CHAPTER VII

THE MECHANISM OF NATURAL IMMUNITY AGAINST MICRO-ORGANISMS

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

[Sidenote: [185]]

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

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

[Sidenote: [186]]

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

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

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

[Sidenote: [187]]

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

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

[Sidenote: [188]]

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

[Sidenote: [189]]

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

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

[Sidenote: [190]]

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

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

[Sidenote: [191]]

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

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

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

[Sidenote: [192]]

[Sidenote: [193]]

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

[Illustration:

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[256]. The bacilli, ingested by these phagocytes, secrete a series of concentric membranes which become impregnated with phosphate of lime (Fig. 5). The process causes the death of the bacilli, of which there remain only the calcified membranes. The precipitation of the lime salt around bacillary membranes itself indicates the alkaline reaction of the medium. The use of certain staining substances fully confirms this conclusion. Thus, with alizarin sulpho-acid the giant cells stain deep violet, this affords clear proof of a very distinct alkaline reaction.

[Sidenote: [194]]

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

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

[Sidenote: [195]]

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

[Sidenote: [196]]

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

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

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

[Sidenote: [197]]

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

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

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

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

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

[Sidenote: [198]]

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

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

[Sidenote: [199]]

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

[Sidenote: [200]]

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

[Sidenote: [201]]

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

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

[Sidenote: [202]]

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

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

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

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

[Sidenote: [203]]

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

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

[Sidenote: [204]]

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

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

[Sidenote: [205]]

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

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

[Sidenote: [206]]

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

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

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

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

[Sidenote: [207]]

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

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

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

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

[Sidenote: [208]]

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

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

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

[Sidenote: [209]]

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

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

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

[Sidenote: [210]]

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

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

[Sidenote: [211]]

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

[Sidenote: [212]]

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

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

[Sidenote: [213]]

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

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

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

[Sidenote: [214]]

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

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

[Sidenote: [215]]

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

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

[Sidenote: [216]]

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

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

[Sidenote: [217]]

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

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

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

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