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.

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 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 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.

A recent investigation, carried out by Bordet 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.

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 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.

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.

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, 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.

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.

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, 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, 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, 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 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 and Cobbett, 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 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.

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.