animal organism against yeasts follows the same rules that hold in the
defence against bacteria.
[Sidenote: [184]]
The animal micro-organisms are much rarer in infective diseases than are
the microphytes; moreover the impossibility of obtaining cultures of
them renders their investigation much more difficult. Yet there exist
facts that are capable of affording us information as to the means made
use of by the refractory organism against certain parasitic Protozoa.
Amongst these latter the _Trypanosomae_ play a most important part. One
species of this genus (_T. lewisi_) produces an infective disease in
rats, especially in the grey rat (_Mus decumanus_), the blood of these
rodents often containing a very large number of them, whilst the small
flagellated organisms flourish well in the serum prepared from the blood
of affected animals. Laveran and Mesnil[247], in their studies on the
_Trypanosomae_, injected defibrinated blood containing numerous
_Trypanosomae_ into the peritoneal cavity of guinea-pigs, which exhibit
a natural immunity against this parasite. The parasites remained alive
for some days and then disappeared completely. Here again it is the
phagocytes of the peritoneal exudation which rid the animal of the
_Trypanosomae_ by ingesting them. Laveran and Mesnil were able, by the
examination of hanging drops of the peritoneal exudation of their
guinea-pigs, to detect leucocytes in the act of devouring _Trypanosomae_
which showed, by their active movements, that they were still alive.
Once the parasites were completely enclosed within the macrophages,
their final disappearance took place with extraordinary rapidity.
In this chapter we have attempted to place before the reader a complete
series of the phenomena observed in natural immunity in animals. We have
passed in review the resistance of the animal organism against the
principal groups of bacteria, and we have dwelt on certain of them which
are most capable of adapting themselves to various media, and on others
which present examples of microorganisms more exacting and more
delicate. We have examined the immunity against Blastomycetes and
parasitic animalcules. Above all, in the lower animals, just as in the
Vertebrata of all classes, we have always observed this general
phenomenon: phagocytic resistance as the principal and constant factor
in natural immunity.
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.
CHAPTER VIII
SURVEY OF THE FACTS BEARING ON ACQUIRED IMMUNITY AGAINST MICRO-ORGANISMS
The discovery of attenuated viruses and its application to vaccination
against infective diseases.—Vaccination by microbial
products.—Vaccination with serums.—The acquired immunity of the
frog against pyocyanic disease.—The acquired immunity against
vibrios.—Extracellular destruction of the cholera vibrio.—Part
played by two substances in Pfeiffer’s phenomenon.—Specificity of
fixatives.—Phagolysis and its relation to the extracellular
destruction of vibrios.—Part played by phagocytosis in the
acquired immunity against vibrios.—Fate of the spirilla of
recurrent fever in the organism of immunised guinea-pigs.—Acquired
immunity against the bacteria of typhoid fever and pyocyanic
disease.—Acquired immunity against swine erysipelas and
anthrax.—Acquired immunity against the streptococcus.—The acquired
immunity of rats against _Trypanosoma_.
[Sidenote: [218]]
Certain of the hypotheses on acquired immunity are of as ancient origin
as are those on natural immunity. For example, it has for long been
known that man is constitutionally refractory to certain diseases which
are very fatal to cattle. It has also been recognised that after a first
attack of a contagious disease, such as small-pox, measles, scarlatina,
typhoid fever, etc., man acquires a lasting immunity; and that the same
rule applies to domestic animals, for example, cattle that have
recovered from cattle plague or sheep that have got better from
sheep-pox, become refractory against these diseases.
[Sidenote: [219]]
The discoveries of variolisation and vaccination, as methods of
conferring on man a resistance to small-pox, have notably advanced our
knowledge upon acquired immunity. The researches on the properties of
vaccine had already led to some important results. But it is only since
the publication of Pasteur’s investigation, carried out with his
collaborators Chamberland and Roux, in the first place, and with
Thuillier later, that we have been able to take up the study of acquired
immunity in a really scientific manner. The first in this series of
discoveries, which have opened up a path so fruitful to science and
medical art, was the discovery of the attenuation of micro-organisms.
The small cocco-bacillus of fowl cholera after several weeks’ culture in
broth was found to have become markedly attenuated in virulence. To
Pasteur the idea occurred of testing whether fowls that had resisted the
inoculation of these attenuated organisms had acquired any real immunity
against virulent fowl cholera. Experiment confirmed his expectation and
led to the discovery of the vaccine against this disease. The method was
at once applied to other infective epizootic diseases and shortly
afterwards Pasteur, Chamberland and Roux found a method of preserving
sheep and cattle from anthrax. To attain this end it was found necessary
to prevent the bacillus from producing spores (in this they succeeded by
cultivating it in broth at a temperature of 42°·5 C.), because these
spores fix the virulence and prevent attenuation. Having overcome this
main obstacle, Pasteur and his collaborators found that their cultures,
thus deprived of spores, become attenuated on exposure to the air and so
become transformed into vaccines. They were thus able to prepare their
two anthrax vaccines which soon found such wide application in practice.
A few years later, Pasteur and Thuillier discovered the vaccines against
swine erysipelas and, in collaboration with Roux and Grancher, Pasteur
made the first application of his discoveries to the vaccination of man
against rabies.
[Sidenote: [220]]
The path thus opened up was traversed by many other investigators and
led to many very remarkable discoveries. Vaccination with
micro-organisms became a recognised method and in the hands of Arloing,
Cornevin and Thomas, soon found its application to symptomatic anthrax.
The next step in this onward progress of science was taken when Salmon
and Smith, working at hog-cholera, demonstrated the possibility of
vaccinating not only with hog-cholera bacilli, but also with culture
fluids in which these organisms had developed. These fluids, when
completely deprived of micro-organisms by filtration, protected the
experimental animals from virulent hog-cholera. This discovery, at first
sceptically received, was soon confirmed and extended by the work of
other investigators. Beumer and Peiper extended it to the experimental
disease set up by the typhoid bacillus in small laboratory animals;
Charrin applied it to the disease that he produced by means of the
bacillus of blue pus; and Chamberland and Roux prepared vaccines from
the soluble products of the septic vibrio and of the bacillus of
symptomatic anthrax. And now, as the result of these investigations,
vaccinations by microbial products are in everyday use in all research
laboratories. The vaccinations now used (anthrax, symptomatic anthrax,
swine erysipelas and rabies) are still being carried out by means of the
inoculation of living viruses.
The comparative history of acquired immunity is still very incomplete.
The facts known concerning the adaptation of unicellular organisms to
all kinds of injurious influences of a physical or chemical nature
enable us to perceive that acquired immunity is just as general in
living beings as is natural immunity; but it is impossible, in the
present state of our knowledge, to confirm this hypothesis by exact and
experimental data. The reason for this lies in the great difficulty we
have in carrying out experiments on the lower animals. The majority of
the Invertebrata in captivity do not remain alive long enough and can
not be sufficiently often inoculated for us to obtain in them a well
marked acquired immunity against micro-organisms. Kowalevsky[306], the
celebrated Russian zoologist, has tried to overcome these various
difficulties by making use of Myriapods. He found first that
_Scolopendrae_, when inoculated with anthrax bacilli, die therefrom
during the heats of summer, the blood containing a number of anthrax
bacilli. But when the temperature does not exceed 17°–18° C., a fairly
large number of these myriapods survive. The same survival was observed
when Pasteur’s first vaccine was injected. Kowalevsky utilised the
_Scolopendrae_ that had resisted the first injection of anthrax bacilli
to ascertain whether they had contracted an acquired immunity. The
results were not absolutely demonstrative and Kowalevsky sums up his
results in the following words: “I cannot say, therefore, that I have
succeeded in solving this question of vaccination, but it appears to me
very probable” (p. 607).
[Sidenote: [221]]
In view of this doubt, I asked Mesnil to make a fresh attempt, making
use of _Scolopendrae_ and inoculating them with anthrax bacilli. These
creatures, however, were so delicate and so little capable of remaining
alive under the artificial conditions of their captivity, that the
attempt soon had to be abandoned. I tried to obtain better results with
the larvae of _Oryctes nasicornis_; here again the difficulties were too
great. These insects exhibit a perfect natural immunity against certain
micro-organisms, but for others they showed an insurmountable
susceptibility. It is very evident, then, that it is not an easy matter
to set up an acquired immunity in the Invertebrata.
It was necessary, therefore, to go higher up the animal scale and have
recourse to cold-blooded vertebrates. The choice naturally fell on the
frog. I asked Dr Gheorghiewski[307], who was working in my laboratory,
to try to vaccinate the Batrachians against pyocyanic disease. I ought
first to state that the bacillus of blue pus is pathogenic for the frog,
which it kills both at the ordinary laboratory temperature, and at that
of the incubator, 30°–37° C. In the first case the fatal dose is much
greater than in the second, but it is always easy to induce a fatal
infection. In this respect, therefore, the _Bacillus pyocyaneus_ is much
better adapted for study than the anthrax bacillus or many other
micro-organisms. Gheorghiewski vaccinated green frogs (_Rana
esculenta_), which had been accustomed to the incubator temperature, 30°
C., by injecting every 4 to 7 days considerable doses of cultures of
_Bacillus pyocyaneus_ heated to 80° C. in order to kill all the
micro-organisms. Some (3–4) weeks afterwards the prepared frogs became
more resistant to the _Bacillus pyocyaneus_ than were the controls
placed under the same conditions. The frogs, inoculated with a fatal
dose of the bacilli, clearly exhibited a certain, though slight, degree
of acquired immunity. They withstood a dose that was always fatal to the
controls or even a dose and a half, but died when injected with double
the fatal dose. The lymphatic fluid of the vaccinated frogs feebly
agglutinated (1 : 20–1 : 30) the _Bacillus pyocyaneus_ although it still
formed an excellent culture medium for this organism. Gheorghiewski
satisfied himself that the agglutination was insufficient to assure
immunity to the frog. The bacilli agglutinated into clumps were very
virulent.
[Sidenote: [222]]
A detailed examination of the phenomena observed in the immunised frogs
revealed the following facts. During the earliest stage the bacilli,
injected into the dorsal lymphatic sac, were found free in the fluid,
retained their form and were not transformed into granules. The bacilli
carried by the lymphatic current spread rapidly throughout the body.
Very shortly after inoculation, however, some of the leucocytes began to
ingest the bacilli which became transformed into spherules within these
cells. Later, the phagocytic reaction increased and at the end of 15 to
20 hours all the bacilli were found inside leucocytes. It was easy to
demonstrate that these bacilli had been ingested in a living condition.
Forty-eight hours after inoculation, no bacilli were to be found in the
lymph of the dorsal sac, either inside or outside the cells. But this
fluid when sown on nutrient media gave colonies of the _Bacillus
pyocyaneus_ up to 15 and even 18 days after inoculation.
We may conclude from these facts that the cold-blooded vertebrata are
capable of acquiring immunity to a slight degree and that, in this
acquired immunity, a marked phagocytosis may be observed, but no
bactericidal action of the fluids.
In order to gain a more complete idea of the mechanism of acquired
immunity, it is necessary to observe it in higher vertebrates in which a
well developed immunity of this type is readily obtained. Here we must
have recourse to mammals and pass in review an ample number of examples,
before we attempt to give to our readers a general summary of the
question.
For long, researches on acquired immunity were confined almost
exclusively to the analysis of the facts observed in animals submitted
to anti-anthrax vaccinations by means of the two vaccines of Pasteur. A
large number of important facts were thus collected, the more weighty of
which must be presented to the reader. But, before entering on the
subject, a general orientation on acquired immunity in laboratory
animals against vibrios is indispensable as this example dominates, so
to speak, the whole of the chapter on acquired immunity against
micro-organisms.
[Sidenote: [223]]
Von Behring and Nissen[308], in their researches on the bactericidal
power of serums, examined, amongst others, several specimens of serums
coming from animals that had been vaccinated against various
micro-organisms. In the majority of the examples given by them the
acquired immunity produced no increase in this power, but the blood
serum of guinea-pigs that had been immunised against Gamaleia’s vibrio
(_Vibrio metchnikovi_) was found to be much more bactericidal as regards
this micro-organism than the serum of normal susceptible guinea-pigs.
These authors came to the conclusion that in acquired immunity, at least
as regards the vibrio mentioned, the chief part is played by a
bactericidal substance which is developed in the fluids of the
vaccinated animals. They were content with the mere demonstration of
this fact without making any attempt to follow the course of events in
the destruction of the vibrios as it occurs in the organism of the
vaccinated guinea-pig. R. Pfeiffer[309] in collaboration with Issaeff
sought to fill this gap. But, instead of taking Gamaleia’s vibrio, these
observers concentrated their attention on the study of the acquired
immunity of guinea-pigs against the cholera vibrio. As this vibrio is as
a rule less virulent than Gamaleia’s vibrio, it was necessary, in order
to obtain a fatal infection, to inject it, not into the subcutaneous
tissue but into the peritoneal cavity. We have already seen (Chapter VI)
that the cholera vibrio when inoculated into the peritoneal cavity of
the guinea-pig, there meets with a vigorous resistance on the part of
the leucocytes which seize the living and virulent vibrios and digesting
them rid the animal of their presence. But when the dose of the vibrios
is increased, they multiply in spite of the phagocytic reaction; they
are found swarming in the peritoneal cavity, whence they invade the
lymphatic and blood vessels and cause the death of the animal. It is
easy, then, to induce a fatal infection of the guinea-pig with the
cholera vibrio. But it is also easy to vaccinate these animals against
this experimental disease. We have only to inoculate them with a
non-fatal quantity of living cholera vibrios, or to inject into them a
culture in which the vibrios have been killed by heat, or some of the
culture fluid from which the vibrios have been removed by filtration.
All these methods soon produce an acquired immunity in guinea-pigs. If,
when this has been brought about, a little blood is withdrawn and to the
serum a small quantity of cholera vibrios is added, _in vitro_, we can
readily demonstrate their disappearance, under the influence of the
bactericidal substance dissolved in the fluid. In this respect there is,
then, a marked analogy with the fact established by v. Behring and
Nissen as regards Gamaleia’s vibrio.
[Sidenote: [224]]
When into the peritoneal cavity of vaccinated guinea-pigs a certain
quantity of cholera culture containing virulent and very motile vibrios
is injected, we find that in the peritoneal fluid drawn off by means of
a fine pipette, the vibrios have undergone profound changes in the
refractory organism. Even a few minutes after the injection of the
vibrios, the leucocytes disappear almost completely from the peritoneal
fluid; and only a few small lymphocytes and a large number of vibrios,
the majority of which are already transformed into granules, are found
(fig. 39); and there is presented a most typical case of Pfeiffer’s
phenomenon. Alongside the round granules may be seen swollen vibrios,
and others which have kept their normal form, but all are absolutely
motionless. Some of these granules are gathered into small clumps,
others remain isolated in the fluid. When to the hanging drop containing
these transformed vibrios a small quantity of a dilute aqueous solution
of methylene blue is added, we observe that certain granules stain very
deeply, whilst others take on merely a very pale tint, scarcely visible.
Many of these granules are still alive, because it is easy to watch them
develop outside the animal and elongate into new vibrios. A large number
of the granules, however, no longer exhibit any sign of life and are
evidently dead. R. Pfeiffer and certain other observers affirm that the
granules may be completely dissolved in the peritoneal fluid just as a
piece of sugar dissolves in water. We have repeatedly sought for this
disappearance of the granules in hanging drops of the peritoneal fluid,
without being able to find any diminution in the number of these
transformed vibrios, even after several days; nor have we been able to
observe the phenomenon of the solution of the granules. It is at any
rate indisputable that this granular transformation is a manifestation
of very profound lesions undergone by the cholera vibrios under the
influence of the peritoneal fluid of the immunised animal.
[Illustration:
FIG. 39. Cholera vibrios in the peritoneal cavity showing Pfeiffer’s
phenomenon.
]
[Sidenote: [225]]
An attempt has been made to define the mechanism of Pfeiffer’s
phenomenon more exactly, and Fischer[310] has sought to refer it to
osmotic action, exercised by the salts of the fluids in which the
vibrios are suspended. These vibrios, under the action of media richer
or poorer in salts than is the fluid in which they had developed, are
said to present an increase of their internal pressure, in consequence
of which the vibrios swell up or allow a spherical droplet of protoplasm
to escape at one of their poles. This explanation was inadequately
supported by its author and cannot be regarded as proved. On the other
hand, one is compelled to the conclusion that the granular
transformation is due, as we shall see later, to a fermentative action
of the peritoneal exudation.
Whilst the vibrios are undergoing this transformation in the peritoneal
cavity of an immunised guinea-pig, the animal recovers from a _malaise_
that is quite transitory and continues to live, whilst normal
unvaccinated guinea-pigs die, an enormous quantity of vibrios swarming
in the peritoneal exudation. The difference between the two animals is
most striking, and we can readily understand that Pfeiffer was so
impressed by it that he was led to attribute the acquired immunity of
his guinea-pigs solely to the granular transformation set up by a
bactericidal substance contained in the fluids of the immunised animals.
[Sidenote: [226]]
The ease with which we can gain an idea of the change of form in the
vibrios under the influence of the fluids of the body, greatly aids the
study of the bactericidal substance. Before passing to the question of
the part played by this substance in acquired immunity we must consider
for a moment the principal properties of this acquired immunity. Very
manifest in the peritoneal fluid, the power of causing Pfeiffer’s
phenomenon is equally evident in the blood serum of immunised
guinea-pigs, as has been demonstrated by Bordet. A drop of this serum,
when quite fresh, readily and rapidly transforms a number of vibrios
into granules. When the serum is kept for several days or has been
heated to 55° C. for an hour, the total disappearance of the substance
which produces Pfeiffer’s phenomenon is brought about. This at once
betrays the presence of microcytase in the fluids of guinea-pigs that
have acquired immunity against the cholera vibrio. Yet the blood serum
and the peritoneal fluid of these animals, having been deprived of their
microcytase by heating to 55° or 56° C., still retain a remarkable power
over the vibrios. These organisms no longer undergo granular
transformation, under the influence of the heated body fluids, but they
are deprived of all power of motion, agglutinate into clumps and acquire
a special susceptibility to the action of cytase. Soon after the
discovery of Pfeiffer’s phenomenon, I[311] was able to demonstrate that
this granular transformation can be obtained _in vitro_ under the
following conditions. Prepare a hanging drop with the blood serum of a
guinea-pig vaccinated against the cholera vibrio, a serum which has lost
the power of transforming, by itself, the vibrios into granules. Add to
it a drop of the peritoneal lymph of a normal unvaccinated guinea-pig;
this lymph contains dead or living leucocytes and is, by itself, also
incapable of producing Pfeiffer’s phenomenon. When, to the mixture of
these two fluids, which are inactive when they are employed separately,
a few cholera vibrios are added, they are quickly transformed into
granules. This transformation, obtained _in vitro_, is remarkably like
that produced in the peritoneal cavity of the vaccinated animal.
Jules Bordet[312], working in my laboratory, made a very complete
investigation of Pfeiffer’s phenomenon outside the animal body and found
that, in my experiment, the peritoneal lymph can be replaced by the
blood serum of the vaccinated guinea-pig without thereby in the least
hindering the granular transformation. After making a specially thorough
study of the question he has come to the conclusion that Pfeiffer’s
phenomenon is the result of the action of two substances. One of these
is found in the blood serum and in the peritoneal fluid of guinea-pigs
vaccinated against cholera, heated to 55°–56° C. or deprived by some
other means of their individual power of transforming vibrios into
granules. This substance resists this temperature and only loses its
activity on being heated to 68°–70° C. The second of the two substances,
that found in the peritoneal lymph or in the blood serum of the normal
guinea-pig, is, on the other hand, destroyed at 55°–56° C. and is
nothing but the ordinary cytase (or alexine) present in the fluids of
normal animals.
[Sidenote: [227]]
The facts we have described with regard to Pfeiffer’s phenomenon in the
body fluids of immunised animals must, then, be interpreted as follows.
The fresh peritoneal exudation or blood serum of these animals readily
produces the granular transformation, because in these fluids both the
two necessary substances are found. But as microcytase is a very
unstable substance which, under the influence of time or heating to
55°–56° C., is destroyed, the fluids of immunised animals are very
readily deprived of it. The blood serum then, after being some time
outside the body, becomes incapable of transforming vibrios into
granules; but when to it is added a small quantity of the cytase, found
in the blood serum or in the peritoneal lymph of the normal guinea-pig,
the transformation takes place with great rapidity. To the serum of the
immunised animal, which has become inactive, is restored its property of
setting up Pfeiffer’s phenomenon. This interpretation, formulated by
Bordet, corresponds to the whole of the known data and is now generally
accepted.
[Sidenote: [228]]
As the fluids of immunised animals, that have become incapable of
transforming vibrios into granules, still retain their power of
rendering these organisms motionless and of uniting them into clumps, it
might be asked whether this agglutinative substance might not be the
substance, stable under heat, which is necessary for the production of
Pfeiffer’s phenomenon. For some time, indeed, it was believed that this
phenomenon is due to the microcytase acting on vibrios which have first
been modified by the agglutinative substance. This latter substance
resists heating to 55°–56° C., is only destroyed at higher temperatures,
and is retained in the blood serum long after the cytase has entirely
disappeared. The analogy between the agglutinative substance of the
fluids of animals that have acquired immunity and the substance in the
same fluids that is stable under heat is undeniable, and yet these two
substances are not identical. A whole series of observations, which we
shall presently describe, demonstrate this thesis clearly. A serum may
be highly agglutinative without being capable of bringing about the
transformation of vibrios into granules; the converse also holds good.
The substance which sets up Pfeiffer’s phenomenon, and which is found in
the fluids of immunised guinea-pigs, is a “fixative substance” analogous
to those we have already met with in the serums of animals so adapted
that they are able to resorb the various cell elements. As in the
resorption of cells, so also in the destruction of micro-organisms, the
fixatives are specific. The substance which aids the transformation into
granules is not only distinct from the fixatives which sensibilise red
blood corpuscles or spermatozoa, but also from the fixatives which
sensibilise bacteria. This specificity has been demonstrated and
carefully studied by Pfeiffer, who has shown that it may even serve to
distinguish species of bacteria. The serum of a guinea-pig which has
been immunised against the cholera vibrio, will render sensitive these
vibrios, and these only, to the action of the microcytase. Even allied
vibrios, such as various water vibrios, for example, are not sensitive
to the fixative of anticholera serum. On the other hand, the serums
obtained after the inoculation of these aquatic vibrios are incapable of
producing granular transformation in the cholera vibrio.
When we inject into one and the same animal several species of vibrios
we obtain a serum or a peritoneal fluid which produces Pfeiffer’s
phenomenon with the vibrios of all the species which have been used to
make the inoculations. This antivibrio serum contains only a single
cytase for the vibrios, but contains as many different fixatives as
there were species inoculated.
[Sidenote: [229]]
The transformation of vibrios into granules, when produced in a high
degree against virulent vibrios, under the influence of the body fluids
of immunised animals, affords a very valuable indication of the
simultaneous presence of cytase and of specific fixative. As we have
already stated, at the commencement of this account of the acquired
immunity of guinea-pigs against the cholera vibrio, Pfeiffer’s
phenomenon is manifested in the peritoneal fluid of these animals in a
very short time (5 to 20 minutes) after the inoculation of the vibrios.
This proves that in this fluid the two substances really occur together,
and that the fixative and the cytase are in solution in the plasma of
the exudation. Is it the same in every part of the body of immunised
guinea-pigs? If, instead of introducing the cholera vibrio into the
peritoneal cavity, we inject it into the subcutaneous tissue or into the
anterior chamber of the eye of these animals, Pfeiffer’s phenomenon does
not make its appearance. The vibrios, isolated or collected into small
clumps, do not undergo granular transformation; they keep their normal
vibrio form and remain alive much longer than in the peritoneal cavity.
Some of them may be found still living 24 hours after subcutaneous
injection and several (4–6) days in the anterior chamber of the eye. Nor
can Pfeiffer’s phenomenon be observed when the cholera vibrio is
introduced into the oedema of the foot, produced in consequence of the
slowing of the circulation, the vibrios remaining alive for a fairly
long time. These facts clearly indicate that in the fluid thrown out in
passive oedema, just as in the aqueous humour of the eye or in the
subcutaneous tissue, the two substances necessary to set up the granular
transformation are not present. Are both of them absent or only one?
This question is easily answered on adding to the fluids mentioned
normal guinea-pig’s serum, a serum which, by itself, is incapable of
producing Pfeiffer’s phenomenon. Bordet[313] has made these experiments
and found that when to the fluid of the passive oedema of the immunised
guinea-pig normal serum is added, this fluid transforms the cholera
vibrio into granules, but does so in less degree than does the serum of
the same immunised guinea-pig, when heated to 55°–56° C., to which
normal serum has likewise been added. There is, then, reason to conclude
that the fluid of the oedema does not contain any cytase, but contains a
certain quantity of cholera fixative, less, however, than that which is
found in the blood serum. As to the aqueous humour of the eye of
immunised animals, analogous experiments have demonstrated that it
contains neither of the two substances necessary for the production of
Pfeiffer’s phenomenon.
With the help of the facts I have here summarised, we arrive at the
following conclusion. In the animal that is immunised against the
cholera vibrio, microcytase is found in the peritoneal exudation; it
does not pass, however, either into the fluid of the passive oedema or
into the aqueous humour of the eye; the cholera fixative is found in the
peritoneal fluid and passes into the oedema, but does not penetrate into
the fluid of the eye. This indicates that microcytase is found in fluids
rich in leucocytes, but is absent from those which contain very few or
none of these cells.
The introduction of vibrios into the peritoneal cavity of immunised
guinea-pigs at once produces Pfeiffer’s phenomenon, and at the same time
causes the disappearance of the majority of the leucocytes from the
peritoneal lymph. We have already had occasion, several times, to speak
of this phagolysis, because it is produced as a sequel to the injection
into the peritoneal cavity of blood, spermatic fluid, and all kinds of
fluids. The greater the quantity of fluid injected and the greater the
difference of the temperature between it and the contents of the normal
peritoneum the more vigorous is phagolysis.
[Sidenote: [230]]
Pierallini[314], working in my laboratory, studying phagolysis in the
peritoneal cavity of the guinea-pig, has obtained several results worthy
of attention. Of all the fluids used by him, such as water, broth,
filtered cultures of micro-organisms and physiological saline solution,
the last of these caused the least intense phagolysis, yet one
sufficiently well marked. Immediately after the injection of any of the
above fluids the number of leucocytes in the peritoneal lymph diminishes
very considerably, the cells being found collected in clumps on the
omentum. Many of them exhibit signs of enfeeblement and of partial
destruction. Alongside the leucocytes are found fibrinous masses, this
affording evidence that some of the leucocytes have been greatly damaged
and have given up the fibrin-ferment which induces coagulation of the
fibrin. When Pierallini injected fluids containing coloured powders in
suspension, such as Indian ink and vermilion, he observed that these
substances accumulated on the greater omentum, which became stained
black or red. Microscopical examination revealed the existence of a not
very intense phagocytosis and a number of free coloured granules in the
midst of filaments of fibrin.
[Sidenote: [231]]
The leucocytes which, during this phagolysis, allowed the fibrin-ferment
to escape might also give up a certain amount of their microcytase. This
microcytase would pass into the peritoneal fluid and give rise to
Pfeiffer’s phenomenon. If this hypothesis be correct, the suppression of
phagolysis would result in the absence of the transformation of the
vibrios into granules. It is not a difficult matter to verify this
hypothesis as we have a means of preventing phagolysis or at least of
reducing it very considerably. Issaeff[315], in an investigation carried
out in Pfeiffer’s laboratory, demonstrated that an intraperitoneal
injection of physiological salt solution, broth, urine, etc., reinforces
the leucocytes and brings them up in large numbers into the peritoneal
cavity. It is easy to foresee that such an injection would serve to
diminish the intensity of the phagolysis. In fact, if we first inject a
few cubic centimetres of physiological salt solution or of fresh broth
into the peritoneal cavity of a guinea-pig, and if, on the following
day, we repeat the same operation, we shall find that after the second
injection phagolysis is much less powerful than after the first.
Pierallini, who repeated these experiments, observed that the
phagocytosis of the coloured granules is much more complete in the
guinea-pigs that were treated by a preliminary injection into the
peritoneal cavity. The amount of fibrin on the omentum is in this case
much less, and the phenomena as a whole show that in these guinea-pigs
the damage to the leucocytes is very considerably attenuated.
We have been able to demonstrate that in the case where phagolysis is
thus diminished, Pfeiffer’s phenomenon is not produced or is manifested
in a very feeble degree. If the experiment succeeds, the fluid taken
from the peritoneal cavity of a guinea-pig prepared the day before and
then injected with a culture of cholera, is opaque and thick, like pus.
It contains a mass of leucocytes in good condition, a large number of
which gorge themselves in a few minutes with a number of vibrios. The
plasma of this exudation contains few vibrios, and these retain their
normal form and do not exhibit, save exceptionally, a granular change. A
little later there remain no free vibrios; they are all contained within
leucocytes. Pfeiffer[316] declared himself against the facts I have just
summarised, because he was never able to prevent the granular
transformation of the vibrios, in spite of the preparatory injection of
sodium chloride. Abel[317], who repeated the experiments, expressed an
intermediate view: in guinea-pigs prepared by injections the day before,
he observed that one portion of the vibrios became transformed into
granules, whilst another became the prey of the leucocytes. The fact is,
the suppression of phagolysis demands special conditions: the broth that
is injected must be freshly prepared, and before its introduction into
the peritoneal cavity it must be heated to 37°–39° C. Even when these
precautions are taken it sometimes happens that the experiment is not
very successful. In making the experiment we must be guided by the
appearance of the peritoneal fluid withdrawn into the small glass
pipettes. If the fluid which enters the tube is clear or scarcely
clouded, it indicates that phagolysis has taken place, in spite of the
preparatory injection. The experiment is successful in those cases where
the peritoneal exudation is very cloudy and resembles pus.
[Sidenote: [232]]
As the demonstration of the suppression of Pfeiffer’s phenomenon as well
as that of phagolysis is of fundamental importance, I asked M.
Garnier[318] to carry out further experiments with the object of setting
the question at rest. Using a whole series of fluids for the preparatory
injection he found that fresh broth gives the best results. In
guinea-pigs in which the phagolysis had been reduced to a minimum,
phagocytosis commenced immediately after the injection of the vibrios.
In from two to five minutes many vibrios are found inside the
leucocytes, the free vibrios now being few in number and not exhibiting
Pfeiffer’s phenomenon. Garnier in his memoir gives photographic
reproductions of leucocytes crammed with vibrios; these should convince
the veriest sceptic. Since the publication of this paper no objection
has been advanced, and this question of the suppression of the granular
transformation of vibrios may now be regarded as definitely settled. I
have since demonstrated this feature to many observers, all of whom have
assured themselves of its accuracy. It must, then, be accepted that
Pfeiffer’s phenomenon is not produced in the peritoneal cavity except
when there is phagolysis. As this fact renders it very probable that the
microcytase, which is necessary for the transformation of the vibrios,
escapes from the injured leucocytes, it becomes necessary to verify this
hypothesis by a series of other experiments. If this hypothesis be well
founded, Pfeiffer’s phenomenon should not be observed in those
situations in the body where there are no, or almost no, leucocytes
already present. These conditions can be realised by injecting cholera
vibrios into the subcutaneous tissue or into the anterior chamber of the
eye of guinea-pigs that are well vaccinated against the cholera vibrio.
Under these conditions, as I have demonstrated in my work on the
extracellular destruction of cholera vibrios, the vibrios retain their
normal form and are never transformed into granules. Pfeiffer has
questioned this result, stating that beneath the skin of vaccinated
guinea-pigs the granular transformation is always produced, though in a
more feeble fashion and after more delay than in the peritoneal cavity.
The contradiction between Pfeiffer’s experiments and my own can,
however, be explained. When inoculating the vibrios into the
subcutaneous tissue, or during the withdrawal of the exudation formed at
the point of infection, small haemorrhages are sometimes produced and a
certain amount of microcytase is set free from the leucocytes found in
the effusion of blood; these cells also give up to the extravasated
blood a portion of their fibrin-ferment. When the experiment is
successful, that is to say when no haemorrhage is produced during the
operations involved, the subcutaneous exudation contains normal vibrios
only, without the appearance of any trace of Pfeiffer’s phenomenon in
the fluid.
[Sidenote: [233]]
If the extracellular transformation of the vibrios into granules were
the real cause of the acquired immunity, the absence of this phenomenon
in the subcutaneous tissue of the vaccinated guinea-pig should lead to
the death of the animal. As a matter of fact this does not take place
and the animal resists the inoculation of the vibrios. This conclusion
is open to one serious objection. As the cholera vibrio in the great
majority of cases is incapable of producing a fatal infection when
inoculated subcutaneously, even in normal unvaccinated guinea-pigs, this
example of immunity must be placed in the category of natural immunity,
a kind of immunity which may depend on causes other than those on which
acquired immunity depends. To answer this objection it was necessary to
select a race of vibrios capable, when injected subcutaneously, of
causing death. Mesnil[319], chief of my laboratory staff, undertook to
carry out experiments with the Massowah vibrio, which is regarded by
some authors as belonging to the true cholera species. When inoculated
subcutaneously into unprotected guinea-pigs, it induces local oedema, in
which the vibrios swarm; the infection rapidly becomes generalised and
causes the death of the animal in 24 hours. Yet this vibrio, when
injected into the subcutaneous tissue of well vaccinated guinea-pigs, is
completely resisted by these animals and not the least trace of
Pfeiffer’s phenomenon is produced. Under these conditions, a certain
number of the vibrios are at first united into masses, but a
considerable number remain isolated and motile. Some hours after
inoculation the number of clumps diminishes and the isolated vibrios
become more numerous, a fact which indicates a certain amount of
adaptation of the vibrio to the medium in which it now finds itself. But
never, so long as the vibrios remain free in the subcutaneous exudation,
do they become transformed into granules.
[Sidenote: [234]]
Salimbeni[320], in an investigation carried out in my laboratory,
endeavoured to satisfy himself whether or no Pfeiffer’s phenomenon is
produced in the subcutaneous tissue of a horse that had been
hyperimmunised against the cholera vibrio. This animal had, during a
period of 14 months, received considerable quantities of these
microorganisms, and the serum of its blood transformed the vibrios into
granules with great rapidity and intensity. In spite of such favourable
conditions for the manifestation of Pfeiffer’s phenomenon, it was never
produced beneath the skin of this horse. The vibrios when injected in
this position became completely motionless in a very short time, but
they kept their vibrio form and remained alive for a number of hours.
The exudation drawn off up to 24 hours after inoculation still gave
growths of the cholera vibrio.
As it is more easy to introduce, without effusion of blood, the cholera
vibrio into the anterior chamber of the eye than beneath the skin, and
as the aqueous humour contains no fixative, the absence of the granular
transformation in the first of these two situations has been observed
even by Pfeiffer himself. The demonstration of this fact presents no
difficulty, and for a considerable period we may observe free and
perfectly motile vibrios moving about in the aqueous humour. The
exudation from the eye contains many of these living organisms, which
when sown on culture media made their appearance as colonies even when
the fluid has been withdrawn from the eye several days after
inoculation.
[Sidenote: [235]]
These carefully established facts show very clearly that the microcytase
is only met with in the fluids of the living animal in those situations
in which there are many pre-existing leucocytes and under conditions in
which the cells undergo a more or less marked phagolysis. This may be
corroborated by a decisive experiment. When we inject a suspension of
the cholera vibrio directly into the veins of a guinea-pig, well
vaccinated against these organisms, and whose serum produces _in vitro_
Pfeiffer’s phenomenon with great rapidity, this phenomenon is not
manifested. This experiment has been performed and described by
Bordet[321]. Having injected a suspension of this vibrio into the
jugular vein of a guinea-pig vaccinated against the cholera vibrio, he
killed the animal an hour later and found, in the blood of the heart,
vibrios that had kept intact their form and their property of staining
with methylene blue. Cultivation of the blood of the heart, liver and
spleen gave growths of vibrios. In another guinea-pig, hypervaccinated
against the same organism and inoculated by the same method, the blood
drawn off shortly (4–15 minutes) afterwards showed, in preparations
treated with methylene blue, well-stained vibrios, of normal form and
quite intact. This is the most direct proof of the absence of Pfeiffer’s
phenomenon in the blood fluid of a living animal that enjoys a very
marked acquired immunity. The intact vibrios were lodged inside the
leucocytes.
Levaditi[322] repeated these experiments in my laboratory and varied the
conditions under which the vibrios were injected into the blood vessels.
He was sometimes able to observe phagolysis of the leucocytes of the
blood and their almost complete disappearance from the peripheral
circulation. In these cases the injured leucocytes accumulated in the
pulmonary capillaries and masses of them were seen surrounding groups of
vibrios that were transformed into granules. It was, however, easy to
exclude phagolysis by preparing the animals by means of injections of
physiological saline solution or broth. Under these conditions the
leucocytes remained in the blood current and very soon ingested the
vibrios. Whilst the vibrios that were still free in the blood plasma
retained their form and staining power intact, those found inside
microphages were already, in great part, transformed into granules. The
rapidity with which these phagocytes ingest the vibrios and set up the
changes in them is really extraordinary.
In this case, which affords a typical example of the reaction of the
animal organism in acquired immunity, we see a very marked and immediate
phagocytosis. It is this same process that has already been described as
occurring in the peritoneal cavity of vaccinated guinea-pigs in which
phagolysis was absent as the result of preparatory injection. In the
subcutaneous tissue and in the anterior chamber of the eye, where
Pfeiffer’s phenomenon is regularly absent, the phagocytosis follows its
ordinary course and causes the destruction of the vibrios. This result
has been confirmed repeatedly—see works by Bordet, Mesnil and Salimbeni
already quoted.
[Sidenote: [236]]
We need only compare the extension of Pfeiffer’s phenomenon and that of
phagocytosis in animals that are immunised against the cholera vibrio,
to satisfy ourselves that the former phenomenon is a limited one whilst
the latter is general. There might be advanced against the latter
conclusion the fact of the absence of any ingestion of the vibrios in
the peritoneal fluid of guinea-pigs that are immunised but are not
preserved against phagolysis. When a little of the peritoneal fluid is
drawn off with small tubes shortly after the injection of vibrios into
the peritoneal cavity, as a matter of fact, only a very intense
Pfeiffer’s phenomenon is seen, phagocytosis being completely or almost
entirely absent. But this procedure is insufficient. If we are to get an
idea of what really takes place in the abdominal cavity, the animal must
be killed and the peritoneum and especially the omentum very carefully
examined. As first demonstrated by Max Gruber[323] and later by
Cantacuzène[324], the greater omentum is, in these cases, covered with a
thick layer which contains a large number of leucocytes, of which some
are filled with vibrios; further, this layer contains a mass of vibrios,
in part transformed into granules, in part agglutinated or isolated and
retaining their vibrionic form intact. As time goes on, the phagocytosis
becomes more and more marked, and it is impossible to deny its existence
or to attribute to it merely a secondary part.
We have seen that the suppression of Pfeiffer’s phenomenon in the
peritoneal cavity and in the blood, or its total absence in the anterior
chamber of the eye, does not in the least deprive the vaccinated
guinea-pig of its acquired immunity. The animal resists the vibrios
perfectly, without these requiring to be transformed into granules in
the body fluids. This transformation does take place undoubtedly, but
only inside the phagocytes. As already stated in the discussion on
natural immunity (Chaps. VI, VII) the vibrios, after being ingested by
the microphages, almost immediately undergo within these cells a change
in form, very similar to that observed in the real Pfeiffer’s
phenomenon. The microphages are often full of a quantity of granules,
derived from the ingested vibrios, which in a short time are completely
digested. This fact, of such constant occurrence in the phagocytosis of
the vibrios, furnishes us with still another proof of the microphagic
origin of microcytase.
[Sidenote: [237]]
If Pfeiffer’s phenomenon is merely a particular instance of the
transformation of vibrios into granules in fluids containing
microcytase, it is quite natural that its suppression should not involve
a fatal infection of the vaccinated animals. On the other hand, if the
phagocytic reaction, so widely different, really plays an important part
in acquired immunity, everything that interferes with phagocytosis must
at the same time compromise the refractory condition. With the object of
solving this question Cantacuzène[325], working in my laboratory,
undertook a detailed investigation of this point. He showed that the
injection of opium, in a non-fatal dose, narcotised the guinea-pig and
at the same time prevented the movements of the leucocytes. Small glass
tubes containing cholera vibrios and introduced beneath the skin of
vaccinated guinea-pigs, became filled with numbers of leucocytes in the
non-narcotised animal; in the guinea-pig that had received tincture of
opium, the tubes left for several hours contained no leucocytes and
later only did they begin to enter the tubes. When a strong dose of
cholera vibrios was injected into the peritoneal cavity of thoroughly
vaccinated guinea-pigs, the animals easily resisted the inoculation.
When, however, similarly vaccinated guinea-pigs were submitted to the
influence of tincture of opium, the same dose of vibrios caused their
death. In these narcotised animals, in spite of the considerable
dilatation and hyperaemia of the vessels and in spite of the marked
hyperleucocytosis of the blood, diapedesis was not produced during the
first few hours after the injection of the opium, and it was not till
later (5 to 6 hours after injection) that the leucocytes began to appear
in the peritoneal cavity. The vibrios profit by the period of inactivity
of the phagocytes to multiply, retaining their motility and also the
property of staining with basic aniline dyes. When the retarded
leucocytes make their appearance in the peritoneal cavity, they find it
already invaded by a multitude of vibrios. In spite of this the
leucocytes, especially the microphages, ingest an enormous number of the
organisms; this does not prevent the death of the guinea-pigs, though it
takes place some hours later than in the unvaccinated control animals.
At the moment of death, free vibrios are no longer found in the
exudation; they have all been ingested by the microphages, inside which
they have undergone granular transformation. On making a post-mortem
examination of the animal a large number of small heaps of vibrios, such
as are never met with in animals that have not been submitted to the
action of opium, are found on the omentum.
[Sidenote: [238]]
All that is necessary, then, is to retard the phagocytic reaction for a
few hours in order to cause well-vaccinated guinea-pigs to succumb to
the action of the vibrios. One can readily understand that, with this
result before us, there can be no hesitation in attributing to
phagocytosis a much more important part in assuring acquired immunity
than to Pfeiffer’s phenomenon.
The study of other diseases produced by vibrios only serves to
corroborate the general conclusions that follow from the detailed study
of the essential processes in acquired immunity against the cholera
vibrio. It is here necessary to recall the discovery by v. Behring and
Nissen of the very marked bactericidal power of the blood serum of
guinea-pigs that have been vaccinated against Gamaleia’s vibrio. When
this fact was first demonstrated we were justified in thinking that the
vibriocidal property of the blood might by itself explain this acquired
immunity; but a comparative study of the phenomena which take place _in
vitro_ with those which take place in the living animal, soon
demonstrated how slight was the foundation for this hypothesis. Whilst
the vibrios, when sown in the blood serum of hypervaccinated
guinea-pigs, there perished in large quantities or even the whole of
them, these same organisms, when inoculated into the subcutaneous tissue
of the same animals, remained alive for several days. Gamaleia’s vibrio
is much less capable of being transformed into granules than is the
cholera vibrio, and we find it retaining its normal form even inside the
leucocytes. There is no occasion in this case, therefore, to look for
Pfeiffer’s phenomenon.
[Sidenote: [239]]
The rapid and marked destruction of Gamaleia’s vibrio, _in vitro_, in
the blood serum of vaccinated guinea-pigs, and the prolonged survival of
these organisms in the living animal, afford additional evidence that
the two groups of phenomena cannot be identical. On the other hand, it
furnishes a further proof that, during the preparation of the serum,
there is produced, parallel with the coagulation, another process which
confers bactericidal power on the serum. It is quite evident that, as in
the case of the cholera vibrio, we have here to do with the liberation
of microcytase at the expense of the destroyed or injured leucocytes.
Acting along with the specific fixative of the body fluids, this cytase
causes the death of the vibrios introduced into the serum. In the living
organism, the microcytase not being free, these vibrios, although
influenced by the fixative, resist until they have become the prey of
the phagocytes. In an investigation which was the subject of a
communication to the International Congress of Hygiene in London in
1891[326], I demonstrated that the phagocytic reaction is produced with
great intensity in guinea-pigs that have been vaccinated against
Gamaleia’s vibrio. The inoculation of this organism into the
subcutaneous tissue, an inoculation which sets up a rapidly fatal
infection in untreated guinea-pigs, gives rise in immunised animals to
the formation of an abundant exudation, in which the numerous vibrios
soon meet with resistance from the phagocytes. These phagocytes ingest
the living vibrios, retaining them for some considerable time in their
interior, but in the long run always digesting them completely. During
the last phase of this struggle, we sometimes find, inside the
leucocytes, vibrios that are transformed into spherical granules. It was
with these cells, filled with ingested vibrios, that I was able first to
carry out an experiment that has since been repeated again and again,
always with the same result. When from a well-vaccinated guinea-pig a
drop of subcutaneous exudation is withdrawn, at a stage when all the
vibrios have for some time been ingested by the leucocytes, and
transferred, in the form of a hanging drop, to the incubator at 35°–37°
C., it is found that the ingested vibrios develop inside the phagocytes
which have died outside the animal. The vibrios first fill the leucocyte
and, continuing to multiply, cause the cell to burst when they
distribute themselves in the fluid of the hanging drop (figs. 40 and
41). This experiment proves, in the first place, that the vibrios have
been ingested alive, and, secondly, that the plasma of the exudation was
incapable of preventing their later development.
[Illustration:
FIG. 40. Vibrios (_V. metchnikovi_) developed inside a microphage from
a vaccinated guinea-pig.
]
[Illustration:
FIG. 41. Vibrios (_V. metchnikovi_) developed in a drop of exudation
from a vaccinated guinea-pig. The vibrios have ruptured the
microphage and scattered themselves in the fluid.
]
[Sidenote: [240]]
[Sidenote: [241]]
Having summarised the principal phenomena exhibited by vibrios in an
animal possessing acquired immunity, we must now enquire whether the
mode of destruction and disappearance taking place in these vibrios is
of general application. Naturally, we commence this study with the
spirilla, which in many respects present a great analogy to the vibrios.
The task is an easy one, thanks to a very careful work recently
published by Sawtchenko[327], on the _Spirochaete obermeyeri_ of
recurrent fever. We know, from what has been said in Chapter VI, that
the spirochaetes found in the serum of persons attacked by this
organism, are, in the peritoneal cavity of guinea-pigs, destroyed by the
intervention of the macrophages. These phagocytes guarantee the natural
immunity of the guinea-pig against the parasite of recurrent fever. In
guinea-pigs, into which blood or serum containing spirilla has been
injected on several occasions, the destruction of these micro-organisms
is effected in a different way. When Sawtchenko introduced a number of
_Spirochaete obermeyeri_ into the peritoneal cavity of guinea-pigs so
prepared, he noted that they underwent a transformation resembling that
observed in Pfeiffer’s phenomenon. In a short time the majority of these
micro-organisms assumed the form of very delicate spirilla to which were
attached round granules. There was not a complete transformation of the
spirilla into granules, but a portion of their contents exuded in the
form of spherical drops. The spirilla that exhibited these changes lost
their motility and collected into clumps. There was undoubtedly an
extracellular transformation of the spirilla, but this took place only
in the peritoneal cavity. When injected into the subcutaneous tissue of
a prepared guinea-pig the spirilla brought about the formation of a firm
but scanty exudation in this situation. In this exudation were found
leucocytes containing spirochaetes which retained their normal form.
These micro-organisms were found exclusively in macrophages and gave no
evidence of the occurrence of Pfeiffer’s phenomenon. A like absence of
this phenomenon was observed in normal guinea-pigs which had been
injected subcutaneously with the same quantity of spirilla. In these
animals, however, the oedema that appeared at the seat of inoculation
was abundant and soft, and the disappearance of the spirilla, that is to
say their ingestion by the macrophages, took place at a very much later
period than in the prepared guinea-pigs. We have, therefore, in this
respect a complete analogy with the vibrios: in both cases there is an
absence of granular transformation below the skin and an ingestion by
the leucocytes of the exudation; on the other hand, we have Pfeiffer’s
phenomenon appearing in the peritoneal fluid. This analogy extends even
further. Thus, in guinea-pigs prepared by repeated injections of human
serum rich in spirilla, Sawtchenko could suppress Pfeiffer’s phenomenon
in the peritoneal cavity just as easily as in the case of the vibrios.
All he had to do was to inject a certain quantity of broth into the
peritoneal cavity of his immunised guinea-pigs. Twenty-four hours later,
on introducing spirilla into the animals at the same site, they retained
their motility for hours, did not exhibit any granular transformation
and were ultimately completely ingested by the macrophages.
[Sidenote: [242]]
These facts lead us to conclude that the fate of the spirochaetes of
recurrent fever in the organism of guinea-pigs prepared by previous
injections is governed by laws the same as those established for
acquired immunity against vibrios. The spirilla are ingested and
destroyed by the phagocytes, except where phagolysis occurs, in which
case the cytase, being set free, attacks the micro-organisms outside the
leucocytes.
[Sidenote: [243]]
After his discovery of the granular transformation of vibrios, R.
Pfeiffer, in collaboration with several of his pupils, set himself to
discover how far this phenomenon was general in acquired immunity. He
directed his attention to the typhoid cocco-bacillus, upon which he had
already published[328] a very detailed account of work carried out in
conjunction with Kolle. These observers availed themselves of the
discovery made by Beumer and Peiper[329], and Chantemesse and Widal[330]
and confirmed by other observers, that laboratory animals, especially
mice and guinea-pigs, could be easily vaccinated against the fatal
disease set up by the micro-organism of typhoid fever. As in the
experimental infection of the guinea-pig by the cholera vibrio, the
vaccination of the animals against the typhoid bacillus could be carried
out very easily, either by using sterilised cultures or the fluids of
cultures deprived of their organisms by filtration. In the small
laboratory animals a most marked acquired immunity may thus be obtained,
and the study of the phenomena which appear in the vaccinated organism
afforded evidence of a general analogy with those which have been
observed when vibrios are used. In the peritoneal cavity of the
immunised guinea-pigs, Pfeiffer’s phenomenon proper does not appear,
that is to say, only a few of the bacilli are transformed into granules,
the large majority retaining their bacillary form; still they are
evidently greatly damaged: they become motionless and agglutinate more
or less completely into clumps. If, however, a few of these
micro-organisms are sown on nutritive media, they multiply freely and
give abundant growths. The peritoneal fluid, then, acts most
unmistakably upon the typhoid bacillus, but in a much less degree than
does the peritoneal exudation of guinea-pigs upon the cholera vibrio
when immunised against that organism. In both cases we have a pronounced
phagolysis which sets free the microcytase, whose action on the vibrio
is more marked than on the bacillus of typhoid fever. This extracellular
action on the typhoid bacillus in the peritoneal cavity can be easily
prevented by a previous injection, twenty-four hours before, of broth,
physiological salt solution, or normal serum. The suppression of
phagolysis is, as in the case of vibrios and spirilla, followed by the
suppression of extracellular action on the typhoid bacilli.
The same analogy is observed in the phenomena which appear beneath the
skin. The bacillus of typhoid fever, when introduced into the
subcutaneous tissue of vaccinated guinea-pigs, although not appreciably
injured by the fluid of the exudation, undergoes some agglutination. The
injurious action of the fluids of the body is here still less effective
than in the peritoneal cavity. But, as in the peritoneal cavity of
vaccinated guinea-pigs previously treated with broth, so in the
subcutaneous exudation it is the phagocytes which destroy the
micro-organisms. In both cases there is a very great afflux of
leucocytes, mainly microphages. These cells ingest and digest the
bacilli, which ultimately disappear. The micro-organisms ingested by the
microphages, once inside these phagocytes are transformed into granules
very like those observed in the cholera vibrio similarly treated. In
this respect the analogy between the two micro-organisms is complete.
[Sidenote: [244]]
Oppel, working in my laboratory, has repeated Cantacuzène’s work on the
retarding action of opium upon the phagocytic process. He obtained the
same results: under the influence of the narcotic, the leucocytes
intervened only at a late stage, with the result that the vaccinated
guinea-pigs succumbed to the typhoid infection. The same conclusion must
be drawn from the experiments made by A. Wassermann[331]. Guinea-pigs
that had been immunised against the bacillus of typhoid fever were
completely resistant to a dose that was always fatal to the control
animals. When, however, along with this dose of bacilli, a certain
quantity (3 c.c.) of a serum which hinders the phagocytic reaction is
injected, the guinea-pigs lose their immunity and die from typhoid
infection. The serum employed by Wassermann was obtained from rabbits
that had been treated with the blood serum of guinea-pigs. Rabbit’s
serum, thus prepared, neutralises the action of the guinea-pig’s cytase,
but, as demonstrated by Besredka[332], it also exercises several other
functions, one especially, that of preventing phagocytosis. In
Wassermann’s experiments it was the antiphagocytic function, then, that
was the important factor in the suppression of the acquired immunity of
the guinea-pigs. These experiments supply a fresh proof of the great
importance of the phagocytic reaction in this kind of immunity, and
afford further confirmation of the analogy between the mechanism of
resistance of the animal’s organism against the typhoid bacillus and
that against the cholera vibrio.