Mesnil has fed the same Actinians with repeated doses of blood with a

view to make out whether the cells, under these conditions, acquire any

special aptitude for the production of the actino-diastase.

Notwithstanding numerous attempts, he could never assure himself that

this takes place; the rapidity with which the red corpuscles were

dissolved by the extract of the mesenterial filaments was the same

whether this was prepared from Actinians that had been several times fed

on blood or from those that had received none at all.

From what I have just described no doubt can exist that intracellular

digestion is not a “protoplasmic” process essentially different from

that which is brought about by the digestive juices secreted in the

intestinal canal. In both cases we have a diastatic action, due to

soluble ferments, produced by living elements. In intracellular

digestion, however, the diastases carry on digestion in the interior of

the cells, principally in the vacuoles, whilst in extracellular

digestion this process goes on outside the cells, in the lumen of the

gastro-intestinal canal.

It cannot be doubted that, in the animal scale, intracellular digestion

represents an earlier and primitive condition for the solution of the

food substances. This follows from the fact that it is widely

distributed amongst the lowest animals, such as the Protozoa, Sponges,

Coelenterata and Turbellaria. Intracellular digestion only gives way

step by step to digestion by secreted juices. The higher Invertebrata

furnish us with conclusive testimony on this point. Thus, among the

gasteropod Mollusca, there are some which exhibit the two modes of

digestion in the same animal. In _Phyllirhoë_, a beautiful mollusk,

without a shell and quite transparent, which floats on the surface of

the sea, the food can be seen passing into the cavity of the digestive

canal, where it undergoes a preliminary digestion by secreted juices;

the result is a magma of small solid particles which are at once seized

by the amoeboid epithelium of the coecal appendages, two on each side of

the body. Intracellular digestion then completes the process and ends by

dissolving the nutritive substances and reducing them to their final

stage previous to absorption. On adding to the food some particles of

carmine these may be found along with the digestible particles in the

interior of the epithelial cells of the coeca.

[Sidenote: [63]]

This example furnishes us with a real link between primitive

intracellular digestion and the perfected and derivative extracellular

digestion. In the same group of Gasteropods may be followed out several

stages of this evolution so that in the higher representatives of the

group, such as the slugs and the snails, we meet with digestion carried

on only by secreted juices in the gastro-intestinal contents. In these

Mollusca a voluminous glandular organ, the liver, which is certainly

derived from coecal appendices similar to those of _Phyllirhoë_, is now

met with. Regarded from this point of view the liver is, as Claude

Bernard has stated, an organ of second digestion. I think that a

detailed study of the liver of the Mollusca, guided by this idea, will

give results of considerable importance.

In the Vertebrata intracellular digestion in the gastro-intestinal canal

almost disappears and is replaced by digestion carried on by means of

ferments contained in secreted juices. We cannot, of course, offer to

the reader anything like a complete account of this extracellular

digestion in the higher animals. It is necessary, however, to draw

attention to several aspects of this function which have been

established, thanks to the progress made during recent years, in

obtaining digestive juices and in the study of their action.

[Sidenote: [64]]

For the study of intracellular digestion the sea-anemone is the most

suitable animal for our purpose; for that of extracellular digestion the

dog. In this latter animal, an omnivorous flesh-eater, the food

substances are treated by digestive juices of great activity which

contain a whole series of soluble ferments. The stomach secretes two of

these: rennet and pepsin. The pancreas elaborates three: trypsin,

amylase and saponase, which act on the three main groups of food

substances. To these the small intestine adds a special ferment,

described by Pawloff[83] under the name of enterokynase. Every one

recognises the proteolytic function of pepsin and trypsin and the

analogies and differences between these two diastases. Nor need I dwell

on amylase or on the ferment which saponifies fats. But enterokynase

merits special attention in connection with the study of immunity.

Pawloff entrusted to his pupil Chépowalnikoff the study of the digestive

_rôle_ of the intestinal juice concerning which, up to this, very little

was known. It was known indeed that this juice contained weak

saccharifying and inverting ferments, but it was generally regarded as a

secretion of little importance. Chépowalnikoff[84] has demonstrated that

this view is absolutely erroneous. The intestinal juice fulfils the very

important function of accelerating the action of the three pancreatic

ferments. The duodenal juice of the dog, especially, contains

enterokynase. When this juice is mixed with a pancreatic juice that by

itself actively digests fibrin and albumen, digestion takes place still

more rapidly, the action being from three to four times as great. The

part played by the intestinal juice becomes even more evident when it is

mixed with a pancreatic juice that has little or almost no activity, as

is the case of that from dogs that have recently been operated upon.

Thus pancreatic juice, which has no action upon albumen, digests it

promptly when a certain quantity of duodenal juice is added. When

Chépowalnikoff took 500 c.c. of inactive pancreatic juice diluted with

500 c.c. of water or soda solution and added to it but a single drop of

intestinal juice, the mixture exerted a manifest digestive action on

coagulated albumen.

If, in place of pancreatic juice, we take the aqueous or glycerinated

extract of the pancreas, which by itself exerts a very insignificant

digestive action on albumen, and add to it intestinal juice, digestion

takes place immediately. If it be admitted, as several physiologists

maintain, that the inactivity of the pancreas is due to the fact that we

have zymogen present in place of trypsin, one might conclude with

Chépowalnikoff that “the intestinal juice possesses the power of

transforming the zymogen into trypsin, and that this transformation

takes place in a much more marked degree than in the presence of acids

or the oxygen of the air” (p. 137).

The intestinal juice, from whatever region of the small intestine it be

derived, exercises an undoubtedly favourable influence on the digestion

of starch by the pancreatic juice, but this action is much more feeble

than that on trypsin digestion. The action of the intestinal juice on

the saponification of fats is even less marked. But here it is to the

bile that the more important _rôle_ is transferred. This fluid also

augments the activity of the pancreatic juice, but in a manner different

from the intestinal juice, for it acts especially by accelerating the

digestion of fatty substances.

[Sidenote: [65]]

The action on the pancreatic digestion is not in any way interfered with

when the bile is heated to boiling point. On the other hand the

intestinal juice, under these conditions, completely loses its

accelerating _rôle_. It follows from this, as has been formulated by

Pawloff, that, in the intestinal juice, the existence of a soluble

ferment which is destroyed by heat must be admitted; to this ferment he

proposes to give the name of enterokynase. Without exercising a

digestive power on any of the alimentary substances, it may act as a

ferment of the pancreatic ferments.

Delezenne, at the Pasteur Institute, has repeated Chépowalnikoff’s

experiments. He has confirmed the accuracy of his results and has added

new data of great importance, not only as regards the physiology of

digestion but also in relation to the study of immunity. Enterokynase

appears from Delezenne’s experiments to be a true ferment; carried down

by the same precipitants (collodion, phosphate of lime, alcohol) which

enable us to obtain the greater number of the known ferments; it is

sensitive to high temperatures, and even that of 65° C. is sufficient to

do away with the greater part of its activity. Yet another property of

enterokynase, which it possesses in common with the soluble ferments and

which has for us a very special interest, is the facility with which it

attaches itself to fibrin. By means of flakes of this substance we can

at any time remove from a fluid the whole of the enterokynase contained

therein. This fixative property is very important in connection with the

part which enterokynase plays in digestion. The fibrin to which it has

become attached absorbs trypsin with great avidity. If we introduce

flakes of fibrin impregnated with enterokynase along with other flakes

which have not been in contact with this ferment into a solution of

trypsin, the former are digested with great rapidity, whilst the latter

do not undergo any change. The fibrin that has fixed enterokynase is

capable of clearing a fluid of its trypsin. On the other hand, that

which has not been acted upon by the intestinal juice leaves it there

almost unaltered.

[Sidenote: [66]]

It is of the utmost importance that we should inform ourselves as to the

origin of the enterokynase of the intestinal fluid. This fluid, when

obtained from a fistulous opening, for example, contains mucus and a

considerable amount of _débris_ of various kinds of cells. What are the

elements which furnish such a remarkable ferment? Delezenne has obtained

a very precise answer to this question. The enterokynase is not

contained in the mucus and is not secreted by the intestinal glands; it

comes from the lymphoid organs.

If the small intestine of a fasting dog be washed carefully with water

all the pre-existing enterokynase is removed from it. The Peyer’s

patches are then removed and treated with chloroform water. The other

parts of the small intestine are similarly treated. This fluid dissolves

the enterokynase, as it does the other soluble ferments. We find that

the Peyer’s patches furnish enterokynase, but that the rest of the

intestine, including Lieberkühn’s glands, give none.

We know that the Peyer’s patches are lymphoid organs in which are a

large number of amoeboid mononucleated cells, and that these elements

are even capable of ingesting foreign bodies and of submitting them to

intracellular digestion. It is therefore not at all astonishing that

Delezenne should have succeeded in finding enterokynase in the

mesenteric glands of several Mammals (dog, pig, rabbit). These glands,

when treated by the method just mentioned, yield a substance which

assists the action of trypsin just as does the intestinal juice. Having

reached this point, Delezenne asked himself whether the mononucleated

white corpuscles, so closely allied to the mononucleated cells of the

lymphoid organs, may not also contain enterokynase. With the object of

settling this point he collected exudates that were rich in

mononucleated leucocytes; in these also he found this same soluble

ferment. Moreover, the leucocytic layer of the blood showed itself

equally capable of increasing, very energetically, the action of

trypsin.

The results of the old experiments carried out by Schiff and by Herzen

on the adjuvant _rôle_ of the extract of the spleen in pancreatic

digestion, must without doubt be ranged alongside those we have just

indicated. In fact the mononucleated cells of the spleen, like those of

Peyer’s patches and of the mesenteric glands, contain a substance which

acts like enterokynase. Delezenne has given us a definite demonstration

of its presence and action.

[Sidenote: [67]]

In intracellular digestion it is the chemical side which has been most

difficult of demonstration. The purely physiological functioning, the

sensitiveness of the digestive cells and the amoeboid movements of their

protoplasmic processes are, on the other hand, so manifest that it has

even been suggested that intracellular digestion should be looked upon

as a protoplasmic phenomenon purely vital in character.

In extracellular digestion through the agency of secreted juices we have

a very different condition. Here the chemical side is the striking

feature, the physiological factor being veiled more or less completely.

Nevertheless, thanks to recent advances and above all to the labours of

Pawloff’s disciples in St Petersburg, this problem has been elucidated

in a very remarkable fashion.

The secretion of digestive fluids follows definite laws, the most potent

factor being the reflex action of the nervous system. To use the

expression of Pawloff, the study of the process of salivary secretion

has revealed a real psychology of these organs. You may fill the mouth

of a dog with small polished pebbles or with snow; you may pour into it

very cold water—the saliva will not flow. But merely allow the animal to

see sand in the distance—the glands at once begin to secrete fluid

saliva. Tempt the dog with flesh—and immediately a thick saliva appears;

show him dry bread—saliva is secreted in abundance, even if the dog has

no great desire to eat.

The same phenomena may be observed in the stomach. Mechanical

stimulation by inert bodies, such as stones, provokes no secretion; but

the suggestion of a meal or the sight of food is sufficient to call

forth a large quantity of gastric juice. The quantity and quality of the

gastric juice are regulated by the quantity and quality of the food.

Bread given to a dog provokes the secretion of a gastric juice endowed

with the greatest digestive power. That which flows after the ingestion

of milk contains only one-fourth as much pepsin.

[Sidenote: [68]]

In spite of these differences in the gastric secretion in relation to

food, Pawloff and his pupils have never been able to assure themselves

that there was any prolonged and chronic adaptation of the gastric

function. They were struck by the uniformity of the digestive power of a

great number of their dogs. Samoïloff[85] had under observation three

dogs placed on different diets. In spite of the very long periods during

which these diets were given, the gastric juice, in all the dogs,

presented the same properties and manifested no appreciable difference.

This result harmonises with that indicated above as obtained in the

Actinians fed with blood by Mesnil. In spite of repeated feedings on

blood from the same species of animal, the extract from the mesenterial

filaments was in no way different from that of the fasting Actinians

used for control.

The pancreatic secretion is, in many respects, a more perfect type. We

have here to do with the principal agent in the digestive function,

without which the organism could not continue to exist. The advances

made in surgery have enabled us to remove the stomach, first in the dog

and then in man, and there are already several persons[86] from whom the

stomach has been removed and who, in spite of this operation, have

continued to live. A portion of the small intestine may also be removed,

but, in order that life may not be endangered, a considerable portion of

it must be left intact. It is evident then that the pancreatic digestion

is an admirably organised function both in animals and in man. One of

the main regulators of this process of digestion consists in the great

sensitiveness of the intestinal mucous membrane. Just as the organs of

the buccal cavity possess in the specific sense of taste an excellent

means of discrimination in the choice of foods, so the mucous membrane

of the small intestine is endowed with a special sensitiveness,

comparable to the chemiotaxis of unicellular organisms and of the cells

of more highly developed organisms. Hirsch and Mehring have satisfied

themselves that the passage of the contents of the stomach through the

pyloric orifice depends on a reflex mechanism which proceeds from the

upper reaches of the small intestine. To the researches of the school of

Pawloff, however, we owe what light has been thrown on this question.

The duodenal mucous membrane is endowed with a well-developed

chemiotaxis for acid substances. The passage of the acid content of the

stomach into the duodenum determines this chemiotaxis and brings about a

secretion of alkaline juice which neutralises the acid. This contest

between acid and alkali forcibly calls to our mind the analogous

phenomena in those plants that defend themselves against the alkaline

secretions of parasites by the production of an acid (see Chapter II).

As in these lower organisms, this battle of the chemical secretions is

regulated by the action of living and sensitive parts.

When the acidity of the mass which passes through the pylorus is too

marked, the reflex contraction starting from the duodenal mucosa arrests

its passage. Then takes place a neutralisation of the acid, thanks to

the alkaline secretion, and the pylorus is again allowed to open. This

mechanism thus regulates the passage of the contents of the stomach into

the duodenum, the passage taking place in instalments.

[Sidenote: [69]]

The sensitive intestinal mucous membrane can estimate not only the

degree of acidity, but also the other chemical characters of the

aliments which pass into the duodenum. This chemiotaxis is, as it

were, the starting-point of the reflex action which excites the

pancreatic secretion with its contained three ferments. The passage of

bread through the pylorus excites the secretion of a juice very rich

in amylase and very poor in saponase. The passage of milk into the

duodenum brings forth, on the other hand, a juice very much richer in

saponase but poorer in amylase and in trypsin. Flesh-meat provokes the

secretion of a pancreatic juice which is less rich in amylase than the

juice poured on bread, but richer in saponase. Fat causes the

secretion of a juice still richer in saponase than is the juice poured

out in the presence of bread or milk. These facts now carefully

established—especially by Walter[87]—demonstrate that the pancreatic

function is carefully regulated as regards its adaptation to the

characters of the food substances on which it is to act. Such

adaptation may even become permanent.

Whilst, as already stated, the stomach, under the influence of a fixed

diet, is incapable of effecting any lasting modification in the

composition of its secreted juice, the pancreas may reach this degree of

perfection. When a dog is fed for several weeks on bread or on milk and

is then placed on flesh diet its pancreatic juice is found to become

progressively richer in trypsin. Whilst this augmentation of the

proteolytic power is being brought about, the juice becomes poorer and

poorer in amylase. Wassilieff[88] has carried out a large number of

experiments on this point and has demonstrated a very remarkable

adaptation of the pancreatic juice to the wants of nutrition, an

adaptation that may become permanent. A dog which has been accustomed to

digest bread and milk adapts itself to this nourishment: its pancreatic

juice contains less and less trypsin, but, on the other hand, becomes

richer in amylase. Pawloff observed that in dogs great variations in the

composition of the pancreatic juice are often present; this he

attributes to the diet to which these animals had been previously

subjected.

[Sidenote: [70]]

Not only does the quality of the digestive juices accommodate itself to

the wants of digestion; their quantity also undergoes variations

according to the part that these juices have to play. Thus, Pawloff has

observed that his dogs secreted a saliva which was very fluid and very

abundant when he gave them acids, bitter substances or other substances

they did not like. On the other hand, the presence of food in the mouth,

or even the sight of it, excited the secretion of a thick saliva

containing a large quantity of mucin. In the first case the part played

by the saliva was that of diluting the injurious substances as much as

possible, in the second that of facilitating the deglutition of the

food.

In general the organism manifests a tendency to produce more digestive

ferments than it actually needs for digestion. It is for this reason

probably that they are often found outside the digestive canal. Among

these ferments pepsin and amylase, especially, have been definitely

proved to be present in the urine of man and of some mammals, notably

the dog. The data as to rennet and trypsin are not so well established.

But, as several of these ferments, such as amylase and trypsin, may be

derived from several sources in the organism, their elimination by the

urine is less important for the thesis I have just formulated than is

that of pepsin.

Pepsin was found in the urine by Brücke exactly forty years ago. It is

more frequently found in the morning urine, but is absent from that

passed immediately after the principal meal. Leo and Senator[89] found

only traces of pepsin during the prolonged fast of the Italian Cetti;

but the day he broke his fast they were able to demonstrate the presence

of a considerable quantity of this ferment in his urine.

[Sidenote: [71]]

Delezenne and Froin, with the object of seeking the source of the

urinary pepsin, extirpated the stomach of a dog. After the animal had

recovered, they fed it well and examined its urine at different periods

of the day. By the methods which had shown the presence of pepsin in all

the normal dogs taken as controls they could never discover the faintest

trace of this diastase in the urine of the dog that had been operated

upon. On the other hand, the urine of a dog whose stomach had simply

been isolated, contained very much the same quantity of pepsin as that

of normal dogs. This experiment proved among other things that the

pepsin, before it could be eliminated by the kidneys, must have been

re-absorbed by the wall of the stomach. From these data, combined, it

must therefore be admitted that the pepsin found in the blood and which

passes thence into the urine can only be of gastric origin. As it serves

no useful purpose in the organism we must conclude that a portion of the

pepsin, secreted by the stomach and not used for digestion, has been

rejected as superfluous.

The study of the digestive function of animals gives us information on a

large number of points of the highest importance for the comprehension

of immunity. Intracellular digestion, a function so widely distributed

in the lower animals, is very intimately connected with the phenomena

which are observed when micro-organisms are destroyed in the animal

organism. Extracellular digestion furnishes us with information

concerning many of the features of progressive adaptation, similar to

those which are observed in connection with acquired immunity.

When we examine the phenomena of intracellular digestion and those of

secretory digestion as a whole, we see that, in both, the chemical

processes are subjected to the influence of the living parts of the

organism. In the lower animals, it is the protoplasm of the amoeboid

cells which regulates the chemical processes in digestion; in the higher

animals, this _rôle_ is taken by a very complicated apparatus, in which

the nervous system plays a predominant part.

CHAPTER IV

RESORPTION OF THE FORMED ELEMENTS

Digestion in the tissues.—Resorption of cells in the

Invertebrata.—Resorption of red corpuscles by the phagocytes

of the Vertebrata.—Phagocytes.—Various categories of these

cells.—Macrophages and microphages.—Part played by macrophages

in the resorption of the formed elements.—Digestive property

of the macrophagic organs.—Solution of the red blood

corpuscles by the blood serums.—The two substances which

operate in haemolysis.—Macrocytase and fixative.—Analogy of

the latter with enterokynase.—Escape of the macrocytase during

phagolysis.—Suppression of phagolysis.—Resorption of the

spermatozoa.—Presence of fixatives in plasmas.—Origin of

fixatives.

[Sidenote: [72]]

It is usually understood that nutritive substances must necessarily be

subjected to the influence of the digestive juices in the

gastrointestinal canal before they can be utilised for the nutrition of

the organism. This is a very old idea. It was based on a well-known

experiment by Schiff who injected several animals intravenously with

solutions of cane sugar and egg albumen and others with the same

substances after they had been artificially digested. In the first case

the food substances passed into the urine, in the second they only

appeared there when injected in large quantities.

[Sidenote: [73]]

At the recent International Congress of Medicine held in Paris in 1900,

the question of extra-buccal nutrition was much discussed[90]. It has

been accepted that fats, when injected into the subcutaneous tissues,

are, at least in part, absorbed by the organism, but that carbo-hydrates

and albuminoids are never absorbed. This is perhaps true from the point

of view of clinical medicine. But, in principle, it must be admitted

that food substances of very diverse natures, when introduced into the

organism by channels other than the gastrointestinal canal, still

undergo profound changes.

When we inject milk, blood serum, or white of egg, that is to say,

materials very rich in albuminoid substances, under the skin or into the

peritoneal cavity of laboratory animals, we find that after a time they

disappear. At the same time they give rise to modifications of the

organism which indicate that these injected substances have there

undergone profound changes.

After injecting eel’s serum into rabbits, Th. Tchistovitch[91] found a

substance in the blood of the injected animals which gave a precipitate

with eel’s serum. Shortly afterwards Bordet[92] observed that the blood

of animals into which he had injected cow’s milk acquired a new

property: it gave a precipitate with this milk, a condition never

observed in the serum of untreated animals.

The injection of white of egg into rabbits, carried out by Myers[93] and

Uhlenhuth[94], brought about the same changes in the blood serum. The

researches of the latter of these two observers have for our present

purpose a special interest. He demonstrated first that the injection of

white of egg into the peritoneal cavity of rabbits was followed by the

appearance in the blood serum of these animals of a substance which

precipitates egg albumen _in vitro_. Uhlenhuth then obtained this same

acquired property of the blood in rabbits which had been made to swallow

a considerable quantity of the white of hens’ eggs. Twenty-four days

after the commencement of this regimen the serum of the rabbits

precipitated white of egg in the test-tube. This example affords a

marked analogy between the results of digestion in the alimentary canal

and those of resorption into the tissues. Uhlenhuth points out, indeed,

that his rabbits which received the injections of white of egg into the

peritoneal cavity flourished under this treatment.

[Sidenote: [74]]

A certain number of similar examples are now recognised. They all

indicate that various nutritive substances, when introduced into the

peritoneal cavity or under the skin of animals, are retained there for a

longer or shorter time and are subjected to certain modifying influences

on the part of the organism. The proof that these substances are not

eliminated intact by the kidneys has been furnished by a large number of

experiments. Recently Lindemann[95] and Néfédieff[96], working in my

laboratory, have established the fact that normal blood serum, when

injected under the skin of animals, does not provoke albuminuria at all,

or at least produces it in a very insignificant and transitory degree.

The mechanism by which the organism modifies these nutritive substances,

introduced by a channel other than the digestive canal, is not as yet

sufficiently known; and is therefore not easy to define. But we know,

very definitely, that each injection of serum, whether of white of egg,

milk or fatty matter, is followed by a rather considerable aseptic

inflammation at the point at which these substances are introduced. We

might conclude from this that the organism digests the food substances

outside the gastro-intestinal canal, by means of an inflammatory

reaction. In order to determine more exactly the phenomena that appear

under these conditions, it may be useful to consider first, not the

fluid substances but the solid elements that are introduced into the

tissues and cavities.

Let us begin with the lower animals in which the anatomical organisation

and all the functions are of a much more simple character than they are

in the Vertebrata. In my _Comparative Pathology of Inflammation_

(Lecture IV) I have directed some attention to the digestion of the

Sponges.

The nutritive substances—small organisms—whether they may have entered

by the small openings, so numerous on the surface of Sponges, or have

been introduced through a rent in the body wall, undergo the same fate.

They are seized by vibratile or amoeboid cells which ingest the food and

digest it by an intracellular digestion. These two kinds of cells, which

come under the category of _Phagocytes_, have a great resemblance to one

another, and we may say that digestion and resorption are two very

closely related phenomena.

[Sidenote: [75]]

When we examine somewhat higher Invertebrata, such as the Medusae or

certain other Coelenterates, we can still trace a close analogy between

the true digestion of the food that goes on within the epithelial cells

of the entoderm and the resorption of certain foreign bodies which make

their way by an extra-buccal channel into the intermediary tissue. Here

these bodies are surrounded by amoeboid cells which fulfil their

function as phagocytes by ingesting and digesting the substances that

have come from outside.

It is, here, unnecessary to go over the whole gamut of the perfecting of

the organisation of the Invertebrata, in its relation to the resorption

of foreign bodies, especially as it has already been treated in my

Lectures on Inflammation. Let us choose merely some of the more common

and better-known representatives of the Invertebrata and dwell for a few

moments on the phenomena manifested in their organism, into the midst of

which have been introduced a few nucleated red blood corpuscles[97].

If a small drop of defibrinated blood from a goose be injected beneath

the skin of a snail and another under the skin of a cockchafer larva,

the red corpuscles are disseminated in the blood fluid which, of itself,

is incapable of modifying them, but at the end of a few hours the

leucocytes of the two invertebrates that we have chosen for the

experiment will have ingested a certain number of the injected red blood

corpuscles. The next day red blood corpuscles are still to be found

intact in the blood plasma, but the great majority have been devoured by

the leucocytes (Fig. 13). Inside these cells the red corpuscles undergo

constant and marked changes. In the snail they become round and their

walls permeable. In the vacuoles that are produced around the ingested

red corpuscles dissolved haemoglobin is found (Fig. 14); a portion of

this colouring matter passes into the nucleus of the red corpuscles, so

that it also has undergone a profound change (Fig. 14). Many of the

nuclei become emptied, only the peripheral layer remaining. This layer

and the membrane of the red corpuscle are the parts that resist the

action of the leucocytes longest and they are found for some time after

their ingestion. The white corpuscles of the snail, having devoured one

or more red corpuscles, may themselves become the prey of their fellows.

[Sidenote: [76]]

[Sidenote: [77]]

In the “ver blanc” (French popular name for the larva of the cockchafer)

the phenomena of resorption of the red corpuscles of the goose resemble

those just described. The blood plasma leaves intact the red corpuscles

which undergo no change until they have been ingested by the leucocytes.

The haemoglobin diffuses into the leucocyte, whilst the nucleus and the

membrane persist for a very considerable period (Fig. 15), though they

lose their normal aspect, shrivel, and become transformed into an

irregular mass of brown pigment which may remain in the substance of the

leucocyte (Fig. 15, _p_) for weeks.

[Illustration:

FIG. 13. Leucocytes of a cockchafer larva containing red blood

corpuscles of a goose.

]

[Illustration:

FIG. 14. Red blood corpuscles of a goose, free, and ingested by

leucocytes of a snail (_Helix pomatia_), 24 hours after their

injection.

]

[Illustration:

FIG. 15. Leucocyte of a cockchafer larva, 7 days after last injection

of goose’s blood.

]

[Illustration:

FIG. 16. Leucocyte from peritoneal cavity of a gold-fish after

ingesting red blood corpuscles of a guinea-pig.

]

Having once injected goose’s blood into snails and “vers blancs,” if we

repeat the injection several times, the phenomena observed are

invariably the same. The red corpuscles are unacted upon by the plasma

and undergo the same changes within the leucocytes. These changes are in

fact comparable to those described in the preceding chapter in

discussing the intracellular digestion of the red corpuscles by the

intestinal cells of the Planarians. In both cases the red corpuscles are

seized by amoeboid cells and subjected to the influence of their

contents. In the intestinal phagocytes of the Planarian, as in the

phagocytes of the blood (leucocytes) of the snail and “ver blanc,” the

haemoglobin diffuses through the wall of the red corpuscle, whose most

resistant parts are the nucleus and the membrane. These resistant

residual fragments, impregnated with haemoglobin, become brown in the

Planarian, in the “ver blanc,” and also, but in a less degree, in the

snail. The most appreciable difference consists in the formation of

excretory vacuoles, containing concretions, in the Planarian, and the

absence of these vacuoles in the blood phagocytes of the other

Invertebrata. We have, however, less right to attribute a fundamental

importance to this difference, in that the phenomena in the Actinians,

which ingest the red blood corpuscles by the amoeboid cells of their

entoderm, are in all respects (with the exception of the presence of

these special excretory vacuoles) comparable to the phenomena observed

in the Planarians. From the fact that in these two examples we have to

do with a true intracellular digestion, it must be admitted that the

modifications of the red blood corpuscles within the phagocytes of the

blood in the snail and in the larva of the cockchafer, must also be

placed in the same category of phenomena.

[Sidenote: [78]]

In order to make a more thorough study of this intracellular digestion

in the phagocytes of the blood, we must direct our attention to larger

and more highly organised animals than the snail and the “ver blanc.”

Let us take, first, an example among the inferior cold-blooded

Vertebrata. The red blood corpuscles of a few drops (0·25 c.c.) of the

blood of a guinea-pig injected into the peritoneal cavity of a gold-fish

(_Cyprinus auratus_) are not appreciably changed by the peritoneal fluid

itself; but the numerous leucocytes that are found in the peritoneal

fluid seize them and ingest them, just as do the phagocytes of the blood

of Invertebrata, or the intestinal phagocytes in the Planarians and

Actinians in the case of the red blood corpuscles of the goose. Each

leucocyte of the _Cyprinus_ ingests several red blood corpuscles and

subjects them to intracellular digestion. The stroma of the red

corpuscles becomes permeable; the haemoglobin diffuses into the

nutritive vacuoles and at the end of a shorter or longer period the

whole is dissolved and decolorised (Fig. 16). Here no brown pigment is

produced and the red corpuscles are completely digested, leaving no

“remains”; in this respect differing from the process in the

Invertebrata mentioned.

This result depends, probably, partly upon the more feeble resistance

offered by the non-nucleated red corpuscles of Mammals, and partly upon

the more active digestive power of the leucocytes of Fishes.

As the result of several injections of guinea-pig’s blood into the

peritoneal cavity of _Cyprinus_, the peritoneal fluid acquires new

properties[98]. If, a fortnight after the first injection, a little of

the peritoneal exudation in the gold-fish be withdrawn, it is found that

a drop of the serum which floats on the surface produces, almost

immediately, well-marked agglutination of the red corpuscles of the

guinea-pig, this being soon followed by the rapid solution of these red

blood corpuscles in the fluid. This new property, which does not exist

in the untreated fish, also makes its appearance in the blood serum of

_Cyprini_ treated with guinea-pig’s blood. The experiment is very

successful at a temperature of 18°–19° C.

As the solution or lysis of the red blood corpuscles in the serum is

exactly like that which takes place within the leucocytes of _Cyprinus_,

we are justified in assuming that, in both cases, it is produced by the

same substance. And, since the solvent or haemolytic power of the serum

is only acquired as the result of the intracellular digestion of the red

blood corpuscles by the leucocytes, it is probable that the solvent

substance represents the intracellular ferment derived from the

leucocytes.

[Sidenote: [79]]

The subject we have just broached is of fundamental importance in

connection with the study of resorption and of the phenomena of immunity

dependent upon it. It is necessary, therefore, that we should go more

fully into its analysis. With this object we must first review the

processes that go on during resorption in the higher animals and

continue our examination of the changes that injected or extravasated

blood undergoes in various positions of the organism.

This study is rendered comparatively easy for us by the numerous

researches that have been carried out by pathological anatomists for the

purpose of ascertaining the fate of effusions or extravasations of blood

so frequently met with in disease. It has long been known that in

subcutaneous, cerebral and other haemorrhages, or in hepatised lungs,

there are found in the escaped blood a great number of cells containing

red corpuscles. As was mentioned in the preceding chapter, these cells

were evidently amoeboid cells that had ingested red blood corpuscles. To

Langhans[99] especially we owe a detailed study of the phenomena that

follow extravasation of blood produced artificially in the subcutaneous

tissue of the pigeon, rabbit and guinea-pig. In all these animals the

haemorrhage is early followed by exudative inflammation, during which

the leucocytes come up in great numbers and ingest the red blood

corpuscles which are modified in the interior of the leucocytes. There

is a formation or deposition of pigment and finally all traces of the

red corpuscles disappear. In Mammals the pigment is brown or brownish,

just as it is in the Planarians and in the “ver blanc”; in the pigeon it

is green and resembles that found in the Actinians. In short there is a

great analogy between the resorption of red corpuscles and the true

intracellular digestion of the red blood corpuscles that goes on in the

intestinal cells of the Invertebrata.

But what is the nature of these amoeboid elements that intervene in the

resorption of the extravasated blood? At the period when Langhans

carried out his investigation, we were unable to differentiate the cells

at all satisfactorily. It is only since the publication of Ehrlich’s

classic researches on the white corpuscles that we have been able to

bring more order into this question. Thanks to the use of various

aniline stains, Ehrlich was able to arrange the leucocytes found in the

Vertebrata into several definite groups.

The question has already been touched upon in our eighth lecture on

inflammation; it is therefore unnecessary to treat it here at length. We

must, however, before entering on the analysis of the essential

phenomena in the resorption of cells, as we now understand them, give a

rapid survey of the different varieties of amoeboid cells that are found

in the Vertebrata.

[Sidenote: [80]]

Beside mobile amoeboid cells, represented by several forms of white

corpuscles, we must distinguish fixed amoeboid cells. These are

permanently fixed in certain situations in the body; this, however, in

no way prevents them from throwing out amoeboid processes in various

directions and seizing foreign bodies or certain elements of the same

organism. The nerve cells, the large cells of the splenic pulp and of

the lymphatic glands, certain endothelial cells, the cells of the

neuroglia, and perhaps some connective tissue cells, belong to the

category of fixed amoeboid cells. All these elements, under certain

conditions, are able to ingest solid bodies; consequently, they act as

phagocytes. With the exception of the cells of the nerve centres, all

these fixed phagocytes are of mesoblastic origin. It has been much

discussed whether certain processes of the nerve cells may not really

serve to seize foreign bodies and carry them into the cell contents. It

appears to us that sometimes they undoubtedly do fulfil this function.

For example, it is only by means of such amoeboid movements that leprosy

bacilli can be introduced into the interior of ganglion cells and cells

of the spinal cord[100]. We must not dwell on this question, as the

phagocytic property of the nerve elements plays no part in the

resorption of cells. On the other hand, the neuroglia cells contribute

largely to this process and their phagocytic function is now admitted by

many observers[101].

For long the large “dust” cells of the respiratory channels were looked

upon as being epithelial cells which were capable of ingesting carbon

particles, micro-organisms and other foreign bodies. The researches of

N. Tchistovitch, carried out in my laboratory more than twelve years

ago, made it evident that these elements are nothing more than white

corpuscles that have immigrated into the alveoli and bronchi.

[Sidenote: [81]]

It is probable that the same is the case as regards the stellate cells

of the liver, known as Kupffer’s cells. First described by Kupffer as

cells of a nervous type, having long processes, they were later

recognised by several observers as belonging to the endothelial tissue

of the blood vessels of the liver. Kupffer[102] himself has accepted

this view and in his recently published monograph on these stellate

cells, he describes them as endothelial cells that have retained their

independence. Some researches on the resorption of blood, of which I

shall speak shortly, have led me to think that these cells are nothing

but white corpuscles that have been arrested in the hepatic capillaries.

I have asked Mesnil, head of my laboratory, to study this question for

me. His investigation is not yet concluded, but the demonstration

already made that the livers of guinea-pig embryos and new-born rabbits

do not possess any Kupffer’s cells is an argument in favour of my

hypothesis.

Certain white corpuscles have undoubtedly been often mistaken for

epithelial or connective tissue cells. We must not conclude from this,

however, that these elements are never capable of sending out amoeboid

processes and of ingesting foreign bodies. It would, however, be useful

to collect new and incontestable proofs of the accuracy of this thesis.

In spite of this uncertainty, it may be accepted as fully demonstrated,

that certain fixed amoeboid cells, such as the large elements of the

splenic pulp, of the lymphatic glands, and of the omentum, play an

important part in the resorption of cells. It is there that elements

filled with red corpuscles and white corpuscles in process of being

destroyed are so often found.

[Sidenote: [82]]

Just as certain fixed cells do not function as true phagocytes, so also

in some leucocytes this function is undoubtedly absent. The suggestion

has been made several times that any cell element, provided it be young,

is capable of ingesting foreign bodies. The examination of white

corpuscles proves exactly the contrary. The smaller white corpuscles

found in fairly large numbers in the blood and the lymph, and which are

commonly known as _lymphocytes_ or _small lymphocytes_, are simply

leucocytes with very little protoplasm which in this state never fulfil

phagocytic functions. It is only when it becomes older, when its

nucleus, single and rich in chromatin, becomes surrounded by an ample

layer of protoplasm, that the lymphocyte becomes capable of ingesting

and resorbing foreign bodies. Several authors, with Ehrlich at their

head, still assign to these larger cells the same name—lymphocytes.

Others, however, give them the name of large mononuclear cells.

Confusion is thus possible, especially as Ehrlich includes under the

same term the large mononucleated leucocyte, a very rare form of cell in

human blood, which is distinguished by the greater staining capacity of

its nucleus. To avoid this inconvenience I propose to designate the

large lymphocytes by the name of blood macrophages and lymph macrophages

(_haemomacrophages_, _lymphomacrophages_). This term is preferable to

that of mononuclear leucocytes, especially as in exudations we

frequently meet with macrophages with two and even several sharply

separated nuclei. Giant cells, moreover, are nothing but polynucleated

macrophages. On the other hand, the leucocytes so often designated by

the name of polynuclear in reality contain but a single nucleus. Even

Ehrlich, who introduced this term, acknowledged its imperfection but he

retained it for some time because it was already very extensively used

and could, he thought, give rise to no misunderstanding. In his

excellent work on anaemia, published jointly with Lazarus[103], he now

agrees that the name of “cells with polymorphous nuclei” would be more

exact.

These polymorpho-nuclear leucocytes are very numerous in the blood and

in many exudations and are distinguished by the greater selective

affinity of their nucleus for basic aniline dyes and by a certain

tendency of the protoplasm to become stained by acid aniline colours,

such as eosin. The true macrophages are without granulations, but the

“polymorpho-nuclear” leucocytes contain many. These granulations are

sometimes “eosinophile,” “pseudo-eosinophile” (or “amphophile”) or even

“neutrophile” (as in man and the horse).

[Sidenote: [83]]

These two main groups of leucocytes are generally distributed in the

Vertebrata; and we already meet with them in one of the lowest

vertebrate forms—the _Ammocoetes_ (the larva of the lamprey). The

macrophages of this fish present all the principal characters of the

group to which they belong (protoplasm without granules, easily stained

with methylene blue, large nucleus rich in nuclear juice). In the

“polynuclear” forms in this lower vertebrate the protoplasm does not

stain with methylene blue, but assumes a faint rosy tint with eosin; the

single nucleus is divided into several lobes. In Vertebrates which are

much higher in the scale these characters change. Thus in the cayman

(_Alligator mississipiensis_), according to the researches of Madame

Podwyssotsky, carried out in my laboratory, the two great varieties of

leucocytes are readily found in the blood, lymph and exudations. The

macrophages, however, especially in the exudations, are very often

furnished with two or several nuclei, whilst the small leucocytes

possess only a single nucleus, which is not divided into lobes. In spite

of this peculiarity the two groups are readily distinguished. The

staining reactions of the macrophages are identical with those of the

corresponding corpuscles in all the other Vertebrata; whilst the small

leucocytes, in spite of the absence of a polymorphous nucleus, are

easily recognised by their eosinophile granulations and by the special

affinity of the nucleus for basic aniline dyes. Under these

circumstances it would be quite inappropriate to designate those

leucocytes, which are really polynuclear, that is to say, possessing two

or several nuclei, by the name of “mononuclear,” and to reserve the name

of “polynuclear” for the small corpuscles which possess only a single

nucleus undivided into lobes. For this reason it is much more rational

to retain for these so-called polynuclear cells my proposed name of

_microphages_. Moreover, the microphages are true phagocytes. It was

formerly thought that the eosinophile leucocytes, such as the “‘overfed’

cells (Mastzellen)” of Ehrlich, which are identical with the

_clasmatocytes_ of Ranvier, never ingested foreign bodies. But,

(especially after the researches of Mesnil[104]), we have been compelled

to change our opinion on this point. The true eosinophile cells are able

to devour foreign bodies, especially micro-organisms, and must therefore

be regarded as phagocytes belonging to the group of microphages.

[Sidenote: [84]]

It is the peculiar merit of Ehrlich and of his school that they have

thoroughly established the fact that, in Mammals at any rate, the two

principal groups of white cells are distinguished, amongst other

characters, by the diversity of their origin. The lymphocytes and the

mononuclear cells are developed in the spleen and lymphatic glands,

whilst the “polynuclear” cells arise from the granular mononucleated

myelocytes of the bone marrow. This is now generally accepted as

applicable in the great majority of cases. In _Ammocoetes_, however, the

two chief varieties of leucocytes arise from one and the same organ,

regarded by several observers as a kind of primitive spleen, which runs

along and in part surrounds the intestine. Mesnil has been good enough

to make sections of this primitive organ in which it may be demonstrated

that the macrophages and the microphages in the larva of the lamprey

have the same seat of origin. Frog tadpoles and Cartilaginous Fishes

also possess microphages which do not arise from the bone marrow, since

in them this tissue is completely absent. But even in Mammals, at least

in certain pathological conditions, Dominici[105], in a research

executed with much care and a perfect technique, has demonstrated the

myelogenous transformation going on in the spleen. Thus in the adult

rabbit affected with septicaemia by the typhoid bacillus, he found in

the spleen developmental centres of amoeboid elements which, normally,

appear to develop in the bone marrow only, _i.e._ the megacaryocytes, or

large cells with budding nuclei, the neutrophile myelocytes

(amphophiles), basophiles and eosinophiles.

The mesoblastic phagocytes of the Vertebrata are divided, then, into

fixed phagocytes—the macrophages of the spleen, endothelia, connective

tissue, neuroglia, and muscle fibres—and free phagocytes. These latter

are sometimes haemo- or lympho-macrophages, sometimes microphages. The

fixed macrophages and the free macrophages resemble one another so

greatly that it is very often extremely difficult, if not impossible, to

differentiate them. For this reason it is often very useful, when the

exact origin of a large phagocyte is not known, simply to name it

“macrophage.”

[Sidenote: [85]]

The two principal groups of phagocytes—(1) fixed and free macrophages,

(2) microphages—are distinguished not only by their morphological

characters; they also give evidence of very marked physiological

differences. All phagocytes are endowed with amoeboid movement which

allows them either to move about freely or merely to put out

protoplasmic processes. These movements are regulated by a very great

sensitiveness, often different in the two groups. Besides a tactile

sense, the phagocytes possess a kind of sense of taste or chemiotaxis

which enables them to distinguish the chemical composition of the

substances with which they come in contact. The existence of this

chemiotaxis could be anticipated from the moment that an important part

in the life of the organism began to be ascribed to the amoeboid cells.

Leber[106], Massart and Charles Bordet[107] have, however, demonstrated

it by rigorous experiment. Following the method used by Pfeffer to

demonstrate the chemiotaxis of the vegetable spermatozoids and of

Bacteria, these investigators introduced into the bodies of higher

(rabbits and guinea-pigs) and lower (frogs) Vertebrates small glass

tubes filled with different solutions (peptone, broth, salts, bacterial

products, etc.). The leucocytes, guided by their positive chemiotaxis,

made their way into the tubes and there formed plugs which were often

very voluminous; when, on the other hand, the chemical composition of

the solutions excited their negative chemiotaxis, the leucocytes avoided

the tubes.

[Sidenote: [86]]

Having acquired information as to the chief characters of the

leucocytes, we may ask, To which group do those amoeboid cells, which,

according to the observations of Langhans and many other investigators,

bring about the resorption of the red corpuscles of the blood, belong?

This resorption goes on more rapidly and is observed much better if,

instead of introducing blood of the same species into any part, we

inject defibrinated blood, or red blood corpuscles from which the serum

has been removed by washing, from another species of Vertebrate. It will

be found best to inject the nucleated red corpuscles of lower

Vertebrates into Mammals, or (as already described above) to introduce

the non-nucleated red blood corpuscles of Mammals into lower

Vertebrates. In all these cases the injection of such blood or

corpuscles sets up an aseptic inflammation which attracts a large number

of free phagocytes to the seat of injection. In subcutaneous, peritoneal

or intraocular exudations produced under these conditions, we find, in

addition to a number of microphages, many macrophages. Whilst the former

ingest the injected red corpuscles merely in isolated cases, the

positive chemiotaxis of the macrophages manifests itself much more

actively. In the resorption of the red blood corpuscles the more

important part is played by the macrophage. To get a clear idea of the

phenomena that accompany this resorption, let us take a concrete

example. Inject defibrinated goose’s blood into the peritoneal cavity of

guinea-pigs[108]. During the first few hours after injection the oval

nucleated red corpuscles are found intact in the fluid of the peritoneal

lymph. The plasma, by itself, exercises no destructive or solvent action

on the red corpuscles of the goose.

Immediately after the injection the lymph of the peritoneal cavity

begins to show important changes. The white corpuscles which, in the

normal condition, are fairly abundant, disappear almost completely; some

small lymphocytes presenting their ordinary aspect may indeed be found,

but the few macrophages and the microphages that remain show signs of

very grave lesions. They lose their mobility, run together into clumps

and become incapable of ingesting foreign bodies. At this moment the

phagocytes undergo a critical change which we have designated by the

name of _phagolysis_. This condition lasts for about an hour, sometimes

it continues longer, according to case and circumstance, but after this

the peritoneal fluid becomes filled with leucocytes that have newly come

on to the scene. These cells make their way, by diapedesis, through the

walls of the congested vessels of the peritoneum. A true aseptic

inflammation is produced which induces an exudation of a large number of

white corpuscles, amongst which are found microphages and still more

numerous macrophages. The latter show a very pronounced positive

chemiotaxis towards the injected red corpuscles of the goose. Soon after

their appearance, that is to say two or three hours after the injection

of the blood, the macrophages send out very small protoplasmic processes

and affix them to the surface of the red corpuscles. There follows an

aggregation of the macrophages of the guinea-pig with the red corpuscles

of the goose and characteristic masses, in which can be recognised both

kinds of cells, are produced. This union with the very small pseudopodia

is the first stage in the ingestion of the red corpuscles by the

macrophages (Fig. 17). The red corpuscle, seized by amoeboid processes,

passes into the interior of the macrophage. This macrophage seldom rests

contented with ingesting a single red corpuscle. Usually it devours a

large number and sometimes enormous macrophages may be seen filled with

a score of red corpuscles.

[Sidenote: [87]]

If the quantity of goose’s blood injected into a guinea-pig is large

(5–7 c.c.), the ingestion of red corpuscles by the macrophages continues

for a considerable period—often for three to four days. During the whole

of this time a certain number of the red corpuscles remain free in the

peritoneal plasma, but, in spite of this prolonged stay, none of them

undergo extracellular solution.

[Illustration:

FIG. 17. Macrophage of guinea-pig in process of devouring and

digesting red blood corpuscles of goose.

]

[Illustration:

FIG. 18. Macrophage of guinea-pig in the act of ingesting and

digesting red corpuscles of goose. _Intra vitam_ staining with

neutral red.

]

[Sidenote: [88]]

The red blood corpuscles, anchored by the amoeboid processes of the

macrophages, at first present a normal appearance. Later their membrane

begins to wrinkle, but as soon as they have passed within the phagocytes

the wrinkles disappear and the corpuscles regain their normal aspect. If

a little neutral red solution be added to a drop of peritoneal exudation

(Fig. 18) we observe that the nucleus of the ingested red corpuscle and

even its contents are stained red, whilst the red corpuscles adherent to

the surface of the phagocytes retain their normal yellow colour. This

reaction enables us to see that the red corpuscles are seized by the

macrophages whilst still in their normal condition, but that they

undergo a change immediately after they have been ingested. Little by

little the devoured corpuscles are digested within the phagocytes. The

haemoglobin diffuses into the contents of the macrophage through the

stroma, which has become permeable; the nucleus of the ingested red

corpuscle also becomes stained by the haemoglobin. Part of this

colouring matter is excreted by the phagocyte. The body of the red

corpuscle is pretty soon digested, but the nucleus, impregnated with

haemoglobin, persists for a much longer period. It divides into several

fragments, recognisable by their yellow colour, and in certain cases

these remnants of red corpuscles may be met with for weeks in the

interior of the macrophages. These macrophages do not remain permanently

in the peritoneal fluid. Some (3–4) days after injection the lymph of

the peritoneum contains only leucocytes that have newly come up and

which contain neither red corpuscles nor their remains. We must open the

guinea-pig to find any macrophages that have devoured red corpuscles.

They are to be met with in large numbers in the glandular portion of the

omentum, in the mesenteric glands, in the liver and in the spleen. They

are fairly easily recognised by the characteristic aspect of the

_débris_ of the red blood corpuscles. Having devoured the red corpuscles

the macrophages leave the peritoneal fluid and the digestion is

completed in the positions just mentioned. In the liver they are seen as

large mononuclear cells often with highly developed processes. In this

condition they remind one of Kupffer’s stellate cells—a fact that

suggested to me the idea that these elements are nothing but white

corpuscles which have immigrated into the vessels of the liver.

Following up the fate of the macrophages that have resorbed the red

blood corpuscles, we find them in the large hepatic vessels, in the vena

cava and even in the blood of the heart. But in these latter situations

they contain merely a few scarcely recognisable traces of their prey.

These phagocytes, which left the blood during the inflammation that

followed the injection of red corpuscles of the goose, re-enter it,

having fulfilled their function, during the final period of the

resorption. This resorption must undoubtedly be regarded as an

intracellular digestion. When we compare the essential phenomena taking

place inside the macrophages containing red blood corpuscles with those

we have described in the intestinal phagocytes of the Planarians or

Actinians after a meal, the analogy between the two becomes very

apparent. In both cases the red blood corpuscles undergo a marked change

which results in a diffusion of the haemoglobin. The membrane and

nucleus of the red blood corpuscles persist longer but they also are

ultimately digested. The excretion of haemoglobin from the phagocytes,

just mentioned in the case of the macrophages of the guinea-pig, is also

observed in the Actinians, whose coelenteric cavity is tinted by a

rose-coloured solution.

[Sidenote: [89]]

We have seen that in the Actinians intracellular digestion takes place

in a distinctly acid medium, whilst in the intestinal cells of the

Planarians it takes place in one that is only weakly acid. The

macrophages of the guinea-pig, during the resorption of red blood

corpuscles of the goose, carry on the digestive process in a medium

which shows a still weaker acidity. When made to ingest granules of blue

litmus there is no change of colour. Nor does alizarin sulpho-acid give

any reaction, probably owing to the fact that it exerts a toxic action

on the protoplasm of the macrophages. If, however, we add to a drop of

the peritoneal exudation of a guinea-pig, containing macrophages filled

with red blood corpuscles of the goose, a little of Ehrlich’s 1%

solution of neutral red, the red brick tint at once makes its appearance

in the content of these phagocytes. This coloration is identical with

that described in the _Amoebae_ which digest Bacteria or in the

intestinal phagocytes of the Planarians. It may, then, be regarded as an

indication of weak acidity. This coloration is maintained for some

hours, after which it gives place to complete decoloration, a phenomenon

that must be attributed, as in many other cases, to the neutralisation

of the acid by the alkaline protoplasm that has been macerated in the

fluid after the death of the macrophages.

The example we have chosen—the destruction of red blood corpuscles of

the goose by the macrophages of the guinea-pig—may serve as a prototype

of the resorption of formed elements in general. If, instead of red

blood corpuscles of the goose, we inject into the guinea-pig’s

peritoneal cavity pigeon’s or fowl’s blood, the essential phenomena will

be the same. The red blood corpuscles will always induce positive

chemiotaxis, especially of the macrophages, which in turn will ingest

the nucleated red corpuscles. It may be that in certain cases, when

fowl’s blood containing red corpuscles that are not very resistant is

injected, a certain number of the corpuscles immediately undergo a

partial solution in the peritoneal fluid[109]. Here also the stromas and

the nuclei of all the red blood corpuscles, as well as many of the

corpuscles unacted upon by the plasma of the phagolysed exudation,

undergo digestion inside the macrophages.

[Sidenote: [90]]

When, instead of blood, we inject white corpuscles from the bone marrow,

spleen or lymphatic glands of animals into the peritoneal cavity, we may

still observe their final disappearance in the macrophages. The

spermatozoa of man or of various mammals (bull, rabbit, guinea-pig,

etc.), when injected into the peritoneal cavity of the guinea-pig or

rabbit, are well adapted for this line of investigation. Here again the

immediate result of injection is the very marked phagolysis of the

leucocytes. This phenomenon gives place to an exudative inflammation

which brings into the peritoneal cavity a number of phagocytes. These,

especially the macrophages and in a much smaller degree the microphages,

devour the spermatozoa which in no case are dissolved, even partially,

in the plasma of the exudation. The macrophage seizes the spermatozoa

which sometimes, by the active movements of their flagella, exhibit

great vitality. At the end of several hours all the spermatozoa are

found inside phagocytes where they are completely destroyed. The

flagellum is digested first, but the head and medial portion soon suffer

the same fate. Neutral red reveals the feebly acid reaction, perhaps

with even more distinctness than in the case of the red blood

corpuscles.

The _résumé_ of Langhans’ investigation given in this chapter would lead

us to expect that resorption in the subcutaneous tissue will follow the

same rules as that going on in the peritoneal cavity. As a matter of

fact, blood injected at this position sets up a diapedesis of phagocytes

which ingest the red blood corpuscles. In some cases only is there a

partial solution of these corpuscles in the fluid of the subcutaneous

exudation. It is for this reason that goose’s blood, injected under the

skin of a guinea-pig, gives rise to a fluid exudation coloured a bright

rose red by the dissolved haemoglobin. This haemoglobin is derived from

red blood corpuscles which are damaged by the goose’s blood serum that

was added to the plasma of the exudation. The stroma and nuclei of the

red blood corpuscles cannot, however, be dissolved in this fluid. They

undergo the same fate as the red corpuscles that have remained intact,

that is to say they are ingested by the macrophages which immigrate into

the subcutaneous tissue and which finally digest all these elements. The

cells, less fragile than certain red corpuscles, are, in the

subcutaneous tissue, as in the peritoneal cavity, destroyed solely in

the interior of the phagocytes.

[Sidenote: [91]]

The analogy between the modifications undergone by the red blood

corpuscles and other cells inside the macrophages and the changes that

take place in the intestinal cells of Planarians and Actinians, suggests

that the resorption of formed elements must undoubtedly be regarded as a

true intracellular digestion. It would, however, be a very important

matter to be able to support this conclusion by even more convincing

proofs. The study of the artificial digestion that is observed _in

vitro_ in the case of the macerated mesenterial filaments of Actinians

has furnished a very valuable argument in favour of the enzymatic nature

of intracellular digestion. Animal exudations are not well adapted for

this special line of study. We can only obtain them as the result of the

injection of different substances, solid or fluid, which are greedily

absorbed by phagocytes. If we collect the exudations at a moment when

the number of these cells is still considerable we must withdraw along

with them many digestive substances which interfere with our

observation. We may therefore with advantage turn our attention to

masses of phagocytes collected in organs. As it is mainly the

macrophages which effect the resorption of cells, it is evident that we

must choose the centres where they are formed in order to investigate

the digestive ferments. Let us take, then, the lymphatic glands of the

mesentery, the glandular portion of the omentum and the spleen, the

three pre-eminently macrophagic organs, and let us see if, with an

extract of them, prepared with physiological salt solution (0·75% of

sodium chloride), any digestive effect is to be obtained.

Macerate the three organs mentioned of a guinea-pig and mix the extracts

thus obtained with red blood corpuscles of the goose, corpuscles that

have already given us information in connection with the phenomena of

resorption in the living organism. In almost all the guinea-pigs a

solution of the red blood corpuscles of the goose by the extract of the

glandular portion of the omentum may be observed. The mesenteric glands

likewise give an extract which in most cases has a solvent action. The

extract from the spleen is only active in a limited number of cases. In

all these examples the extracts from macrophagic organs bring about the

solution of the haemoglobin, but leave intact the membrane and nucleus

of the corpuscles. In this respect there exists, then, a certain

difference between this and the digestion of red corpuscles in the

macrophages of exudations, where the membrane and even the nucleus are

in the end completely dissolved. This difference may be explained by the

fact that in the preparation of the extract in physiological salt

solution, one part only of the soluble digestive ferment may be set at

liberty.

[Sidenote: [92]]

The solvent action of extracts of macrophagic organs must in fact be

attributed to the presence of a soluble ferment in the cells of which

these organs are made up. As the diastases are distinguished, in

general, by their great sensitiveness to heat, we tried the action of

our extracts after a preliminary heating, when it was found that a

temperature of 56° C., applied for three quarters of an hour, completely

abolished the solvent action of the extracts upon the red blood

corpuscles of the goose. The soluble ferment of macrophagic organs, to

which we propose to give the name of _macrocytase_[110] or macrophage

ferment, is in many respects analogous to the actino-diastase of Mesnil,

described in the preceding chapter.

With a view to obtain more complete information on the cytases I

suggested to Tarassewitch that he should make a detailed study of them;

this he has carried out in my laboratory. He has demonstrated that the

macrophagic organs of other mammals than the guinea-pig, especially

those of the rabbit and dog, exert the same solvent action on the red

blood corpuscles. He has also established the fact that this action

applies not only to the red corpuscles of the goose but extends also to

those of several other birds and mammals. Tarassewitch succeeded in

confirming the injurious action of heat on macrocytase. Extracts of

macrophagic organs which contain much _debris_ in suspension, when

heated for an hour at 55°·5 C. in certain cases lose their solvent

property for red blood corpuscles; sometimes this temperature brings

about merely a weakening of the macrocytase. In order to destroy it

surely and completely, the suspensions must be heated at 58°·5–62° C.

for an hour. If, however, instead of heating the entire suspension, we

first pass it through filter paper, the clear fluid filtrate is deprived

of its diastatic action even after it has been heated at 55°·5 C. for

three quarters of an hour.

[Sidenote: [93]]

Of all the other organs of which extracts have been kept in prolonged

contact with the red blood corpuscles of birds, the pancreas alone has

shown a very well-marked digestive action. Extracts of the salivary

glands exerted a feeble solvent action on a certain quantity of the red

corpuscles. The other organs, such as the liver, kidneys, brain, spinal

cord, ovary, testicles, suprarenal capsules and placenta, exercised no

such action. Even bone marrow, in agreement with my results published

some years ago, showed itself quite inactive.

The blood serum of guinea-pigs which I employed in my researches, as

well as that of the animals examined by Tarassewitch, has not shown

itself capable of dissolving the red blood corpuscles of the goose,

although the macrophagic organs dissolve them easily. It has long been

known, however, that the serum of the blood of many animals will destroy

the red corpuscles of a different species. This demonstration was

afforded during the period when attempts were being made to transfuse

the defibrinated blood of mammals, especially of the sheep, into man.

This practice had to be abandoned, in consequence of the difficulties

resulting from the solution of the human red corpuscles. Later,

Daremberg[111] and Buchner[112] set themselves to study this haemolytic

action of serums systematically. They found that it was due to a

particular substance to which Buchner gave the name of _alexine_ or

protective substance. Of indeterminate chemical composition, this

substance is allied to albuminoid substances. It is destroyed when

heated to 55°–56° C. and only acts in the presence of certain salts.

When these salts are removed from the serum by dialysis, it loses its

haemolytic power; but as soon as the salts are replaced in proper

proportion this power reappears. Later, Buchner[113] compared the action

of alexine to that of soluble ferments and referred it to the category

of the digestive diastases. According to him the same alexine is capable

of dissolving the red blood corpuscles of several species of

Vertebrates. Bordet[114], in a series of researches made in the Pasteur

Institute, confirmed this view. He came to the conclusion that the

alexines of the various species of animals differ from one another.

Thus, the alexine of the blood serum of the rabbit is not the same as

that found in the serum of the guinea-pig or dog. Nevertheless each of

these alexines is capable of exerting a solvent action on the red blood

corpuscles of several species.

[Sidenote: [94]]

[Sidenote: [95]]

Ehrlich and Morgenroth[115], in a series of memoirs on the solution of

red blood corpuscles, have combated the idea that there is only a single

alexine in one and the same serum. Moreover, they state that alexine

always requires for its action the aid of another substance, and that

matters are much more complicated than at first sight appears. They

maintain that in each normal serum a number of different substances are

found, each one of which only attacks a single species of red blood

corpuscle. They point out that the solution of the red corpuscles by the

normal serum takes place through the combined action of two different

substances and cite several cases where a normal serum, after being

heated to 55° C. and so deprived of its haemolytic power, again becomes

capable of dissolving the red corpuscles when some normal serum from

another species, which of itself is destitute of the solvent property,

is added to it. Let us quote an example from Ehrlich and Morgenroth. The

normal serum of the goat readily dissolves the red blood corpuscles of

the rabbit and guinea-pig, but if heated for half-an-hour at 55° C., it

loses this power. On the other hand, the normal serum of many horses

shows itself powerless to dissolve the red corpuscles of these rodents.

Here, then, are two serums, equally incapable of effecting the solution

of the red corpuscles of the rabbit and guinea-pig. Yet, when they are

mixed together and to them a few drops of blood from one of the rodents

cited is added, haemolysis takes place readily. The heated goat’s serum

then, has, retained in it something that resists a temperature of 55°

C., a substance which, by itself, leaves the red blood corpuscles

intact; but which, when combined with a second substance present in the

horse’s serum, causes their solution. Ehrlich gives to the first

substance, that is to say that found in the heated goat’s serum, the

name of _intermediary body_ (“Zwischenkörper”). The second substance,

present in the unheated horse’s serum, is designated by him the

_complement_. In order that a normal serum may dissolve the red

corpuscles, it is not sufficient that it should possess a single

substance, the alexine of Buchner. It must, to exert this action,

contain two distinct substances which are very often found together in

the same normal serum. Unheated goat’s serum was only capable of

dissolving the red blood corpuscles of the rabbit because a particular

complement and intermediary substance were both present. Deprived of its

complement at 55° C., the serum is solvent only when we add to it

another substance that is contained in the normal serum of a different

species (horse). Continuing their researches in this direction, Ehrlich

and Morgenroth have come to the conclusion that the normal serum of a

single species may contain several intermediary substances, each one

acting on a single species of red blood corpuscles. Further, that normal

serum must contain several or even many different complements.

[Sidenote: [96]]

Ehrlich and Morgenroth carried on researches on the intermediary

substances in normal serums and found several in addition to that

already mentioned. The serum of the normal dog readily dissolves the red

blood corpuscles of the guinea-pig. When heated to 57° C. it loses this

property; but with the addition of normal guinea-pig’s serum the

property is regained. In the serum of the normal dog there exists, then,

besides the complement, at least one intermediary substance. The same

result can be obtained with several combinations of serums of normal

mammals, heated or unaltered[116]. Yet it often happens, as Ehrlich and

Morgenroth themselves point out, that the demonstration of the presence

of the intermediary substance in normal serums is accompanied with

marked difficulties. Bordet, also, who has studied this question very

thoroughly, often failed completely in his attempts to make normal

serums, that were incapable of producing haemolysis, active by the

addition of heated serums of other species of animals. Thus he observed

that normal fowl’s serum readily dissolves the red corpuscles of the

rabbit. When heated to 55°–56° C. this serum loses its haemolytic power

which cannot be restored by the addition of any normal serum. He thinks

therefore that, in this example, haemolysis is produced solely by the

alexine, without the assistance of any intermediary substance in the

serum of the normal fowl. P. Müller[117], whilst confirming Bordet’s

experimental results, considers that, in this case also, there is the

intervention of an intermediary substance. When he mixed heated fowl’s

serum with a small quantity of unaltered fowl’s serum the solution of

the red corpuscles of the rabbit is not brought about. When, however,

instead of adding a little unheated normal fowl’s serum, he added the

same quantity of serum from a fowl previously treated with physiological

salt solution, the red corpuscles of the rabbit were dissolved without

any difficulty. Müller explains this difference as due to the fact that

the serum of the treated fowl contains more complementary substance than

does that of the normal fowl.

We see, then, from this example that the analysis of the phenomena

taking place in the solution of the red corpuscles by normal serums is

beset with very great difficulties. For this reason it is much more

profitable to make researches in this direction, using more active

serums, where the demonstration of the two substances can be made simply

and exactly. This desideratum has been supplied by J. Bordet, when

_preparateur_ in our laboratory; he described an easy method of

increasing the haemolytic power of serums.

As stated above, guinea-pigs that have received an intraperitoneal

injection of goose’s blood digest the corpuscles, although the

peritoneal fluid exerts no haemolytic action. _In vitro_, the extract of

their macrophagic organs certainly dissolves the red corpuscles, whilst

the blood serum usually fails to do so. Now, if a second or a third

injection of goose’s blood be made into the peritoneal cavities of the

same guinea-pigs, partial solution of the corpuscles takes place in the

peritoneal plasma and the serum of the blood acquires new properties: it

becomes capable of clumping the red corpuscles, that is to say of

agglutinating them; afterwards it dissolves them _in vitro_.

[Sidenote: [97]]

J. Bordet[118] has shown that the injection of the blood of one species

of Vertebrate (mammal or bird) into the peritoneal cavity or under the

skin of an animal of a different species, always produces in the blood

serum of the latter the haemolysing substance. This haemolysing

substance is specific or nearly so, that is to say it dissolves the red

corpuscles of the species which has furnished the injected blood and

also, but more feebly, the red corpuscles of allied species.

Consequently, with guinea-pig’s serum, treated with goose’s blood, we

obtain the greatest solvent action on the red corpuscles of the goose,

though there is a certain haemolysis of the red corpuscles of some other

birds. This rule, thoroughly established by Bordet, has been the

starting-point for a large number of researches on haemolysis and

amongst others of those which bear on the intermediary substance of

normal bloods.

Bordet demonstrated very definitely a fact of fundamental

importance—that in the blood serums of animals treated with blood from a

different species, there exist two distinct substances which only

dissolve the red blood corpuscles when they are combined. Here the

duality of the haemolytic agent cannot be doubted, as it may in certain

examples of normal serums. Each time that we deprive the serum of a

treated animal of its solvent action by heating at 55°–56° C., this

property can be restored to it with certainty by the addition of a

little normal serum which, by itself, is incapable of bringing about

haemolysis. The heated serum of these injected animals loses the power

of dissolving the corresponding red corpuscles, but it retains its other

acquired property—the agglutination of the corpuscles. The red

corpuscles, aggregated into voluminous masses quite visible to the naked

eye, remain intact indefinitely, if left in the prepared and heated

serum. But as soon as we add to them a trace of normal blood (taken from

one of a number of species of Vertebrates), the solution of the red

corpuscles is not long in taking place. Under these conditions an action

of two substances is set up; one of these substances is found in the

heated serum of the injected animal, and the other in unheated normal

serum. The first of these substances which not only resists a

temperature of 55°–56° C., but stands, without alteration, heating to

60°–65° C., corresponds to the intermediary substance of Ehrlich. By

Bordet it has been termed “substance sensibilisatrice[119].” The second

substance, a common one, found in normal serums and destroyed at 55°–56°

C., is the alexine of Buchner and of Bordet, or the complement of

Ehrlich.

[Sidenote: [98]]

The ease with which one can demonstrate the co-operation of two

substances in the haemolysis by the serums of animals treated with the

blood of a different species, is due to the fact, that during the course

of this treatment the animal organism produces a quantity of an

intermediary or sensibilising substance. In fresh animals which have not

been treated, it is often very difficult to demonstrate the presence of

this substance. Bordet has established the fact that the serum of

animals which have been injected several times with the blood of a

different species, contains almost the same amount of alexine as does

untreated serum. On the other hand, the sensibilising substance makes

its appearance in large quantity as the result of these injections. Von

Dungern[120] has confirmed this result and contributes the interesting

fact that the sensibilising substance is found even in great excess in

the serum of treated animals. When he adds to this serum blood that has

not been heated, he produces a haemolysis that is more than thirty times

as active as when the serum of the prepared animal alone is used. From

the quantitative point of view, then, there is no relation between the

amount of the two substances in the serum of prepared animals.

It may be suggested that the sensibilising or intermediary substance is

the same as that which produces the agglutination of the red corpuscles.

But careful researches have thoroughly demonstrated the difference

between the two substances that have this character in common, both

resist heating to 55°–60° C. and even beyond this point.

[Sidenote: [99]]

Having established this co-operation of two substances in haemolysis the

intimate mechanism of their action was next studied. Here I must give

pride of place to the discovery by Ehrlich and Morgenroth that the

intermediary (or sensibilising) substance links itself to its

corresponding red corpuscles. A serum, capable of dissolving the red

corpuscles of a different species, is heated to 56° C. which causes it

to lose this solvent property. When a certain number of these corpuscles

are added to it, such corpuscles remain intact although they are

agglutinated. It is sufficient, after some hours of contact, to

centrifugalise the mixture in order to separate a limpid serum from the

mass of red corpuscles, the former being now entirely deprived of its

intermediary substance, that is to say it has become incapable of

dissolving the red corpuscles even with the addition of a large quantity

of the “complement” (normal serum, unheated). On the other hand, the red

corpuscles, having fixed (linked) all the intermediary substance,

dissolve very rapidly when placed in contact with normal serum which

contains the necessary quantity of the complement (or alexine). This

fundamental experiment has been confirmed and repeated by many observers

and has now become classic. The idea that the intermediary (or

sensibilising) substance links itself to the red corpuscle, without

dissolving it, is generally accepted and may be regarded as permanently

settled. We should do well, then, instead of designating by all sorts of

synonyms the substance in serums which resists the action of a

temperature of 55°–65° C., to apply to it, once for all, the name of

_fixative substance_ or simply that of _fixative_. This name is short,

expresses the essential character of the substance and gives rise to no

misunderstanding, as do the other names proposed up to the present

(amongst them that of _philocytase_ employed by myself in some of my

earlier publications).

Another of Ehrlich and Morgenroth’s experiments has furnished the proof

that the complement does not fix itself to the red corpuscles only. A

normal serum, unheated, which, by itself, is quite as incapable of

dissolving the red corpuscles as the fixative alone, is mixed with some

defibrinated blood. After the centrifugalisation of this mixture, it is

easy to demonstrate that the supernatant fluid has lost none of its

complement (alexine), whilst the red corpuscles have fixed none.

If, instead of an inactive serum, we take a serum which is capable of

dissolving the red corpuscles and which consequently contains the two

haemolysing substances, and if we place it in contact with the

corresponding red corpuscles, at a temperature between 0° and 3° C., the

solution will not take place (Ehrlich and Morgenroth). Under these

conditions the fixative certainly attaches itself to the red corpuscles,

but the alexine remains in solution, unused. It is only necessary,

however, to heat the mixture up to 30° C. to bring about rapid

haemolysis.

[Sidenote: [100]]

From their very ingenious experiments, as a whole, Ehrlich and

Morgenroth conclude that the fixative possesses two different

affinities: one for the red corpuscle and another for the complement. Of

these two affinities the stronger is that which links it to the red

corpuscle, for this is manifested at a very low temperature. In order

that the fixative may combine with the complement a much higher

temperature is requisite. Ehrlich comes to the conclusion that the

molecule of the fixative possesses two haptophore groups, or groups

capable of chemical combination. The first of these links it to a

corresponding molecule of the red corpuscle to which he gives the name

of receptor; the second combines the fixative with the molecule of the

complement and in this way introduces the latter into the red corpuscle.

These investigators give a diagram which greatly facilitates the

understanding of their hypothesis (Fig. 19). They seek to prove that the

combinations of the fixative with the red blood corpuscle and with the

complement follow the law of definite multiples and that these phenomena

must, in consequence, be looked upon as being of a purely chemical

character.

[Illustration:

FIG. 19. Schema of Ehrlich’s theory.

_c_, complement (alexine, cytase)—_am_, amboceptor (fixative)—_r_,

receptor of the red corpuscle.

(After Levaditi in the _Presse médicale_.)

]

The hypothesis advanced by J. Bordet does not accord very well with the

theory we have just set forth. He could never convince himself that the

fixative combines with the complement. He was of opinion rather that the

fixative, retained by the corpuscle, exercises upon it a mordant action

which enables it to absorb the alexine. The alexine is supposed to

attach itself to the sensibilised red blood corpuscle as a dye attaches

itself to a mordanted element. Bordet rests his interpretation mainly on

the fact that the absorption of alexine by the sensibilised corpuscles

does not follow the elementary laws of chemical combination, especially

those of definite multiples.

Nolf[121] has sought to define more accurately the part played by these

two substances in the solution of the red blood corpuscles. He agrees

with Bordet, that in this phenomenon the fixative plays the same part

that the mordants do in dyeing. Linked to the red corpuscle the fixative

renders it more greedy for alexine, exactly as the mordant facilitates

the fixation of the dye on the fibre of the textile fabric. Under these

conditions the alexine (complement), finding itself in large quantity

inside the red corpuscle, exercises upon it its hydrating action, thus

bringing about the diffusion of the haemoglobin and often even the

solution of the corpuscular stroma.

Nolf compares the solvent action of alexine upon the red corpuscle to

that of certain mineral salts, such as ammonium chloride. He passes in

review the various properties of alexines and finds them very similar to

the solvent action of certain salts. Even the peculiarity of alexine, of

remaining inactive at a temperature of 0°–3° C., is shared by ammonium

chloride which, alone of all the salts studied by Nolf, exercises no

solvent action under these conditions. But Nolf found it impossible to

push these analogies further, and especially to sensibilise, by the

fixative, the red corpuscles to the action of quantities, which were of

themselves inactive, either of ammonium chloride or of any other salt.

[Sidenote: [101]]

London[122] hoped by fresh experiments to solve the problem of the mode

of action of the two substances which act in haemolysis. He pronounced

in favour of the theory that they entered into chemical combination with

the red corpuscles. But the facts accumulated up to the present do not

enable us to make a positive statement as to the exact nature of the

reaction which is set up during the solution of the red blood

corpuscles; this is not astonishing in view of the fact that it is

impossible to isolate the haemolysing substances in a pure state.

It may, however, be admitted that the action of alexine (complement)

comes under the category of phenomena that are produced by soluble

ferments. Buchner[123] maintains that there is an analogy between this

substance and the diastases (or enzymes); Bordet[124], from the

appearance of his first publications on haemolysis, has expressed

himself in favour of this view. Ehrlich and Morgenroth[125], in their

two first memoirs, very distinctly put forward the same idea. “We shall

not deceive ourselves”—they say—“if we attribute to the addiment (syn.

complement, or alexine) the character of a digestive ferment.” In one of

their last memoirs[126] they no longer express themselves in so decided

a fashion. Nevertheless we are still quite justified in maintaining this

proposition. The substance which dissolves the red blood corpuscles of

Mammals or a portion only of those of Birds, undoubtedly presents very

great analogies to the digestive ferments. As has been mentioned

repeatedly, it is very sensitive to the action of heat and is completely

destroyed by heating for one hour at 55° C. In this respect it closely

resembles the macrocytase of macrophagic organs which also dissolves red

corpuscles. As it is the macrophages which ingest and digest the red

blood corpuscles in the organism, it is evident that alexine is nothing

but the macrocytase which has escaped from the phagocytes during the

preparation of the serums.

[Sidenote: [102]]

We know that the leucocytes contain quite a series of soluble ferments

of which some are set at liberty after the blood has been drawn from the

vessels. It is thus that plasmase, or fibrin-ferment, is set free from

the leucocytes to combine with fibrinogen to produce the clot. This is

not the only soluble ferment of leucocytic origin. It has been known for

some time that in addition to this coagulating ferment the leucocytes

contain ferments which are especially digestive or decoagulating. Thus

Rossbach[127] has demonstrated the presence of amylase in the leucocytes

of different organs, especially the tonsils. Arthus has confirmed this

discovery and Zabolotny[128] has completed it by his observations on the

phenomena which appear in the peritoneal cavity of animals into which

wheat flour or starch were injected. He observed that the small granules

are quickly ingested by isolated leucocytes, whilst the large granules

are surrounded by quite a layer of phagocytes. He agrees with several

other writers, that the amylase found in defibrinated blood has its

origin in leucocytes.

[Sidenote: [103]]

Leber[129], in the course of his researches on inflammation, made the

observation that the pus of a hypopyon that was absolutely aseptic

digests coagulated fibrin at a temperature of 25° C. and liquefies

gelatine very readily. Achalme[130] has confirmed this and has added

several other interesting data. He investigated the soluble ferments of

pus and directed his attention amongst others to experimental pus, set

up by the injection of spirit of turpentine. In addition to amylase and

a ferment which liquefies gelatine, Achalme has discovered in pus,

saponase (lipase), casease, and a ferment closely allied to trypsin.

This last readily digests fibrin and also attacks coagulated white of

egg; in the products of this digestion Achalme found peptone but could

not always obtain leucin and tyrosin. He never succeeded in

demonstrating the presence of sucrase, inulase, emulsin or lactase in

pus. On the other hand he found large quantities of oxydase, thus

confirming the discovery of Portier[131] who was the first to

demonstrate that these ferments met with in the blood are, in the living

animal, found inside leucocytes. By a large number of experiments,

carried out on most diverse representatives of the animal kingdom,

Portier was able to establish the important fact that the oxydases which

are found in many organs or in the fluid of blood withdrawn from the

organism really originate in leucocytes as they deteriorate and break

up. In this respect, then, they resemble fibrin-ferment very closely.

To complete the list, already considerable, of leucocytic ferments, I

must further cite the anticoagulating soluble ferment whose existence in

Mammals has been so well demonstrated by Delezenne.

All this evidence encourages us, then, to support the thesis that

alexine is one of the numerous intraleucocytic soluble ferments and that

it only passes into the fluids as the result of rupture or of damage to

the phagocytes. Nolf (_l.c._) has recently pronounced against this view;

we must therefore examine his arguments closely. In the first place he

takes his stand on the analogies between the solution of the red blood

corpuscles by the serums and by certain salts. It must not be forgotten,

in connection with his theory, that haemolysis is but one example, out

of many, of the action of alexines. Of all the formed elements the red

corpuscles are the most delicate; they are readily broken up by all

sorts of agents (moderate heat, water, salts, etc.). Further, there are

numerous other cells (white corpuscles, spermatozoa, and inferior

organisms) which resist the action of salts much better, which,

nevertheless, are very injuriously affected by the action of the

alexines.

Nolf lays special stress on the experiments in which, after keeping red

blood corpuscles in prolonged contact with active serums, he has looked

in vain for the peptone reaction. He prepared his mixtures in sealed

tubes or flasks, and kept them in an incubator at 37° C. for 24–48

hours, or even for weeks. Under these conditions the haemoglobin is

transformed into metahaemoglobin, but peptones never appear. Nolf

concludes therefrom “with confidence, that the alexines do not exert the

slightest peptonising effect on the albuminoids of the corpuscle”

(_l.c._ p. 672).

[Sidenote: [104]]

To this conclusion it must be objected that peptone is not the only

product of the digestion of albuminoids by soluble ferments. Under

certain conditions the disintegration is carried much further, in others

it is arrested at an earlier stage. Thus human urine which contains

pepsin, never gives the peptone reaction with fibrin; the digestion of

the latter only goes on up to the stage of protalbumose. When, however,

the urinary pepsin is fixed on flakes of heated fibrin which are

submitted to digestion in acidulated water the digestion proceeds

further and gives as final products deuteroalbumose and peptone[132].

Now, under the conditions in Nolf’s experiments the digestion would be

very quickly stopped, because, at the temperature of 37° C., alexine

very soon loses its strength. Investigators who have experimented with

haemolytic serums know well that, even when kept at a low temperature,

alexine may lose its activity within 24 hours.

It has been mentioned above that Nolf sought in vain for a parallel

between haemolysis by salts and that by serums, in what relates to the

action of the fixative. He was unable to find anything comparable to

this action amongst salts, although digestion by soluble ferments offers

undoubted analogies. I need only recall further the discovery of

enterokynase, the soluble ferment of the digestive juice of the dog,

which actively stimulates the action of pancreatic ferments, and

especially that of trypsin. The recent researches of Delezenne

(communicated to the International Congress of Physiology held at Turin

in September 1901) support this conclusion in a very important fashion.

As already pointed out in Chapter III the enterokynase of the intestinal

juice exerts an action comparable with that of the fixatives of

haemolytic serums. Alone, it does not act as a solvent ferment, but when

it attaches itself to the fibrin it aids the action of the trypsin in a

marked degree. In pancreatic digestion enterokynase plays the part of

the fixatives in the solution of red corpuscles.

[Sidenote: [105]]

The analogy between the resorption of formed elements and intestinal

digestion extends even beyond this. When we inject, into the peritoneal

cavity or under the skin of various animals, blood from a different

species, the blood serum of the former becomes haemolytic for the red

corpuscles of the latter. The solution of these red corpuscles is

effected by the alexine of the serum, whose activity is rendered very

great owing to the presence of a quantity of specific fixative. This

same fixative appears also in the fluids of animals to whom, instead of

injecting blood, we simply give it by the mouth. This fact has been

established by Metalnikoff[133].

Another fact in favour of the close relationship between the fixatives

and enterokynase consists in the presence of both in the lymphatic

(lymphopoietic) organs. The fixatives which aid the solution of red

corpuscles are found specially in the mesenteric glands. Enterokynase,

as demonstrated by Delezenne, is found not only in the intestinal juice,

but also in Peyer’s patches, the solitary glands, the mesenteric glands,

and the leucocytes of exudations and of the blood.

Supported by these various facts we are quite justified in regarding the

haemolysing substance of serum as containing two soluble ferments, of

which one, alexine, corresponds to trypsin, the other, the fixative,

resembling enterokynase. The alexine, whose nature is gradually

disclosing itself with more precision, should bear the name of _cytase_

or cell-ferment. The cytase of the macrophagic organs, or _macrocytase_,

comes under this category. According to the researches of Tarassewitch

it also acts more actively when there is added to it some of the

fixative found in the serum (heated to 56° C.) of prepared animals.

[Sidenote: [106]]

We have said that in the living animal the macrocytase is localised in

the phagocytes of the organs and of the blood. Thus, when goose’s blood

is injected into the peritoneal cavity of the guinea-pig the red blood

corpuscles are digested within the macrophage and not in the fluid of

the peritoneal exudation. When, however, the same kind of blood is

injected a second or a third time, it is found that a certain number of

the red corpuscles become permeable and lose their haemoglobin, which

they give up to the fluid of the exudation, and only the membrane and

the nucleus remain. These are at once ingested by the macrophages which

under these conditions manifest a real excess of activity. Instead of

sending out small processes, as they do after the first injection of

blood, these phagocytes move about like true Amoebae, sending out broad

pseudopodia, and ingest not only the remains of the red corpuscles but

also those still intact[134] (Fig. 20). Under these conditions

macrocytase must undoubtedly be found in the peritoneal plasma. It is,

however, easily demonstrable that this ferment was not preformed in the

fluid but has escaped from the leucocytes that have undergone

phagolysis. After the rapid injection of alien blood the phagocytes of

the peritoneal lymph gather into clumps, become immobile, and for a time

lose their phagocytic power. It is only after the lapse of a longer or

shorter period that the leucocytes recover from the phagolysis, arrive

in great numbers in the peritoneal cavity and display their phagocytic

energy.

[Illustration:

FIG. 20.—Rapid ingestion of red corpuscles of the goose by

macrophages.

]

If the damage to the phagocytes—the phagolysis—is the actual cause of

the setting free of the intraleucocytic ferment, we have only to prevent

this phagolysis in order to inhibit the solution of red blood corpuscles

in the fluid of the exudation. For this purpose it is sufficient to

prepare guinea-pigs (which have already received several injections of

goose’s blood) by means of an injection of fresh broth, of physiological

salt solution, or of carbonic acid into the peritoneal cavity on the eve

of the decisive experiment. Such injection at once provokes phagolysis,

which is then followed by an abundant exudation of leucocytes. When,

next day, a dose of red blood corpuscles of the goose (deprived of serum

by centrifugalising) is introduced into the peritoneal cavity thus

prepared phagolysis is no longer produced, or very feebly, and is of

very short duration. Under these conditions the solution of the red

corpuscles by the peritoneal fluid is reduced to a minimum, and in its

place an extremely rapid and considerable ingestion of red corpuscles by

the macrophages may be observed. In order that the experiment may be

completely successful it is advisable to use goose’s blood heated to 37°

C. or thereabouts for the injection.

[Sidenote: [107]]

Even when the red corpuscles of the goose are introduced, not into the

peritoneal cavity but into the subcutaneous tissue of guinea-pigs that

have received several injections of goose’s blood, we can easily prevent

the extracellular solution of the red corpuscles which takes place, as

already indicated, in the normal guinea-pig. As in this case the goose’s

serum which is mixed with the corpuscles contributes to the haemolysis,

it must be suppressed by centrifugalising the defibrinated goose’s blood

and by washing the corpuscles with normal saline solution.

Collectively, the facts I have just described clearly indicate that the

phagocytes must be regarded as the source of the haemolytic ferment. The

macrocytase remains in the body of these cells so long as they are in a

normal condition; but immediately they are injured, in consequence of

the sudden introduction of foreign substances into the peritoneal

cavity, a portion of the macrocytase escapes and acts on the red

corpuscles as if it had been employed _in vitro_.

As the conclusion I have just formulated is of fundamental importance in

the study of resorption and immunity it is necessary to support it by as

many arguments as possible. For this reason, therefore, I feel obliged

to draw the attention of the reader to another example of the resorption

of formed elements.

We have already spoken of the resorption of spermatozoa in the

peritoneal cavity, and of the part played by the macrophages in this

phenomenon. As a result of this resorption, just as after that of red

blood corpuscles, the organism acquires new properties of the same

character. Landsteiner[135] and the writer[136] have shown that the

blood serum and the peritoneal fluid of animals that have been injected

with the spermatic fluid of bull, rabbit, or man, become spermotoxic,

that is to say, they render the corresponding spermatozoa motionless and

kill them. These fluids, however, never acquire the power of dissolving,

even partially, these elements. The disappearance and final solution of

the spermatozoa is only effected within phagocytes, and almost

exclusively in the macrophages.

[Sidenote: [108]]

Moxter[137] has demonstrated that the spermotoxin which appears in the

serum of prepared animals consists of two substances, corresponding to

those present in the haemolytic serums. These are the macrocytase

(alexine, complement) and the fixative (intermediary or sensibilising

substance). For him they are identical with those which dissolve the red

corpuscles. Without dwelling on the subject we may say that the

macrocytase which dissolves the red corpuscles and that which arrests

the motion of the spermatozoa are really identical in the same species

of animal, as is accepted and developed by Bordet. On the other hand, it

is impossible to accept Moxter’s theory of the identity of the two

fixatives. They must be regarded as different; this we have attempted to

prove in one of our memoirs[138] and is in accordance with the law of

the specificity of fixatives in general.

The question which interests us more especially at this moment is where

are these two constituent substances of the spermotoxin to be found and

how do they behave in the living organism? This question has been very

thoroughly studied by Metalnikoff[139] in my laboratory. His experiments

have been closely followed by me, and in presenting their principal

results I can bear witness to their correctness.

The spermotoxin obtained by Metalnikoff is distinguished from the

haemotoxins we have discussed up to the present in that they develop,

not as a result of the injection of cell elements from a different

species, but as a result of the introduction into the organism of

spermatozoa from the same species, the guinea-pig. We have here, then,

to deal with what has been termed autospermotoxin.

The serum of the normal guinea-pig acts but feebly on the spermatozoa of

this species, which, under its influence, remain motile for several

hours. When, however, guinea-pigs have received one or several

injections of the spermatozoa of their own species, their serum and

peritoneal lymph become distinctly toxic and render the spermatozoa

motionless in a few minutes. In male guinea-pigs so prepared the serum

acquires this toxic property not only for the spermatozoa of other male

guinea-pigs, but likewise for those of the individual itself which

furnishes the serum. This latter, then, becomes distinctly

autospermotoxic.

[Sidenote: [109]]

If the spermotoxin were diffused in the plasma and other fluids of the

guinea-pig which furnishes it, it ought to render motionless the

spermatozoa contained in the genital organs. Experiment demonstrates,

however, that this is not the case. If the male organs be removed from a

guinea-pig whose serum is very autospermotoxic _in vitro_, we find,

especially in the epididymis, a mass of very virile spermatozoa which

for a long time retain their motility in physiological salt solution.

The macrocytase, then, has not reached the spermatozoa in the living

animal; this is because it is not found in the plasmas. Let us inject

into a guinea-pig, whose serum is strongly autospermotoxic, one portion

of sperm into the subcutaneous tissue and another portion into the

peritoneal cavity. In the first site a soft oedema, filled with

transuded fluid, in which the very active spermatozoa retain their

motility for a couple of hours, is produced. In the peritoneal fluid the

same spermatozoa become motionless in a few minutes. This great

difference is explained by the fact that, under the skin, there are no,

or almost no pre-existing leucocytes, whilst in the peritoneal fluid

they are abundant. The phagocytes injured by the introduction of sperm

into the peritoneal cavity, abandon a portion of their macrocytase,

sufficient to render the spermatozoa motionless. But when Metalnikoff

injected physiological salt solution into the peritoneal cavity of his

autospermotoxic guinea-pigs, and then, on the following day, a quantity

of sperm, the spermatozoa continued very active for more than an hour.

In this case phagolysis is very transitory and insignificant; it is soon

followed by a great afflux of leucocytes which bring about a rapid

ingestion of the spermatozoa. Many of these elements are devoured in a

living state; for even when their body is enclosed in the macrophage,

their tail, left outside, continues to move very actively.

[Sidenote: [110]]

All these experiments demonstrate that in the normal state the

macrocytase remains within the phagocytes and only escapes during

phagolysis, or at the moment when the blood, after it has been withdrawn

from the organism, coagulates. Is it the same for the fixative? It is

easy to prove that this soluble ferment circulates in the plasmas of the

living organism. We have already said that the spermatozoa of a

guinea-pig whose serum is very autospermotoxic, remain alive for some

time in the physiological salt solution. But if we introduce them, _in

vitro_, into the serum of a normal guinea-pig they remain motile but a

short time (some 10–20 minutes), whilst the spermatozoa of a normal

guinea-pig will live in the same serum for several hours. This

difference is explained by the fact that the spermatozoa of the

autospermotoxic guinea-pig, although very active, have absorbed the

fixative during the life of the animal. This fixative is, as we have

stated, found in the body fluids and has been able to penetrate to the

male organs. Here the spermatozoa become charged with the fixative and,

once transported into the serum of the normal guinea-pig, rich in

macrocytase, they lose their movements very quickly. At the same time

the spermatozoa used for control, not having absorbed any fixative, are

able to live for a long time in the same serum.

As the macrocytase remains fixed to the phagocytes there can be no doubt

as to its origin; it is elaborated by these cells. Whence however comes

the fixative which is free in the body fluids and which is precisely the

substance that is developed in so large a quantity in the treated

animals? The exact solution of this question is not easy; nevertheless

there are many facts which indicate that this fixative is also of

phagocytic origin. We know already that the serums of normal animals

contain only small quantities or sometimes, perhaps, none of the

fixative. This fixative only appears abundantly as the result of the

resorption of the corresponding elements, red corpuscles or spermatozoa.

This resorption, as we have said, is almost exclusively the work of the

macrophages. It is just in those cases where the red corpuscles,

injected into the peritoneal cavity of an animal of the same species,

pass directly into the lymph, without being injured or, save

exceptionally, ingested by the phagocytes, that the fixative is not

formed. When the red blood corpuscles of the goose, introduced with

defibrinated blood below the skin of a guinea-pig, undergo there a

partial solution in the fluid of the exudation, and where the

phagocytosis is more limited than in the peritoneal cavity, the

production of fixative is small. When the injection of the same goose’s

blood is made into the peritoneal cavity of a guinea-pig and is followed

by complete phagocytosis, the fixative is produced in greater abundance.

There exists, then, in all these cases a constant relation between the

degree of phagocytosis and the amount of the fixative produced. As this

fixative facilitates the access of the cytase to the cells and as the

resorption of these elements takes place specially in the macrophages,

we are bound to come to the conclusion that the fixative is a second

phagocytic ferment which is produced in abundance during the process of

intracellular digestion. Only, instead of remaining in the substance of

the phagocytes, this fixative is in part thrown out from these elements.

It passes into the plasma of the blood and into the other fluids and

ends by disappearing from the organism, probably being eliminated by the

excretory channels.

[Sidenote: [111]]

In the Invertebrata, where, as we have seen, the alien red blood

corpuscles are also digested within the phagocytes, we have never been

able to demonstrate any haemolytic property of the blood fluid, even

after repeated injections of blood. We must conclude from this that in

these animals the quantity of fixative is merely sufficient to bring

about the solution of the red corpuscles which are within the

phagocytes. In the case of fishes and higher animals (we may recall the

example of the red corpuscles of the guinea-pig when resorbed into the

organism of the gold-fish) the production of the fixative is much more

abundant, and this ferment can be easily demonstrated by its action _in

vitro_.

This over-production of a ferment which acts in the phagocytic

resorption, finds its analogue in the passage of certain digestive

ferments, such as amylase and pepsin in man and the dog, into the blood

and urine, as mentioned in the preceding chapter.

One of the best arguments in favour of the thesis here developed, has

been furnished to us by the analysis of the phenomena observed in

connection with the autospermotoxic serums of the guinea-pig. This idea

of autotoxins was originally put forward by Ehrlich in his memoirs,

published in conjunction with Morgenroth and already repeatedly cited.

Ehrlich asked himself whether the organism which resorbs, not red

corpuscles of an alien species, but red corpuscles of its own species,

would also be capable of developing haemolytic substances. With this

object he injected blood obtained from goats into these same goats or

into other individuals of the same species. He and Morgenroth[140] were,

under these conditions, able to obtain isotoxic serums, that is to say

serums which dissolve the red corpuscles of the goat, coming from other

individuals than those which had been treated by the blood and which

furnished the serum. In order to obtain this result, however, they had

to inject, not unaltered blood but blood mixed with water. The red

corpuscles of the unaltered blood pass readily into the circulation of

the animal of the same species, without being attacked by the

phagocytes. Now, we know from the experiments of Bordet that the stromas

of the red corpuscles suffice for the production of the fixative, whilst

the haemoglobin does not incite to the development of this ferment by

the organism. As the stromas, injected with a mixture of blood and

water, must be devoured by the macrophages, we can readily understand

that these phagocytes may serve for the elaboration of the fixative.

[Sidenote: [112]]

The resorption of the red corpuscles and that of spermatozoa which we

have presented as examples, may serve as types for the resorption

phenomena of formed elements in general. When other species of cells are

introduced into the organism, the resulting process always reveals the

same character: inflammatory reaction with preponderant intervention of

the macrophages; intraphagocytic digestion of the introduced elements;

excessive production and excretion of the fixatives. Whilst the

macrocytase is always the same in the same species of animal, the

fixatives are different and specific. In addition to the haemofixatives

and spermofixatives already described, we may obtain, as the result of

the injection of the corresponding cells, leucofixatives,

nephrofixatives, hepatofixatives, trichofixatives, etc. It does not

enter into our programme to treat the subject here[141]. We wish simply

to insist on those aspects of the resorption of cells which are closely

connected with the problem of Immunity. In the next chapter we must,

however, recur to certain features of the phenomena of resorption.

CHAPTER V

RESORPTION OF ALBUMINOID FLUIDS.

Resorption of albuminoid substances.—The precipitins of blood serum

which appear as a result of the absorption of serums and of

milk.—Absorption of gelatine.—Leucocytic origin of the ferment

which digests gelatine.—Anti-enzymes.—Antirennet.—The

anticytotoxins.—Antihaemotoxic serums.—Their two constituent

parts: anticytase and antifixative.—Action of anticytase.—The

antispermotoxins.—Origin of anticytotoxins.—Ehrlich’s theory on

this question.—Origin of antihaemotoxin.—Origin of

antispermotoxin.—Production of this antibody by castrated

males.—The antispermofixative produced when the spermatozoa are

excluded.—Distribution of spermotoxin and antispermotoxin in the

organism.

[Sidenote: [113]]

We stated at the beginning of the last chapter that various fluid

substances of very complicated chemical composition may be absorbed by

the tissues and utilised by the organism without requiring to be

modified by the digestive juices of the intestinal canal. We must now

describe, exactly, the phenomena observed in these cases and endeavour

to establish the mechanism of the absorption of fluids in the living

organism.

[Sidenote: [114]]

We have already cited the examples of blood serum, milk, and white of

egg, all of which are readily utilised by the organism which receives

them directly into the peritoneal cavity or below the skin. The proof

that these substances are modified—digested by the tissues, is furnished

by the observation that their injection necessarily brings about

appreciable changes in the properties of the blood. Th.

Tchistovitch[142], in a research carried out in the Pasteur Institute,

was the first to demonstrate that the resorption of the blood serums of

the eel and horse by the organism of the rabbit, excites in the blood of

the latter animal the production of specific precipitates. The blood

serum of rabbits that have been vaccinated against the toxic eel’s serum

gives a precipitate with eel’s serum; the serum of rabbits treated with

horse’s blood gives a similar precipitate with horse’s serum, etc. This

property has since been confirmed and studied by several observers, who

have made use of it for the recognition of human blood in medico-legal

investigations[143].

Bordet[144] has made the discovery that intraperitoneal injections of

the milk of cows into rabbits provokes in the blood serum of the latter

the property of giving a specific precipitate with cow’s milk only. This

precipitation bears a great resemblance to the coagulation of casein;

which, however, does not justify us in identifying the precipitating

substance with rennet. This fact has been confirmed for several other

species of milk, and Schütze[145], in an investigation carried on in the

Berlin Institute, essayed to apply it to the differentiation of the

various kinds of milk. In the same order of ideas, researches have been

made on the artificial precipitins that develop in the blood as the

result of injection of white of egg and other albuminoids[146].

Leclainche and Vallée[147] have prepared animals in such a fashion that

their serum produces a precipitate with urinary albumen. The biological

precipitin reactions are more sensitive than any of the chemical

reagents properly so called. These specific substances in the serums

must be looked upon as belonging to the group of soluble ferments,

approximating to the fixatives rather than to the cytases, since they

are unaltered by being heated to 56° C. Their action gradually declines

after passing 60° C. but is only destroyed at a temperature beyond 70°

C.

[Sidenote: [115]]

An analogous soluble ferment has been discovered in the blood serum of

animals treated with injections of gelatine. We owe to Delezenne, who

has studied this question in his laboratory at the Pasteur Institute,

the most important and most complete data on the resorption of gelatine.

The blood serum of normal animals possesses only a very feeble power,

sometimes even none, of liquefying gelatine. When however this substance

is injected several times, the serum, as is the rule for the formed

elements and quite a series of fluid substances, acquires a much more

pronounced activity. The gelatine, without giving any precipitate, is

simply dissolved and will no longer solidify when it is cooled. The

ferment of the serum that produces this effect resembles the precipitins

in that it withstands the action of a temperature of 56° C. and is only

destroyed beyond 60° C. Like the trypsins it acts in a weakly alkaline,

neutral, or weakly acid medium; but digestion takes place best in a

slightly alkaline medium.

The question of especial interest to us is that of the origin of this

ferment which digests gelatine. If several c.c. of a 10% solution of

this substance be injected into the peritoneal cavity of a laboratory

animal, there is provoked with certainty, within a few hours, a marked

leucocytosis of the peritoneal fluid. A considerable afflux of

leucocytes, amongst which the microphages are even more numerous than

the macrophages, takes place. When to a hanging drop of such an

exudation a trace of Ehrlich’s neutral red solution is added, there

appears almost at once an intense coloration of the numerous droplets

inside the two kinds of leucocytes. It is, therefore, manifest that the

gelatine excites a powerful positive chemiotaxis of the mobile

phagocytes and that it is absorbed by these cells. This experiment

demonstrates that the phagocytes can not only ingest solid bodies, such

as the various formed elements, coloured granules, etc., but that they

are also capable of absorbing fluid substances introduced into the

tissues or cavities of the organism.

The data brought forward by Delezenne demonstrate very clearly the part

played by the mobile phagocytes in the digestion of gelatine. He

obtained his best results in the dog. We know that it is easy in this

animal to provoke an aseptic exudation, very rich in leucocytes. This

exudation when deprived of its serum and washed with physiological salt

solution gives a solution which exerts a feeble digestive action on

gelatine. If the exudation be produced in a dog that has previously

received several injections of this substance, we obtain leucocytes

whose extract, obtained by the same method, will digest gelatine much

more actively. The digestive power of the leucocytes of the treated dog

is sometimes five times greater than that of the leucocytes of the

normal dog. Here, then, we undoubtedly have an acquired digestive power

which reveals a great reinforcement of the phagocytic activity.

[Sidenote: [116]]

In the prepared dogs the leucocytes have a much greater digestive action

on gelatine than has the blood serum of the same animals, a fact which

indicates that the source of the soluble ferment must be sought for in

the phagocytes themselves. The results of these researches are of great

service to us in the study of immunity properly so called.

For some time past attempts have been made to show that the soluble

ferments, diastases, or enzymes, are closely allied to albuminoid

substances. Nencki and Mme Sieber[148] support this view by their recent

researches on the chemical composition of pepsin. In all the above cases

there is this in common between the two categories of substances, their

absorption by the organism is followed by the appearance in the blood of

antagonistic ferments. Just as after the injection of milk, white of

egg, serums, etc. into the cavities or tissues, specific precipitins are

produced, so the injection of certain enzymes provokes the formation in

the organism of antienzymes or antidiastases.

[Sidenote: [117]]

It has been known for some time that the blood serum of many animals

prevents the action of certain enzymes. Thus Röden has shown that normal

horse’s serum retards or even completely prevents the coagulation of

milk by rennet. It has often been observed, too, that normal serums

hinder, more or less, the digestion of albuminoids by trypsin. It is

only quite recently, however, that we have begun to prepare antienzymes

by the injection into animals of corresponding enzymes. Thus,

Hildebrand[149] has succeeded in obtaining an antiemulsin in the serum

of rabbits, into which he had injected several separate doses of

emulsin. Fermi and Pernossi[150] have prepared an antitrypsin, and von

Dungern[151] has obtained an antidiastase against the proteolytic

enzymes of some bacteria. But of all the antienzymes, the one that has

been best studied up to the present is indisputably antirennet, obtained

independently by Morgenroth[152] and Briot[153]. The former of these

investigators treated goats with increasing quantities of rennet and was

able to assure himself, by comparative detailed researches, of the

appearance and increase in quantity of antirennet in the blood serum.

The goat which gave the best result ceasing to develop antirennet it was

impossible to make the antirennet potency go beyond a certain point.

Briot also obtained antirennet in rabbits into which he had injected

fluid rennet on several occasions. He was able to convince himself that

the antirennet of horse’s serum is a non-dialysable substance which is

precipitated by alcohol and certain salts. Like the precipitins and the

diastase which digests gelatine, antirennet resists a temperature of

55°–56° C.; even heating to 58° C. has no effect on the antirennet

serum. At 60° C. however, the heat begins to exert an injurious effect,

and after three hours at 62° C. the serum has lost all power to prevent

the coagulation of the casein by antirennet. Morgenroth and Briot both

state that the antirennet neutralises the rennet by a direct action.

The cell poisons, or cytotoxins, of animal origin which were treated in

the preceding chapter, likewise set up the production of special

antibodies, or anticytotoxins. The consideration of these latter has a

very special interest for those who study the question of immunity from

a general point of view. The first discovery of these anticytotoxins was

made in connection with the study of the toxic power of the blood serum

of eels. Camus and Gley[154] and, independently of them, H. Kossel[155]

demonstrated that animals when treated with increasing doses of eel’s

serum acquire an antitoxic property which protects their corpuscles

against the haemolytic action of ichthyotoxin, or the toxic substance of

the blood of eels. Th. Tchistovitch[156] has not only confirmed this

discovery, but has added to it new and interesting data.

[Sidenote: [118]]

When antitoxic serum is mixed _in vitro_ with red blood corpuscles of

the species which furnished the serum and there is added to it some

haemolytic eel’s serum, it will be found that the red corpuscles remain

quite unaltered. In the control tubes, however, in which the antitoxic

serum is replaced by normal serum of the same species, the red

corpuscles are very readily dissolved under the toxic influence of the

eel’s serum. In animals (rabbits) that are treated with this latter

fluid, there is established not only an antitoxic power of the blood,

but the red corpuscles acquire a resisting power more or less pronounced

against the ichthyotoxin of eel’s serum. When the red corpuscles are

separated from the serum of rabbits (treated with eel’s serum) and some

ichthyotoxin is added to them, solution very often does not take place

at all. According to the experiments of Tchistovitch there is no direct

relation between this acquired resistance and the antitoxic power of the

blood. Sometimes even a kind of antagonism is observed between the two

properties; that is to say, the red corpuscles of a rabbit whose serum

is very antitoxic may be extremely sensitive to the poison of the eel

whilst the converse may also hold good [cf. _infra_ p. 120].

The toxic action of the eel’s serum upon the red corpuscles of a great

number of Vertebrates is a natural property which demands no previous

treatment of the eel. It is the antitoxic power, directed against the

ichthyotoxin, which is developed only as a result of the preparation of

the animals by the administration of increasing doses of eel’s serum.

Nevertheless we also find natural antitoxins present in the blood of man

or animals that have not been treated and which act against the cell

poisons, cytotoxins, so widely distributed in the blood of a large

number of species of animals.

Besredka[157] has demonstrated that the blood serum of Man and many

Vertebrates contains a substance which prevents the solution of red

corpuscles under the influence of blood serums of a different species.

To reveal the presence of these antitoxins it is useful to heat the

serums to 56° C. and then to add to them red corpuscles of the same

species and some haemolytic serum of a different species. Under these

conditions the solution of the red corpuscles does not take place,

whilst their mixture with haemolytic serum alone, inevitably provokes

haemolysis.

[Sidenote: [119]]

Along with these natural antihaemolysins there exist a number of

artificial antihaemolysins or antihaemotoxins. Jules Bordet[158] was the

first to draw attention to this important subject. He first obtained

these antihaemolysins by injecting blood serum of the fowl, which

possesses a very great haemolytic power on the red corpuscles of the

rabbit, into individuals of this latter species. After some injections,

the serum of these treated rabbits was found to be antihaemotoxic

against the fowl’s serum. Later[159], Bordet obtained a serum against an

artificial haemotoxin. The serum of the guinea-pig is innocuous to the

red corpuscles of the rabbit. But when rabbit’s blood was injected

several times into guinea-pigs the serum of the latter became very

solvent for the red corpuscles of the rabbit. To prevent this action it

is sufficient to inject the haemotoxin of treated guinea-pigs several

times into rabbits. The serum of these rabbits becomes antihaemotoxic

and protects the red corpuscles of the rabbit against the solvent action

of guinea-pig’s serum.

In the normal haemolytic serums, such as the serums of the eel and fowl,

the presence of two substances which act by combining could not be

demonstrated. On the other hand, in the serums that were obtained as a

result of the treatment of animals by the injection of blood from a

different species, it was easy to demonstrate, as we have shown in the

preceding chapter, the presence of two constituent substances which are:

the macrocytase (alexine, complement) and the fixative (amboceptor of

Ehrlich, sensibilising substance of Bordet). For this reason the study

of the antihaemotoxins obtained against artificial haemotoxins is

endowed with special interest. As the solution of the red corpuscles, in

this case, can be prevented either by an antitoxic action directed

against the cytase, or by a neutralisation of the fixative (for the

concurrence of these two substances is indispensable in order that the

solution may take place), Bordet asked whether the antitoxic serum,

obtained by him in rabbits, is anticytatic or antifixative, or whether

it contains both properties. Before resolving this problem it was

necessary to establish some of the essential characters of artificial

antihaemotoxic serums. The principal one amongst them is the resistance

of these antihaemotoxins to a temperature of 55–60° C.; even when heated

to 70° C. the antihaemotoxins retain, at least in part, their

fundamental property. In this respect these substances differ from the

cytases and approach the precipitins, fixatives and agglutinins.

[Sidenote: [120]]

[Sidenote: [121]]

The very exact experiments carried out by Bordet have demonstrated that

in the serum of rabbits, treated with the specific haemotoxic serum of

guinea-pigs, two substances, an anticytase and an antifixative, are

found in combination. The former of these antitoxins is found in

abundance, but the amount of antifixative is very small. Bordet was led

to this result in the following way. To prevent the solution of the red

corpuscles of the rabbit in the haemotoxic serum of the guinea-pig, it

was necessary for him to add a considerable dose (10 to 20 times) of the

antitoxic serum. When, however, he heated the latter to 55° C. the

quantity of this serum necessary to prevent haemolysis could be reduced

very considerably. In place of its being necessary to add to the

haemotoxic serum 10 or 20 volumes of antitoxic serum, it was sufficient

to add three or sometimes only two volumes of this heated serum. As we

know already, heating to 55° C. destroys the macrocytase which should be

found in the antitoxic blood of the rabbit. This cytase by itself is

incapable of dissolving the red corpuscles of the same species; but when

it is added to the fixative of the haemotoxic serum of the guinea-pig

the macrocytase of the rabbit’s serum dissolves them very readily. Hence

the conclusion that in the haemotoxic serum of the guinea-pig there must

be present a quantity of fixative sufficient to allow of the solution of

the red corpuscles by the macrocytase of the rabbit’s serum. This

antitoxic serum, therefore, which only prevents the haemolysis on the

condition of being added in comparatively large quantity, contains very

little antifixative. When, by heating this serum to 55° C. we destroy

the rabbit’s macrocytase, the mixture of antitoxic serum of the rabbit

and haemotoxic serum of the guinea-pig, which ordinarily dissolves the

red corpuscles of the rabbit, now leaves them intact. The reason is that

the free fixative contained in this mixture does not find any available

macrocytase: that of the rabbit being destroyed by the heating, and that

of the guinea-pig neutralised by the antitoxic serum. The experiment I

have just described proves that this antitoxic serum contains specific

anticytase. This anticytase is capable of neutralising the guinea-pig’s

macrocytase, but is altogether powerless against that of the rabbit.

This last circumstance allows us to investigate whether the antitoxic

serum of the rabbit contains, in addition to anticytase, a specific

antifixative. Bordet prepared a mixture of antitoxic serum of the

rabbit, heated to 55° C., with haemotoxic serum of the guinea-pig, also

heated to 55° C. In this mixture the two macrocytases (that of rabbit

and that of guinea-pig) have been destroyed by heat, but the antitoxins

of the rabbit’s serum and the fixative of the haemotoxic serum have

remained intact. This mixture owing to its want of macrocytases was

incapable of dissolving the red corpuscles of the rabbit. By adding to

it some fresh unheated serum from a normal rabbit the rabbit’s

macrocytase was introduced. As the latter could not be neutralised by

the anticytase of the antitoxic serum and was incapable, by itself, of

dissolving the red corpuscles of the rabbit, it was unable to produce

haemolysis except on the condition that there is in the mixture a

sufficient quantity of unneutralised free specific fixative. As a matter

of fact, the red corpuscles of the rabbit are not dissolved in the

mixture described; this proves that the fixative had become inactive in

consequence of the presence of an antifixative in the antitoxic serum of

the rabbit. I need not enter into further details of Bordet’s

experiments, which have fully demonstrated the fact that in the

antitoxic serum of his rabbits there were really two antitoxins; an

anticytase abundant in quantity, and an antifixative present in much

smaller amount.

Ehrlich and Morgenroth[160] quite independently of Bordet have shown

that an antihaemotoxic serum is very rich in anticytase. After making a

number of injections of normal horse’s serum (very rich in cytase) into

a goat, they obtained in the blood serum of the latter an anticytase

very active against the cytase of the horse. This antitoxic serum of the

goat, as might be anticipated, contains no antifixative, the horse’s

serum that served for the injections coming from normal horses which

contained no, or very little, fixative. Even in another case, where

these investigators[161] injected a dog with sheep’s serum very rich in

fixative specific for the red corpuscles of the dog, they did not

succeed in obtaining any antifixative. These observations in no way

diminish the value of the discovery of the antifixative by Bordet,

though they demonstrate that this antitoxin cannot, in certain cases, be

found in the serum. Ehrlich and Morgenroth themselves throw out, in this

connection, the suggestion that in these cases the antifixative remains

linked to the cell which produces it, without being thrown off into the

blood.

[Sidenote: [122]]