Fig. 13). Inside these cells the red corpuscles undergo constant and marked changes. In the snail they become round and their walls permeable. In the vacuoles that are produced around the ingested red corpuscles dissolved haemoglobin is found (Fig. 14); a portion of this colouring matter passes into the nucleus of the red corpuscles, so that it also has undergone a profound change (Fig. 14). Many of the nuclei become emptied, only the peripheral layer remaining. This layer and the membrane of the red corpuscle are the parts that resist the action of the leucocytes longest and they are found for some time after their ingestion. The white corpuscles of the snail, having devoured one or more red corpuscles, may themselves become the prey of their fellows.
[Sidenote: [76]]
[Sidenote: [77]]
In the “ver blanc” (French popular name for the larva of the cockchafer) the phenomena of resorption of the red corpuscles of the goose resemble those just described. The blood plasma leaves intact the red corpuscles which undergo no change until they have been ingested by the leucocytes. The haemoglobin diffuses into the leucocyte, whilst the nucleus and the membrane persist for a very considerable period (Fig. 15), though they lose their normal aspect, shrivel, and become transformed into an irregular mass of brown pigment which may remain in the substance of the leucocyte (Fig. 15, p) for weeks.
Having once injected goose’s blood into snails and “vers blancs,” if we repeat the injection several times, the phenomena observed are invariably the same. The red corpuscles are unacted upon by the plasma and undergo the same changes within the leucocytes. These changes are in fact comparable to those described in the preceding chapter in discussing the intracellular digestion of the red corpuscles by the intestinal cells of the Planarians. In both cases the red corpuscles are seized by amoeboid cells and subjected to the influence of their contents. In the intestinal phagocytes of the Planarian, as in the phagocytes of the blood (leucocytes) of the snail and “ver blanc,” the haemoglobin diffuses through the wall of the red corpuscle, whose most resistant parts are the nucleus and the membrane. These resistant residual fragments, impregnated with haemoglobin, become brown in the Planarian, in the “ver blanc,” and also, but in a less degree, in the snail. The most appreciable difference consists in the formation of excretory vacuoles, containing concretions, in the Planarian, and the absence of these vacuoles in the blood phagocytes of the other Invertebrata. We have, however, less right to attribute a fundamental importance to this difference, in that the phenomena in the Actinians, which ingest the red blood corpuscles by the amoeboid cells of their entoderm, are in all respects (with the exception of the presence of these special excretory vacuoles) comparable to the phenomena observed in the Planarians. From the fact that in these two examples we have to do with a true intracellular digestion, it must be admitted that the modifications of the red blood corpuscles within the phagocytes of the blood in the snail and in the larva of the cockchafer, must also be placed in the same category of phenomena.
[Sidenote: [78]]
In order to make a more thorough study of this intracellular digestion in the phagocytes of the blood, we must direct our attention to larger and more highly organised animals than the snail and the “ver blanc.” Let us take, first, an example among the inferior cold-blooded Vertebrata. The red blood corpuscles of a few drops (0·25 c.c.) of the blood of a guinea-pig injected into the peritoneal cavity of a gold-fish (Cyprinus auratus) are not appreciably changed by the peritoneal fluid itself; but the numerous leucocytes that are found in the peritoneal fluid seize them and ingest them, just as do the phagocytes of the blood of Invertebrata, or the intestinal phagocytes in the Planarians and Actinians in the case of the red blood corpuscles of the goose. Each leucocyte of the Cyprinus ingests several red blood corpuscles and subjects them to intracellular digestion. The stroma of the red corpuscles becomes permeable; the haemoglobin diffuses into the nutritive vacuoles and at the end of a shorter or longer period the whole is dissolved and decolorised (Fig. 16). Here no brown pigment is produced and the red corpuscles are completely digested, leaving no “remains”; in this respect differing from the process in the Invertebrata mentioned.
This result depends, probably, partly upon the more feeble resistance offered by the non-nucleated red corpuscles of Mammals, and partly upon the more active digestive power of the leucocytes of Fishes.
As the result of several injections of guinea-pig’s blood into the peritoneal cavity of Cyprinus, the peritoneal fluid acquires new properties[98]. If, a fortnight after the first injection, a little of the peritoneal exudation in the gold-fish be withdrawn, it is found that a drop of the serum which floats on the surface produces, almost immediately, well-marked agglutination of the red corpuscles of the guinea-pig, this being soon followed by the rapid solution of these red blood corpuscles in the fluid. This new property, which does not exist in the untreated fish, also makes its appearance in the blood serum of Cyprini treated with guinea-pig’s blood. The experiment is very successful at a temperature of 18°–19° C.
As the solution or lysis of the red blood corpuscles in the serum is exactly like that which takes place within the leucocytes of Cyprinus, we are justified in assuming that, in both cases, it is produced by the same substance. And, since the solvent or haemolytic power of the serum is only acquired as the result of the intracellular digestion of the red blood corpuscles by the leucocytes, it is probable that the solvent substance represents the intracellular ferment derived from the leucocytes.
[Sidenote: [79]]
The subject we have just broached is of fundamental importance in connection with the study of resorption and of the phenomena of immunity dependent upon it. It is necessary, therefore, that we should go more fully into its analysis. With this object we must first review the processes that go on during resorption in the higher animals and continue our examination of the changes that injected or extravasated blood undergoes in various positions of the organism.
This study is rendered comparatively easy for us by the numerous researches that have been carried out by pathological anatomists for the purpose of ascertaining the fate of effusions or extravasations of blood so frequently met with in disease. It has long been known that in subcutaneous, cerebral and other haemorrhages, or in hepatised lungs, there are found in the escaped blood a great number of cells containing red corpuscles. As was mentioned in the preceding chapter, these cells were evidently amoeboid cells that had ingested red blood corpuscles. To Langhans[99] especially we owe a detailed study of the phenomena that follow extravasation of blood produced artificially in the subcutaneous tissue of the pigeon, rabbit and guinea-pig. In all these animals the haemorrhage is early followed by exudative inflammation, during which the leucocytes come up in great numbers and ingest the red blood corpuscles which are modified in the interior of the leucocytes. There is a formation or deposition of pigment and finally all traces of the red corpuscles disappear. In Mammals the pigment is brown or brownish, just as it is in the Planarians and in the “ver blanc”; in the pigeon it is green and resembles that found in the Actinians. In short there is a great analogy between the resorption of red corpuscles and the true intracellular digestion of the red blood corpuscles that goes on in the intestinal cells of the Invertebrata.
But what is the nature of these amoeboid elements that intervene in the resorption of the extravasated blood? At the period when Langhans carried out his investigation, we were unable to differentiate the cells at all satisfactorily. It is only since the publication of Ehrlich’s classic researches on the white corpuscles that we have been able to bring more order into this question. Thanks to the use of various aniline stains, Ehrlich was able to arrange the leucocytes found in the Vertebrata into several definite groups.
The question has already been touched upon in our eighth lecture on inflammation; it is therefore unnecessary to treat it here at length. We must, however, before entering on the analysis of the essential phenomena in the resorption of cells, as we now understand them, give a rapid survey of the different varieties of amoeboid cells that are found in the Vertebrata.
[Sidenote: [80]]
Beside mobile amoeboid cells, represented by several forms of white corpuscles, we must distinguish fixed amoeboid cells. These are permanently fixed in certain situations in the body; this, however, in no way prevents them from throwing out amoeboid processes in various directions and seizing foreign bodies or certain elements of the same organism. The nerve cells, the large cells of the splenic pulp and of the lymphatic glands, certain endothelial cells, the cells of the neuroglia, and perhaps some connective tissue cells, belong to the category of fixed amoeboid cells. All these elements, under certain conditions, are able to ingest solid bodies; consequently, they act as phagocytes. With the exception of the cells of the nerve centres, all these fixed phagocytes are of mesoblastic origin. It has been much discussed whether certain processes of the nerve cells may not really serve to seize foreign bodies and carry them into the cell contents. It appears to us that sometimes they undoubtedly do fulfil this function. For example, it is only by means of such amoeboid movements that leprosy bacilli can be introduced into the interior of ganglion cells and cells of the spinal cord[100]. We must not dwell on this question, as the phagocytic property of the nerve elements plays no part in the resorption of cells. On the other hand, the neuroglia cells contribute largely to this process and their phagocytic function is now admitted by many observers[101].
For long the large “dust” cells of the respiratory channels were looked upon as being epithelial cells which were capable of ingesting carbon particles, micro-organisms and other foreign bodies. The researches of N. Tchistovitch, carried out in my laboratory more than twelve years ago, made it evident that these elements are nothing more than white corpuscles that have immigrated into the alveoli and bronchi.
[Sidenote: [81]]
It is probable that the same is the case as regards the stellate cells of the liver, known as Kupffer’s cells. First described by Kupffer as cells of a nervous type, having long processes, they were later recognised by several observers as belonging to the endothelial tissue of the blood vessels of the liver. Kupffer[102] himself has accepted this view and in his recently published monograph on these stellate cells, he describes them as endothelial cells that have retained their independence. Some researches on the resorption of blood, of which I shall speak shortly, have led me to think that these cells are nothing but white corpuscles that have been arrested in the hepatic capillaries. I have asked Mesnil, head of my laboratory, to study this question for me. His investigation is not yet concluded, but the demonstration already made that the livers of guinea-pig embryos and new-born rabbits do not possess any Kupffer’s cells is an argument in favour of my hypothesis.
Certain white corpuscles have undoubtedly been often mistaken for epithelial or connective tissue cells. We must not conclude from this, however, that these elements are never capable of sending out amoeboid processes and of ingesting foreign bodies. It would, however, be useful to collect new and incontestable proofs of the accuracy of this thesis. In spite of this uncertainty, it may be accepted as fully demonstrated, that certain fixed amoeboid cells, such as the large elements of the splenic pulp, of the lymphatic glands, and of the omentum, play an important part in the resorption of cells. It is there that elements filled with red corpuscles and white corpuscles in process of being destroyed are so often found.
[Sidenote: [82]]
Just as certain fixed cells do not function as true phagocytes, so also in some leucocytes this function is undoubtedly absent. The suggestion has been made several times that any cell element, provided it be young, is capable of ingesting foreign bodies. The examination of white corpuscles proves exactly the contrary. The smaller white corpuscles found in fairly large numbers in the blood and the lymph, and which are commonly known as lymphocytes or small lymphocytes, are simply leucocytes with very little protoplasm which in this state never fulfil phagocytic functions. It is only when it becomes older, when its nucleus, single and rich in chromatin, becomes surrounded by an ample layer of protoplasm, that the lymphocyte becomes capable of ingesting and resorbing foreign bodies. Several authors, with Ehrlich at their head, still assign to these larger cells the same name—lymphocytes. Others, however, give them the name of large mononuclear cells. Confusion is thus possible, especially as Ehrlich includes under the same term the large mononucleated leucocyte, a very rare form of cell in human blood, which is distinguished by the greater staining capacity of its nucleus. To avoid this inconvenience I propose to designate the large lymphocytes by the name of blood macrophages and lymph macrophages (haemomacrophages, lymphomacrophages). This term is preferable to that of mononuclear leucocytes, especially as in exudations we frequently meet with macrophages with two and even several sharply separated nuclei. Giant cells, moreover, are nothing but polynucleated macrophages. On the other hand, the leucocytes so often designated by the name of polynuclear in reality contain but a single nucleus. Even Ehrlich, who introduced this term, acknowledged its imperfection but he retained it for some time because it was already very extensively used and could, he thought, give rise to no misunderstanding. In his excellent work on anaemia, published jointly with Lazarus[103], he now agrees that the name of “cells with polymorphous nuclei” would be more exact.
These polymorpho-nuclear leucocytes are very numerous in the blood and in many exudations and are distinguished by the greater selective affinity of their nucleus for basic aniline dyes and by a certain tendency of the protoplasm to become stained by acid aniline colours, such as eosin. The true macrophages are without granulations, but the “polymorpho-nuclear” leucocytes contain many. These granulations are sometimes “eosinophile,” “pseudo-eosinophile” (or “amphophile”) or even “neutrophile” (as in man and the horse).
[Sidenote: [83]]
These two main groups of leucocytes are generally distributed in the Vertebrata; and we already meet with them in one of the lowest vertebrate forms—the Ammocoetes (the larva of the lamprey). The macrophages of this fish present all the principal characters of the group to which they belong (protoplasm without granules, easily stained with methylene blue, large nucleus rich in nuclear juice). In the “polynuclear” forms in this lower vertebrate the protoplasm does not stain with methylene blue, but assumes a faint rosy tint with eosin; the single nucleus is divided into several lobes. In Vertebrates which are much higher in the scale these characters change. Thus in the cayman (Alligator mississipiensis), according to the researches of Madame Podwyssotsky, carried out in my laboratory, the two great varieties of leucocytes are readily found in the blood, lymph and exudations. The macrophages, however, especially in the exudations, are very often furnished with two or several nuclei, whilst the small leucocytes possess only a single nucleus, which is not divided into lobes. In spite of this peculiarity the two groups are readily distinguished. The staining reactions of the macrophages are identical with those of the corresponding corpuscles in all the other Vertebrata; whilst the small leucocytes, in spite of the absence of a polymorphous nucleus, are easily recognised by their eosinophile granulations and by the special affinity of the nucleus for basic aniline dyes. Under these circumstances it would be quite inappropriate to designate those leucocytes, which are really polynuclear, that is to say, possessing two or several nuclei, by the name of “mononuclear,” and to reserve the name of “polynuclear” for the small corpuscles which possess only a single nucleus undivided into lobes. For this reason it is much more rational to retain for these so-called polynuclear cells my proposed name of microphages. Moreover, the microphages are true phagocytes. It was formerly thought that the eosinophile leucocytes, such as the “‘overfed’ cells (Mastzellen)” of Ehrlich, which are identical with the clasmatocytes of Ranvier, never ingested foreign bodies. But, (especially after the researches of Mesnil[104]), we have been compelled to change our opinion on this point. The true eosinophile cells are able to devour foreign bodies, especially micro-organisms, and must therefore be regarded as phagocytes belonging to the group of microphages.
[Sidenote: [84]]
It is the peculiar merit of Ehrlich and of his school that they have thoroughly established the fact that, in Mammals at any rate, the two principal groups of white cells are distinguished, amongst other characters, by the diversity of their origin. The lymphocytes and the mononuclear cells are developed in the spleen and lymphatic glands, whilst the “polynuclear” cells arise from the granular mononucleated myelocytes of the bone marrow. This is now generally accepted as applicable in the great majority of cases. In Ammocoetes, however, the two chief varieties of leucocytes arise from one and the same organ, regarded by several observers as a kind of primitive spleen, which runs along and in part surrounds the intestine. Mesnil has been good enough to make sections of this primitive organ in which it may be demonstrated that the macrophages and the microphages in the larva of the lamprey have the same seat of origin. Frog tadpoles and Cartilaginous Fishes also possess microphages which do not arise from the bone marrow, since in them this tissue is completely absent. But even in Mammals, at least in certain pathological conditions, Dominici[105], in a research executed with much care and a perfect technique, has demonstrated the myelogenous transformation going on in the spleen. Thus in the adult rabbit affected with septicaemia by the typhoid bacillus, he found in the spleen developmental centres of amoeboid elements which, normally, appear to develop in the bone marrow only, i.e. the megacaryocytes, or large cells with budding nuclei, the neutrophile myelocytes (amphophiles), basophiles and eosinophiles.
The mesoblastic phagocytes of the Vertebrata are divided, then, into fixed phagocytes—the macrophages of the spleen, endothelia, connective tissue, neuroglia, and muscle fibres—and free phagocytes. These latter are sometimes haemo- or lympho-macrophages, sometimes microphages. The fixed macrophages and the free macrophages resemble one another so greatly that it is very often extremely difficult, if not impossible, to differentiate them. For this reason it is often very useful, when the exact origin of a large phagocyte is not known, simply to name it “macrophage.”
[Sidenote: [85]]
The two principal groups of phagocytes—(1) fixed and free macrophages, (2) microphages—are distinguished not only by their morphological characters; they also give evidence of very marked physiological differences. All phagocytes are endowed with amoeboid movement which allows them either to move about freely or merely to put out protoplasmic processes. These movements are regulated by a very great sensitiveness, often different in the two groups. Besides a tactile sense, the phagocytes possess a kind of sense of taste or chemiotaxis which enables them to distinguish the chemical composition of the substances with which they come in contact. The existence of this chemiotaxis could be anticipated from the moment that an important part in the life of the organism began to be ascribed to the amoeboid cells. Leber[106], Massart and Charles Bordet[107] have, however, demonstrated it by rigorous experiment. Following the method used by Pfeffer to demonstrate the chemiotaxis of the vegetable spermatozoids and of Bacteria, these investigators introduced into the bodies of higher (rabbits and guinea-pigs) and lower (frogs) Vertebrates small glass tubes filled with different solutions (peptone, broth, salts, bacterial products, etc.). The leucocytes, guided by their positive chemiotaxis, made their way into the tubes and there formed plugs which were often very voluminous; when, on the other hand, the chemical composition of the solutions excited their negative chemiotaxis, the leucocytes avoided the tubes.
[Sidenote: [86]]
Having acquired information as to the chief characters of the leucocytes, we may ask, To which group do those amoeboid cells, which, according to the observations of Langhans and many other investigators, bring about the resorption of the red corpuscles of the blood, belong? This resorption goes on more rapidly and is observed much better if, instead of introducing blood of the same species into any part, we inject defibrinated blood, or red blood corpuscles from which the serum has been removed by washing, from another species of Vertebrate. It will be found best to inject the nucleated red corpuscles of lower Vertebrates into Mammals, or (as already described above) to introduce the non-nucleated red blood corpuscles of Mammals into lower Vertebrates. In all these cases the injection of such blood or corpuscles sets up an aseptic inflammation which attracts a large number of free phagocytes to the seat of injection. In subcutaneous, peritoneal or intraocular exudations produced under these conditions, we find, in addition to a number of microphages, many macrophages. Whilst the former ingest the injected red corpuscles merely in isolated cases, the positive chemiotaxis of the macrophages manifests itself much more actively. In the resorption of the red blood corpuscles the more important part is played by the macrophage. To get a clear idea of the phenomena that accompany this resorption, let us take a concrete example. Inject defibrinated goose’s blood into the peritoneal cavity of guinea-pigs[108]. During the first few hours after injection the oval nucleated red corpuscles are found intact in the fluid of the peritoneal lymph. The plasma, by itself, exercises no destructive or solvent action on the red corpuscles of the goose.
Immediately after the injection the lymph of the peritoneal cavity begins to show important changes. The white corpuscles which, in the normal condition, are fairly abundant, disappear almost completely; some small lymphocytes presenting their ordinary aspect may indeed be found, but the few macrophages and the microphages that remain show signs of very grave lesions. They lose their mobility, run together into clumps and become incapable of ingesting foreign bodies. At this moment the phagocytes undergo a critical change which we have designated by the name of phagolysis. This condition lasts for about an hour, sometimes it continues longer, according to case and circumstance, but after this the peritoneal fluid becomes filled with leucocytes that have newly come on to the scene. These cells make their way, by diapedesis, through the walls of the congested vessels of the peritoneum. A true aseptic inflammation is produced which induces an exudation of a large number of white corpuscles, amongst which are found microphages and still more numerous macrophages. The latter show a very pronounced positive chemiotaxis towards the injected red corpuscles of the goose. Soon after their appearance, that is to say two or three hours after the injection of the blood, the macrophages send out very small protoplasmic processes and affix them to the surface of the red corpuscles. There follows an aggregation of the macrophages of the guinea-pig with the red corpuscles of the goose and characteristic masses, in which can be recognised both kinds of cells, are produced. This union with the very small pseudopodia is the first stage in the ingestion of the red corpuscles by the macrophages (Fig. 17). The red corpuscle, seized by amoeboid processes, passes into the interior of the macrophage. This macrophage seldom rests contented with ingesting a single red corpuscle. Usually it devours a large number and sometimes enormous macrophages may be seen filled with a score of red corpuscles.
[Sidenote: [87]]
If the quantity of goose’s blood injected into a guinea-pig is large (5–7 c.c.), the ingestion of red corpuscles by the macrophages continues for a considerable period—often for three to four days. During the whole of this time a certain number of the red corpuscles remain free in the peritoneal plasma, but, in spite of this prolonged stay, none of them undergo extracellular solution.
[Sidenote: [88]]
The red blood corpuscles, anchored by the amoeboid processes of the macrophages, at first present a normal appearance. Later their membrane begins to wrinkle, but as soon as they have passed within the phagocytes the wrinkles disappear and the corpuscles regain their normal aspect. If a little neutral red solution be added to a drop of peritoneal exudation (Fig. 18) we observe that the nucleus of the ingested red corpuscle and even its contents are stained red, whilst the red corpuscles adherent to the surface of the phagocytes retain their normal yellow colour. This reaction enables us to see that the red corpuscles are seized by the macrophages whilst still in their normal condition, but that they undergo a change immediately after they have been ingested. Little by little the devoured corpuscles are digested within the phagocytes. The haemoglobin diffuses into the contents of the macrophage through the stroma, which has become permeable; the nucleus of the ingested red corpuscle also becomes stained by the haemoglobin. Part of this colouring matter is excreted by the phagocyte. The body of the red corpuscle is pretty soon digested, but the nucleus, impregnated with haemoglobin, persists for a much longer period. It divides into several fragments, recognisable by their yellow colour, and in certain cases these remnants of red corpuscles may be met with for weeks in the interior of the macrophages. These macrophages do not remain permanently in the peritoneal fluid. Some (3–4) days after injection the lymph of the peritoneum contains only leucocytes that have newly come up and which contain neither red corpuscles nor their remains. We must open the guinea-pig to find any macrophages that have devoured red corpuscles. They are to be met with in large numbers in the glandular portion of the omentum, in the mesenteric glands, in the liver and in the spleen. They are fairly easily recognised by the characteristic aspect of the débris of the red blood corpuscles. Having devoured the red corpuscles the macrophages leave the peritoneal fluid and the digestion is completed in the positions just mentioned. In the liver they are seen as large mononuclear cells often with highly developed processes. In this condition they remind one of Kupffer’s stellate cells—a fact that suggested to me the idea that these elements are nothing but white corpuscles which have immigrated into the vessels of the liver.
Following up the fate of the macrophages that have resorbed the red blood corpuscles, we find them in the large hepatic vessels, in the vena cava and even in the blood of the heart. But in these latter situations they contain merely a few scarcely recognisable traces of their prey. These phagocytes, which left the blood during the inflammation that followed the injection of red corpuscles of the goose, re-enter it, having fulfilled their function, during the final period of the resorption. This resorption must undoubtedly be regarded as an intracellular digestion. When we compare the essential phenomena taking place inside the macrophages containing red blood corpuscles with those we have described in the intestinal phagocytes of the Planarians or Actinians after a meal, the analogy between the two becomes very apparent. In both cases the red blood corpuscles undergo a marked change which results in a diffusion of the haemoglobin. The membrane and nucleus of the red blood corpuscles persist longer but they also are ultimately digested. The excretion of haemoglobin from the phagocytes, just mentioned in the case of the macrophages of the guinea-pig, is also observed in the Actinians, whose coelenteric cavity is tinted by a rose-coloured solution.
[Sidenote: [89]]
We have seen that in the Actinians intracellular digestion takes place in a distinctly acid medium, whilst in the intestinal cells of the Planarians it takes place in one that is only weakly acid. The macrophages of the guinea-pig, during the resorption of red blood corpuscles of the goose, carry on the digestive process in a medium which shows a still weaker acidity. When made to ingest granules of blue litmus there is no change of colour. Nor does alizarin sulpho-acid give any reaction, probably owing to the fact that it exerts a toxic action on the protoplasm of the macrophages. If, however, we add to a drop of the peritoneal exudation of a guinea-pig, containing macrophages filled with red blood corpuscles of the goose, a little of Ehrlich’s 1% solution of neutral red, the red brick tint at once makes its appearance in the content of these phagocytes. This coloration is identical with that described in the Amoebae which digest Bacteria or in the intestinal phagocytes of the Planarians. It may, then, be regarded as an indication of weak acidity. This coloration is maintained for some hours, after which it gives place to complete decoloration, a phenomenon that must be attributed, as in many other cases, to the neutralisation of the acid by the alkaline protoplasm that has been macerated in the fluid after the death of the macrophages.
The example we have chosen—the destruction of red blood corpuscles of the goose by the macrophages of the guinea-pig—may serve as a prototype of the resorption of formed elements in general. If, instead of red blood corpuscles of the goose, we inject into the guinea-pig’s peritoneal cavity pigeon’s or fowl’s blood, the essential phenomena will be the same. The red blood corpuscles will always induce positive chemiotaxis, especially of the macrophages, which in turn will ingest the nucleated red corpuscles. It may be that in certain cases, when fowl’s blood containing red corpuscles that are not very resistant is injected, a certain number of the corpuscles immediately undergo a partial solution in the peritoneal fluid[109]. Here also the stromas and the nuclei of all the red blood corpuscles, as well as many of the corpuscles unacted upon by the plasma of the phagolysed exudation, undergo digestion inside the macrophages.
[Sidenote: [90]]
When, instead of blood, we inject white corpuscles from the bone marrow, spleen or lymphatic glands of animals into the peritoneal cavity, we may still observe their final disappearance in the macrophages. The spermatozoa of man or of various mammals (bull, rabbit, guinea-pig, etc.), when injected into the peritoneal cavity of the guinea-pig or rabbit, are well adapted for this line of investigation. Here again the immediate result of injection is the very marked phagolysis of the leucocytes. This phenomenon gives place to an exudative inflammation which brings into the peritoneal cavity a number of phagocytes. These, especially the macrophages and in a much smaller degree the microphages, devour the spermatozoa which in no case are dissolved, even partially, in the plasma of the exudation. The macrophage seizes the spermatozoa which sometimes, by the active movements of their flagella, exhibit great vitality. At the end of several hours all the spermatozoa are found inside phagocytes where they are completely destroyed. The flagellum is digested first, but the head and medial portion soon suffer the same fate. Neutral red reveals the feebly acid reaction, perhaps with even more distinctness than in the case of the red blood corpuscles.
The résumé of Langhans’ investigation given in this chapter would lead us to expect that resorption in the subcutaneous tissue will follow the same rules as that going on in the peritoneal cavity. As a matter of fact, blood injected at this position sets up a diapedesis of phagocytes which ingest the red blood corpuscles. In some cases only is there a partial solution of these corpuscles in the fluid of the subcutaneous exudation. It is for this reason that goose’s blood, injected under the skin of a guinea-pig, gives rise to a fluid exudation coloured a bright rose red by the dissolved haemoglobin. This haemoglobin is derived from red blood corpuscles which are damaged by the goose’s blood serum that was added to the plasma of the exudation. The stroma and nuclei of the red blood corpuscles cannot, however, be dissolved in this fluid. They undergo the same fate as the red corpuscles that have remained intact, that is to say they are ingested by the macrophages which immigrate into the subcutaneous tissue and which finally digest all these elements. The cells, less fragile than certain red corpuscles, are, in the subcutaneous tissue, as in the peritoneal cavity, destroyed solely in the interior of the phagocytes.
[Sidenote: [91]]
The analogy between the modifications undergone by the red blood corpuscles and other cells inside the macrophages and the changes that take place in the intestinal cells of Planarians and Actinians, suggests that the resorption of formed elements must undoubtedly be regarded as a true intracellular digestion. It would, however, be a very important matter to be able to support this conclusion by even more convincing proofs. The study of the artificial digestion that is observed in vitro in the case of the macerated mesenterial filaments of Actinians has furnished a very valuable argument in favour of the enzymatic nature of intracellular digestion. Animal exudations are not well adapted for this special line of study. We can only obtain them as the result of the injection of different substances, solid or fluid, which are greedily absorbed by phagocytes. If we collect the exudations at a moment when the number of these cells is still considerable we must withdraw along with them many digestive substances which interfere with our observation. We may therefore with advantage turn our attention to masses of phagocytes collected in organs. As it is mainly the macrophages which effect the resorption of cells, it is evident that we must choose the centres where they are formed in order to investigate the digestive ferments. Let us take, then, the lymphatic glands of the mesentery, the glandular portion of the omentum and the spleen, the three pre-eminently macrophagic organs, and let us see if, with an extract of them, prepared with physiological salt solution (0·75% of sodium chloride), any digestive effect is to be obtained.
Macerate the three organs mentioned of a guinea-pig and mix the extracts thus obtained with red blood corpuscles of the goose, corpuscles that have already given us information in connection with the phenomena of resorption in the living organism. In almost all the guinea-pigs a solution of the red blood corpuscles of the goose by the extract of the glandular portion of the omentum may be observed. The mesenteric glands likewise give an extract which in most cases has a solvent action. The extract from the spleen is only active in a limited number of cases. In all these examples the extracts from macrophagic organs bring about the solution of the haemoglobin, but leave intact the membrane and nucleus of the corpuscles. In this respect there exists, then, a certain difference between this and the digestion of red corpuscles in the macrophages of exudations, where the membrane and even the nucleus are in the end completely dissolved. This difference may be explained by the fact that in the preparation of the extract in physiological salt solution, one part only of the soluble digestive ferment may be set at liberty.
[Sidenote: [92]]
The solvent action of extracts of macrophagic organs must in fact be attributed to the presence of a soluble ferment in the cells of which these organs are made up. As the diastases are distinguished, in general, by their great sensitiveness to heat, we tried the action of our extracts after a preliminary heating, when it was found that a temperature of 56° C., applied for three quarters of an hour, completely abolished the solvent action of the extracts upon the red blood corpuscles of the goose. The soluble ferment of macrophagic organs, to which we propose to give the name of macrocytase[110] or macrophage ferment, is in many respects analogous to the actino-diastase of Mesnil, described in the preceding chapter.
With a view to obtain more complete information on the cytases I suggested to Tarassewitch that he should make a detailed study of them; this he has carried out in my laboratory. He has demonstrated that the macrophagic organs of other mammals than the guinea-pig, especially those of the rabbit and dog, exert the same solvent action on the red blood corpuscles. He has also established the fact that this action applies not only to the red corpuscles of the goose but extends also to those of several other birds and mammals. Tarassewitch succeeded in confirming the injurious action of heat on macrocytase. Extracts of macrophagic organs which contain much debris in suspension, when heated for an hour at 55°·5 C. in certain cases lose their solvent property for red blood corpuscles; sometimes this temperature brings about merely a weakening of the macrocytase. In order to destroy it surely and completely, the suspensions must be heated at 58°·5–62° C. for an hour. If, however, instead of heating the entire suspension, we first pass it through filter paper, the clear fluid filtrate is deprived of its diastatic action even after it has been heated at 55°·5 C. for three quarters of an hour.
[Sidenote: [93]]
Of all the other organs of which extracts have been kept in prolonged contact with the red blood corpuscles of birds, the pancreas alone has shown a very well-marked digestive action. Extracts of the salivary glands exerted a feeble solvent action on a certain quantity of the red corpuscles. The other organs, such as the liver, kidneys, brain, spinal cord, ovary, testicles, suprarenal capsules and placenta, exercised no such action. Even bone marrow, in agreement with my results published some years ago, showed itself quite inactive.
The blood serum of guinea-pigs which I employed in my researches, as well as that of the animals examined by Tarassewitch, has not shown itself capable of dissolving the red blood corpuscles of the goose, although the macrophagic organs dissolve them easily. It has long been known, however, that the serum of the blood of many animals will destroy the red corpuscles of a different species. This demonstration was afforded during the period when attempts were being made to transfuse the defibrinated blood of mammals, especially of the sheep, into man. This practice had to be abandoned, in consequence of the difficulties resulting from the solution of the human red corpuscles. Later, Daremberg[111] and Buchner[112] set themselves to study this haemolytic action of serums systematically. They found that it was due to a particular substance to which Buchner gave the name of alexine or protective substance. Of indeterminate chemical composition, this substance is allied to albuminoid substances. It is destroyed when heated to 55°–56° C. and only acts in the presence of certain salts. When these salts are removed from the serum by dialysis, it loses its haemolytic power; but as soon as the salts are replaced in proper proportion this power reappears. Later, Buchner[113] compared the action of alexine to that of soluble ferments and referred it to the category of the digestive diastases. According to him the same alexine is capable of dissolving the red blood corpuscles of several species of Vertebrates. Bordet[114], in a series of researches made in the Pasteur Institute, confirmed this view. He came to the conclusion that the alexines of the various species of animals differ from one another. Thus, the alexine of the blood serum of the rabbit is not the same as that found in the serum of the guinea-pig or dog. Nevertheless each of these alexines is capable of exerting a solvent action on the red blood corpuscles of several species.
[Sidenote: [94]]
[Sidenote: [95]]
Ehrlich and Morgenroth[115], in a series of memoirs on the solution of red blood corpuscles, have combated the idea that there is only a single alexine in one and the same serum. Moreover, they state that alexine always requires for its action the aid of another substance, and that matters are much more complicated than at first sight appears. They maintain that in each normal serum a number of different substances are found, each one of which only attacks a single species of red blood corpuscle. They point out that the solution of the red corpuscles by the normal serum takes place through the combined action of two different substances and cite several cases where a normal serum, after being heated to 55° C. and so deprived of its haemolytic power, again becomes capable of dissolving the red corpuscles when some normal serum from another species, which of itself is destitute of the solvent property, is added to it. Let us quote an example from Ehrlich and Morgenroth. The normal serum of the goat readily dissolves the red blood corpuscles of the rabbit and guinea-pig, but if heated for half-an-hour at 55° C., it loses this power. On the other hand, the normal serum of many horses shows itself powerless to dissolve the red corpuscles of these rodents. Here, then, are two serums, equally incapable of effecting the solution of the red corpuscles of the rabbit and guinea-pig. Yet, when they are mixed together and to them a few drops of blood from one of the rodents cited is added, haemolysis takes place readily. The heated goat’s serum then, has, retained in it something that resists a temperature of 55° C., a substance which, by itself, leaves the red blood corpuscles intact; but which, when combined with a second substance present in the horse’s serum, causes their solution. Ehrlich gives to the first substance, that is to say that found in the heated goat’s serum, the name of intermediary body (“Zwischenkörper”). The second substance, present in the unheated horse’s serum, is designated by him the complement. In order that a normal serum may dissolve the red corpuscles, it is not sufficient that it should possess a single substance, the alexine of Buchner. It must, to exert this action, contain two distinct substances which are very often found together in the same normal serum. Unheated goat’s serum was only capable of dissolving the red blood corpuscles of the rabbit because a particular complement and intermediary substance were both present. Deprived of its complement at 55° C., the serum is solvent only when we add to it another substance that is contained in the normal serum of a different species (horse). Continuing their researches in this direction, Ehrlich and Morgenroth have come to the conclusion that the normal serum of a single species may contain several intermediary substances, each one acting on a single species of red blood corpuscles. Further, that normal serum must contain several or even many different complements.
[Sidenote: [96]]
Ehrlich and Morgenroth carried on researches on the intermediary substances in normal serums and found several in addition to that already mentioned. The serum of the normal dog readily dissolves the red blood corpuscles of the guinea-pig. When heated to 57° C. it loses this property; but with the addition of normal guinea-pig’s serum the property is regained. In the serum of the normal dog there exists, then, besides the complement, at least one intermediary substance. The same result can be obtained with several combinations of serums of normal mammals, heated or unaltered[116]. Yet it often happens, as Ehrlich and Morgenroth themselves point out, that the demonstration of the presence of the intermediary substance in normal serums is accompanied with marked difficulties. Bordet, also, who has studied this question very thoroughly, often failed completely in his attempts to make normal serums, that were incapable of producing haemolysis, active by the addition of heated serums of other species of animals. Thus he observed that normal fowl’s serum readily dissolves the red corpuscles of the rabbit. When heated to 55°–56° C. this serum loses its haemolytic power which cannot be restored by the addition of any normal serum. He thinks therefore that, in this example, haemolysis is produced solely by the alexine, without the assistance of any intermediary substance in the serum of the normal fowl. P. Müller[117], whilst confirming Bordet’s experimental results, considers that, in this case also, there is the intervention of an intermediary substance. When he mixed heated fowl’s serum with a small quantity of unaltered fowl’s serum the solution of the red corpuscles of the rabbit is not brought about. When, however, instead of adding a little unheated normal fowl’s serum, he added the same quantity of serum from a fowl previously treated with physiological salt solution, the red corpuscles of the rabbit were dissolved without any difficulty. Müller explains this difference as due to the fact that the serum of the treated fowl contains more complementary substance than does that of the normal fowl.
We see, then, from this example that the analysis of the phenomena taking place in the solution of the red corpuscles by normal serums is beset with very great difficulties. For this reason it is much more profitable to make researches in this direction, using more active serums, where the demonstration of the two substances can be made simply and exactly. This desideratum has been supplied by J. Bordet, when preparateur in our laboratory; he described an easy method of increasing the haemolytic power of serums.
As stated above, guinea-pigs that have received an intraperitoneal injection of goose’s blood digest the corpuscles, although the peritoneal fluid exerts no haemolytic action. In vitro, the extract of their macrophagic organs certainly dissolves the red corpuscles, whilst the blood serum usually fails to do so. Now, if a second or a third injection of goose’s blood be made into the peritoneal cavities of the same guinea-pigs, partial solution of the corpuscles takes place in the peritoneal plasma and the serum of the blood acquires new properties: it becomes capable of clumping the red corpuscles, that is to say of agglutinating them; afterwards it dissolves them in vitro.
[Sidenote: [97]]
J. Bordet[118] has shown that the injection of the blood of one species of Vertebrate (mammal or bird) into the peritoneal cavity or under the skin of an animal of a different species, always produces in the blood serum of the latter the haemolysing substance. This haemolysing substance is specific or nearly so, that is to say it dissolves the red corpuscles of the species which has furnished the injected blood and also, but more feebly, the red corpuscles of allied species. Consequently, with guinea-pig’s serum, treated with goose’s blood, we obtain the greatest solvent action on the red corpuscles of the goose, though there is a certain haemolysis of the red corpuscles of some other birds. This rule, thoroughly established by Bordet, has been the starting-point for a large number of researches on haemolysis and amongst others of those which bear on the intermediary substance of normal bloods.
Bordet demonstrated very definitely a fact of fundamental importance—that in the blood serums of animals treated with blood from a different species, there exist two distinct substances which only dissolve the red blood corpuscles when they are combined. Here the duality of the haemolytic agent cannot be doubted, as it may in certain examples of normal serums. Each time that we deprive the serum of a treated animal of its solvent action by heating at 55°–56° C., this property can be restored to it with certainty by the addition of a little normal serum which, by itself, is incapable of bringing about haemolysis. The heated serum of these injected animals loses the power of dissolving the corresponding red corpuscles, but it retains its other acquired property—the agglutination of the corpuscles. The red corpuscles, aggregated into voluminous masses quite visible to the naked eye, remain intact indefinitely, if left in the prepared and heated serum. But as soon as we add to them a trace of normal blood (taken from one of a number of species of Vertebrates), the solution of the red corpuscles is not long in taking place. Under these conditions an action of two substances is set up; one of these substances is found in the heated serum of the injected animal, and the other in unheated normal serum. The first of these substances which not only resists a temperature of 55°–56° C., but stands, without alteration, heating to 60°–65° C., corresponds to the intermediary substance of Ehrlich. By Bordet it has been termed “substance sensibilisatrice[119].” The second substance, a common one, found in normal serums and destroyed at 55°–56° C., is the alexine of Buchner and of Bordet, or the complement of Ehrlich.
[Sidenote: [98]]
The ease with which one can demonstrate the co-operation of two substances in the haemolysis by the serums of animals treated with the blood of a different species, is due to the fact, that during the course of this treatment the animal organism produces a quantity of an intermediary or sensibilising substance. In fresh animals which have not been treated, it is often very difficult to demonstrate the presence of this substance. Bordet has established the fact that the serum of animals which have been injected several times with the blood of a different species, contains almost the same amount of alexine as does untreated serum. On the other hand, the sensibilising substance makes its appearance in large quantity as the result of these injections. Von Dungern[120] has confirmed this result and contributes the interesting fact that the sensibilising substance is found even in great excess in the serum of treated animals. When he adds to this serum blood that has not been heated, he produces a haemolysis that is more than thirty times as active as when the serum of the prepared animal alone is used. From the quantitative point of view, then, there is no relation between the amount of the two substances in the serum of prepared animals.
It may be suggested that the sensibilising or intermediary substance is the same as that which produces the agglutination of the red corpuscles. But careful researches have thoroughly demonstrated the difference between the two substances that have this character in common, both resist heating to 55°–60° C. and even beyond this point.
[Sidenote: [99]]
Having established this co-operation of two substances in haemolysis the intimate mechanism of their action was next studied. Here I must give pride of place to the discovery by Ehrlich and Morgenroth that the intermediary (or sensibilising) substance links itself to its corresponding red corpuscles. A serum, capable of dissolving the red corpuscles of a different species, is heated to 56° C. which causes it to lose this solvent property. When a certain number of these corpuscles are added to it, such corpuscles remain intact although they are agglutinated. It is sufficient, after some hours of contact, to centrifugalise the mixture in order to separate a limpid serum from the mass of red corpuscles, the former being now entirely deprived of its intermediary substance, that is to say it has become incapable of dissolving the red corpuscles even with the addition of a large quantity of the “complement” (normal serum, unheated). On the other hand, the red corpuscles, having fixed (linked) all the intermediary substance, dissolve very rapidly when placed in contact with normal serum which contains the necessary quantity of the complement (or alexine). This fundamental experiment has been confirmed and repeated by many observers and has now become classic. The idea that the intermediary (or sensibilising) substance links itself to the red corpuscle, without dissolving it, is generally accepted and may be regarded as permanently settled. We should do well, then, instead of designating by all sorts of synonyms the substance in serums which resists the action of a temperature of 55°–65° C., to apply to it, once for all, the name of fixative substance or simply that of fixative. This name is short, expresses the essential character of the substance and gives rise to no misunderstanding, as do the other names proposed up to the present (amongst them that of philocytase employed by myself in some of my earlier publications).
Another of Ehrlich and Morgenroth’s experiments has furnished the proof that the complement does not fix itself to the red corpuscles only. A normal serum, unheated, which, by itself, is quite as incapable of dissolving the red corpuscles as the fixative alone, is mixed with some defibrinated blood. After the centrifugalisation of this mixture, it is easy to demonstrate that the supernatant fluid has lost none of its complement (alexine), whilst the red corpuscles have fixed none.
If, instead of an inactive serum, we take a serum which is capable of dissolving the red corpuscles and which consequently contains the two haemolysing substances, and if we place it in contact with the corresponding red corpuscles, at a temperature between 0° and 3° C., the solution will not take place (Ehrlich and Morgenroth). Under these conditions the fixative certainly attaches itself to the red corpuscles, but the alexine remains in solution, unused. It is only necessary, however, to heat the mixture up to 30° C. to bring about rapid haemolysis.
[Sidenote: [100]]
From their very ingenious experiments, as a whole, Ehrlich and Morgenroth conclude that the fixative possesses two different affinities: one for the red corpuscle and another for the complement. Of these two affinities the stronger is that which links it to the red corpuscle, for this is manifested at a very low temperature. In order that the fixative may combine with the complement a much higher temperature is requisite. Ehrlich comes to the conclusion that the molecule of the fixative possesses two haptophore groups, or groups capable of chemical combination. The first of these links it to a corresponding molecule of the red corpuscle to which he gives the name of receptor; the second combines the fixative with the molecule of the complement and in this way introduces the latter into the red corpuscle. These investigators give a diagram which greatly facilitates the understanding of their hypothesis (Fig. 19). They seek to prove that the combinations of the fixative with the red blood corpuscle and with the complement follow the law of definite multiples and that these phenomena must, in consequence, be looked upon as being of a purely chemical character.
The hypothesis advanced by J. Bordet does not accord very well with the theory we have just set forth. He could never convince himself that the fixative combines with the complement. He was of opinion rather that the fixative, retained by the corpuscle, exercises upon it a mordant action which enables it to absorb the alexine. The alexine is supposed to attach itself to the sensibilised red blood corpuscle as a dye attaches itself to a mordanted element. Bordet rests his interpretation mainly on the fact that the absorption of alexine by the sensibilised corpuscles does not follow the elementary laws of chemical combination, especially those of definite multiples.
Nolf[121] has sought to define more accurately the part played by these two substances in the solution of the red blood corpuscles. He agrees with Bordet, that in this phenomenon the fixative plays the same part that the mordants do in dyeing. Linked to the red corpuscle the fixative renders it more greedy for alexine, exactly as the mordant facilitates the fixation of the dye on the fibre of the textile fabric. Under these conditions the alexine (complement), finding itself in large quantity inside the red corpuscle, exercises upon it its hydrating action, thus bringing about the diffusion of the haemoglobin and often even the solution of the corpuscular stroma.
Nolf compares the solvent action of alexine upon the red corpuscle to that of certain mineral salts, such as ammonium chloride. He passes in review the various properties of alexines and finds them very similar to the solvent action of certain salts. Even the peculiarity of alexine, of remaining inactive at a temperature of 0°–3° C., is shared by ammonium chloride which, alone of all the salts studied by Nolf, exercises no solvent action under these conditions. But Nolf found it impossible to push these analogies further, and especially to sensibilise, by the fixative, the red corpuscles to the action of quantities, which were of themselves inactive, either of ammonium chloride or of any other salt.
[Sidenote: [101]]
London[122] hoped by fresh experiments to solve the problem of the mode of action of the two substances which act in haemolysis. He pronounced in favour of the theory that they entered into chemical combination with the red corpuscles. But the facts accumulated up to the present do not enable us to make a positive statement as to the exact nature of the reaction which is set up during the solution of the red blood corpuscles; this is not astonishing in view of the fact that it is impossible to isolate the haemolysing substances in a pure state.
It may, however, be admitted that the action of alexine (complement) comes under the category of phenomena that are produced by soluble ferments. Buchner[123] maintains that there is an analogy between this substance and the diastases (or enzymes); Bordet[124], from the appearance of his first publications on haemolysis, has expressed himself in favour of this view. Ehrlich and Morgenroth[125], in their two first memoirs, very distinctly put forward the same idea. “We shall not deceive ourselves”—they say—“if we attribute to the addiment (syn. complement, or alexine) the character of a digestive ferment.” In one of their last memoirs[126] they no longer express themselves in so decided a fashion. Nevertheless we are still quite justified in maintaining this proposition. The substance which dissolves the red blood corpuscles of Mammals or a portion only of those of Birds, undoubtedly presents very great analogies to the digestive ferments. As has been mentioned repeatedly, it is very sensitive to the action of heat and is completely destroyed by heating for one hour at 55° C. In this respect it closely resembles the macrocytase of macrophagic organs which also dissolves red corpuscles. As it is the macrophages which ingest and digest the red blood corpuscles in the organism, it is evident that alexine is nothing but the macrocytase which has escaped from the phagocytes during the preparation of the serums.
[Sidenote: [102]]
We know that the leucocytes contain quite a series of soluble ferments of which some are set at liberty after the blood has been drawn from the vessels. It is thus that plasmase, or fibrin-ferment, is set free from the leucocytes to combine with fibrinogen to produce the clot. This is not the only soluble ferment of leucocytic origin. It has been known for some time that in addition to this coagulating ferment the leucocytes contain ferments which are especially digestive or decoagulating. Thus Rossbach[127] has demonstrated the presence of amylase in the leucocytes of different organs, especially the tonsils. Arthus has confirmed this discovery and Zabolotny[128] has completed it by his observations on the phenomena which appear in the peritoneal cavity of animals into which wheat flour or starch were injected. He observed that the small granules are quickly ingested by isolated leucocytes, whilst the large granules are surrounded by quite a layer of phagocytes. He agrees with several other writers, that the amylase found in defibrinated blood has its origin in leucocytes.
[Sidenote: [103]]
Leber[129], in the course of his researches on inflammation, made the observation that the pus of a hypopyon that was absolutely aseptic digests coagulated fibrin at a temperature of 25° C. and liquefies gelatine very readily. Achalme[130] has confirmed this and has added several other interesting data. He investigated the soluble ferments of pus and directed his attention amongst others to experimental pus, set up by the injection of spirit of turpentine. In addition to amylase and a ferment which liquefies gelatine, Achalme has discovered in pus, saponase (lipase), casease, and a ferment closely allied to trypsin. This last readily digests fibrin and also attacks coagulated white of egg; in the products of this digestion Achalme found peptone but could not always obtain leucin and tyrosin. He never succeeded in demonstrating the presence of sucrase, inulase, emulsin or lactase in pus. On the other hand he found large quantities of oxydase, thus confirming the discovery of Portier[131] who was the first to demonstrate that these ferments met with in the blood are, in the living animal, found inside leucocytes. By a large number of experiments, carried out on most diverse representatives of the animal kingdom, Portier was able to establish the important fact that the oxydases which are found in many organs or in the fluid of blood withdrawn from the organism really originate in leucocytes as they deteriorate and break up. In this respect, then, they resemble fibrin-ferment very closely.
To complete the list, already considerable, of leucocytic ferments, I must further cite the anticoagulating soluble ferment whose existence in Mammals has been so well demonstrated by Delezenne.
All this evidence encourages us, then, to support the thesis that alexine is one of the numerous intraleucocytic soluble ferments and that it only passes into the fluids as the result of rupture or of damage to the phagocytes. Nolf (l.c.) has recently pronounced against this view; we must therefore examine his arguments closely. In the first place he takes his stand on the analogies between the solution of the red blood corpuscles by the serums and by certain salts. It must not be forgotten, in connection with his theory, that haemolysis is but one example, out of many, of the action of alexines. Of all the formed elements the red corpuscles are the most delicate; they are readily broken up by all sorts of agents (moderate heat, water, salts, etc.). Further, there are numerous other cells (white corpuscles, spermatozoa, and inferior organisms) which resist the action of salts much better, which, nevertheless, are very injuriously affected by the action of the alexines.
Nolf lays special stress on the experiments in which, after keeping red blood corpuscles in prolonged contact with active serums, he has looked in vain for the peptone reaction. He prepared his mixtures in sealed tubes or flasks, and kept them in an incubator at 37° C. for 24–48 hours, or even for weeks. Under these conditions the haemoglobin is transformed into metahaemoglobin, but peptones never appear. Nolf concludes therefrom “with confidence, that the alexines do not exert the slightest peptonising effect on the albuminoids of the corpuscle” (l.c. p. 672).
[Sidenote: [104]]
To this conclusion it must be objected that peptone is not the only product of the digestion of albuminoids by soluble ferments. Under certain conditions the disintegration is carried much further, in others it is arrested at an earlier stage. Thus human urine which contains pepsin, never gives the peptone reaction with fibrin; the digestion of the latter only goes on up to the stage of protalbumose. When, however, the urinary pepsin is fixed on flakes of heated fibrin which are submitted to digestion in acidulated water the digestion proceeds further and gives as final products deuteroalbumose and peptone[132]. Now, under the conditions in Nolf’s experiments the digestion would be very quickly stopped, because, at the temperature of 37° C., alexine very soon loses its strength. Investigators who have experimented with haemolytic serums know well that, even when kept at a low temperature, alexine may lose its activity within 24 hours.
It has been mentioned above that Nolf sought in vain for a parallel between haemolysis by salts and that by serums, in what relates to the action of the fixative. He was unable to find anything comparable to this action amongst salts, although digestion by soluble ferments offers undoubted analogies. I need only recall further the discovery of enterokynase, the soluble ferment of the digestive juice of the dog, which actively stimulates the action of pancreatic ferments, and especially that of trypsin. The recent researches of Delezenne (communicated to the International Congress of Physiology held at Turin in September 1901) support this conclusion in a very important fashion. As already pointed out in Chapter III the enterokynase of the intestinal juice exerts an action comparable with that of the fixatives of haemolytic serums. Alone, it does not act as a solvent ferment, but when it attaches itself to the fibrin it aids the action of the trypsin in a marked degree. In pancreatic digestion enterokynase plays the part of the fixatives in the solution of red corpuscles.
[Sidenote: [105]]
The analogy between the resorption of formed elements and intestinal digestion extends even beyond this. When we inject, into the peritoneal cavity or under the skin of various animals, blood from a different species, the blood serum of the former becomes haemolytic for the red corpuscles of the latter. The solution of these red corpuscles is effected by the alexine of the serum, whose activity is rendered very great owing to the presence of a quantity of specific fixative. This same fixative appears also in the fluids of animals to whom, instead of injecting blood, we simply give it by the mouth. This fact has been established by Metalnikoff[133].
Another fact in favour of the close relationship between the fixatives and enterokynase consists in the presence of both in the lymphatic (lymphopoietic) organs. The fixatives which aid the solution of red corpuscles are found specially in the mesenteric glands. Enterokynase, as demonstrated by Delezenne, is found not only in the intestinal juice, but also in Peyer’s patches, the solitary glands, the mesenteric glands, and the leucocytes of exudations and of the blood.
Supported by these various facts we are quite justified in regarding the haemolysing substance of serum as containing two soluble ferments, of which one, alexine, corresponds to trypsin, the other, the fixative, resembling enterokynase. The alexine, whose nature is gradually disclosing itself with more precision, should bear the name of cytase or cell-ferment. The cytase of the macrophagic organs, or macrocytase, comes under this category. According to the researches of Tarassewitch it also acts more actively when there is added to it some of the fixative found in the serum (heated to 56° C.) of prepared animals.
[Sidenote: [106]]
We have said that in the living animal the macrocytase is localised in the phagocytes of the organs and of the blood. Thus, when goose’s blood is injected into the peritoneal cavity of the guinea-pig the red blood corpuscles are digested within the macrophage and not in the fluid of the peritoneal exudation. When, however, the same kind of blood is injected a second or a third time, it is found that a certain number of the red corpuscles become permeable and lose their haemoglobin, which they give up to the fluid of the exudation, and only the membrane and the nucleus remain. These are at once ingested by the macrophages which under these conditions manifest a real excess of activity. Instead of sending out small processes, as they do after the first injection of blood, these phagocytes move about like true Amoebae, sending out broad pseudopodia, and ingest not only the remains of the red corpuscles but also those still intact[134] (Fig. 20). Under these conditions macrocytase must undoubtedly be found in the peritoneal plasma. It is, however, easily demonstrable that this ferment was not preformed in the fluid but has escaped from the leucocytes that have undergone phagolysis. After the rapid injection of alien blood the phagocytes of the peritoneal lymph gather into clumps, become immobile, and for a time lose their phagocytic power. It is only after the lapse of a longer or shorter period that the leucocytes recover from the phagolysis, arrive in great numbers in the peritoneal cavity and display their phagocytic energy.
If the damage to the phagocytes—the phagolysis—is the actual cause of the setting free of the intraleucocytic ferment, we have only to prevent this phagolysis in order to inhibit the solution of red blood corpuscles in the fluid of the exudation. For this purpose it is sufficient to prepare guinea-pigs (which have already received several injections of goose’s blood) by means of an injection of fresh broth, of physiological salt solution, or of carbonic acid into the peritoneal cavity on the eve of the decisive experiment. Such injection at once provokes phagolysis, which is then followed by an abundant exudation of leucocytes. When, next day, a dose of red blood corpuscles of the goose (deprived of serum by centrifugalising) is introduced into the peritoneal cavity thus prepared phagolysis is no longer produced, or very feebly, and is of very short duration. Under these conditions the solution of the red corpuscles by the peritoneal fluid is reduced to a minimum, and in its place an extremely rapid and considerable ingestion of red corpuscles by the macrophages may be observed. In order that the experiment may be completely successful it is advisable to use goose’s blood heated to 37° C. or thereabouts for the injection.
[Sidenote: [107]]
Even when the red corpuscles of the goose are introduced, not into the peritoneal cavity but into the subcutaneous tissue of guinea-pigs that have received several injections of goose’s blood, we can easily prevent the extracellular solution of the red corpuscles which takes place, as already indicated, in the normal guinea-pig. As in this case the goose’s serum which is mixed with the corpuscles contributes to the haemolysis, it must be suppressed by centrifugalising the defibrinated goose’s blood and by washing the corpuscles with normal saline solution.
Collectively, the facts I have just described clearly indicate that the phagocytes must be regarded as the source of the haemolytic ferment. The macrocytase remains in the body of these cells so long as they are in a normal condition; but immediately they are injured, in consequence of the sudden introduction of foreign substances into the peritoneal cavity, a portion of the macrocytase escapes and acts on the red corpuscles as if it had been employed in vitro.
As the conclusion I have just formulated is of fundamental importance in the study of resorption and immunity it is necessary to support it by as many arguments as possible. For this reason, therefore, I feel obliged to draw the attention of the reader to another example of the resorption of formed elements.
We have already spoken of the resorption of spermatozoa in the peritoneal cavity, and of the part played by the macrophages in this phenomenon. As a result of this resorption, just as after that of red blood corpuscles, the organism acquires new properties of the same character. Landsteiner[135] and the writer[136] have shown that the blood serum and the peritoneal fluid of animals that have been injected with the spermatic fluid of bull, rabbit, or man, become spermotoxic, that is to say, they render the corresponding spermatozoa motionless and kill them. These fluids, however, never acquire the power of dissolving, even partially, these elements. The disappearance and final solution of the spermatozoa is only effected within phagocytes, and almost exclusively in the macrophages.
[Sidenote: [108]]
Moxter[137] has demonstrated that the spermotoxin which appears in the serum of prepared animals consists of two substances, corresponding to those present in the haemolytic serums. These are the macrocytase (alexine, complement) and the fixative (intermediary or sensibilising substance). For him they are identical with those which dissolve the red corpuscles. Without dwelling on the subject we may say that the macrocytase which dissolves the red corpuscles and that which arrests the motion of the spermatozoa are really identical in the same species of animal, as is accepted and developed by Bordet. On the other hand, it is impossible to accept Moxter’s theory of the identity of the two fixatives. They must be regarded as different; this we have attempted to prove in one of our memoirs[138] and is in accordance with the law of the specificity of fixatives in general.
The question which interests us more especially at this moment is where are these two constituent substances of the spermotoxin to be found and how do they behave in the living organism? This question has been very thoroughly studied by Metalnikoff[139] in my laboratory. His experiments have been closely followed by me, and in presenting their principal results I can bear witness to their correctness.
The spermotoxin obtained by Metalnikoff is distinguished from the haemotoxins we have discussed up to the present in that they develop, not as a result of the injection of cell elements from a different species, but as a result of the introduction into the organism of spermatozoa from the same species, the guinea-pig. We have here, then, to deal with what has been termed autospermotoxin.
The serum of the normal guinea-pig acts but feebly on the spermatozoa of this species, which, under its influence, remain motile for several hours. When, however, guinea-pigs have received one or several injections of the spermatozoa of their own species, their serum and peritoneal lymph become distinctly toxic and render the spermatozoa motionless in a few minutes. In male guinea-pigs so prepared the serum acquires this toxic property not only for the spermatozoa of other male guinea-pigs, but likewise for those of the individual itself which furnishes the serum. This latter, then, becomes distinctly autospermotoxic.
[Sidenote: [109]]
If the spermotoxin were diffused in the plasma and other fluids of the guinea-pig which furnishes it, it ought to render motionless the spermatozoa contained in the genital organs. Experiment demonstrates, however, that this is not the case. If the male organs be removed from a guinea-pig whose serum is very autospermotoxic in vitro, we find, especially in the epididymis, a mass of very virile spermatozoa which for a long time retain their motility in physiological salt solution. The macrocytase, then, has not reached the spermatozoa in the living animal; this is because it is not found in the plasmas. Let us inject into a guinea-pig, whose serum is strongly autospermotoxic, one portion of sperm into the subcutaneous tissue and another portion into the peritoneal cavity. In the first site a soft oedema, filled with transuded fluid, in which the very active spermatozoa retain their motility for a couple of hours, is produced. In the peritoneal fluid the same spermatozoa become motionless in a few minutes. This great difference is explained by the fact that, under the skin, there are no, or almost no pre-existing leucocytes, whilst in the peritoneal fluid they are abundant. The phagocytes injured by the introduction of sperm into the peritoneal cavity, abandon a portion of their macrocytase, sufficient to render the spermatozoa motionless. But when Metalnikoff injected physiological salt solution into the peritoneal cavity of his autospermotoxic guinea-pigs, and then, on the following day, a quantity of sperm, the spermatozoa continued very active for more than an hour. In this case phagolysis is very transitory and insignificant; it is soon followed by a great afflux of leucocytes which bring about a rapid ingestion of the spermatozoa. Many of these elements are devoured in a living state; for even when their body is enclosed in the macrophage, their tail, left outside, continues to move very actively.
[Sidenote: [110]]
All these experiments demonstrate that in the normal state the macrocytase remains within the phagocytes and only escapes during phagolysis, or at the moment when the blood, after it has been withdrawn from the organism, coagulates. Is it the same for the fixative? It is easy to prove that this soluble ferment circulates in the plasmas of the living organism. We have already said that the spermatozoa of a guinea-pig whose serum is very autospermotoxic, remain alive for some time in the physiological salt solution. But if we introduce them, in vitro, into the serum of a normal guinea-pig they remain motile but a short time (some 10–20 minutes), whilst the spermatozoa of a normal guinea-pig will live in the same serum for several hours. This difference is explained by the fact that the spermatozoa of the autospermotoxic guinea-pig, although very active, have absorbed the fixative during the life of the animal. This fixative is, as we have stated, found in the body fluids and has been able to penetrate to the male organs. Here the spermatozoa become charged with the fixative and, once transported into the serum of the normal guinea-pig, rich in macrocytase, they lose their movements very quickly. At the same time the spermatozoa used for control, not having absorbed any fixative, are able to live for a long time in the same serum.
As the macrocytase remains fixed to the phagocytes there can be no doubt as to its origin; it is elaborated by these cells. Whence however comes the fixative which is free in the body fluids and which is precisely the substance that is developed in so large a quantity in the treated animals? The exact solution of this question is not easy; nevertheless there are many facts which indicate that this fixative is also of phagocytic origin. We know already that the serums of normal animals contain only small quantities or sometimes, perhaps, none of the fixative. This fixative only appears abundantly as the result of the resorption of the corresponding elements, red corpuscles or spermatozoa. This resorption, as we have said, is almost exclusively the work of the macrophages. It is just in those cases where the red corpuscles, injected into the peritoneal cavity of an animal of the same species, pass directly into the lymph, without being injured or, save exceptionally, ingested by the phagocytes, that the fixative is not formed. When the red blood corpuscles of the goose, introduced with defibrinated blood below the skin of a guinea-pig, undergo there a partial solution in the fluid of the exudation, and where the phagocytosis is more limited than in the peritoneal cavity, the production of fixative is small. When the injection of the same goose’s blood is made into the peritoneal cavity of a guinea-pig and is followed by complete phagocytosis, the fixative is produced in greater abundance. There exists, then, in all these cases a constant relation between the degree of phagocytosis and the amount of the fixative produced. As this fixative facilitates the access of the cytase to the cells and as the resorption of these elements takes place specially in the macrophages, we are bound to come to the conclusion that the fixative is a second phagocytic ferment which is produced in abundance during the process of intracellular digestion. Only, instead of remaining in the substance of the phagocytes, this fixative is in part thrown out from these elements. It passes into the plasma of the blood and into the other fluids and ends by disappearing from the organism, probably being eliminated by the excretory channels.
[Sidenote: [111]]
In the Invertebrata, where, as we have seen, the alien red blood corpuscles are also digested within the phagocytes, we have never been able to demonstrate any haemolytic property of the blood fluid, even after repeated injections of blood. We must conclude from this that in these animals the quantity of fixative is merely sufficient to bring about the solution of the red corpuscles which are within the phagocytes. In the case of fishes and higher animals (we may recall the example of the red corpuscles of the guinea-pig when resorbed into the organism of the gold-fish) the production of the fixative is much more abundant, and this ferment can be easily demonstrated by its action in vitro.
This over-production of a ferment which acts in the phagocytic resorption, finds its analogue in the passage of certain digestive ferments, such as amylase and pepsin in man and the dog, into the blood and urine, as mentioned in the preceding chapter.
One of the best arguments in favour of the thesis here developed, has been furnished to us by the analysis of the phenomena observed in connection with the autospermotoxic serums of the guinea-pig. This idea of autotoxins was originally put forward by Ehrlich in his memoirs, published in conjunction with Morgenroth and already repeatedly cited. Ehrlich asked himself whether the organism which resorbs, not red corpuscles of an alien species, but red corpuscles of its own species, would also be capable of developing haemolytic substances. With this object he injected blood obtained from goats into these same goats or into other individuals of the same species. He and Morgenroth[140] were, under these conditions, able to obtain isotoxic serums, that is to say serums which dissolve the red corpuscles of the goat, coming from other individuals than those which had been treated by the blood and which furnished the serum. In order to obtain this result, however, they had to inject, not unaltered blood but blood mixed with water. The red corpuscles of the unaltered blood pass readily into the circulation of the animal of the same species, without being attacked by the phagocytes. Now, we know from the experiments of Bordet that the stromas of the red corpuscles suffice for the production of the fixative, whilst the haemoglobin does not incite to the development of this ferment by the organism. As the stromas, injected with a mixture of blood and water, must be devoured by the macrophages, we can readily understand that these phagocytes may serve for the elaboration of the fixative.
[Sidenote: [112]]
The resorption of the red corpuscles and that of spermatozoa which we have presented as examples, may serve as types for the resorption phenomena of formed elements in general. When other species of cells are introduced into the organism, the resulting process always reveals the same character: inflammatory reaction with preponderant intervention of the macrophages; intraphagocytic digestion of the introduced elements; excessive production and excretion of the fixatives. Whilst the macrocytase is always the same in the same species of animal, the fixatives are different and specific. In addition to the haemofixatives and spermofixatives already described, we may obtain, as the result of the injection of the corresponding cells, leucofixatives, nephrofixatives, hepatofixatives, trichofixatives, etc. It does not enter into our programme to treat the subject here[141]. We wish simply to insist on those aspects of the resorption of cells which are closely connected with the problem of Immunity. In the next chapter we must, however, recur to certain features of the phenomena of resorption.
V. RESORPTION OF ALBUMINOID FLUIDS
Resorption of albuminoid substances.—The precipitins of blood serum which appear as a result of the absorption of serums and of milk.—Absorption of gelatine.—Leucocytic origin of the ferment which digests gelatine.—Anti-enzymes.—Antirennet.—The anticytotoxins.—Antihaemotoxic serums.—Their two constituent parts: anticytase and antifixative.—Action of anticytase.—The antispermotoxins.—Origin of anticytotoxins.—Ehrlich’s theory on this question.—Origin of antihaemotoxin.—Origin of antispermotoxin.—Production of this antibody by castrated males.—The antispermofixative produced when the spermatozoa are excluded.—Distribution of spermotoxin and antispermotoxin in the organism.
[Sidenote: [113]]
We stated at the beginning of the last chapter that various fluid substances of very complicated chemical composition may be absorbed by the tissues and utilised by the organism without requiring to be modified by the digestive juices of the intestinal canal. We must now describe, exactly, the phenomena observed in these cases and endeavour to establish the mechanism of the absorption of fluids in the living organism.
[Sidenote: [114]]
We have already cited the examples of blood serum, milk, and white of egg, all of which are readily utilised by the organism which receives them directly into the peritoneal cavity or below the skin. The proof that these substances are modified—digested by the tissues, is furnished by the observation that their injection necessarily brings about appreciable changes in the properties of the blood. Th. Tchistovitch[142], in a research carried out in the Pasteur Institute, was the first to demonstrate that the resorption of the blood serums of the eel and horse by the organism of the rabbit, excites in the blood of the latter animal the production of specific precipitates. The blood serum of rabbits that have been vaccinated against the toxic eel’s serum gives a precipitate with eel’s serum; the serum of rabbits treated with horse’s blood gives a similar precipitate with horse’s serum, etc. This property has since been confirmed and studied by several observers, who have made use of it for the recognition of human blood in medico-legal investigations[143].
Bordet[144] has made the discovery that intraperitoneal injections of the milk of cows into rabbits provokes in the blood serum of the latter the property of giving a specific precipitate with cow’s milk only. This precipitation bears a great resemblance to the coagulation of casein; which, however, does not justify us in identifying the precipitating substance with rennet. This fact has been confirmed for several other species of milk, and Schütze[145], in an investigation carried on in the Berlin Institute, essayed to apply it to the differentiation of the various kinds of milk. In the same order of ideas, researches have been made on the artificial precipitins that develop in the blood as the result of injection of white of egg and other albuminoids[146]. Leclainche and Vallée[147] have prepared animals in such a fashion that their serum produces a precipitate with urinary albumen. The biological precipitin reactions are more sensitive than any of the chemical reagents properly so called. These specific substances in the serums must be looked upon as belonging to the group of soluble ferments, approximating to the fixatives rather than to the cytases, since they are unaltered by being heated to 56° C. Their action gradually declines after passing 60° C. but is only destroyed at a temperature beyond 70° C.
[Sidenote: [115]]
An analogous soluble ferment has been discovered in the blood serum of animals treated with injections of gelatine. We owe to Delezenne, who has studied this question in his laboratory at the Pasteur Institute, the most important and most complete data on the resorption of gelatine. The blood serum of normal animals possesses only a very feeble power, sometimes even none, of liquefying gelatine. When however this substance is injected several times, the serum, as is the rule for the formed elements and quite a series of fluid substances, acquires a much more pronounced activity. The gelatine, without giving any precipitate, is simply dissolved and will no longer solidify when it is cooled. The ferment of the serum that produces this effect resembles the precipitins in that it withstands the action of a temperature of 56° C. and is only destroyed beyond 60° C. Like the trypsins it acts in a weakly alkaline, neutral, or weakly acid medium; but digestion takes place best in a slightly alkaline medium.
The question of especial interest to us is that of the origin of this ferment which digests gelatine. If several c.c. of a 10% solution of this substance be injected into the peritoneal cavity of a laboratory animal, there is provoked with certainty, within a few hours, a marked leucocytosis of the peritoneal fluid. A considerable afflux of leucocytes, amongst which the microphages are even more numerous than the macrophages, takes place. When to a hanging drop of such an exudation a trace of Ehrlich’s neutral red solution is added, there appears almost at once an intense coloration of the numerous droplets inside the two kinds of leucocytes. It is, therefore, manifest that the gelatine excites a powerful positive chemiotaxis of the mobile phagocytes and that it is absorbed by these cells. This experiment demonstrates that the phagocytes can not only ingest solid bodies, such as the various formed elements, coloured granules, etc., but that they are also capable of absorbing fluid substances introduced into the tissues or cavities of the organism.
The data brought forward by Delezenne demonstrate very clearly the part played by the mobile phagocytes in the digestion of gelatine. He obtained his best results in the dog. We know that it is easy in this animal to provoke an aseptic exudation, very rich in leucocytes. This exudation when deprived of its serum and washed with physiological salt solution gives a solution which exerts a feeble digestive action on gelatine. If the exudation be produced in a dog that has previously received several injections of this substance, we obtain leucocytes whose extract, obtained by the same method, will digest gelatine much more actively. The digestive power of the leucocytes of the treated dog is sometimes five times greater than that of the leucocytes of the normal dog. Here, then, we undoubtedly have an acquired digestive power which reveals a great reinforcement of the phagocytic activity.
[Sidenote: [116]]
In the prepared dogs the leucocytes have a much greater digestive action on gelatine than has the blood serum of the same animals, a fact which indicates that the source of the soluble ferment must be sought for in the phagocytes themselves. The results of these researches are of great service to us in the study of immunity properly so called.
For some time past attempts have been made to show that the soluble ferments, diastases, or enzymes, are closely allied to albuminoid substances. Nencki and Mme Sieber[148] support this view by their recent researches on the chemical composition of pepsin. In all the above cases there is this in common between the two categories of substances, their absorption by the organism is followed by the appearance in the blood of antagonistic ferments. Just as after the injection of milk, white of egg, serums, etc. into the cavities or tissues, specific precipitins are produced, so the injection of certain enzymes provokes the formation in the organism of antienzymes or antidiastases.
[Sidenote: [117]]
It has been known for some time that the blood serum of many animals prevents the action of certain enzymes. Thus Röden has shown that normal horse’s serum retards or even completely prevents the coagulation of milk by rennet. It has often been observed, too, that normal serums hinder, more or less, the digestion of albuminoids by trypsin. It is only quite recently, however, that we have begun to prepare antienzymes by the injection into animals of corresponding enzymes. Thus, Hildebrand[149] has succeeded in obtaining an antiemulsin in the serum of rabbits, into which he had injected several separate doses of emulsin. Fermi and Pernossi[150] have prepared an antitrypsin, and von Dungern[151] has obtained an antidiastase against the proteolytic enzymes of some bacteria. But of all the antienzymes, the one that has been best studied up to the present is indisputably antirennet, obtained independently by Morgenroth[152] and Briot[153]. The former of these investigators treated goats with increasing quantities of rennet and was able to assure himself, by comparative detailed researches, of the appearance and increase in quantity of antirennet in the blood serum. The goat which gave the best result ceasing to develop antirennet it was impossible to make the antirennet potency go beyond a certain point.
Briot also obtained antirennet in rabbits into which he had injected fluid rennet on several occasions. He was able to convince himself that the antirennet of horse’s serum is a non-dialysable substance which is precipitated by alcohol and certain salts. Like the precipitins and the diastase which digests gelatine, antirennet resists a temperature of 55°–56° C.; even heating to 58° C. has no effect on the antirennet serum. At 60° C. however, the heat begins to exert an injurious effect, and after three hours at 62° C. the serum has lost all power to prevent the coagulation of the casein by antirennet. Morgenroth and Briot both state that the antirennet neutralises the rennet by a direct action.
The cell poisons, or cytotoxins, of animal origin which were treated in the preceding chapter, likewise set up the production of special antibodies, or anticytotoxins. The consideration of these latter has a very special interest for those who study the question of immunity from a general point of view. The first discovery of these anticytotoxins was made in connection with the study of the toxic power of the blood serum of eels. Camus and Gley[154] and, independently of them, H. Kossel[155] demonstrated that animals when treated with increasing doses of eel’s serum acquire an antitoxic property which protects their corpuscles against the haemolytic action of ichthyotoxin, or the toxic substance of the blood of eels. Th. Tchistovitch[156] has not only confirmed this discovery, but has added to it new and interesting data.
[Sidenote: [118]]
When antitoxic serum is mixed in vitro with red blood corpuscles of the species which furnished the serum and there is added to it some haemolytic eel’s serum, it will be found that the red corpuscles remain quite unaltered. In the control tubes, however, in which the antitoxic serum is replaced by normal serum of the same species, the red corpuscles are very readily dissolved under the toxic influence of the eel’s serum. In animals (rabbits) that are treated with this latter fluid, there is established not only an antitoxic power of the blood, but the red corpuscles acquire a resisting power more or less pronounced against the ichthyotoxin of eel’s serum. When the red corpuscles are separated from the serum of rabbits (treated with eel’s serum) and some ichthyotoxin is added to them, solution very often does not take place at all. According to the experiments of Tchistovitch there is no direct relation between this acquired resistance and the antitoxic power of the blood. Sometimes even a kind of antagonism is observed between the two properties; that is to say, the red corpuscles of a rabbit whose serum is very antitoxic may be extremely sensitive to the poison of the eel whilst the converse may also hold good [cf. infra p. 120].
The toxic action of the eel’s serum upon the red corpuscles of a great number of Vertebrates is a natural property which demands no previous treatment of the eel. It is the antitoxic power, directed against the ichthyotoxin, which is developed only as a result of the preparation of the animals by the administration of increasing doses of eel’s serum. Nevertheless we also find natural antitoxins present in the blood of man or animals that have not been treated and which act against the cell poisons, cytotoxins, so widely distributed in the blood of a large number of species of animals.
Besredka[157] has demonstrated that the blood serum of Man and many Vertebrates contains a substance which prevents the solution of red corpuscles under the influence of blood serums of a different species. To reveal the presence of these antitoxins it is useful to heat the serums to 56° C. and then to add to them red corpuscles of the same species and some haemolytic serum of a different species. Under these conditions the solution of the red corpuscles does not take place, whilst their mixture with haemolytic serum alone, inevitably provokes haemolysis.
[Sidenote: [119]]
Along with these natural antihaemolysins there exist a number of artificial antihaemolysins or antihaemotoxins. Jules Bordet[158] was the first to draw attention to this important subject. He first obtained these antihaemolysins by injecting blood serum of the fowl, which possesses a very great haemolytic power on the red corpuscles of the rabbit, into individuals of this latter species. After some injections, the serum of these treated rabbits was found to be antihaemotoxic against the fowl’s serum. Later[159], Bordet obtained a serum against an artificial haemotoxin. The serum of the guinea-pig is innocuous to the red corpuscles of the rabbit. But when rabbit’s blood was injected several times into guinea-pigs the serum of the latter became very solvent for the red corpuscles of the rabbit. To prevent this action it is sufficient to inject the haemotoxin of treated guinea-pigs several times into rabbits. The serum of these rabbits becomes antihaemotoxic and protects the red corpuscles of the rabbit against the solvent action of guinea-pig’s serum.
In the normal haemolytic serums, such as the serums of the eel and fowl, the presence of two substances which act by combining could not be demonstrated. On the other hand, in the serums that were obtained as a result of the treatment of animals by the injection of blood from a different species, it was easy to demonstrate, as we have shown in the preceding chapter, the presence of two constituent substances which are: the macrocytase (alexine, complement) and the fixative (amboceptor of Ehrlich, sensibilising substance of Bordet). For this reason the study of the antihaemotoxins obtained against artificial haemotoxins is endowed with special interest. As the solution of the red corpuscles, in this case, can be prevented either by an antitoxic action directed against the cytase, or by a neutralisation of the fixative (for the concurrence of these two substances is indispensable in order that the solution may take place), Bordet asked whether the antitoxic serum, obtained by him in rabbits, is anticytatic or antifixative, or whether it contains both properties. Before resolving this problem it was necessary to establish some of the essential characters of artificial antihaemotoxic serums. The principal one amongst them is the resistance of these antihaemotoxins to a temperature of 55–60° C.; even when heated to 70° C. the antihaemotoxins retain, at least in part, their fundamental property. In this respect these substances differ from the cytases and approach the precipitins, fixatives and agglutinins.
[Sidenote: [120]]
[Sidenote: [121]]
The very exact experiments carried out by Bordet have demonstrated that in the serum of rabbits, treated with the specific haemotoxic serum of guinea-pigs, two substances, an anticytase and an antifixative, are found in combination. The former of these antitoxins is found in abundance, but the amount of antifixative is very small. Bordet was led to this result in the following way. To prevent the solution of the red corpuscles of the rabbit in the haemotoxic serum of the guinea-pig, it was necessary for him to add a considerable dose (10 to 20 times) of the antitoxic serum. When, however, he heated the latter to 55° C. the quantity of this serum necessary to prevent haemolysis could be reduced very considerably. In place of its being necessary to add to the haemotoxic serum 10 or 20 volumes of antitoxic serum, it was sufficient to add three or sometimes only two volumes of this heated serum. As we know already, heating to 55° C. destroys the macrocytase which should be found in the antitoxic blood of the rabbit. This cytase by itself is incapable of dissolving the red corpuscles of the same species; but when it is added to the fixative of the haemotoxic serum of the guinea-pig the macrocytase of the rabbit’s serum dissolves them very readily. Hence the conclusion that in the haemotoxic serum of the guinea-pig there must be present a quantity of fixative sufficient to allow of the solution of the red corpuscles by the macrocytase of the rabbit’s serum. This antitoxic serum, therefore, which only prevents the haemolysis on the condition of being added in comparatively large quantity, contains very little antifixative. When, by heating this serum to 55° C. we destroy the rabbit’s macrocytase, the mixture of antitoxic serum of the rabbit and haemotoxic serum of the guinea-pig, which ordinarily dissolves the red corpuscles of the rabbit, now leaves them intact. The reason is that the free fixative contained in this mixture does not find any available macrocytase: that of the rabbit being destroyed by the heating, and that of the guinea-pig neutralised by the antitoxic serum. The experiment I have just described proves that this antitoxic serum contains specific anticytase. This anticytase is capable of neutralising the guinea-pig’s macrocytase, but is altogether powerless against that of the rabbit. This last circumstance allows us to investigate whether the antitoxic serum of the rabbit contains, in addition to anticytase, a specific antifixative. Bordet prepared a mixture of antitoxic serum of the rabbit, heated to 55° C., with haemotoxic serum of the guinea-pig, also heated to 55° C. In this mixture the two macrocytases (that of rabbit and that of guinea-pig) have been destroyed by heat, but the antitoxins of the rabbit’s serum and the fixative of the haemotoxic serum have remained intact. This mixture owing to its want of macrocytases was incapable of dissolving the red corpuscles of the rabbit. By adding to it some fresh unheated serum from a normal rabbit the rabbit’s macrocytase was introduced. As the latter could not be neutralised by the anticytase of the antitoxic serum and was incapable, by itself, of dissolving the red corpuscles of the rabbit, it was unable to produce haemolysis except on the condition that there is in the mixture a sufficient quantity of unneutralised free specific fixative. As a matter of fact, the red corpuscles of the rabbit are not dissolved in the mixture described; this proves that the fixative had become inactive in consequence of the presence of an antifixative in the antitoxic serum of the rabbit. I need not enter into further details of Bordet’s experiments, which have fully demonstrated the fact that in the antitoxic serum of his rabbits there were really two antitoxins; an anticytase abundant in quantity, and an antifixative present in much smaller amount.
Ehrlich and Morgenroth[160] quite independently of Bordet have shown that an antihaemotoxic serum is very rich in anticytase. After making a number of injections of normal horse’s serum (very rich in cytase) into a goat, they obtained in the blood serum of the latter an anticytase very active against the cytase of the horse. This antitoxic serum of the goat, as might be anticipated, contains no antifixative, the horse’s serum that served for the injections coming from normal horses which contained no, or very little, fixative. Even in another case, where these investigators[161] injected a dog with sheep’s serum very rich in fixative specific for the red corpuscles of the dog, they did not succeed in obtaining any antifixative. These observations in no way diminish the value of the discovery of the antifixative by Bordet, though they demonstrate that this antitoxin cannot, in certain cases, be found in the serum. Ehrlich and Morgenroth themselves throw out, in this connection, the suggestion that in these cases the antifixative remains linked to the cell which produces it, without being thrown off into the blood.
[Sidenote: [122]]
The very precise data that we have just summarised do not seem to agree with the statements of certain other investigators. Thus Schütze[162], from his researches on the antihaemotoxic serum of guinea-pigs, directed against the rabbit’s haemotoxin, has arrived at the conclusion that in the former an antifixative only is produced. As he merely injected into his guinea-pigs haemotoxic rabbit’s serum that had been heated to 60° C. and consequently deprived of the macrocytase, he concluded that in this serum there remained only the specific fixative capable of provoking the formation of an antitoxin. This must consequently be an antifixative. Paul Müller[163] came to a similar conclusion, after injecting rabbits with the heated haemotoxic serum of fowls. These injections caused the formation in the rabbit’s serum of an antitoxin that Müller regarded as an antifixative.
Ehrlich and Morgenroth[164] objected to this interpretation, taking their stand on experiments made with the serums of normal animals. They were able to show that these serums, when injected in the fresh state or after being heated to 60° C., caused the production of a corresponding antihaemotoxin which is nothing but an anticytase. When Schütze and Paul Müller concluded that by heating the serums they had entirely deprived them of cytase elements they did not take into account the possibility of the cytases being transformed, under the influence of heat, into other bodies unable to produce haemolysis, but quite capable of provoking the formation of anticytases. Ehrlich and Morgenroth give to these new bodies, derived from cytases under the influence of temperatures between 55°–60° C., the name of complementoids; and these complementoids appear in the experiments of Schütze and Müller to have caused the production of antitoxins—anticytases.
In all the investigations just summarised the anticytases have been obtained by the injection into animals of various blood serums, fresh or heated. Wassermann[165] has discovered another method of arriving at the same result. He injected into guinea-pigs the leucocytes of rabbits, carefully deprived of all traces of serum. After some time the blood serum of guinea-pigs thus treated became weakly but distinctly anticytatic. From this experiment Wassermann draws the conclusion that, as has been often affirmed by several observers, the leucocytes really contain cytases.
[Sidenote: [123]]
How do the anticytases act upon the cytases? On this point all observers who have studied this question have but one answer, the action of the anticytases is direct. Bordet thinks that the two substances combine so intimately that they cannot be again separated by heat. We know that the cytases are very sensitive to heat and that their haemolytic property is destroyed at 55° C. The anticytases, on the other hand, as already noted, are much more resistant to the action of heat. Bordet has prepared mixtures of haemolytic cytase serum and of antihaemolytic serum, neutral mixtures, that is to say, inactive for red corpuscles or with a very feeble action upon red corpuscles that have been sensibilised by the specific fixative. These mixtures no longer exhibit antihaemotoxic properties or they exercise this power in a very feeble degree. If in these mixtures the cytases remain uncombined alongside the anticytases, it is to be expected that heating them to 55° C. will restore the antihaemotoxic function of the anticytases; the cytases being destroyed at 55° C. there will remain in the mixtures only active anticytase. The experiments made on this point have demonstrated that the heating of these mixtures does not restore the antihaemotoxic action, that is to say, the anticytase is definitely combined with the cytase.
Ehrlich and Morgenroth have satisfied themselves that their antihaemotoxin exerts no influence, either upon the red corpuscles or upon the fixative, and is only capable of preventing the action of the cytase. They introduced red corpuscles of the rabbit into a mixture of goat’s serum, heated to 56° C. and thus only retaining its fixative, and anticytase serum. The fluid bathing the red corpuscles was then removed by centrifugalisation and the corpuscles were mixed with normal haemolytic horse’s serum. Solution of the red corpuscles took place at once as the anticytase had been completely removed during centrifugalisation, being combined with neither the red corpuscles nor the fixative.
These investigators have obtained various anticytases by injecting serum of various species of animals into other mammals. They observed, however, that injections of the serum of an allied species did not bring about the formation of anticytases. Thus the injection of goat’s serum into sheep, or of that of sheep into goats, never produced anticytase serum.
[Sidenote: [124]]
In addition to antihaemotoxic serums several other analogous anticytotoxic serums have now been obtained. Thus Delezenne has prepared serums which prevent the action of neurotoxin and of the cell poison which destroys the liver cells. We[166] have been able to obtain a rabbit’s serum which prevents the spermatozoa of this rodent being rendered motionless by the specific spermotoxin of the guinea-pig. More recently Metalnikoff[167], working in my laboratory, has prepared another antispermotoxic serum which prevents the specific spermotoxin of the rabbit from arresting the movement of the guinea-pig’s spermatozoa.
[Sidenote: [125]]
As the history of these antispermotoxins presents certain interesting general features we may with advantage, perhaps, dwell on some of their characters. The two antispermotoxins mentioned above are distinguished by certain peculiarities. When Metalnikoff set to work to inject rabbit’s spermotoxin into guinea-pigs, he thought that he had an easy task before him and that after a few injections the guinea-pig’s serum would become antispermotoxic. This, however, was not the case. The serum from these animals when mixed with spermotoxic serum was powerless to prevent the immobilisation of the spermatozoa of the guinea-pig. It was only when he heated the serum of his treated guinea-pigs to 56° C. that the antispermotoxic power appeared with the greatest distinctness. The inefficacy of the unheated serum must therefore depend on the toxic action of the guinea-pig’s macrocytase, because it is this substance alone that can have been destroyed by the heating process. Now, in order that this macrocytase may act, the presence of the fixative is necessary, which leads us to the conclusion that the serum of the guinea-pigs injected by Metalnikoff contained no antifixative. This hypothesis was fully confirmed by experiment. Metalnikoff introduced a drop of guinea-pig’s serum into a mixture of antispermotoxic serum, heated to 56° C., with spermotoxic serum. The spermatozoa continued their movements in normal fashion. But when afterwards he added a few drops of unheated serum from a normal guinea-pig the motions of the spermatozoa were arrested almost instantaneously. Consequently there was present in the mixture rabbit’s macrocytase which had been neutralised by the anticytase of the prepared guinea-pig’s serum and for that reason the spermatozoa remained motile. But in the same mixture we had also the specific fixative, coming from the rabbit’s spermotoxic serum, which remained free and not neutralised. The motile spermatozoa had become impregnated with this fixative and a little guinea-pig’s macrocytase (against which the anticytase was powerless) was sufficient to make them suddenly cease their movements.
There is no doubt, then, that the serum of guinea-pigs that have been treated with spermotoxin contains anticytase only and no, or almost no, antifixative. Such is not the case with the antispermotoxin obtained by us in rabbits that were treated with spermotoxic toxin of guinea-pigs. Several consecutive injections were sufficient to render the serum of the rabbits so treated capable of preventing the action of the spermotoxic serum of the guinea-pig on the motility of the rabbit’s spermatozoa. In the mixture of antispermotoxic serum and spermotoxic serum these spermatozoa continue to move for a considerable time, whilst in the control mixture prepared with normal rabbit’s serum and spermotoxic serum they become motionless at the end of a few minutes. To obtain this marked effect it was not necessary to heat the antispermotoxic serum as in Metalnikoff’s case. Indeed I have performed almost all my experiments with fresh serums, unheated. As the rabbit’s serum contains macrocytase capable of rendering the spermatozoa, sensibilised by the fixative, motionless and as this macrocytase cannot be neutralised by the anticytase that is active against the guinea-pig’s macrocytase, the fact I have just pointed out indicates that the antispermotoxic serum of my rabbits contains antifixative. The difference between the antispermotoxic serum obtained by Metalnikoff and that prepared by me is similar to that observed between the antihaemotoxic serums. Some contain only anticytase but others undoubtedly contain antifixative also.
[Sidenote: [126]]
As this result appeared to me to be of far-reaching importance I felt bound to verify it by another method. I injected certain rabbits with spermotoxic serum of the guinea-pig and others with normal guinea-pig’s serum. The amount of cytases being about the same in both, the strength of the serums obtained as the result of injections of normal serum and of specific serum should be the same if the antispermotoxic serums contain anticytase only. Experiment demonstrates just the contrary. The antispermotoxic serum of rabbits treated with normal guinea-pig’s serum was on every occasion much less active than the serum of rabbits injected with the spermotoxic serum of prepared guinea-pigs. The former contains anticytase only, whilst the latter contains in addition antifixative. Weichhardt’s[168] experiments carried out in my laboratory corroborated the conclusion I have just formulated.
Having made ourselves acquainted with the constitution of the anticytotoxins we may now pass to the question of the origin of these bodies and of analogous ferments which act in the resorption of albuminoid substances in the blood and in the tissues.
We have already mentioned that the leucocytes are charged with a soluble ferment which digests gelatine, and that in animals treated with injections of gelatine these cells elaborate a much larger amount of the ferment. Here we have evidence of a kind of education of the leucocytes to produce a greater amount of digestive ferment, in a manner quite analogous to that which has been described in Chapter III in connection with the augmentation of the pancreatic ferments in intestinal digestion. It is, then, quite permissible to look upon leucocytes, and probably phagocytes in general, as the source of the soluble ferment that digests gelatine.
[Sidenote: [127]]
Is this the case with the other substances which take an active part in the resorption of albuminoid substances in the fluids and tissues of the organism? Up to the present the origin of precipitins and antiferments, such as antirennet, has not been studied. The problem being very complex and difficult, it appears to be impossible at present to solve it. It is known indeed that the introduction of these substances into the organism provokes a reaction similar to the one we have described in the case of the injection of gelatine into the peritoneal cavity of guinea-pigs. Thus Morgenroth[169] observed that in his goats the subcutaneous injection of sterile rennet caused the formation of extensive infiltration at the seat of inoculation, this being accompanied by fever; we are justified in concluding from this that rennet provokes a marked leucocytic reaction. Hildebrandt[170] has demonstrated by direct experiment that rennet, when enclosed in capillary glass tubes and introduced below the skin of rabbits, induces a marked positive chemiotaxis. This led to the formation of a leucocytic plug several millimetres long. Now we know from Briot that the rabbit is capable of producing antirennet. Hildebrandt has further shown that several other diastases, or hydrolytic ferments, such as sucrase and emulsin, give rise to a similar chemiotactic phenomenon. The leucocytic reaction is consequently a general phenomenon following the introduction into the tissues of substances of complex chemical composition capable of provoking the formation of antibodies. We are tempted from this fact to accept it as a law that the leucocytes are capable of producing these latter substances. Although this hypothesis may be very probable, the number of facts at our disposal is not yet sufficient to justify the statement that its truth is demonstrated.
Since it is the red corpuscles which are affected by the haemotoxins it might be asked whether it may not be that these elements defend themselves by the production of antihaemotoxins the overplus of which is thrown into the blood and fluids in general? The researches that have been made on this point relate especially to the antihaemotoxin of the blood serum of rabbits in relation to the ichthyotoxin of eel’s serum.
We must therefore examine the collected evidence bearing on anticytotoxins and analogous bodies and endeavour to form some idea as to their probable origin. A large accumulation of exact data bearing on the antihaemotoxins does not afford us sufficient information as to the source of these substances.
Let us first examine the question, is it possible to attribute to the red corpuscles the function of producing the antihaemotoxins? If these elements are really the source of the antihaemotoxins it is probable that the red corpuscles of animals whose serum is antihaemotoxic will exhibit marked resistance to the toxins; thus we know that the white corpuscles which produce gelatinase digest gelatine much better than does the serum of the same animals. From the experiments of Tchistovitch (l. c. supra p. 110) on rabbits that have been immunised against eel’s ichthyotoxin, it must be accepted that the red corpuscles of these animals are often very sensitive to the action of the poison at a period when the blood serum of the same rabbits exhibits a marked antihaemotoxic power. It is not until later in the process of immunisation, when the serum loses a great part of this power, that the red corpuscles become resistant to the ichthyotoxin.
[Sidenote: [128]]
But before we abandon the hypothesis of the production of antihaemotoxins by the red corpuscles we must see if it cannot be reconciled with the facts, by the application of Ehrlich’s side-chain theory[171]. This theory was evolved with the object of explaining the production of antitoxins and their action on bacterial and vegetable toxins. Later, Ehrlich has extended it to the cytotoxins, anticytotoxins and bactericidal substances.
[Sidenote: [129]]
According to Ehrlich the complex molecule of albuminoid substances contains, besides the central stable nucleus, a number of side-chains, or “receptors,” which fulfil various accessory functions and serve especially for the nutrition of the cell. These receptors have a great affinity for the various substances necessary for the maintenance of the life of the cell. Under normal conditions these receptors seize nutritive molecules, as a leaf of Dionaea seizes the fly that serves it as food. Under special conditions these receptors lay hold of complex molecules of albuminoid substances, such as the various toxins. In this case the receptor, instead of combining with a molecule which supports life, fixes a molecule which poisons the cell. According to Ehrlich’s theory on the constitution of toxins their molecules contain an atomic group which poisons—the toxophore, and another group which combines with the receptor—the haptophore. The toxic group of a complex poison, such as ichthyotoxin, cannot penetrate into a red corpuscle except by the help of the haptophore group and of the corresponding receptor. When a red corpuscle has absorbed a large number of molecules of ichthyotoxin, the united action of the toxophore groups renders life impossible and the corpuscle is dissolved. But when a red corpuscle has been touched by only a few toxic molecules, too few to compromise life, there is merely immobilisation of the receptors which are combined with the haptophore groups of the ichthyotoxin. As these receptors fulfil an important function in the nutrition of the red corpuscles, the latter reproduce them in larger numbers than were originally present. We know that in the phenomena of repair an over-production of the new-formed parts often takes place and, according to Ehrlich, to this over-production the presence of antitoxins in the fluids of the body is due. The receptors, developed in excess by the red corpuscles, fill these cells, and no longer finding room therein are extruded from them and overflow into the blood and other fluids of the organism. When a fresh injection of toxin makes its way to the blood it there meets with a number of free receptors, endowed with an affinity for the haptophore group of the molecule of the toxic substance. The chemical combination between the two substances takes place at once in the plasmas, a fact which prevents the haptophore group of the toxin from uniting with the receptor of the red corpuscles and so injuring these cells by introducing the toxophore group into them. According to this theory the same receptors which, in the free state in the fluids, fulfil the antitoxic function become in the interior of the red corpuscles the vehicles of intoxication and consequently fulfil a philotoxic function. This opposite rôle of the receptors has often been compared to a lightning-conductor; so long as the receptors are attached to the molecule of the living protoplasm they attract the toxin just as a lightning-conductor attracts the lightning when it is badly insulated.
[Sidenote: [130]]
So interpreted, it is easy to conceive that the red corpuscles of animals whose fluids are antihaemotoxic may be sensitive to the toxic action of eel’s serum, as has been observed by Tchistovitch. As soon as the protective fluids have been removed from the red corpuscles of the immunised organism, the corpuscles when placed in contact with ichthyotoxin (eel’s serum) attract the haptophore groups of the poison by means of their numerous receptors. These haptophores in their turn introduce the toxophore groups which dissolve the red corpuscles without the slightest difficulty. This theory does not explain the cases, which are numerous, in which the red corpuscles of rabbits that are vaccinated against eel’s poison resist this poison. Camus, Gley, and Kossel, working independently, have arrived at the result that the red corpuscles of immunised rabbits, from which the serum has been carefully removed, are not dissolved when submitted to the action of ichthyotoxin, whilst the red corpuscles of untreated rabbits placed under the same conditions, undergo a rapid solution. Tchistovitch confirming this fact has added to it the observation that the resistance of the red corpuscles of the rabbit is most often found when the serum loses its antitoxic power. If the receptors of the red corpuscles of immunised rabbits, owing to their great affinity for the haptophore group of the ichthyotoxin molecule, only attract the toxophore group of this poison, as the lightning-conductor when badly insulated attracts the lightning, the red corpuscles should never manifest resistance. To explain this contradiction we must not suppose that the red blood corpuscles which have become resistant have got rid of their receptors. In fact, if these receptors are so necessary to the nutrition of the cell that their absence has set up this extraordinary over-production which has inundated the fluids, it is evident that one cannot admit the existence of red corpuscles entirely deprived of corresponding receptors.
When examined from different points of view the hypothesis of the production of antihaemotoxin by the red corpuscles is surrounded with very great difficulties. It appears to be probable, therefore, that the source of this antitoxin must be sought for in other cell elements, and we may be allowed to recall to mind those cells which manifest a general and local reaction of the most constant kind after each injection of ichthyotoxin. Tchistovitch has observed that eel’s serum when introduced into rabbits in non-fatal but immunising doses excites a marked hyperleucocytosis.
[Sidenote: [131]]
The question of the origin of anticytotoxins being so complicated, it has been necessary for its elucidation to seek an experimental method of excluding the organ in which the antibody is supposed to have its origin. As we cannot think of eliminating the red or white corpuscles, nor the greater part of the tissues and organs, there remains only one way of bringing about this result. It is the suppression of the male genital organs. We know already that the injection of semen readily excites the production of a spermotoxin, and that this spermotoxin gives rise to the development of a corresponding antispermotoxin. If it is the spermatozoa, that is to say the elements having a particular affinity for the spermotoxin, which elaborate the antitoxin we must conclude that castrated males would be incapable of producing it. With this in view we have carried out a great number of experiments which have amply proved to us that male rabbits when deprived of their sexual organs are fully as capable of developing antispermotoxin in their fluids as are control rabbits in which the male genital apparatus remains intact. Doe-rabbits, and young, sexually immature rabbits of both sexes, also react to injections of spermotoxin by producing the corresponding antispermotoxin. The specific elements which are sensitive to the action of a cytotoxin undoubtedly are not indispensable for the development of the corresponding anticytotoxin. This result is in complete harmony with the hypothesis above put forward, that the red corpuscles cannot be regarded as the source of the antihaemotoxin. In the case of antispermotoxin this fact can be rigorously established by experiment.
Here arises the following question. We have seen that the anticytotoxins are composed of two different substances: an anticytase and an antifixative. The former is an antitoxin capable of neutralising macrocytase, the soluble ferment which will attack indifferently all kinds of cell elements. It is not to be wondered at, then, that the exclusion of the spermatozoa in no way prevents the production of anticytase by an organism which receives injections of cytotoxins. These latter, as we have already said, contain cytase along with the specific fixative; the macrocytase can attack any kind of animal cell provided that it can find some fixative or any other means to penetrate into the interior of these formed elements. We have seen that the antispermotoxin, obtained by Metalnikoff in guinea-pigs, does not contain any anticytase. Amongst his animals treated with spermotoxin was a castrated male guinea-pig which also produced anticytase. There is nothing astonishing in this fact, the injected cytase must have linked itself to many other cells which were able to develop anticytase.
[Sidenote: [132]]
But the example of the antispermotoxin of the rabbits in my own experiments is very different. In order that it might manifest its action the serum of these rabbits did not need to be heated to 56° C.; it was not necessary to rid it of its own macrocytase which could have acted under the influence of the fixative, if this latter for want of antifixative had remained free in the added spermotoxin. This antifixative, then, is undoubtedly found in the serum of castrated males which have shown themselves capable of producing not only anticytase, but also antifixative. This result has been further verified by comparative experiments on castrated male rabbits, some of which received spermotoxic guinea-pig’s serum whilst the others received only normal guinea-pig’s serum. It has been demonstrated that the amount of cytases remains almost constant in both normal and vaccinated animals[172]. If, then, the antispermotoxins contain only anticytase, the injection of specific guinea-pig’s serum and that of normal guinea-pig’s serum should produce the same result, that is to say the serums of castrated rabbits, when treated by these two kinds of guinea-pig’s serum, should exhibit the same antispermotoxic power. Experiments have, however, proved that this is not the case. The serum of castrated rabbits that have been injected several times with normal guinea-pig’s serum becomes distinctly antispermotoxic, but its power to protect the spermatozoa of the rabbit against being deprived of motility by the guinea-pig’s spermotoxin is greatly inferior to that which is developed in the serum of other castrated rabbits that I injected with spermotoxic guinea-pig’s serum. Of course all the other conditions of the experiment were the same for the two groups of rabbits.
[Sidenote: [133]]
Several series of facts, then, focus to this fundamental point, that the organism of an animal that has been deprived of its male sexual organs is in a condition to produce antispermofixative. Against the argument that we have drawn from the fact that the antispermotoxic serum of castrated rabbits that have been treated with spermotoxic serum acts without being heated, might be cited certain experiments made by Ehrlich and Morgenroth. The antispermotoxic action in this case, as already stated, demonstrates that the serum of prepared rabbits contains antifixative. Otherwise, had the fixative not been neutralised, it would have allowed the macrocytase of the rabbit’s serum to arrest the movements of the spermatozoa. Now the two above-named observers have demonstrated[173] that the injection of different serums into animals is capable of exciting in their blood the development of anticytases. The macrocytase of castrated rabbits which, before treatment with the spermotoxin, was capable of arresting the movements of rabbits’ spermatozoa acted upon by a fixative, might become inert after the injections of spermotoxic serum of guinea-pigs. To clear up this point I asked M. Weichardt[174], who has carried out work on this subject in my laboratory, to try by means of unheated serums of normal animals, to restore the activity of spermotoxin that had been mixed with antispermotoxic serum. Spermatozoa of rabbits were put into a definite mixture of spermotoxic guinea-pig’s serum, heated to 56° C., and antispermotoxic serum, also heated to 56° C., obtained from castrated rabbits that had been treated with spermotoxin. The spermatozoa remained very active in this mixture which contains specific fixative (in the spermotoxic guinea-pig’s serum) and antispermotoxin. To this mixture is added a little normal rabbit’s or horse’s serum, unheated. These serums contain cytases and would be quite capable of arresting the movements of the spermatozoa if there was found in the mixture any free fixative that would enable the macrocytase to be linked to the spermatozoa. Under these conditions the spermatozoa remain motile for a long time. The fixative, then, was no longer active; it was neutralised by the antifixative of the antispermotoxic serum of castrated rabbits. A control experiment was made with the same substances; but the castrated rabbits’ serum that had been treated with spermotoxic serum was replaced by the serum of other castrated rabbits treated with normal guinea-pig’s serum. In these latter mixtures the spermatozoa became motionless at the end of a very short time; the fixative, not being neutralised, readily allowed the rabbit’s and horse’s cytases to affect the spermatozoa.
It follows from all this that the antispermotoxic serum of castrated male rabbits, when treated with normal guinea-pig’s serum, contains anticytase only; whilst the serum of castrated male rabbits, treated with specific and spermotoxic guinea-pig’s serum, contains anticytase and antifixative. The latter, then, has been produced independently of the sensitive elements,—the spermatozoa.
[Sidenote: [134]]
Having established the fact that antispermotoxin does not come from the male organs, it was necessary to try to ascertain its true source. With this object in view we injected spermotoxic serum into young rabbits (quite capable of producing antispermotoxin) and tried to follow the fate of the spermotoxin in the organism. When spermotoxic guinea-pig’s serum is injected into the peritoneal cavity of the rabbit a notable amount of spermotoxin is found in the thickened portion of the omentum made up of lymphoid tissue. But the greater portion of the poison passes into the circulation whence it goes to fix itself in various organs, especially the spleen. At the moment when the spermotoxin is found in the blood a certain quantity of this fluid was drawn off into tubes containing some drops of extract of leeches’ heads. After the blood thus treated had been centrifugalised the plasma was decanted and its power of arresting the movements of spermatozoa was compared with that of serum of the same blood prepared in the usual way. From these researches it results that the plasma is always richer in spermotoxin than is the corresponding serum. Sometimes the difference in favour of the plasma is very great.
A part of the spermotoxin passes into the kidneys and the suprarenal capsules. It is probable that, as is the case with so many soluble poisons, a certain proportion of the spermotoxin may be eliminated by the uropoietic organs. A small quantity of this poison is found also in the male and female sexual glands of young non-castrated rabbits.
The search for some main centre of origin for the production of antispermotoxin has as yet led to no positive result. The power of arresting the movements of spermatozoa first appears in the blood plasma, and it is this same fluid which, later, is more antispermotoxic than is any organ. Amongst the tissues which fix spermotoxin the genital organs play not the slightest part in the production of antispermotoxin. The experiments with castrated rabbits afford sufficient proof of this. On the other hand it becomes more and more probable that the phagocytic system, disseminated in many organs, and especially the leucocytes, furnish the antispermotoxic substance. The fixation of the spermotoxin by the leucocytes of the blood, such as the cells of the omentum and of the spleen, already offers us a valuable indication. The absence of any particular organ that might have the monopoly of fixing the spermotoxin and which should later be found charged with a predominant amount of antispermotoxin also speaks in favour of the phagocytic origin of this antitoxin.
[Sidenote: [135]]
After a single intraperitoneal injection of spermotoxic guinea-pig’s serum into young rabbits, the blood of the latter is distinctly spermotoxic for several days; later it becomes indifferent, but eight or ten days after the commencement of the experiment the blood begins to exhibit an antispermotoxic power. In these cases the plasma shows itself more active than the serum. When the rabbits are killed at this stage of commencing antitoxic production, it is found that an extract of the organs is not antispermotoxic or only feebly so. In all cases this power, when it exists, is more feeble than that of the blood fluid. The results obtained with extracts of organs are not constant. Sometimes the spleen possesses more antitoxic activity, whilst the liver, thymus, omentum, lymphatic glands and genital glands exhibit none of this property. In other cases the survival of the spermatozoa that are influenced by the spermotoxin has been longest in the extract of the suprarenal capsules. Sometimes the extract of the omentum exhibits the greatest antispermotoxic power. This great variability in the development of the property of protecting the spermatozoa accords well with the idea that the elements which produce antispermotoxin are wandering cells which, under diverse influences, may be localised in very diverse points of the organism.
We must not deceive ourselves. The facts which have been collected up to the present do not allow us as yet to form a final opinion on the origin of anticytotoxins, but we are quite justified in regarding as very probable the hypothesis that the phagocytes play a most important part in the process. It is in all cases beyond doubt that the amoeboid cells which resorb the formed elements play a very important part in the resorption of fluids of very complex molecular composition.