Pfeiffer's experiments, 1895--The Buda-Pest Congress--Extracellular
destruction of microbes--Reaction of the organism against toxins--
Dr. Besredka's researches--Macrophages--The Moscow Congress, 1897--
Bordet's experiments.
Metchnikoff had scarcely recovered from all the emotions caused by his
experiments on cholera, which he was still studying, when, in 1894, a
work appeared by a well-known German scientist, Pfeiffer, bringing out
new facts in favour of the extracellular destruction of microbes.
Whilst studying the influence of the blood serum within the organism
and not outside it as his predecessors had done, he had found that
cholera vibriones, injected into the peritoneum of a guinea-pig
vaccinated against cholera, were nearly all killed in a few minutes
and that they then presented the form of motionless granules in
the peritoneal liquid. This granular degenescence, said Pfeiffer,
took place apart from the phagocytes and therefore without their
intervention. Metchnikoff repeated the experiment at once and
ascertained that it was perfectly accurate.
The complexity of biological phenomena being very great, he fully
admitted the possibility of other means of defence in the organism
besides that of the phagocytic reaction. However, this new fact
disagreed so much with his own observation, and seemed so isolated,
that Metchnikoff supposed an error of interpretation must have been
made and tried to throw light upon it. He spent sleepless nights
seeking the conclusive experiment which might explain Pfeiffer's
phenomenon.
His excitement was all the greater that he was very soon going to the
International Congress at Buda-Pest, where he intended to expose the
results of his new researches, and he feared that he should not have
time to make all the experiments which he required in support of his
arguments. However, the general impression of the Congress was clearly
favourable to the phagocyte theory. This is how M. Roux picturesquely
described the scene at Metchnikoff's Jubilee in 1915:
"I can see you now at the Buda-Pest Congress in 1894, disputing
with your antagonists; with your fiery face, sparkling eyes, and
dishevelled hair, you looked like the Dæmon of Science, but your
words, your irresistible arguments raised the applause of your
audience.
"The new facts, which had at first sight seemed to contradict the
phagocyte theory, now entered into harmony with it. It was found to
be sufficiently comprehensive to reconcile the holders of the humoral
theory with the partisans of the cellular theory."
This is how Metchnikoff had reconciled the apparent disagreement of
Pfeiffer's phenomenon with the phagocyte doctrine: he demonstrated,
by a series of experiments, that the extracellular destruction of
the cholera vibriones in the peritoneum of a guinea-pig vaccinated
against cholera, did in no wise depend on the _chemical_ properties
of the blood serum, but was simply due to the digestive juices which
had escaped from the inside of the leucocytes, damaged by the
intraperitoneal injection. Those digestive juices, or _cytases_, poured
into the peritoneal liquid were what killed the injected cholera
vibriones and transformed them into "Pfeiffer's granulations." On the
other hand, if by means of various precautions the phagocytes were left
unmolested, the extracellular destruction did not take place and the
vibriones were digested within the phagocytes.
Metchnikoff used other experiments to prove that the bactericidal
property of blood juices did not exist without intervention from the
phagocytes. For instance, in a guinea-pig vaccinated against cholera,
the bacilli are not destroyed if they are injected into parts of the
organism that are devoid of pre-existing phagocytes, such as in the
subcutaneous tissue, in the anterior chamber of the eye or in an
aseptically-obtained oedema. On the other hand, if, in the same
medium, some exudate is injected containing damaged leucocytes from
which the digestive juice is leaking, the vibriones introduced are
destroyed. The same results are obtained _in vitro_.
All these experiments proved that the extracellular destruction of
the cholera vibrio was accomplished by the digestive juices which had
passed from the phagocytes into the humors and not at all through a
special property of those humors. Once again the phagocyte theory rose
triumphant from the test.
After having finally proved that it is by means of its phagocytes that
the organism fights _microbes_, Metchnikoff wished to find out whether
it was by the same process that it struggled with their poisons, or
_toxins_. This problem, far more difficult to solve, took him many
years' study. Whilst every phase of the phagocytes' struggle against
microbes can be followed with the eyes, it is impossible to do so
where poisons are concerned, since they are invisible; it is necessary
to proceed by a different road.
Faithful to his method of taking as a starting-point the simplest
expression of the phenomenon to be studied, Metchnikoff began by
lower beings. Unicellular organisms, such as myxomycetes, amoebæ,
and infusoria, sometimes manifest a natural immunity to certain
poisons. It is also possible to endow them with artificial immunity
by accustoming them gradually to substances which, ingested straight
away, would infallibly have killed them. Such phenomena, seen in
unicellular beings, could only be ascribed to the reaction of the cell
itself. Therefore Metchnikoff supposed _a priori_ that the phagocytes,
being similar primitive cells of multicellular beings, would also
react against poisons. And, in fact, he ascertained that the number
of phagocytes in a rabbit's blood diminished considerably under the
influence of a fatal dose of arsenic, whilst it increased under the
influence of small doses of the poison, to which it was possible to
accustom the animal.
Dr. Besredka, a disciple of Metchnikoff, made some very interesting
researches, which entirely confirmed the share of the phagocytes in the
reaction against sulphides of arsenic. He had chosen the trisulphide,
a very slightly soluble salt of an orange colour, in order to find it
again easily within the organism. After having injected non-fatal doses
of it into the peritoneal cavity, he obtained an exudate in which all
the orange granules of the salt were to be found included within those
leucocytes which have a large, non-lobed nucleus--the _macrophages_.
These cells gradually digested the salt they had englobed, which ended
by disappearing entirely within them, and the rabbit remained safe and
sound. On the other hand, it died if the same doses of the same salt
had been protected from the leucocytes by an elderberry bag, or when
the leucocytes had been attracted elsewhere by a previous injection of
carmine for instance. Those experiments removed all doubts as to the
share of the phagocytes in the destruction of mineral poisons.
Certain experiments on _microbian_ poisons spoke in the same sense.
Thus MM. Roux and Borrel had observed that the diphtheritic toxin,
which is inoffensive to rats even in large doses, kills that animal
if a small quantity of it is introduced into the brain, the probable
explanation being that, in cases of subcutaneous injections, the
poison, "phagocyted" on the way, was destroyed before it reached the
nerve cells.
Thus experiments seemed to plead in favour of the view that the part
played by phagocytosis is not limited to the struggle against microbes,
but also extends to the defence against poisons and toxins.
* * * * *
After having studied the mode of destruction of these, Metchnikoff
wished to elucidate the origin of the counter-poisons, the specific
antitoxins discovered by Behring in the humors of immunised organisms,
a question of which the study was even more difficult.
Metchnikoff began by asking himself whether the microbes themselves
did not produce antitoxins in order to defend themselves against enemy
micro-organisms. He made many experiments but only obtained negative
results, and concluded that the antitoxins must be manufactured by the
organism itself.
The origin of this property must be more recent than that of the
phagocytic reaction, for it does not exist in plants or in inferior
animals. It was only from superior cold-blooded vertebrates, such as
the crocodile--and that only in artificial conditions--and upwards,
that Metchnikoff succeeded in finding a specific antitoxic power in the
humors.
He ascertained that the vaccination of animals by toxins conferred,
after a time, antitoxic powers to the blood and humors _which contained
leucocytes_. He concluded therefrom that the presence of antitoxins
depended on that of the phagocytes. Experiments on divers higher
animals having proved that, in them also, antitoxins were localised
in _humors containing phagocytes_, Metchnikoff concluded that the
antitoxins were manufactured by the cells themselves. As toxins are
absorbed and digested chiefly by _macrophages_, it is probable that
it is the latter also which manufacture specific antitoxins, or the
final product of the digestion of corresponding toxins. Metchnikoff
could only propound this idea as an hypothesis, for the complexity and
difficulty of a material demonstration did not yet allow of a definite
solution of the problem. However, certain observations on toxins and
antitoxins pleaded in favour of this thesis.
For instance, working in collaboration with MM. Roux and Salimbeni, he
had found that it is by soluble poisons that the cholera vibrions harm
the organism or kill it, but that small doses of the same _poisons_
are vaccines and make the blood of the vaccinated animal _antitoxic_.
On the other hand, a _microbian_ vaccination is preventive against
_microbes_ only but not against toxins and the blood does not become
antitoxic. This is explained by the fact that it is not the same cells
which digest cholera microbes and cholera toxins: the _microphages_
digest the vibriones whilst the _macrophages_ digest the poisons and,
probably, manufacture as products of this digestion, the corresponding
antibody, the cholera antitoxins.
On the contrary, in cases of the inclusion of _microbes_ by
_macrophages_, as, for instance, in plague, the blood acquires an
_antitoxic_ power by injection of the microbes themselves and not by
their toxins, as was demonstrated by M. Roux and his collaborators. The
same fact was observed by Metchnikoff on the alligator, in whom also
microbes are digested by _macrophages_. In those cases, when microbes
and toxins are digested by the same cells, the latter manufacture
antibodies against both.
These facts rendered legitimate the supposition of the macrophagic
origin of antitoxins.
* * * * *
In 1897 an International Congress took place in Moscow. Metchnikoff
read a paper on the phagocytic reaction against toxins and another
dealing with the whole of the knowledge acquired concerning human
plague. He ended this by a plea in favour of Science, so often accused
of having contributed nothing to the solution of the most important
human problems, particularly ethical ones, and of having, on the
contrary, sanctioned the law of Might by tabulating the laws of the
struggle for existence. Metchnikoff objected that, far from doing
so, Science, by revealing the laws of Nature, applied to humanity
the benefits derived from them, whilst striving to counterbalance
their cruel or harmful effects. The struggle against plague and other
diseases was a concrete example of this, for here medical science
opposed itself to the cruelty of "natural selection." He wound up
his speech by the following words, "Just as, in order to satisfy his
æsthetic tastes, Man revolts against the laws of Nature which creates
races of sterile and fragile flowers, he does not hesitate to defend
the weak against the laws of natural selection. Science has been
faithful to her mission and to her generous traditions. Let her, then,
progress unhindered."
Metchnikoff's friend and companion, M. Nocard, wrote to me concerning
Metchnikoff's paper:
Do not believe a word that Metchnikoff tells you. He had tremendous
success. The somewhat free form of his paper contributed to its
success, as it only made his conviction and enthusiasm more apparent.
Thus the Sibyl on her tripod.
Metchnikoff had at this period a very talented disciple, M. I. Bordet,
who opened a new path by a series of researches of the greatest
importance. He found, among other things, that "the figured elements"
can be destroyed outside the cells, in the humors. Thus, if red blood
corpuscles from one animal are injected into an animal of a different
kind, these globules are destroyed, not within the phagocytes,
but outside them, in the ambient humors. Metchnikoff studied this
phenomenon and proved that the explanation was the same that he had
previously given of Pfeiffer's phenomenon in the case of cholera
vibrions. In Bordet's experiments, the leucocytes which were already
existing in the humors were also damaged by the experimental shock;
but, if this was carefully avoided, the phagocytes, remaining intact,
englobed and digested the injected red corpuscles and no phenomenon
similar to Pfeiffer's took place.
These observations led Metchnikoff to a thorough study of the
destruction of cellular elements by the phagocytes. He had already
observed that, whilst the struggle with microbes is chiefly undertaken
by small leucocytes with a lobed nucleus--the _microphages_--it is
the great leucocytes with a single large nucleus--the so-called
_macrophages_--which undertake the destruction of cells, "figured"
elements, as well as that of toxins. The _macrophages_ are to be
found not only in the blood but also in different organs such as the
liver, spleen, kidneys, etc.; they seize upon living cells by means
of mobile protoplasmic prolongations with which they draw them in and
end by ingesting them completely. Not only do they thus absorb foreign
cellular elements such as red corpuscles, spermatozoa, etc., but also
all the weakened cells of the organism itself.
This weakening may be due to normal phenomena such as the metamorphosis
of insects or tadpoles, when certain organs, as they weaken,
become useless or inactive. But, oftener, this weakening is due to
pathological causes, as in morbid atrophies or poisoning by microbian
toxins. In any case, the enfeeblement of cells exposes them to be
devoured by macrophages, which brings about the atrophy of the cells or
even of the organs which contain them.
These observations suggested to Metchnikoff the idea that senile
atrophy might be due to the same mechanism, and his thoughts turned
towards the problem of the causes of old age.
But, before undertaking researches in a new direction, he wished to
conclude those he had been pursuing for twenty years on the phenomenon
of phagocytosis. He therefore started to complete his investigations on
immunity in order to epitomise them and to give a definite form to his
doctrine on that subject.