Metchnikoff had scarcely recovered from all the emotions caused by his experiments on cholera, which he was still studying, when, in 1894, a work appeared by a well-known German scientist, Pfeiffer, bringing out new facts in favour of the extracellular destruction of microbes.

Whilst studying the influence of the blood serum within the organism and not outside it as his predecessors had done, he had found that cholera vibriones, injected into the peritoneum of a guinea-pig vaccinated against cholera, were nearly all killed in a few minutes and that they then presented the form of motionless granules in the peritoneal liquid. This granular degenescence, said Pfeiffer, took place apart from the phagocytes and therefore without their intervention. Metchnikoff repeated the experiment at once and ascertained that it was perfectly accurate.

The complexity of biological phenomena being very great, he fully admitted the possibility of other means of defence in the organism besides that of the phagocytic reaction. However, this new fact disagreed so much with his own observation, and seemed so isolated, that Metchnikoff supposed an error of interpretation must have been made and tried to throw light upon it. He spent sleepless nights seeking the conclusive experiment which might explain Pfeiffer’s phenomenon.

His excitement was all the greater that he was very soon going to the International Congress at Buda-Pest, where he intended to expose the results of his new researches, and he feared that he should not have time to make all the experiments which he required in support of his arguments. However, the general impression of the Congress was clearly favourable to the phagocyte theory. This is how M. Roux picturesquely described the scene at Metchnikoff’s Jubilee in 1915:

This is how Metchnikoff had reconciled the apparent disagreement of Pfeiffer’s phenomenon with the phagocyte doctrine: he demonstrated, by a series of experiments, that the extracellular destruction of the cholera vibriones in the peritoneum of a guinea-pig vaccinated against cholera, did in no wise depend on the chemical properties of the blood serum, but was simply due to the digestive juices which had escaped from the inside of the leucocytes, damaged by the intraperitoneal injection. Those digestive juices, or cytases, poured into the peritoneal liquid were what killed the injected cholera vibriones and transformed them into “Pfeiffer’s granulations.” On the other hand, if by means of various precautions the phagocytes were left unmolested, the extracellular destruction did not take place and the vibriones were digested within the phagocytes.

Metchnikoff used other experiments to prove that the bactericidal property of blood juices did not exist without intervention from the phagocytes. For instance, in a guinea-pig vaccinated against cholera, the bacilli are not destroyed if they are injected into parts of the organism that are devoid of pre-existing phagocytes, such as in the subcutaneous tissue, in the anterior chamber of the eye or in an aseptically-obtained oedema. On the other hand, if, in the same medium, some exudate is injected containing damaged leucocytes from which the digestive juice is leaking, the vibriones introduced are destroyed. The same results are obtained in vitro.

All these experiments proved that the extracellular destruction of the cholera vibrio was accomplished by the digestive juices which had passed from the phagocytes into the humors and not at all through a special property of those humors. Once again the phagocyte theory rose triumphant from the test.

After having finally proved that it is by means of its phagocytes that the organism fights microbes, Metchnikoff wished to find out whether it was by the same process that it struggled with their poisons, or toxins. This problem, far more difficult to solve, took him many years’ study. Whilst every phase of the phagocytes’ struggle against microbes can be followed with the eyes, it is impossible to do so where poisons are concerned, since they are invisible; it is necessary to proceed by a different road.

Faithful to his method of taking as a starting-point the simplest expression of the phenomenon to be studied, Metchnikoff began by lower beings. Unicellular organisms, such as myxomycetes, amoebÆ, and infusoria, sometimes manifest a natural immunity to certain poisons. It is also possible to endow them with artificial immunity by accustoming them gradually to substances which, ingested straight away, would infallibly have killed them. Such phenomena, seen in unicellular beings, could only be ascribed to the reaction of the cell itself. Therefore Metchnikoff supposed a priori that the phagocytes, being similar primitive cells of multicellular beings, would also react against poisons. And, in fact, he ascertained that the number of phagocytes in a rabbit’s blood diminished considerably under the influence of a fatal dose of arsenic, whilst it increased under the influence of small doses of the poison, to which it was possible to accustom the animal.

Dr. Besredka, a disciple of Metchnikoff, made some very interesting researches, which entirely confirmed the share of the phagocytes in the reaction against sulphides of arsenic. He had chosen the trisulphide, a very slightly soluble salt of an orange colour, in order to find it again easily within the organism. After having injected non-fatal doses of it into the peritoneal cavity, he obtained an exudate in which all the orange granules of the salt were to be found included within those leucocytes which have a large, non-lobed nucleus—the macrophages. These cells gradually digested the salt they had englobed, which ended by disappearing entirely within them, and the rabbit remained safe and sound. On the other hand, it died if the same doses of the same salt had been protected from the leucocytes by an elderberry bag, or when the leucocytes had been attracted elsewhere by a previous injection of carmine for instance. Those experiments removed all doubts as to the share of the phagocytes in the destruction of mineral poisons.

Certain experiments on microbian poisons spoke in the same sense. Thus MM. Roux and Borrel had observed that the diphtheritic toxin, which is inoffensive to rats even in large doses, kills that animal if a small quantity of it is introduced into the brain, the probable explanation being that, in cases of subcutaneous injections, the poison, “phagocyted” on the way, was destroyed before it reached the nerve cells.

Thus experiments seemed to plead in favour of the view that the part played by phagocytosis is not limited to the struggle against microbes, but also extends to the defence against poisons and toxins.

After having studied the mode of destruction of these, Metchnikoff wished to elucidate the origin of the counter-poisons, the specific antitoxins discovered by Behring in the humors of immunised organisms, a question of which the study was even more difficult.

Metchnikoff began by asking himself whether the microbes themselves did not produce antitoxins in order to defend themselves against enemy micro-organisms. He made many experiments but only obtained negative results, and concluded that the antitoxins must be manufactured by the organism itself.

The origin of this property must be more recent than that of the phagocytic reaction, for it does not exist in plants or in inferior animals. It was only from superior cold-blooded vertebrates, such as the crocodile—and that only in artificial conditions—and upwards, that Metchnikoff succeeded in finding a specific antitoxic power in the humors.

He ascertained that the vaccination of animals by toxins conferred, after a time, antitoxic powers to the blood and humors which contained leucocytes. He concluded therefrom that the presence of antitoxins depended on that of the phagocytes. Experiments on divers higher animals having proved that, in them also, antitoxins were localised in humors containing phagocytes, Metchnikoff concluded that the antitoxins were manufactured by the cells themselves. As toxins are absorbed and digested chiefly by macrophages, it is probable that it is the latter also which manufacture specific antitoxins, or the final product of the digestion of corresponding toxins. Metchnikoff could only propound this idea as an hypothesis, for the complexity and difficulty of a material demonstration did not yet allow of a definite solution of the problem. However, certain observations on toxins and antitoxins pleaded in favour of this thesis.

For instance, working in collaboration with MM. Roux and Salimbeni, he had found that it is by soluble poisons that the cholera vibrions harm the organism or kill it, but that small doses of the same poisons are vaccines and make the blood of the vaccinated animal antitoxic. On the other hand, a microbian vaccination is preventive against microbes only but not against toxins and the blood does not become antitoxic. This is explained by the fact that it is not the same cells which digest cholera microbes and cholera toxins: the microphages digest the vibriones whilst the macrophages digest the poisons and, probably, manufacture as products of this digestion, the corresponding antibody, the cholera antitoxins.

On the contrary, in cases of the inclusion of microbes by macrophages, as, for instance, in plague, the blood acquires an antitoxic power by injection of the microbes themselves and not by their toxins, as was demonstrated by M. Roux and his collaborators. The same fact was observed by Metchnikoff on the alligator, in whom also microbes are digested by macrophages. In those cases, when microbes and toxins are digested by the same cells, the latter manufacture antibodies against both.

These facts rendered legitimate the supposition of the macrophagic origin of antitoxins.

In 1897 an International Congress took place in Moscow. Metchnikoff read a paper on the phagocytic reaction against toxins and another dealing with the whole of the knowledge acquired concerning human plague. He ended this by a plea in favour of Science, so often accused of having contributed nothing to the solution of the most important human problems, particularly ethical ones, and of having, on the contrary, sanctioned the law of Might by tabulating the laws of the struggle for existence. Metchnikoff objected that, far from doing so, Science, by revealing the laws of Nature, applied to humanity the benefits derived from them, whilst striving to counterbalance their cruel or harmful effects. The struggle against plague and other diseases was a concrete example of this, for here medical science opposed itself to the cruelty of “natural selection.” He wound up his speech by the following words, “Just as, in order to satisfy his Æsthetic tastes, Man revolts against the laws of Nature which creates races of sterile and fragile flowers, he does not hesitate to defend the weak against the laws of natural selection. Science has been faithful to her mission and to her generous traditions. Let her, then, progress unhindered.”

Metchnikoff’s friend and companion, M. Nocard, wrote to me concerning Metchnikoff’s paper:

Metchnikoff had at this period a very talented disciple, M. I. Bordet, who opened a new path by a series of researches of the greatest importance. He found, among other things, that “the figured elements” can be destroyed outside the cells, in the humors. Thus, if red blood corpuscles from one animal are injected into an animal of a different kind, these globules are destroyed, not within the phagocytes, but outside them, in the ambient humors. Metchnikoff studied this phenomenon and proved that the explanation was the same that he had previously given of Pfeiffer’s phenomenon in the case of cholera vibrions. In Bordet’s experiments, the leucocytes which were already existing in the humors were also damaged by the experimental shock; but, if this was carefully avoided, the phagocytes, remaining intact, englobed and digested the injected red corpuscles and no phenomenon similar to Pfeiffer’s took place.

These observations led Metchnikoff to a thorough study of the destruction of cellular elements by the phagocytes. He had already observed that, whilst the struggle with microbes is chiefly undertaken by small leucocytes with a lobed nucleus—the microphages—it is the great leucocytes with a single large nucleus—the so-called macrophages—which undertake the destruction of cells, “figured” elements, as well as that of toxins. The macrophages are to be found not only in the blood but also in different organs such as the liver, spleen, kidneys, etc.; they seize upon living cells by means of mobile protoplasmic prolongations with which they draw them in and end by ingesting them completely. Not only do they thus absorb foreign cellular elements such as red corpuscles, spermatozoa, etc., but also all the weakened cells of the organism itself.

This weakening may be due to normal phenomena such as the metamorphosis of insects or tadpoles, when certain organs, as they weaken, become useless or inactive. But, oftener, this weakening is due to pathological causes, as in morbid atrophies or poisoning by microbian toxins. In any case, the enfeeblement of cells exposes them to be devoured by macrophages, which brings about the atrophy of the cells or even of the organs which contain them.

These observations suggested to Metchnikoff the idea that senile atrophy might be due to the same mechanism, and his thoughts turned towards the problem of the causes of old age.

But, before undertaking researches in a new direction, he wished to conclude those he had been pursuing for twenty years on the phenomenon of phagocytosis. He therefore started to complete his investigations on immunity in order to epitomise them and to give a definite form to his doctrine on that subject.