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.
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.
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.
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.
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.
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.
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.