CHAPTER VII. The Mechanism of Natural Immunity Against Micro-Organisms

THE MECHANISM OF NATURAL IMMUNITY AGAINST MICRO-ORGANISMS

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

FIG. 38.—Peritoneal macrophage of guinea-pig that has ingested a number of Bacilli coli. Stained intra vitam with neutral red. ]

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

It is as yet very difficult to establish whether, besides the cytases, there exist other endo-enzymes within phagocytes, that is to say, soluble ferments which do not pass into the serums on the destruction of the phagocytes, but continue within these cells. Our present methods of investigation do not enable us to come to any conclusion on this point. We know only that the digestion of the formed elements is more complete inside the phagocytes than in the serums. Thus, as we have seen in