Chapter XII: Artificial Immunity Against Toxins (3)

These observers extended their researches to the protective action of fats and demonstrated that olive oil when emulsified and neutralised with soda and mixed with twice and even four times the lethal dose of botulinic toxin, prevented the contraction of a fatal poisoning by mice. Tyrosin also protected mice against this intoxication, not only when injected simultaneously with the poison, but even when introduced into the animal 24 hours before the poison was administered. Kempner and Schepilewsky conclude “that not only with the substance of the nerve centres, but also with various other substances, they were able to obtain a certain protective effect against the toxin of botulism” (p. 221). Their experiments with cholesterin and tyrosin were suggested to them by the previous researches of Phisalix[628] who demonstrated that the bile salts, as well as the two substances I have just mentioned, would protect animals against the venom of the viper.

Bearing all these facts in mind, it appears to be probable that in the above cases it is principally the fatty matters of the nerve centres that temporarily fix these toxins, and allow the animal organism to divert the poisons from their morbific action. From this point of view, it is interesting to note that the toxic action of the tetanus poison can also be prevented by other substances than the emulsion of the nerve centres. Thus Stoudensky[629] demonstrated, in an investigation carried out in Roux’s laboratory, that carmine fixes the tetanus toxin and prevents its action on the guinea-pig. As in the case of the cerebral substance, this fixation by carmine is very unstable. When the carmine that has fixed the tetanotoxin is macerated in distilled water it gives up the poison to the water which is then capable of producing tetanus. Such fixation does not end, any more than in the case of the cerebral substance, in the destruction or disappearance of the toxin. Carmine if first dissolved or macerated in water (especially if heated) loses its fixative power and can no longer prevent tetanus poisoning. Sterilisation, at 120°, 100° and even at 60° C., of the carmine, suspended in physiological salt solution, caused it to lose its protective action, although dry heat applied to it in closed tubes did not destroy this power.

[Sidenote: [408]]

In many respects carmine, which is derived especially from the adipose body of the cochineal insect, exerts an antitoxic influence analogous to that of maceration with the nerve centres. If fats play a special part in this action, we can readily understand how a brain, such as that of the frog, poor in fatty matters, cannot fix the tetanus toxin and prevent its morbific action. In any case the fact that certain substances of diverse nature, acting on toxins, exert an influence similar to that of the pounded mass of the nerve centres, does not allow us to accept Wassermann and Takaki’s experiment as proving the nervous origin of tetanus antitoxin. The analogy with the facts bearing on the anticytotoxins, collected and described in the fifth chapter, also tells against this hypothesis. We would here remind the reader that the two constituent parts of the antispermotoxin, the anticytase and the antispermofixative, develop in castrated animals and are consequently produced outside the spermatozoa, elements susceptible to the spermotoxin. The facts collected concerning the antihaemotoxins indicate also that these substances have some other origin than the red blood corpuscles.

[Sidenote: [409]]

[Sidenote: [410]]

This latter supposition appears to be in contradiction to Ransom’s[630] very interesting researches on the haemolytic action of saponin, carried out in Meyer’s laboratory at Marburg. This glucoside, owing to its property of fixing itself on the stroma of these corpuscles dissolves the red corpuscles of many vertebrates. The cholesterin of this stroma combines with the saponin, as the result of which the red corpuscles become altered and allow the haemoglobin to diffuse. But this same substance, cholesterin, which causes the poison to penetrate into the red blood corpuscles, prevents the solution of these elements when they are bathed in blood-serum. This fluid, in fact, acts as the antitoxin to saponin and does so just because it contains cholesterin. The cholesterin of the serum, fixing the saponin, prevents it from affecting the red corpuscles, thus fulfilling the function of a well fitted lightning conductor. On the other hand, when the cholesterin of the stroma of these corpuscles is linked on to the saponin, it renders them the disservice of a defective lightning conductor. The accord between these facts and the postulates of Ehrlich’s theory led Ransom to suppose that in the haemolysins and antihaemolysins, cholesterin perhaps played a similar part. His experiments convinced him that this was not the case. As it is generally accepted, after Calmette’s[631] experiments and according to Ehrlich’s view, that the alkaloids and the glucosides in general are incapable of setting up the formation of antitoxins, we might regard the attempts to find an antisaponin and to settle whether it is identical with cholesterin as useless. But in regard to these delicate questions we must be careful not to give too great weight to _a priori_ arguments. It was believed until quite recently that substances with very complex molecules, such as the albuminoids, toxins and soluble ferments, must always give rise to the production of antibodies in the animal; whilst the simpler substances whose chemical nature was better defined could never lead to this. Facts acquired in recent years have led to a modification of this view. In our fifth chapter we have already spoken of the fruitless attempts of Ehrlich and Morgenroth to obtain certain antifixatives. And yet the fixatives, as is shown by the results of the researches of Bordet and myself, belong to the category of substances which are quite capable of setting up the formation of antibodies. Again, certain mineral poisons, quite unexpectedly, gave rise to the development of the counterpoison in the animal body. This fact forced itself upon Besredka[632] in his researches on the adaptation to arsenic made in my laboratory. His experiments were undertaken for the purpose of studying the mechanism of the refractory condition against a poison, apart from any antitoxic action whatever, which, according to previous investigations, seemed excluded. This action, however, was exhibited in such a degree that it could not be ignored. The serum of animals immunised against arsenious acid was found to possess both protective and antitoxic properties against a dose of this poison killing a rabbit in 48 hours. It is true that Morishima[633], in a research carried out in Heyman’s laboratory at Ghent, has thrown doubt upon these results. His objections, however, cannot refute the statements of Besredka which rest on very precise and numerous experiments which I witnessed. Morishima left out of account several important circumstances and carried out his experiments without any continuous check by means of control animals. It must be said also that the resistance of the rabbit against arsenic depends on many different factors and that, at certain seasons, it is much more difficult to adapt them to the poison than at others. It is only by numerous researches extending over a very long period that we can arrive at precise and conclusive results.

From these observations there is every inducement for us to attempt to ascertain whether, by subjecting animals to repeated injections of saponin, it is possible to augment the antisaponic power of their blood-serum and whether, if this takes place, the antitoxic action is due to a rise in the amount of cholesterin in this serum. I therefore requested Besredka to carry out some experiments bearing on this point. Guinea-pigs, injected with progressive doses of saponin for more than two months, at the end of this period showed no increase in the antisaponic power of their serum. They followed the rule established by Ehrlich; they developed no antitoxin against a glucoside. Moreover, they gave us no new information as to the origin of these antibodies.

[Sidenote: [411]]

In his first memoir in which the theory of side-chains is treated, Ehrlich insists on the nervous origin of antitetanin as an example of the production of antitoxins by animals susceptible to poisons. Now, however, that he has come to distinguish haptophore and toxophore groups in the toxic molecule, it is to the side-chain, which fixes the first group, that Ehrlich attributes prime importance. “The formation of antitoxins”—he says[634] in the opening address at his Institute at Frankfort—“would, therefore, be absolutely independent of the action of the toxophore elements.” In other words, for a cell to be capable of producing antitoxin, it is not at all necessary that it should be susceptible to the toxic influence of the poison; it is only necessary that it should possess receptors, or side-chains, capable of combining with the haptophore group of the toxin. Thus it is possible, as we have described above, to produce antitoxins, with modified toxins whose toxic action is _nil_ or almost so, but which have retained their power of combining with antitoxic substances. According to Ehrlich, these modified toxins are _toxoids_, in which the toxophore group is completely destroyed; “whilst the haptophore group, the producer of immunising substances, is retained in its integrity.” It is evident then that, under such conditions, the tetanus antitoxin might be developed elsewhere than in the nerve centres. For that it would be sufficient that outside the nerve cells there should be other living elements capable of fixing the tetanus toxin, or, to use Ehrlich’s phraseology, elements, possessing side-chains, having an affinity for the haptophore group of the tetanus poison.

Dönitz[635] has already expressed the view that in the rabbit the tetanus toxin may be fixed not only by the nerve elements but also by the various other cells.

[Sidenote: [412]]

[Sidenote: [413]]

The existence of such cells, outside the nervous system, is not merely hypothetical. It is shown very clearly in Roux and Borrel’s experiments on cerebral tetanus. In order to produce this disease in the rabbit, it is sufficient to introduce a very small dose of toxin directly into the brain. When inoculated subcutaneously with much larger quantities of the same tetanus poison, the rabbit remains in good health or exhibits merely a slight and transient tetanus. “The resistance of the rabbit against the tetanus toxin, injected under the usual conditions”—conclude Roux and Borrel[636]—“is not due, then, to a relative insusceptibility of the nerve centres, but to the fact that much of the poison introduced does not reach the nerve cells and is destroyed in some part of the animal.” In the guinea-pig, as shown by the same investigators, the difference of the dose of tetanus poison, necessary to produce fatal tetanus by intracerebral or by subcutaneous injection, is minimal or nil, from which it may be argued that in this very susceptible animal there is no destruction of toxin outside the nerve centres and that the whole of the poison introduced makes its way without hindrance as far as these organs. Ehrlich, in his report to the International Congress of Medicine in Paris (August, 1900), accepted these results, as seen from his tenth and eleventh propositions: “The receptors exist, sometimes in certain tissues only, sometimes in the majority of the organs (action of tetanus poison in the guinea-pig and in the rabbit),” “... the presence of numerous receptors in the organs of less vital importance may bring about—thanks to a kind of diversion of the toxin molecules—a diminution in the susceptibility of the animal to this toxin[637].” We must here recall the differences between the susceptibility of the guinea-pig and that of the rabbit to small doses of tetanus toxin frequently repeated as in Knorr’s experiments already referred to. The guinea-pig, subjected to these injections, dies in a tetanic condition long before it has received the minimal lethal dose for this species when injected in a single dose. The rabbit, on the other hand, is very tolerant of repeated doses and even rapidly acquires an immunity against five minimal lethal doses for the rabbit (injected at once). Knorr explained this difference as due to the hypersusceptibility of the nerve centres in the guinea-pig and to their acquired insusceptibility in the rabbit. The experiments of Roux and Borrel on the cerebral tetanus of rabbits vaccinated against tetanus, have demonstrated that this insusceptibility is not produced in these animals. We must, therefore, seek some other explanation. In rabbits subjected to small repeated doses, the poison is more and more prevented by certain living elements from reaching the nerve centres. Further, it is neutralised by the antitoxin which is rapidly produced. We find from Knorr’s[638] researches that in rabbits antitoxin appears in the blood in cases where, affected with a transitory tetanus, their limbs remain contracted for weeks. In guinea-pigs, affected with the same form of tetanus, antitoxin in appreciable quantity is never found, even after complete recovery. All these facts accord with the hypothesis that there exist, outside the nervous system, certain living cells which absorb the tetanus toxin and produce antitoxin. Rabbits and fowls possess this property in a much greater degree than do guinea-pigs. The fowl, according to Knorr, develops “a large quantity of antitoxin, whilst the tetanic symptoms are still augmenting.” In this animal, as we have been able to show[639], a portion of the tetanus toxin is absorbed by the leucocytes. By exciting aseptic exudations in fowls into which I had previously injected this toxin, I was able to convince myself that these exudations, much richer in leucocytes than was the blood, were also much more tetanigenic than was the blood. I observed also a more or less pronounced leucocytosis after the injection of non-lethal doses of tetanus toxin into fowls. It is possible that the leucocytes were actual agents in protecting the animal against the penetration of this poison to the susceptible nerve centres.

The great susceptibility of leucocytes to microbial toxins serves to indicate that these cells are of some importance in the struggle of the animal against these poisons. Their injection usually sets up a marked hyperleucocytosis of the blood. On this point Chatenay[640], working in my laboratory, has carried out a series of experiments on animals poisoned by bacterial (tetanus and diphtheria), phanerogamic (ricin and abrin) and animal (snake venom) toxins. He was able to demonstrate a striking analogy between them and the phenomena which occur in bacterial infections. When death takes place at the end of a very short period, the number of leucocytes markedly diminishes; if the animal lives beyond 24 hours or resists completely, a hyperleucocytosis, often of very marked character, is produced. In the guinea-pig, which is so susceptible to tetanus, the leucocytosis observed occurs even after injections of quantities of tetanus toxin equal to several lethal doses, and it is only after the introduction of an amount equal to one hundred times the lethal dose that the number of leucocytes remains stationary or shows a diminution. Here we have something analogous to what takes place against the anthrax bacillus in the same animal. The penetration of this deadly organism sets up a marked leucocytosis, but the accumulated leucocytes are incapable of seizing the bacilli or of preventing their noxious action. In other species of animals, such as the rabbit and the fowl, the intervention of the leucocytes against the anthrax bacillus, as well as against the tetanus toxin, is more effective.

[Sidenote: [414]]

If this toxin, instead of being injected in solution, be introduced along with the bodies of the micro-organisms which contain it, the struggle on the part of the animal takes place under more favourable conditions and even very susceptible animals may afford evidence that they offer a high resistance. Vaillard and Vincent[641] have shown that if we inject living tetanus bacilli, or the spores of these bacilli, deprived of free toxin, into guinea-pigs a great accumulation of leucocytes, which prevent the production of infection and intoxication by devouring the bacilli and their spores, takes place. The toxin contained in the ingested bacilli remains innocuous; this affording evidence of the protective part played by the leucocytes against the toxin. The same interpretation may be offered to explain the survival of animals very susceptible to tetanus, when the tetanus poison, mixed with pounded cerebral substance or with carmine powder, is injected. In these mixtures the toxin, as mentioned above, becomes attached to certain substances of the triturated brain or to the grains of carmine. This fixation is very unstable, the toxin is readily set free; but, when introduced into the body of the animal, the mixture induces a great accumulation of leucocytes which seize the cerebral particles and the grains of carmine and along with them take possession of the toxin. Wassermann and Takaki’s experiments and those of Stoudensky are easily explained if we assume two protective acts: the first of these consists in fixing the toxin, thus preventing it from diffusing and rapidly reaching the living nerve cells; the second is the absorption of the toxin fixed by the leucocytes,—cells endowed with receptors for the haptophore group of the toxin, but insusceptible to its toxophore group. When one of the two factors is absent, tetanus cannot be prevented. It is for this reason that in Courmont and Doyon’s experiments with emulsion of the frog’s brain, mixed with tetanus toxin, the inoculated animals died from tetanus in spite of an accumulation of leucocytes. This fact affords additional proof that, under these conditions, the toxin does not become anchored to the particles of the pounded cerebral substance, this anchoring being a condition necessary for the effective reaction of the leucocytes.

[Sidenote: [415]]

[Sidenote: [416]]

The absorption of the tetanus toxin becomes evident when we study in detail the phenomena produced in the experiments carried out according to Vaillard’s methods with tetanus spores and those of Wassermann and Takaki with poison to which cerebral emulsion has been added, or according to Stoudensky’s method with grains of carmine. When, however, it is desired to bring forward rigorous proof of the presence of the tetanus toxin inside the leucocytes charged with spores, with granules of cerebral substance or with grains of carmine, very great difficulties are encountered. How, indeed, is it possible to demonstrate this poison fixed upon these various bodies, a poison, the presence of which cannot be demonstrated except by its injection into the animal? For this, in the study of the reaction of the organism of the animal against the poisons, it is very important to have recourse to substances, whose presence can be demonstrated more easily than can the microbial toxins. We must first have recourse to the alkaloids, especially atropin, which, in this respect, present numerous advantages. We know that rabbits resist considerable doses of sulphate of atropin, even when this poison is injected directly into the blood. On the other hand, when it is introduced into the brain, according to Roux and Borrel’s method, even small quantities are quite sufficient, as demonstrated by Calmette[642], to produce a fatal poisoning. The intracerebral injection of the one-hundredth part of a dose which, when introduced into the circulation of the rabbit, produces no disturbance, in the same animal at the end of a few minutes sets up an enormous pupillary dilatation with symptoms of very lively excitation, increase of the reflexes, and general anaesthesia. These phenomena are succeeded by paralysis and death, which supervenes three or four hours after the injection. The natural immunity of the rabbit against atropin falls therefore into the same category as that against morphin. It is not due to the innate insusceptibility of the nerve cells, but to something which prevents the alkaloid from reaching these living elements. With the object of ascertaining the mechanism of this immunity, Calmette injected into the veins of rabbits a fairly large quantity of sulphate of atropin (0·2), he then bled these animals and collected from their blood the plasma and the white corpuscles, separating them by centrifugalisation. When injected into the brain of other rabbits, these constituents of the blood did not act in the same way. Whilst large doses of plasma set up merely a short period of excitation and a very transitory pupillary dilatation, corresponding quantities of leucocytes caused grave disturbances, sometimes followed by death in from seven to twelve hours. Calmette concludes from his researches that the atropin does not remain in the fluid part of the blood, since mere traces of it are found in the serum, but that it is seized and absorbed almost immediately by the leucocytes[643]. This result has been confirmed by Lombard[644] by another series of experiments. After injecting very large quantities of sulphate of atropin into rabbits and guinea-pigs, he bled these animals and separated out the elements of their blood. Instead of introducing these elements into the brain of rabbits, he injected them into cats, animals very sensitive to atropin. The cats which received the red corpuscles and the plasma exhibited very insignificant symptoms of poisoning. Those, on the other hand, which were injected with a corresponding quantity of leucocytes, had much graver symptoms of intoxication, such as photophobia with maximal pupillary dilatation, dysphagia and persistent diarrhoea.

It is, therefore, to the absorption of the atropin by the leucocytes that naturally refractory animals owe their immunity, an immunity which is very marked in spite of the susceptibility of the nervous elements of these animals. We have been able to obtain this result thanks to the delicate physiological reactions obtained with certain alkaloids. As regards arsenic the demonstration could be pushed even further, for the absorption of this mineral poison by the leucocytes has been established by chemical analysis.

[Sidenote: [417]]

When engaged in my researches on the leucocytic phenomena in intoxications I succeeded[645] in showing that in rabbits subjected to rapidly fatal doses of arsenious acid, there is a marked diminution in the number of white corpuscles in the blood. On the other hand, in rabbits habituated to arsenic, the same doses which brought about hypoleucocytosis and death of the control rabbits, induced a considerable rise in the number of leucocytes. Later, Besredka[646] made continuous and detailed researches upon this subject and obtained most interesting results. In order to simplify the conditions of experiment, he studied the reaction of the organism of the animal after the introduction of a red trisulphide of arsenic[647], a not very soluble salt, easily recognisable by its colour and markedly toxic. When non-lethal doses of this salt were injected into the peritoneal cavity of the guinea-pig, there was, first a transitory fall in the number of the white corpuscles in the peritoneal fluid, followed by a hyperleucocytosis of the most marked character. Of the leucocytes accumulated in the exudation the macrophages almost exclusively seized the yellowish-red granules of the trisulphide of arsenic. Very shortly, the whole of the salt injected was found within the peritoneal leucocytes, and the animals in which this marked phagocytosis occurred remained in good health. The ingested granules could be observed for several days in the macrophages; but in course of time, these arsenical particles were broken up into very small granules and ultimately disappeared. Here, then, we have an intraphagocytic solution of the trisulphide of arsenic and very probably a transformation of this salt into some other arsenical combination, innocuous to the animal. This soluble substance escapes from the macrophages and is finally excreted by the urinary passages.

[Sidenote: [418]]

Since the phagocytes ingest the trisulphide of arsenic and render it innocuous, it was to be anticipated that the elimination of these protective cells would lead to a fatal poisoning by doses which, under normal conditions, are readily withstood by guinea-pigs. When Besredka used sacs of reed pith containing non-fatal quantities of the red trisulphide and introduced them into the peritoneal cavity of guinea-pigs these animals were not long in exhibiting symptoms of poisoning and died at the end of a longer or shorter period, this varying with the amount of poison introduced. Even when the phagocytic reaction had been impaired as the result of a previous injection of carmine powder, the guinea-pigs died after doses of trisulphide of arsenic which, under ordinary conditions, did not kill them. The phagocytes in this experiment devoured numerous grains of carmine and were rendered incapable of ingesting enough of the trisulphide of arsenic to save the animal. On the other hand, when Besredka set up a previous accumulation of macrophages in the peritoneal cavity of his guinea-pigs, he succeeded in rendering these animals resistant to doses of trisulphide of arsenic that, under normal conditions, were fatal. The whole of these facts converge to show that the phagocytes, thanks to their power of seizing the trisulphide of arsenic and of modifying it within them, exercise a beneficent and immunising action on the organism of the animal. The analogy of the main facts concerning this protective influence with that observed in the immunity against infective micro-organisms is indeed very considerable.

Having determined the part played by the macrophages in the resistance of the organism of the animal against a not very soluble salt of arsenic, Besredka proceeded to study the leucocytic phenomena in poisoning by soluble arsenical compounds. In his experiments he made use of potassium arsenite and he found that when lethal doses were injected the guinea-pigs showed a diminution of leucocytes in the blood in less than 24 hours, whilst with non-lethal doses, he produced a marked hyperleucocytosis. When he injected lethal doses into rabbits accustomed to arsenic, these animals manifested an increase of white corpuscles, just as in animals injected with non-lethal doses. These oscillations in the number of leucocytes, like those which have been observed after poisoning by trisulphide of arsenic, certainly indicate that the organism and its defensive cells behave in the same way to both slightly soluble and very soluble salts of arsenic. In the first case it was easy to demonstrate that the accumulation of leucocytes in the blood and in the peritoneal exudation terminated in the ingestion of the granules of trisulphide. With potassium arsenite, it was not so easy to prove the point; a chemical analysis of the elements of the blood, however, has given a decisive answer. After injecting the lethal dose of this soluble salt into rabbits accustomed to arsenic, Besredka bled them in order to separate the plasma, leucocytes and red corpuscles. Several experiments made on these rabbits gave a concordant result which this observer sums up thus: “Although the bulk of plasma and of red corpuscles was much greater than that of the leucocytes, it was in the latter only that arsenic was found” by chemical analysis. It was only in those cases where the animals survived, and manifested hyperleucocytosis, that Besredka succeeded in demonstrating the presence of arsenic in the white corpuscles.

[Sidenote: [419]]

These experiments, excluding any doubt as to the protective part played by the leucocytes against arsenical intoxication, of course suggested the idea of investigating whether the nerve elements, submitted to the direct influence of potassium arsenite, exhibit any real susceptibility to this poison. The injection of solutions of this salt into the brain demonstrated that the one-hundredth part of an ordinary lethal subcutaneous dose was sufficient to cause fatal poisoning. This fact, then, falls into line with other facts, already numerous, as to the susceptibility of the nerve centres to microbial toxins, alkaloids and other poisons. But in the case of potassium arsenite, it was even more easily demonstrated than in the other cases that immunity natural or acquired, is connected with the absorption of the poison by the leucocytes. These cells, themselves much less susceptible to the toxic action than are the nerve elements, protect them from contact with the poison.

It is manifest that arsenic is not the only mineral substance capable of being absorbed by the phagocytes, and there are already on record well established facts in support of this thesis. Some time previous to the researches on arsenical poisoning just summarised, Kobert, then in Dorpat, set his pupils, Stender, Samoïloff, Lipsky and others[648] to make systematic researches on the fate of iron in the animal organism. For this purpose these observers made use of a very soluble preparation of iron—or better expressed, as soluble as possible—Dr Hornemann’s _ferrum oxydatum saccharatum solubile_, which does not precipitate in alkaline media. They proved that a small quantity of the iron introduced into the animal is eliminated by the kidneys and the wall of the intestine, but that the greater part of the metal is arrested in the organs, especially the liver, spleen and bone marrow. The iron is there absorbed by the leucocytes which hold it for some time and then throw it into the intestine.

[Sidenote: [420]]

I have had the opportunity of observing this circulation of Dr Hornemann’s soluble salt in the organism of several species of vertebrates. Some time after its introduction into the organism by the blood vessels, peritoneally or subcutaneously, the iron may be found (by means of the microchemical reaction with potassium ferrocyanide) accumulated in the various phagocytes, especially the leucocytes, the stellate Kupffer’s cells of the liver and the macrophages of the splenic pulp. The non-phagocytic cells, as, for example, Ehrlich’s basophile leucocytes, so abundant in the lymph of rats, take up very little of this iron, although the macrophages and microphages are full of it[649]. Against these facts Weigert[650] has advanced the objection that the leucocytes absorb only the iron precipitated in the form of granules, but my own researches allow of no doubt that not only granular but dissolved iron is absorbed. This discussion, however, loses much of its importance in view of the results obtained with potassium arsenite.

According to Samoïloff[651], soluble salts of silver in the animal organism undergo a fate similar to that of Hornemann’s soluble iron salt and are absorbed by the phagocytic elements. It must be noted, further, that according to the experiments of Arnozan and Montel[652], the leucocytes absorb such drugs as calomel and salicylate of soda.

[Sidenote: [421]]

These observations all clearly show that the phagocytes must not be looked upon as cells capable of seizing merely the dead bodies of micro-organisms and of animal cells, always fearing and avoiding poisons and only able to come forward when protected by some other antitoxic function. The phagocytes no doubt often exhibit a negative susceptibility for many poisons, when these are introduced into the animal organism in too large a quantity. But these cells are most resistant to toxic substances and protect the higher elements from the poison. Under these conditions, it is quite natural to assign to the phagocytes the rôle of the fighting agents of the animal organism against poisons and we may even enquire whether these elements do not produce the antitoxins. It has been pointed out that it is very difficult to attribute this function to the cells susceptible to the toxic action,—the spermatozoa in the production of antispermotoxin, the red blood corpuscles in the development of antihaemotoxin, or the nerve cells in the production of tetanus antitoxin. Moreover since, according to Ehrlich’s theory, it is only the haptophore group which excites the formation of antitoxins on the part of the elements which possess the corresponding receptors, it is quite possible that the phagocytes, thanks to the facility with which they absorb the poisons, occupy an important place as producers of antitoxins. I have already formulated this hypothesis, and several investigators, amongst whom may be cited Gautier[653] and Courmont[654], have received it favourably, though in the imperfect state of our knowledge, it cannot, as yet, be fully demonstrated. It might perhaps be objected against this hypothesis that in many instances, after the injection of micro-organisms living or dead, in spite of a vigorous leucocytic reaction the organism of the animal does not produce any antitoxin. In such cases, there is clearly a development of antibodies, such as the fixatives, whose phagocytic origin may reasonably be claimed, but no true antitoxins. It must not be forgotten, however, that the various kinds of phagocytes present, amongst themselves, great differences, and that perhaps certain only of these elements are capable of producing antitoxins. When micro-organisms, living or dead, are introduced into an animal it is found that antitoxins do not as a rule appear in the fluids; in these cases the reaction is set up mainly by the microphages. The macrophages represent the principal source of antitoxins. In cases where these phagocytes ingest the micro-organism the blood exhibits an undoubted antitoxic power. Such is the case with bubonic plague in the human subject, where the micro-organism is readily ingested by the macrophages. Here we obtain antitoxic serums even after the introduction of living or dead organisms into the animal, a fact observed by Roux and his collaborators. Another fact in favour of the hypothesis I am defending is furnished to us by the cayman. As noted above, this reptile, of all known animals, supplies antitoxins most quickly and easily. In the cayman the leucocytic system is composed of eosinophile microphages filled with granules, and of macrophages. As the eosinophile cells are only very weakly phagocytic, it is the macrophages almost exclusively which intervene in the reaction against the micro-organisms. It is probable, then, that in the cayman and in animals inoculated with the plague bacillus the exclusion of the microphages from the struggle constitutes a factor favourable to the production of antitoxins and at the same time favourable to the manifestation of the activity of the macrophages.

[Sidenote: [422]]

If these latter phagocytes play the primary rôle in the excretion of antitoxins in the fluids of the body we should expect to find this function exercised not only by the motile macrophages of the blood and lymph, but also by the fixed macrophages, so widely diffused through almost all the organs.

I advance this hypothesis for what it is worth, simply as a guiding idea for new researches in this field, of which so much is still unknown[655]. The brief account of the actual state of the question of artificial immunity against toxins, has indicated to us that this is a problem far more difficult of solution than is that of acquired immunity against micro-organisms. The mere fact that these latter can still be found some hours or even days after their entry into the refractory animal, affords a great advantage in these researches as compared with those on toxins which are lost, often almost immediately, after their injection. Consequently our knowledge of antimicrobial immunity is more advanced than is that on immunity against the soluble products of micro-organisms.

The facts narrated in this chapter support the thesis I have defended on the subject of immunity against micro-organisms—that antimicrobial immunity in no way depends on a previous resistance against the toxins. As a general rule the immunity against micro-organisms is developed more readily than the immunity against their toxic products and at an earlier stage.

Although much still remains to be done in the elucidation of the mechanism of antitoxic immunity, the principal data acquired on the subject of this immunity have undoubtedly led to applications of the highest importance, as will be set forth in one of the following chapters.