III. ON THE DEMONSTRATION OF THE CELL-GRANULES, AND THEIR SIGNIFICANCE.
During the last ten years a large amount of valuable work has been done on the cell-granules from histological, biological and clinical sides. This has particularly assisted hæmatology, where a number of problems remain whose solution is only possible by the aid of a knowledge of the granules. We must therefore consider the history, methods, and results of this work.
Ehrlich was undoubtedly the first to insist on the importance of the cell-granules, and to obtain practical results in this direction. We are obliged to mention this, since Altmann has, in spite of express corrections, repeatedly asserted the contrary. In 1891[20] Ehrlich refuted Altmann's claim to priority, nevertheless, Altmann in the 2nd edition of his Elementary Organisms (1894) stated that before him no one had recognised the specific importance of the granules, though some authors had viewed them as "rare and isolated phenomena."
We may quote a passage published by Ehrlich in 1878[21], that is, ten years before Altmann's papers. "Since the beginning of histology the word 'granular' has been used to describe the character of cellular forms. This term is not a very happy one, since many circumstances produce a granular appearance of the protoplasm. Modern work has shewn that many cells, formerly described as granular, owe this appearance to a reticular protoplasmic framework. And we have no more right to call cells granular in which proteid precipitates occur, either spontaneously as in coagulation, or from reagents (alcohol). The name should be kept exclusively for cells in which during life substances, chemically distinct from normal proteid, are embedded in a granular form. We can readily distinguish but few of these substances, such as fat and pigment; most of them we can at present characterise but imperfectly, or not at all."
"Earlier observations, especially on the mast cells, led me to expect that these granulations, though they had long been inaccessible to chemical analysis, could be distinguished by their behaviour with certain stains. I found, in fact, granules of this kind, characterised by their affinity for certain dyes, and which could thereby be easily followed through the animal series and in various organs. I further found that certain granules only occurred in particular cells, for which they were characteristic, as pigment is for pigment cells, and glycogen for cartilage cells (Neumann) and so forth. We can diagnose the variously shaped mast cells only by the staining of their granules in dahlia solution, that is by a microchemical test. And in the same way we can separate tinctorially other granulated cells, morphologically indistinguishable, into definite sub-groups. And for this reason, I propose to call these granulations specific."
"The investigations were performed after Koch's method in the following manner. The fluid (blood) or the parenchyma of the organs (bone-marrow, spleen, etc.) was spread on cover-slips in as thin a layer as possible, dried at room temperature, and after a convenient length of time stained. I had chosen this apparently coarse method for the special reason that for the histological recognition of new, possibly definite chemical combinations, corresponding to the granulations, all substances must be avoided that might act as solvents, e.g. water or alcohol, or as oxydising agents, such as osmic acid. In this instance only such procedures may be employed as will leave the simple drying of each single chemical substance as much as possible unchanged."
A more detailed study of the process of staining, and of the relation between chemical constitution and staining power, enabled a further advance to be made. And the first result in this previously unworked direction, was the sharp distinction between acid, basic, and neutral dyes, and between the corresponding, oxy-, baso-, and neutrophil granulations. The triacid solution was only found after trial of many hundred combinations; and up to the present day this stain in its original form or in slight modifications has played a prominent part in various provinces of histology.
The classification of the cell-granules of the blood according to their various chemical affinities which was drawn up by this method is accepted to-day as the most valuable, and the only practical means of grouping the leucocytes. From the first Ehrlich has insisted, that different kinds of cells possess different granules, distinguished not only by their tinctorial properties, but also by their various reactions to solvents.
It is in this connection indeed, that Altmann's method, consisting of a complicated hardening process, and the use of a single, always similar stain, constitutes a retrograde step, in as much as it tends to obscure the principle of the specificity of each kind of granulation.
A further disadvantage of Altmann's hardening method lies in the circumstance, that the cell proteids are precipitated by it in a spherical form, and stain in the subsequent treatment. Hence it is extremely difficult to distinguish what is preformed, and what is artefact. Since A. Fischer's publication, where the formation of granule-like precipitates under the influence of various reagents is experimentally demonstrated, grave doubts as to the reality of Altmann's forms have been raised from various quarters. Ehrlich's dry process, on the contrary, is entirely free from error. Granules cannot be artificially produced, by desiccation, and the stained appearances correspond precisely to what is seen in fresh living blood. The greatest value of the dry method is that the chemical nature of the single granules remains unchanged, so that attempts at differentiation are made on a nearly unaltered object[22].
Another means of studying the nature of the granules depends on the principle of vital staining. The "vital methylene blue staining" (Ehrlich) that has since become so important, especially in neurology, led to the first attempts at staining the granules in living animals. One of the first publications on this subject is that of O. Schultze, who placed the larvæ of frogs in dilute methylene blue solution, and after a short period found the granules of the stomach, the red blood corpuscles and other cells stained blue. This method, however, cannot pass as entirely free from error, as Ehrlich frequently found that when the experiment lasts some time the methylene blue often forms granular precipitates that may be confused with the granules. Teichmann directs a detailed analysis to this point, and regards most of the granules described by Schultze as artificial products.
Neutral red is highly suitable for the study of vital granule-staining, a dye recommended by Ehrlich, and employed successfully since that time by Przesmycki, Prowazek, S. Mayer, Solger, Friedmann, Pappenheim and others. This dye was prepared by O. N. Witt from nitrosodimethylamin and metatoluylendiamin, and is the hydrochloric acid salt of a base which is soluble in pure water, yielding a fuchsin red colour, but which in weak alkaline solution—the alkalinity of mineral water suffices—is a yellow-orange hue.
Now neutral red is characterised by a really maximal affinity for the 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 larvæ 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 larvæ 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° 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 anæmia (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, Polychætes, 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 Müller, these results of Hirschfeld's must be described as inaccurate. After many vain endeavours, Dr Müller 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 lævulose 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 hæmorrhagic 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.