majority of the granules. Ehrlich was able by the aid of this dye to
demonstrate granules, even in some vegetable cells. Moreover the method
of using it is the simplest conceivable, as subcutaneous or intravenous
injection, or even feeding, in the higher animals stains the granules;
with frog's larvae and invertebrates, to allow them to swim in a dilute
solution of the dye is often sufficient. The staining also succeeds in
"surviving" organs, and is best effected by allowing small pieces to
float in physiological salt solution, to which a trace of neutral red is
added, under plentiful access of air. When the object is macroscopically
red it is ready for examination.
The finest results are naturally given by organs that are easily teased
out, _e.g._ flies' eggs, or the Malpighian canals of insects. The
staining solution is to be chosen so that the act of staining does not
last too long, but on the other hand too high a concentration must not
be used. About 1/50000 to 1/100000 is recommended, so that the
protoplasm and nucleus remain quite uncoloured. Artificial products with
this method cannot entirely be excluded, and, _e.g._ in plant-cells
containing tannin, are to be explained by the production and
precipitation of the salt of tannic acid. However it is not difficult
for the experienced to recognise artificial products as such in
individual cases. The kind of granulation, the typical distribution, a
comparison with neighbouring cells, the combination of various methods,
the comparison of the same object under vital and "=survival=" staining,
facilitate judgment and obviate mistakes of this kind.
The majority of the granules of vertebrates are stained orange-red by
neutral red, corresponding with the weakly alkaline reaction of these
forms. Granules staining in pure fuchsin colour and which hence possess
a weak acid reaction are much more rarely found.
Combination staining may be recommended as a valuable aid to the neutral
red method. Ehrlich has used a double stain with neutral red and
methylene blue. Frog's larvae were allowed to remain in a solution of
neutral red, to which a trace of methylene blue had been added. He then
found red granulations almost exclusively, only the granules of the
smooth musculature of the stomach were stained intensely blue. With the
aid of a threefold combination Ehrlich obtained a still further
differentiation of the living cell-granules. There is no doubt whatever
that a thorough study of this neutral red method would lead to
important conclusions as to the nature and function of the granules, and
lead us to the most real problems of cell life. With our present
information even we can get definite conceptions founded upon facts, as
to the =biological importance of the cell-granules=.
* * * * *
In his first publication Ehrlich described the =granules as products of
the metabolism of the cells=, deposited within the protoplasm in a solid
form, in part to serve as reserve material, in part to be cast off from
the cell. On the ground of observations on the liver cells, described in
detail in a paper of Frerichs (1883, page 43), Ehrlich gave up this
position, though only temporarily. Ehrlich shewed that the liver cells
of a rabbit's liver, rich in glycogen, appear in dry preparations as
bulky polygonous elements, of a uniform homogeneous brown colour,
surrounded by a thin, well-defined yellow membrane. In cells that were
not too rich in glycogen, small roundish bodies, clearly of a
protoplasmic nature, of a pure yellow, can be seen embedded in the
homogeneous cells that are coloured brown with glycogen. "The hyaline
cellular ground substance, carrying the glycogen, could not under any
circumstances be stained, but the cell-granules above mentioned stained
easily with all kinds of dyes. It was further possible to shew by
staining that the membrane was chemically different from the granules,
since with eosin-aurantia-indulin-glycerine, the membrane stained black,
but the granules orange-red."
To these observations Ehrlich added the following conclusion, "that the
cells of the liver after food really possess a thin =protoplasmic=
membrane, and a homogeneous glycogen-bearing substance, in which the
nucleus and =round granules= (? functionally active) of protoplasm are
embedded.
"On comparing these results with those of more recent investigation of
the cells, it is easy to determine the location of the glycogen very
accurately. Kupffer has shewn, first for the liver cells--and this is
now recognised as generally valid--that their contents do not represent
a microscopically single substance. In the 'survival' preparation he
found, in addition to the nucleus, two clearly distinct substances: a
hyaline ground substance in preponderating amount, and a more scanty,
finely granular, fibrillary substance embedded in it. Kupffer calls the
first paraplasm, the latter protoplasm. On warming the preparation to
about 22 deg. C. manifest though feeble movements appeared in the network.
It can hardly be doubted, that of these two substances the granular
reticulated one--the protoplasm--is the more important; and it should
not be erroneous to suppose that the granulations of the network form
the =centre of the particular (specific) cell function=. In any case, it
is desirable to give a special name, such as microsomes (Hanstein) to
these forms, which in the liver cells are recognisable as distinct,
round or oval granules, colouring yellow with iodine, and easily and
deeply staining in other ways."
It was necessary to quote in full from this older paper, to shew that
Ehrlich regarded the granules as the special carriers of the cell
function so long ago as 1883, a view that Altmann advocated many years
later, under the name "theory of bioblasts." Altmann's ever repeated
assertion that no one before him had allotted so high an importance to
the granules is consequently in disagreement with the facts we have
above made sufficiently clear.
The importance Altmann ultimately gave to the granules, which he also
calls by the name "=Ozonophores=" is shewn by his own words (_Elementary
Organisms_, 1st edit., p. 39):
"Our conception of the ozonophores may therefore replace that of the
living protoplasm, at least so far as vegetative function is concerned;
and may serve us as an explanation of complicated organic processes.
Once again, shortly summarising the properties of the ozonophores; as
oxygen carriers they can perform reduction and oxydation, and can thus
effect the decompositions and syntheses of the body, without losing
their own individuality."
In the meantime Ehrlich had made various observations which could not be
completely brought into line with his own earlier hypothesis or the
far-reaching conclusions of Altmann. Studies in particular on the oxygen
requirements of the organism, shewed that the "ozonophores" could
certainly not be an important part of the cell. In addition it was found
that normally cells occur in which no granules can be recognised by
ordinary methods. Finally a pathological observation made untenable the
view that the granules are the bearers of the cell function. In a case
of pernicious anaemia (cp. _Farbenanalytische untersuchungen_) Ehrlich
found the polynuclear cells of the blood and bone-marrow and their early
forms free from all neutrophil granulation. On the grounds of this
observation Ehrlich returned to his original assumption that the
granules are secretory products of the cells, and defined his standpoint
at that time as follows:
"Did the neutrophil granulations really represent the bodies which
supply these cells with oxygen, as Altmann supposes, a condition such as
we have here brought forward would be impossible, since with the
disappearance of the granules death of the cells must follow. But from
the point of view of the secretion theory the condition described is
easily explainable. Just as under certain conditions fat-cells may
completely lose their contents without dying, so the bone-marrow cell,
if the blood fails to yield to it the necessary substances, may
occasionally be unable to produce more neutrophil granules. And thus it
becomes non-granular."
The view, that the granules are special metabolic products of the
specific cellular activity, is strongly supported by the great chemical
differences between them. Ehrlich made these peculiarities clear for the
blood-cells, and found that their granulations differ from one another,
not only in their colour reactions, but also in their =shape= and
=solubility=; so that they must be sharply distinguished.
Whilst for instance the majority of the granules are more or less
rounded forms, in some classes of animals, _e.g._ in birds, the
analogues of the granules of mammalian blood are characterised by a
decided crystalline form, and a strong oxyphilia. The substance of the
mast cell granulations is also crystalline in some species of animals.
The size of the individual granules is constant in any animal species
for every kind of granule--excepting only the mast cells. The eosinophil
granulation reaches its greatest size in the horse, where really
gigantic examples are found.
The presence of granulated colourless blood-cells has been demonstrated
in the most various classes of animals, and even in the blood of many
invertebrates, particularly, as Knoll has shewn, in the
Lamellibranchiates, Polychaetes, Pedates, Tunicates and Cephalopods.
Concerning vertebrates, especially the higher classes, accurate and
ample researches are to hand. In birds we recognise two oxyphil
granulations, of which one is embedded in the cells in the crystalline,
the other in the usual granular form. Amongst the vertebrates most
investigated classes possess granulated polynuclear cells. To this
circumstance Hirschfeld has recently devoted a thorough paper containing
many details worthy of note. In the majority of the animals observed, he
found too that the polynuclear cells contained neutrophil granules; in
only one animal, the white mouse, did he find them, or granulations
analogous to them, completely wanting.
According to the investigations carried out some years back in Ehrlich's
laboratory by Dr Franz Mueller, these results of Hirschfeld's must be
described as inaccurate. After many vain endeavours, Dr Mueller was able
to find a method by which numerous though very minute granules could be
found in the polynuclear cells of the mouse. The case shews that it is
not permissible to assume the absence of granules, when the ordinary
staining methods are not at once successful. There is no universal
method for the staining of granules, any more than for the staining of
various kinds of bacteria. Indeed all granules, that are easily soluble,
vanish when the triacid method is used, and so a homogeneous cell
protoplasm is simulated.
But naturally, the occurrence of non-granulated polynuclear cells in
certain classes of animals is not to be denied from these
considerations. Hirschfeld asserts that such cells occur side by side
with granulated cells, for instance in the dog; and draws from them
far-reaching conclusions as to the meaning of the granules. From
Kurloff's work (see p. 85) we must insist, on the contrary, that there
is no evidence that the non-granulated polynuclears are identical with
the granulated cells. Kurloff has shewn, at least for guinea-pig's
blood, that these two heterogeneous elements are to be sharply separated
one from the other, and that they have an entirely different origin.
Specially important for a theory of the nature of the granules is the
circumstance, that generally speaking in all species of animals =they are
present in those cells of the blood only which are adapted to and
capable of emigration=. That a certain nutritive function is to be
ascribed to the emigration of the granulated cells is a very obvious
supposition, scarcely to be denied; and naturally cells with a plentiful
store of reserve material are eminently suited for this purpose. The
lymphocytes on the contrary, incapable of emigration, are almost totally
devoid of specific granulations.
A further indication that =the granulations really are connected with a
specific cell activity lies in the fact, that one cell bears but one
specific granulation=. The contrary assertions that neutrophil and
eosinophil, or eosinophil and mast cell granulations occur in the same
cell Ehrlich regards as unfounded, from extensive researches specially
directed to this point. Nor has Ehrlich seen a pseudoeosinophil cell of
the rabbit change to a true eosinophil[23]. That such a transition does
not occur is most distinctly shewn by the fact that the various
granulations behave entirely differently towards solvents. With the aid
of acids, for example, the pseudoeosinophil granules can be completely
extracted from the cells, whilst the eosinophil granules remain whole
under this process, and can now be stained by themselves.
The clearest proof that the neutrophil, eosinophil, and mast cells are
entirely separated from one another by the fundamental diversity of
their protoplasm, of which the granulation is but a specially striking
expression, is afforded by the study of the various forms of
leucocytosis. As will be shewn in detail in the following chapter,
neutrophil and eosinophil leucocytes behave quite differently in their
susceptibility to chemiotactic stimulation. Substances strongly
positively or negatively chemiotactic for one cell group are as a rule
indifferent for the other; frequently indeed there is an exactly opposed
relationship, inasmuch as substances which attract the one kind repel
the other. Still greater is the difference between the mast cells and
the other two cell groups; for so far as present investigations go, they
are quite uninfluenced by substances chemiotactic for the neutrophil or
eosinophil cells.
As specific cellular secretions, various kinds of granules must also be
sharply marked off from each other by their chemical properties. The
granules of the blood corpuscles seem to be of very simple chemical
constitution. We have special grounds for the assumption that the
crystalline granulations are for the most part composed of a single
chemical compound, not necessarily highly complex even, but which seems
to be a relatively simple body such as guanin, fat, melanin, etc.
Doubtless other granulations have a more complicated constitution, and
very often are a mixture of various chemical substances. The most
complicated granules of the blood are the eosinophil, which are, as has
elsewhere already been mentioned, of a more complex histological
structure. For a peripheral layer is plainly distinguishable from the
central part of the granule. It should be mentioned that according to
Barker the eosinophil granulations appear to contain iron.
The key-stone of the hypothesis of the secretory nature of the granules
is the direct observation of a secretory process in the cells bearing
the granules. Naturally these researches offer extraordinary
difficulties since only the coincidence of a number of lucky
circumstances would allow the passage of dissolved granule substance
into the neighbourhood to be followed. Kanthack and Hardy have succeeded
in demonstrating the secretory nature of the eosinophil granules of the
frog. When, for example, anthrax bacilli are introduced into the dorsal
lymph sac of the frog they exert a positive chemiotaxis on the
eosinophil cells. The latter come in contact with the bacilli, and
remain for some time attached to them. During this period Kanthack and
Hardy observed a discharge of granules from these cells, which now
possess a protoplasm relatively homogeneous. Afterwards these cells move
away from the bacilli, and are succeeded by the polynuclear neutrophil
cells, as will be mentioned later. These authors were further able to
observe gradual accumulation of granules in eosinophil cells in lymph
kept under microscopic observation as a hanging drop, and thus
demonstrated that they undergo the two stages characteristic of
secretion, (1) appearance of granules within the cells, (2) discharge of
these granules externally.
The mast cells too seem suited for this purpose since their specific
substance is strongly characterised by its peculiar metachromatic
staining, and is further especially readily recognisable, since by its
great affinity for basic dyes it remains plainly stained, even in
preparations that are almost quite decolorised. In fact appearances of
the mast cells are not infrequently found, which must be referred to
excretory processes of this kind.
In the first place it is occasionally seen that the mast cell
granulation is dissolved within the cell, and diffuses in solution into
the nucleus. In place of the well-known picture of the mast cell (see
page 76) of a colourless nucleus, surrounded by a deeply stained
metachromatic granulation, a nucleus is present intensely and
homogeneously stained in the tint of the mast cell granulation,
surrounded by a protoplasm shewing but traces of granules.
Still more convincing is the presence of a peculiar halo of the mast
cells, described by various authors. Ehrlich first shortly mentioned
this halo in his book on the oxygen requirements of the organism. A few
years ago, Unna, whose notice Ehrlich's remark had no doubt escaped,
described an analogous condition as follows: "in some nodules the mast
cells appeared in part twice as large as usual, especially with the new
mast cell stain (polychrome methylene blue, glycerine ether mixture).
This was caused by the staining of a large round halo, in the centre of
which lay the peculiar long-known mast cell, consisting of blue nucleus,
and an areola of deep red granules. Higher magnification shewed that the
halo was not granular, but very finely reticular; although it exhibited
exactly the same red colour as the granules. It was consequently a
spongioplasm peculiar to these mast cells."
The appearance of the mast cells described by Unna may also be
artificially produced, by allowing a preparation that is stained with
the oxygen containing analogue of thionin, oxamine, to remain for some
time in laevulose syrup or watery glycerine. Evidently part of the dyed
mast cell substance is dissolved and retained in the immediate
neighbourhood. But as Unna possesses great experience of the mast cells
and is a complete master of the methods of their demonstration, one must
suppose that the halos described by him were preformed, and did not
arise during the preparation of the specimen.
It must hence be concluded that an analogous process may go on during
life, that these halos are the expression of a vital secretion of the
substance of the mast cells externally[24].
A condition that Prus has brought forward in the so-called purpura of
the horse, is also to be interpreted as a secretory process of the mast
cells. He describes young mast cells from the haemorrhagic foci of the
wall of the gut, on the margins of which bodies of various sizes
appeared, and which differ essentially from the mast cells themselves by
their staining. Nevertheless from their whole configuration and position
it is evident that these bodies have arisen in the mast cells
themselves; and Prus comes to the conclusion "=that the degenerating
young mast cells secrete a fluid or semi-fluid substance, which as a
rule sets on the surface of the cells, but also, more rarely, in their
interior=."
Evidence that the substance of the granules is given off externally may
sometimes be seen in the polynuclear neutrophil or their analogues. Thus
in rabbit's blood in which he had experimentally produced leucocytosis,
Hankin found a distinct progressive decrease of the pseudoeosinophil
granules on allowing the samples of blood to remain some time in the
thermostat. Further in suppurating foci in man, especially when
suppuration has lasted long, or the pus has remained for some time in
the place in question (Janowski) a rarefaction almost to complete
disappearance of the polynuclear neutrophil granules occurs, and is to
be explained by a giving up of the granulations to the exterior.
These facts and considerations, on the whole, lead then to the
conclusion, =that in general the granules of the wandering cells are
destined for excretion. This elimination of the granules is probably one
of the most important functions of the polynuclear leucocytes.=
FOOTNOTES:
[20] _Farbenanalytische Untersuchungen_ XII. _zur Geschichte der
Granula_, p. 134.
[21] _loc. cit._ pp. 5, 6.
[22] Altmann's freezing process would be similar to the advance always
insisted on by Ehrlich. It offers such great technical difficulties,
however, that it has up to now been little used.
[23] The cause of these misunderstandings is the tinctorially different
stages of development of the granules, as we have fully explained above.
How little adequate tinctorial differences by themselves are to settle
the chemical identity of a granulation, is at once evident on
consideration of the granules of other organs. No one surely would
assert, that a liver, muscle, or brain cell could occasionally secrete
trypsin, simply because the granules of the pancreas stain similarly and
analogously to those of the cells mentioned. We would here expressly
insist that we only assume a distinct character for each kind of
granulation, in the strict sense of the term for the cells of the blood,
since they possess a relatively simple function. In very complex
glandular cells, however, with various simultaneous functions, several
kinds of granules may be contained.
[24] From a paper of Calleja we learn that Ramon y Cajal recognised the
halos of the mast cells, and interpreted them in the manner we have
above. Calleja also describes these halos and the method of
demonstrating them in detail (thionin staining, and mounting the
sections in glycerine). We must mention, however, that we do not
consider this method suitable for the recognition of preformed halos,
for the reasons above mentioned.
IV. LEUCOCYTOSIS.
The problem of leucocytosis is one of the most keenly debated questions
of modern medicine. An exhaustive account of the various works devoted
to it, of the methods and results, could fill by itself a whole volume,
and would widely exceed the limits of an account of the histology of the
blood. We can only deal fully therefore with the purely haematological
side of the subject.
Virchow designated by the name "=Leucocytosis=," a transient increase in
the number of the leucocytes in the blood; and taught that it occurred
in many physiological and pathological conditions. In the period that
followed particular attention was paid to the leucocytosis in infectious
diseases, and to the investigators of the last 15 years in this province
we owe very important conclusions as to the ~biological meaning~ of this
symptom. Above all Metschnikoff has done pioneer service in this
direction by his theory of phagocytes, and though his theory has been
shaken in many essential points, yet it has exercised a stimulating and
fruitful influence on the whole field of investigation.
To sketch Metschnikoff's doctrine in a few strokes is only possible by a
paraphrase of the very pregnant words "Phagocytes, digestive cells."
These words express the view, that the leucocytes defend the organism
against bacteria by imprisoning them by the aid of their pseudopodia,
taking them up into their substance, and so depriving them of the power
of external influence. The issue of an infectious disease would chiefly
depend on whether the number of leucocytes in the blood is sufficient
for this purpose.