ROLE OF ENDOTHELIUM IN THE PRODUCTION OF POLYBLASTS (MONONUCLEAR WANDERING CELLS) IN INFLAMMATION(*).
The dominant idea at the present time ascribes to the nongranulated leukocytes a modest role in this respect. A certain amount of emigration of lymphocytes and monocytes is admitted, but the emigrated cells are believed to be incapable of progressive development.
According to the majority of investigators, the chief source of the exudate cells, "the macrophages," as they were called many years ago by Metchnikoff,(5) are the local fixed cells of the connective tissue. However, this role is not attributed to the common fibroblasts. It is well known at the present time that there are different types of fixed cells in the connective tissue. It is believed that the sources of the mononuclear exudate cells are, on the one hand, the "resting wandering cells" of Maximow(6) [the "clasmatocytes" of Ranvier, the "histiocytes" of Aschoff and Kiyono(7) and Kiyono(8)]; on the other hand, the endothelium of the common blood vessels, to which an important r61e is attributed. Recently, Foot tried to give decisive evidence for the active participation of the endothelium in inflammation. He used the method devised by McJunkin(9)--the intravenous injection of a "colloidal" carbon suspension (Higgins India ink). The endothelium of the blood vessels phagocytizes carbon particles, whereas other cells are supposed to remain free of carbon. If, after the injection of India ink, inflammation is experimentally provoked in the same animal, the transformations of the endothelium, "labeled" with carbon, can easily be followed. The agent used to call forth the macrophages in the first series of the experiments of Foot was sterile melted agar injected subcutaneously. Under the influence of this stimulus the endothelial cells of the adjacent blood vessels, containing carbon particles in their protoplasm, were seen to separate from each other, to become free and to transform themselves into ameboid cells, typical mononuclear exudate cells or macrophages. In the following series of experiments, Foot used various other types of inflammatory stimuli; but the results regarding the active participation of the blood vessel endothelium in the formation of macrophages were essentially the same.
In Germany the school of Marchand (G. Herzog (10)) also emphasizes the active role played by the endothelium and the "cells of the blood vessel walls" in the production of mononuclear phagocytic exudate cells. Under this rather indefinite term, "cells of the blood vessel walls" (Gefasswandzellen), evidently different cell types can be understood: the endothelium proper, certain cells of embryonic character, adjacent to the outer surface of the endothelium, the resting wandering cells or histiocytes, etc. According to Herzog and Marchand,(11) the so-called adventitial clasmatocytes (histiocytes) are the most important elements. However, they are supposed also to originate from the endothelium.
A different opinion was expressed by Maximow.(2) During the period from 1902 to 1909, he published a series of papers on the histogenesis of inflammatory lesions. He distinguished three cell types in the field of inflammation: (1) Special granulocytes (polymorphonuclear granular leukocytes). They emigrate out of the blood vessels in the early stages of inflammation. They do not give origin to any permanent elements and sooner or later degenerate and disappear. (2) Fibroblasts--the local common connective tissue cells. They respond to the inflammatory stimulation by mitotic proliferation and form the basis of the granulation tissue. In the later stages they elaborate the collagenous intercellular substance of the scar. (3) Polyblasts--the ameboid phagocytic mononuclear exudate cells. Maximow gave this name to the macrophages of Metchnikoff, because they display during the course of the inflammatory process a surprising amount of developmental potencies and appear in various forms, such as large ameboid mononuclear phagocytes (macrophages proper), as epithelioid cells, as multinuclear giant cells, as pus phagocytes, etc. The polyblasts, according to Maximow, have a double origin. A part arises through the mobilization of the local resting wandering cells (clasmatocytes, histiocytes) of the connective tissue; another part comes from the blood and represents hematogenous, emigrated lymphocytes and monocytes. After their emigration out of the blood vessels into the tissue, they rapidly hypertrophy and join the polyblasts of local origin in their further transformations. In the later stages they remain scattered in the scar tissue as peculiar fixed cells, the resting polyblasts, between the fibroblasts.
No evidence could be found in the experiments of Maximow of a participation of the endothelium of the blood vessels in the formation of polyblasts. The endothelium in inflammation forms new capillary sprouts; it may also proliferate and give off cells into the tissue which at once assume the characters of fibroblasts; but its elements never become transformed into ameboid cells.
Tschaschin,(12) a student of Maximow, confirmed these results in a series of experiments with inflammation in vitally stained animals. He also could find no indication of a transformation of the endothelium of blood vessels into ameboid elements.
Hence, in the presence of two clearly contradictory opinions, new experiments for the elucidation of the histogenesis of the mononuclear exudate cells became necessary. The technic of McJunkin and Foot seemed to offer the best means for following the transformations of the endothelium of the blood vessels throughout the whole inflammatory process.
The present investigation has been undertaken and conducted at the suggestion and under the direction of Professor Alexander A. Maximow in the anatomical laboratory of the University of Chicago.
MATERIAL AND METHODS
Adult rabbits were chosen as experimental animals. Five cubic centimeters of Higgins India ink, diluted with an equal amount of distilled water, was injected intravenously according to the method of McJunkin and Foot. From thirty to forty-five minutes after the first injection, foreign bodies were introduced into the subcutaneous tissue of the abdominal wall (experimental series a). Small (from 4 to 5 mm.) particles of sponge filled with agar and lecithin(13) were used as foreign bodies. They were prepared in the following way: Small dried bits of a thoroughly washed sponge were autoclaved together with a mixture of bacteriologic agar and lecithin in the proportion of 5 to 1. The melted agar-lecithin mixture could thus penetrate and fill the meshes of the sponge, and at the same time the whole was sterilized. After cooling, the portions of sponge were pulled out of the jelly with a sterile forceps and were introduced into the subcutaneous tissue through a small incision; the latter was closed with one or two silk sutures.
Foreign bodies saturated with lecithin-agar were used because Bergel(14) claims to have found that lipoid substances and especially lecithin have an elective positive chemotactic influence on lymphocytes.
At different time intervals after the introduction of the foreign body the animals were killed. If the interval was longer than twenty-four hours, a second intravenous injection of the same quantity of diluted India ink was given the next day. The stages obtained were three; six, twenty-four, thirty, forty-eight and seventy-one hours.
In a second series of experiments (experimental series b), on two successive days the animals received an intravenous injection of India ink. Immediately after the second injection 1 to 2 cc. of fresh egg yolk was injected into the subcutaneous tissue by means of a syringe with a needle of large caliber. Simultaneously 0.5 to 1 cc. of a 1: 300 aqueous solution of silver nitrate was injected into the peritoneal cavity. These animals were killed ten, fifteen, eighteen and thirty hours after the injection of the egg yolk.
As we were interested in the origin of the polyblasts (mononuclear exudate cells) in acute aseptic inflammation, only early stages of the process were to be cared for.
From all animals the usual dry blood smears were prepared at various stages of the experiment. They were stained with May-Grunwald and Giemsa stain according to the panoptic method of Pappenheim.
After the animal was killed, the skin covering the foreign bodies or the place where egg yolk was injected was carefully separated and the denuded layer of loose subcutaneous tissue, together with the underlying muscle sheet. was stretched in natural position on a cork frame by means of cactus needles and fixed in warm Zenker-formol solution. Besides, from every animal pieces of liver, spleen and lymph nodes were taken. The fixed material was embedded in celloidin; the subcutaneous tissue was cut in serial sections parallel to its broad surface. The sections were stained with eosin-azure according to the method devised by Maximow;(15) recently the method has been slightly modified and improved by staining the sections previous to the eosin-azure mixture with a very weak solution of Delafield's hematoxylin for twenty-four hours. The chromatin of the nuclei acquires after this hematoxylin-eosin-azure stain a particularly deep blue color, and the slides do not fade.
Pieces of the omentum of the animals in the second series, after fixation in a stretched position, were stained in the same way as the sections, and mounted in toto.
FATE OF THE INDIA INK INJECTED INTRAVENOUSLY
If a drop of Higgins India ink--diluted with distilled water or undiluted--is placed on a slide, covered with a coverslip and focused with a high power immersion lens, innumerable dark gray particles, just within the limits of microscopic visibility, are seen going through the typical Brownian movement against a light gray background. If a drop of the same ink is mixed with a drop of fresh blood, the carbon precipitates at once in the form of rather coarse black particles, assembling in small irregular clusters. I did not find this phenomenon mentioned in the papers of the other authors who used intravenous injections of India ink. This point is of outstanding importance, however, because it shows that after India ink enters the circulation, it ceases to be a "colloidal" carbon suspension, and turns into a common suspension of rather coarse carbon particles.
When the blood was examined under the microscope a few minutes after the intravenous injection of India ink, distinct black particles of carbon were seen floating among the blood corpuscles. They were of a size fairly easily visible even under medium power lenses and did not by far approach the limit of visibility. In many cases they, were seen agglutinated and forming smaller or larger black clusters. This could easily be confirmed on sections, on which in the lumen of the blood vessels a similar picture was seen.
The fate of India ink injected intravenously has been followed by many investigators. Of the more recent workers, Wislocki(16) may be mentioned; in his paper an extensive bibliography on this subject can be found. According to Wislocki, India ink injected intravenously is deposited in the lumen of the blood sinuses or capillaries of the liver, the spleen, the bone marrow and the lung; one hour after the injection, phagocytosis of the coal particles is manifest; the active role in this process in the liver, the spleen and the bone marrow is played by the so-called "reticulo-endothelial" elements. In the lung, to which Wislocki paid special attention, one hour after the injection he found numerous interalveolar capillaries with tiny plugs of carbon in the lumen. Clasmatocyte-like cells, located in the partitions between the alveoli--their origin was not followed--begin to phagocytize the carbon. In this way a part of the originally solid mass of carbon is being converted into a mass of mononuclear cells loaded with granules of carbon. On the surface of the endothelial cells carbon granules also can be seen: later they may be found even in their cytoplasm. However, Wislocki emphasizes that these endothelial cells never show any sign of active phagocytosis. In the later stages, the India ink is carried by the phagocytes into the lymph channels and finally into the lymph nodes.
The relations of India ink, injected intravenously, to the cells of the vascular walls and especially to the endothelium are certainly by themselves of great interest. However, we repeated the experiments of Foot with the special purpose of solving the question of the origin of the mononuclear exudate cells, the polyblasts, in inflammation.
Regarding the first problem, the behavior of the endothelium toward the particles of carbon circulating in the blood, we obtained practically the same results, as McJunkin(9) and Foot(4) and, later, Wislocki.(16) The "endothelium," or better, the histiocytes, lining the sinusoids of the liver, the spleen and the bone marrow, accumulates large quantities of carbon particles immediately following the injection. In the vessels of the loose subcutaneous connective tissue of the abdominal wall--as well as probably in most of the other vascular regions of the body, except perhaps the central nervous system and the leptomeninges, in which the amount of carbon was always minimal--carbon particles were also easily found (Figs. 3 and 4). Their quantity was incomparably smaller than in the vessels of the spleen, the liver, etc.--the organs which Wislocki found to be the chief places of carbon deposits. Besides, the distribution of the carbon was uneven, and vessels with large quantities alternated with vessels which contained very little carbon or none. Nevertheless, in the earliest stages observed, capillaries with India ink can be found in many microscopic fields of a section. This is also the case in the immediate proximity of the foreign bodies; thus, the inflammatory reaction of carbon containing cells could easily be followed.
[Figures 3-4 ILLUSTRATION OMITTED]
In some capillaries the clusters of precipitated carbon particles form emboli, sometimes apparently plugging and completely obstructing the lumen (Fig. 5 Emb). In the majority of the capillaries and capillary veins the endothelium in the earliest stages shows various quantities of unevenly sized carbon particles, single or in clusters, sticking to the free surface of the protoplasm. In slightly later stages, after six hours and more, the same black particles, some of them of large size, are seen partly embedded in the interior of the endothelial protoplasm (Figs. 3 and 4). If large particles are embedded in the endothelial protoplasm. the corresponding part of the cell may bulge distinctly into the lumen of the vessel (Fig. 3 Ed'). Thus, not only the histiocytes of the special organs mentioned above, but the endothelial cells of the common blood vessels also prove able to engulf fine particulate matter. The same has been observed recently in the vessels of the tongue of the living frog by F. Herzog(17) and Stilwell.(18) It is certain that this phenomenon is not flue to active phagocytosis, and that it is not connected with ameboid movement and formation of pseudopodia. It is merely the result of the physical properties of the free surface of the endothelial protoplasm and is due to peculiar conditions of the surface tension of the latter.
[Figure 5 ILLUSTRATION OMITTED]
The carbon particles circulating in the blood are taken up not only by the fixed histiocytes lining the sinusoids of the liver, spleen and bone marrow and by the common endothelium, lining the other vessels, but beginning with the earliest stages of the experiment, after from three to six hours, white blood corpuscles containing carbon particles are found everywhere in the circulation. This refers, first, to the polymorphonuclear special granular (pseudo-eosinophilic in the rabbit) leukocytes. On dry blood smears (Fig. 2 Lkc) and in the lumen of the blood vessels in sections (Fig. 1 Lkc) many of them are seen containing a few small carbon particles. Second, the majority of the monocytes (Figs. 2 and 3 Mon) also show carbon particles in their protoplasm. The particles here are often somewhat larger and more numerous than in the special granular leukocytes. Simpson(19) saw the number of monocytes increase in the blood of animals subjected to repeated intravenous injections of various kinds of colloidal substances and particulate matter. Although our experiments were of an acute character and although we did not make leukocyte counts in the blood, we also received the impression that in our animals the quantity of monocytes was larger than usual. However, it is known that of all the leukocytes the monocytes show a particularly uneven distribution in the vascular bed.
[Figures 1-2 ILLUSTRATION OMITTED]
Finally, large free phagocytic cells were occasionally found in the lumen of blood vessels in sections (Fig. 3 Hist'). In blood smears they could not be found, probably because of their insignificant quantity. Their pale, vacuolated protoplasm contained large quantities of finer and coarser carbon granules, of which bulky, angular lumps occasionally obscured the nucleus. The latter seemed always to be very lightly stained and showed a wrinkled membrane.
These were the free histiocytes of Kiyono,(8) the "macrophages" of Simpson,(19) the hemohistiocytes of Ferrata.(20) In the blood of our animals the differences between these free histiocytes and the monocytes were usually distinct, and no transitional forms between them were seen, at least in the stages we observed.
It may be added that in many of our animals the blood after the injections of India ink contained a small quantity of special metamyelocytes. This may be looked on as the symptom of a slight irritation of the bone marrow.
CHANGES OF CONNECTIVE TISSUE SURROUNDING FOREIGN BODY
Earliest Stages (Three to Six Hours).--The tissue shows a slight edema, its texture is distinctly loosened and its elements are pushed apart. As a result of direct traumatic injury, a varying quantity of fixed cells in the immediate proximity of the foreign body--fibroblasts and histiocytes (resting wandering cells)--are seen undergoing necrosis; they contain disfigured, shrunken and darkly staining nuclei, whereas the protoplasm shows vacuolization and disintegration.
The vessels appear enlarged and tortuous, especially the venous capillaries. Their endothelium as yet does not show any distinct changes; its surface in many places is sprinkled with granules of India ink, as described above; in some cells the granules have already entered the protoplasm. The lumen shows an accumulation of leukocytes, special polymorphonuclear, as well as monocytes and lymphocytes. All, with the exception of the smallest lymphocytes, may contain particles of India ink.
The fibroblasts in the surrounding tissue as yet do not show any distinct changes: They are merely pushed apart from each other by the edematous liquid. The histiocytes (resting wandering cells) show the first signs of inflammatory reaction--they begin to contract and their protoplasm contains a varying quantity of clear vacuoles, which are especially numerous when egg yolk is injected into the tissue.
Special (pseudo-eosinophilic) granular leukocytes with polymorphous nuclei are present in the tissue. Their quantity rapidly increases; a part of them contain a few small carbon particles. They creep about in the tissue and are seen accumulating especially in the vicinity of the sponge; when yolk is injected, their distribution in the tissue is more diffuse. A varying quantity of these cells undergoes degeneration.
The origin of these leukocytes is obvious--they emigrate out of the blood vessels. However, typical pictures of their penetration through the endothelial vascular wall are not common in the fixed preparation. In the earliest stages now under consideration lymphoid cells are scarce in the tissue; pictures of their emigration out of the vessels are still more difficult to find than for the polymorphonuclear special leukocytes.
No signs of proliferation, mitotic or amitotic, can be found in any of the cells.
Medium Stages (Ten to Eighteen Hours).--Edema: The edema is extensive in these stages. The collagenous fibers appear partly torn and swollen, and are pushed widely apart by a liquid accumulated in the spaces between them; in some places scarce, delicate fibrin precipitates are seen. The degenerating cells, mentioned in the earlier stages, seem to be more numerous.
Blood Vessels: The blood vessels are seen in large quantities on the surface of the muscular aponeuroses. They all appear enlarged and have a tortuous course, especially the capillary veins. The endothelial cells are swollen; their oval, vesicular, clear nuclei bulge in many places into the lumen; their protoplasm acquires a certain degree of basophilia. They show some rare mitotic figures; signs of amitotic division could never be found. The general distribution of particles of India ink on the whole remains the same as described previously; in some places the swollen endothelium shows a varying, sometimes large, amount of black particles (Fig. 4 Ed); in other places, the wall of the vessel is free from India ink. Only a few of the carbon particles seem to keep their original position on the inner surface of the endothelial cells; the intracellular position of the majority is certain. In exceptional cases an endothelial cell may contain, besides small particles, a large lump of carbon; the protoplasm is distinctly seen to cover this foreign body on the inner surface of the cell and to bulge into the lumen of the vessel (Fig. 3 Ed'). Here and there a capillary may be found with a lumen Completely obstructed by a large carbon embolus (Fig. 5). In such cases the endothelial cells adjacent to the carbon mass always contain small black particles apparently detached from the embolus and taken up by the protoplasm.
Special attention was paid to the presumed loosening of the connection between the endothelial cells and their isolation and transformation into ameboid elements. The nature of the preparations was such that a phenomenon of this type could not possibly remain unnoticed. However, we failed to find anything suggesting such a possibility in the places in which the endothelium contained carbon as well as in the vessels completely free from India ink.
In the lumen of the dilated blood vessels--especially the capillary veins--a large accumulation of granulated and nongranulated white blood corpuscles is seen. In many places both types of leukocytes display a typical marginal position, and both, except the small lymphocytes, may contain a few small carbon particles in their protoplasm. In the monocytes this is especially often the case. The presence of carbon containing monocytes can be easily shown on dry smears of blood taken from the ear veins of the respective animals (Fig. 2 Mon).
Both lymphocytes and monocytes retained in the enlarged vessels show distinct signs of hypertrophy; this causes a gradual effacement of their differences. Transitional forms between lymphocytes and monocytes, ordinarily missing or rare in the circulating blood, seem to become common in the stagnant blood of the vessels described. The nucleus of the lymphocyte becomes larger, stains less dark and acquires a one-sided indentation and an excentric position. The protoplasm gradually accumulates on the indented side of the nucleus and may show ingestion of small particles of India ink.
In some cases exceptionally large free ameboid cells with a pale nucleus, a wrinkled membrane and a large amount of fine and coarse carbon particles, completely filling the protoplasm, can be found in the lumen of the enlarged vessels (Fig. 3 Hist'). The cells are the free histiocytes of Aschoff(7) and Kiyono,(8) which, as mentioned in the foregoing, can be found in the general circulation on careful examination, and are known to originate in the sinusoids of the bone marrow, the liver, the spleen, etc.
As in the general circulation, in the enlarged vessels of the inflamed area, no distinct transition forms between them and the true monocytes can be found.
All the described white blood cells and the free histiocytes often can be found closely adjacent to the inner surface of the endothelium in the dilated blood vessels (Fig. 3). No difficulty was encountered in distinguishing them from the carbon containing or empty endothelial cells.
PENETRATION OF PARTICLES OF INDIA INK THROUGH WALLS OF VESSELS
During the stages now under consideration, an important phenomenon can be noticed. The carbon particles contained, as has been described, in the protoplasm of the endothelial cells of the capillaries, are seen passing through the unchanged, intact endothelium into the tissue surrounding the vessels. Some of them, leaving the protoplasm of the endothelium on its outer surface, seem to lie freely between the cells and the thin collagenous fibers (Fig. 8 II). Most of them, however, are transferred from the protoplasm of the endothelium directly into the protoplasm of cells intimately adjacent to the outer surface of the capillaries (Figs. 4 to 6 Per).
[Figures 6,8 ILLUSTRATION OMITTED]
These elements contain an elongated or oval nucleus, almost similar in its inner structure to the endothelial nuclei; their protoplasm is pale and not distinctly defined; in the majority of the cases it is stretched parallel to the axis of the capillary, thus displaying a spindle-shaped form on longitudinal sections of the latter. In the subcutaneous tissue--with the technic used in this investigation--these cells rarely show distinct transverse processes encircling the capillary.
It is obvious that these elements are the adventitial cells of the capillaries or the pericytes of Zimmermann(21)--a cell type which recently has received much attention and which by some investigators is made responsible for the contractility of the capillaries. The recent experiments of Stilwell,(18) who repeated the work of F. Herzog(17) on the blood vessels of the living tongue of the frog after intravenous injection of India ink, did not give convincing evidence of the contractility of these elements. They have shown instead that the pericytes actively collect particles of India ink, which, after having been engulfed by the endothelial protoplasm, leave the latter on its outer surface.
The same is true for the pericytes of the capillaries in the inflamed subcutaneous connective tissue of the rabbit. It is possible, of course, that the same phenomenon--the transportation of particulate matter from the endothelium into the pericytes--might also be observed, perhaps on a smaller scale, in the absence of inflammation. Our special aim was, however, to elucidate the changes of the endothelium under the influence of the inflammatory stimulus, and the migration of the particles of India ink under the conditions of our experiments manifested itself with a peculiar clearness.
Whether pericytes and the endothelial cells are connected with each other by means of protoplasmic processes and the carbon particles flow through the protoplasm of these connections front one cell to another, or whether they are simply eliminated from the endothelial cell on its outer surface and then immediately enter the protoplasm of the pericytes, cannot be decided. The occasional presence of seemingly free carbon particles outside the endothelium suggests the second explanation. On the other hand, in many cases an apparently uninterrupted row of black particles seems to connect the endothelial cell with a pericyte.
The thin protoplasmic layer of a pericyte adjacent to the endothelium may contain single, tiny carbon particles, arranged longitudinally one behind the other. Often the endothelium also contains similarly arranged particles, and in such a case the wall of the vessel is lined by two parallel rows of black granules. Sometimes the black granules in the protoplasm of the pericyte are numerous and may form an angular irregular cluster, partly or completely concealing the nucleus.
We were trying to find evidence of the possible migration or recession of the carbon containing endothelial cells into the surrounding tissue, but in this we were unsuccessful. The mitoses of the endothelium are rare and cannot explain the appearance of numerous carbon containing cells paralleling the vascular wall. The endothelial tube everywhere remains intact and continuous.
EMIGRATION OF LEUKOCYTES
The classical pictures of emigration of leukocytes with distinctly constricted nuclei from the blood vessels into the tissue are present but are by no means numerous (Fig. 1 x). This fact, which seems to be in striking contradiction to the large quantity of leukocytes found in the tissue, has been discussed by Maximow in several of his papers. He has also pointed out that this scarcity of the emigration pictures of nongranular leukocytes, which has been looked on as one of the strong proofs against the hematogenous origin of the polyblasts, pertains equally to the special granulated leukocytes whose hematogenous origin cannot be doubted. The explanation can be sought, on the one hand, in the rapidity of the process, and, on the other hand, in the peculiar position of the emigrating cells when they pass through the endothelial wall. In most cases the leukocytes traverse the wall of the capillary in a decidedly oblique or even parallel direction, sometimes causing a distinct splitting of the endothelial protoplasm into two layers and undergoing for a while a considerable compression (Fig. 3 x). Such pictures of emigration can easily be overlooked. This pertains to the special granular (pseudoeosinophilic) leukocytes with the polymorphous nucleus and to the nongranulated leukocytes--the lymphocytes and monocytes as well. Many of the emigrating special granular leukocytes and monocytes contain small granules of India ink in their protoplasm (Fig. 3 x).
The hypertrophy of the emigrating lymphocytes and monocytes, which began, as we have seen, while the cells were still in the lumen, continues after their migration into the tissue; in this way the emigrated nongranulated blood leukocytes transform themselves into polyblasts (Figs. 1 and 4 Plb').
The fibroblasts in the edematous tissue are scattered at considerable distances from each other (Figs. 1 and 4 Fbl). They keep their general structure, and the outlines of their long, wing-shaped or spear-shaped processes are particularly well distinguishable in the tissue spaces filled with clear liquid. In many places, especially in the proximity of the foreign body, their protoplasm appears swollen and hypertrophied, and assumes a distinct basophilic staining property; it also often contains a variable number of vacuoles. The nucleus remains unchanged in its typical inner structure and always enables one to identify the cell. Rounding off, formation of ameboid pseudopodia and transformation into wandering cells could never be detected. Mitoses begin to appear in many fibroblasts. Carbon particles seem to enter their protoplasm only rarely and in small quantities.
HISTIOCYTES OR RESTING WANDERING CELLS (CLASMATOCYTES)
The histiocytes of the loose subcutaneous tissue near the foreign body are mobilized (Fig. 5 Hist). For the most part, they keel) their former position, but appear enlarged. They are rounding off and display ameboid pseudopodia of varying form and size. Their protoplasm has a distinct reticular structure and contains--especially in the experiments with the introduction of egg yolk into the subcutaneous tissue--numerous clear vacuoles and granular inclusions. Their phagocytic activity is clearly manifested by the presence of normal or degenerating special granular leukocytes in their protoplasm. Occasional small particles of India ink can be found in some of the histiocytes which are closely adjacent to the capillaries. The nucleus of these cells keeps its characteristic features and can be easily distinguished from the nucleus of the fibroblasts by its smaller size, its irregular outlines and the darker stain. In somewhat later stages one can follow the gradual transformation of these mobilized histiocytes into large ameboid phagocytic cells, the histogenous polyblasts (Fig. 4 Plb).
SPECIAL GRANULAR POLYMORPHONUCLEAR LEUKOCYTES
This type of cell is found in large quantities in the tissue surrounding the foreign body. These cells accumulate markedly in the proximity of the sponge and penetrate into its cavities, resorbing the agar. Huge masses of them are scattered also between the adjacent striated muscle fibers. Many of them contain granules of India ink, and many show various stages of degeneration and disintegration.
It is needless to discuss the origin of these elements--they have all emigrated from the blood vessels, although, as we have stressed in the foregoing, the pictures of their emigration are not much more common than for the nongranulated leukocytes.
Lymphoid, nongranulated wandering cells (with round compact, nonpolymorphous nucleus), polyblasts or mononuclear exudate cells, are now also numerous in the inflamed tissue, and their number continues to increase (Figs. 1 and 4 Plb, Plb'). The stages now under consideration are especially important for the decisive solution of the problem of their origin and histogenesis. It is distinctly characteristic of the early stages of inflammation that the polyblasts, infiltrating the tissue, are represented by cells of different size and structure, so that two extreme types connected by an uninterrupted series of transitional forms are easily demonstrable (Fig. 4 Plb').
Many of the round ameboid cells are morphologically identical with common small and medium sized lymphocytes, as described in the foregoing for the blood, filling the enlarged capillaries and capillary veins. The only difference detected is the distinct ameboism of the cells in the tissue, while most of the intravascular specimens are spherical.
The other extreme is represented by cells of the size of blood monocytes or slightly larger. However, even the largest among them does not as yet reach the size of the local histiocytes. They have an abundant, slightly basophilic protoplasm, accumulated on one side of the excentrically located, usually slightly folded and indented, nucleus; the quantity of chromatin is smaller than in the nucleus of the lymphocytes, and the nucleus therefore stains lighter than in the latter. Sometimes a distinct cytocentrum is seen at the indented surface of the nucleus. The protoplasm, whose ameboid character is more manifest than in the small cells. frequently contains a few small carbon granules and clear vacuoles. The largest cells may also contain debris of degenerated special leukocytes.
Between the two extremes described every possible transition can easily be found at any place in the tissue.
Mitoses in the polyblasts are extremely rare. The distribution of the polyblasts in the edematous tissue is irregular. They are scattered in small groups or singly everywhere around the enlarged blood vessels in the vicinity of the sponge. Some of them are seen, together with the leukocytes, penetrating into the cavities of the foreign body.
According to Foot,(4) in the stages under consideration we should expect to find the transformation of the endothelial cells of the blood capillaries into polyblasts in full swing. However, our preparations failed to furnish us any facts supporting this assumption.
The endothelial cells, as I have already mentioned, show a distinct swelling (Fig. 4 Ed), and during these stages just begin to enter the period of active mitotic proliferation. But no trace of loosening of the endothelial membrane into single cells and of their isolation and rounding off as free ameboid elements and of their movement into the tissue could be found. There was, moreover, no bulging of these cells into the lumen of the vessel which could be supposed to result in the production of free intravascular cells in the form of the so-called "endothelial leukocytes."
As we have pointed out in the foregoing, the hypertrophying lymphoid cells in the enlarged capillaries and capillary veins, i. e., the lymphocytes and monocytes, on one hand, and the polyblasts, the "mononuclear exudate cells" outside the vessels, in the tissue, on the other hand, are morphologically similar in every respect (Figs. 1 and 4). The only difference is the further advancement of a part of the extravascular cells in their progressive development and enlargement. Cells from either group may contain a variable number of carbon particles. Pictures of emigration of similar cells can be demonstrated, as also has been stated. The only possible conclusion, therefore, seems to be that the ameboid mononuclear exudate cells rapidly accumulating in these early stages in the tissue are emigrated nongranular blood leukocytes, lymphocytes and monocytes.
It is important, from the general hematologic point of view, that (luring this process of emigration and transformation no sharp line of distinction can be drawn between the lymphocytes and monocytes. Both cell types, intravascularly as well as extravascularly, are connected by a continuous series of gradual transitions. Both take indiscriminately an active part in the production of hematogenous polyblasts.
Later Stages (Twenty-Four to Forty-Eight to Seventy-One Hours).--Of the phenomena shown by the tissue surrounding the foreign body in these stages, only the facts concerning the polyblasts need a more detailed discussion.
The edema still persists partly, but the cellular elements of the tissue, due to their continued increase in number, appear arranged much closer to each other. The tissue is overflowed with special granular leukocytes; they accumulate in increasing quantities in the sponge and there undergo degeneration. The fibroblasts all show distinct hypertrophy, an increase of the basophilia of their protoplasm, and are found everywhere in active mitotic proliferation. As in the earlier stages, they do not become transformed into ameboid exudate cells.
The vessels are still enlarged; their endothelium is considerably swollen and occasionally contains mitotic figures. Here again the most attentive search fails to show any signs of their supposed transformation into free ameboid cells either in the lumen or on the outer surface of the wall of the vessel.
Many of the endothelial cells still contain a varying quantity of carbon particles. But in the cells, surrounding the capillaries and adjacent to their endothelium--the pericytes, described in the foregoing, and the histiocytes (resting wandering cells), with occasional transitions between the two--the carbon particles are found in larger numbers than in the preceding stage.
These perivascular cells are seen to recede from the immediate vicinity of the capillaries farther into the tissue carrying the carbon particles with them (Fig. 4 Per). It is possible that the carbon may also be transmitted from cell to cell without an actual changing of the position of the cells themselves.
Sometimes small vessels, capillary veins, are found, whose endothelium, as the result of mitotic proliferation, becomes double layered. It is possible to assume that in such cases the outer layer of endothelial cells recedes into the tissue, acquiring the properties of fibroblasts, as has been shown by Maximow(2) in his early papers on inflammation. There are no indications of a transformation of such receding endothelial cells into ameboid elements. They are to be differentiated, from the pericytes. This process may well be compared with the active participation of endothelium in the production of fibroblasts during the organization of an intravascular thrombus.
The emigration of the special leukocytes continues for a while, and our experiments gave us the impression that the presence of lecithin in the foreign body stimulates this process and increases the number of the emigrating cells. The carbon particles in the newly migrated cells are becoming more and more scarce.
The quantity of the ameboid mononuclear exudate cells in the tissue during the present stages reaches its climax. In many places they nearly crowd out the other cell types. Emigration of lymphocytes and monocytes is still going on. An important change has taken place.
Whereas in the preceding stages there was a distinct gap between the local, large, mobilizing histiocytes (resting wandering cells) and the smaller, round (lymphocyte-like and monocyte-like) polyblasts of hematogenous origin, now the local histiocytes seem to have disappeared almost completely and the tissue contains, instead, great quantities of large ameboid, phagocytic cells, among which no distinction can be made as to their local or hematogenous origin. There can be only one explanation of this fact. The local histiocytes have all been mobilized and are all transformed into large polyblasts or macrophages; but the latter are so numerous and show such convincing transitional forms to the smaller lymphoid exudate cells that there cannot be any doubt as to the origin of a considerable part of the largest polyblasts from the hematogenous nongranulated leukocytes, which in the former stage were small and could be differentiated sharply, from the awakening histiocytes which in the meantime have developed progressively.
The large polyblasts or macrophages need not be described in detail. Their excentrically located, usually kidney-shaped nucleus, which is always darker than in the fibroblasts, is a secure criterion for their identification. Mitoses can be found occasionally, but are rare. The protoplasm is sometimes vacuolated. Engulfed and partly digested special granulocytes are common. If their granules scatter in the protoplasm of the phagocytic polyblast, such cells can easily be mistaken for myelocytes. Some of the polyblasts contain carbon granules (Fig. 4 Plb). In the experiments with the agar sponges, the method of Ciaccio(22) in the majority of the polyblasts reveals the presence of "lecithin" granules. In the experiments with egg yolk they contain yolk granules and numerous vacuoles.
In places in which lumps of agar have been pressed out of the sponge and lie freely in the tissue, the polyblasts assemble in large groups, surround the agar and fuse together, forming multinucleated giant cells.
As the emigration of new lymphocytes and monocytes still continues. the tissue always contains transition forms from the largest polyblasts of the macrophage type to the smallest, lymphocyte-like polyblasts, which have just left the blood vessels.
On the contrary, transition forms between endothelial cells and polyblasts, which could be made responsible for the endothelial origin of at least a part of the latter, are never found.
The tissue of the omentum of our animals that received intravenous injections with India ink and intraperitoneal injections with a small amount of silver nitrate, proved to be especially favorable for the study of the transformations of the endothelial cells of the small blood vessels. The capillaries in the rabbit's omentum are extremely long tubes, branching slightly and only in special places, as for instance in the vascular milky spots. They show a distinct so-called adventitia capillaris, a thin collagenous membrane, surrounding the endothelial tube. With this membrane are connected numerous pericytes--elongated spindle-shaped cells, whose nuclei and thin cell bodies everywhere are seen, with the adventitia capillaries closely adjacent to the outer surface of the endothelium (Figs. 7, 9 and 10 Per). Often transversely arranged processes of these cells are seen encircling a smaller or larger part of the periphery of the capillary.
[Figures 7, 9-10 ILLUSTRATION OMITTED]
These pericytes, especially in the omentum, seem to play an important role in the production of new cells and in the reactions of the tissue toward irritations of various kinds. Marchand,(23) who previously in his papers dealing with the inflammatory changes of the omentum described only one type of "adventitial cells" or "clasmatocytes" near the capillaries, elements which are now known to belong to the phagocytic and dye-storing cell type of the histiocytes, at the present time,(11) under the influence of the investigations of his student Herzog,(24) distinguishes two cell types accompanying the capillaries. This view is now also adopted by Maximow.(25) The one type of cell seen best in animals intravitally stained with lithium carmine or trypan blue is the common histiocytes or clasmatocyte (Figs. 9 and 10 Hist). These cells react in inflammation by rounding off and transforming themselves into large ameboid, phagocytic, wandering cells, the polyblasts of Maximow. The others are the cells intimately adjacent to the endothelium of the capillaries (Figs. 7, 9 and 10 Per). They do not store vital dyes and, in the structure of their nucleus and the behavior of their protoplasm, resemble mesenchymal cells. They are elements of embryonic type, keeping the various potencies of development which are characteristic of the mesenchymal cells of the embryo. They can give rise--provided there is adequate stimulus--to various other types of cells, and may become differentiated in several ways. The two most common results of their differentiation are, on the one hand, the phagocytic and dye-storing histiocytes (resting wandering cells, clasmatocytes) and, on the other hand, common fibroblasts. In inflammation they are usually seen to move away from the vessel (Fig. 8 Per, Fig. 10 Per) and to become transformed into histiocytes, which at once, under the influence of the inflammatory stimulation, develop further into ameboid polyblasts.
In the animals that received intravenous injections with India ink, the capillaries of the omentum showed carbon particles in many places. The stages of ten to fifteen hours proved to be the most interesting. The microscopic study is highly facilitated by the possibility of preparing whole mounts of the fixed and stained transparent membrane without the necessity of recurring to the section method. This implies another advantage--the capillaries are seen with all their parts intact and arranged in the plane of the microscopic stage, whereas in the sections they are often cut off at the most important place, so that one has to resort to the difficult and not always satisfactory comparison of the serial sections.
In the capillaries of the omentum, besides the usual sprinkling of the endothelial surface with smaller or larger, isolated or clustered carbon particles (Figs. 8-10 Ed), emboli in the form of large lumps of agglutinated carbon particles are especially common (Figs. 7-10 Emb). They plug and obstruct the lumen, sometimes causing a distinct bulging on the outer surface of the thin endothelial membrane. The proximal part of the obstructed capillary in such cases is usually enlarged, while the distal part sometimes appears collapsed.
In many places large carbon filled free histiocytes, occurring more frequently than in the vessels of the subcutaneous tissue, are seen in the lumen of the capillaries, either together with the free lumps of carbon or as isolated cells (Fig. 7 Hist'). Monocytes and special granular leukocytes containing small carbon particles and small lymphocytes can also be found in the capillaries, although their number here, on the contrary, is smaller than in the enlarged tortuous capillaries of the subcutaneous tissue (Fig. 10 Mon). In the omentum the emigration of these cells can also be seen, sometimes in the immediate proximity of carbon emboli (Fig. 7 x). However, this phenomenon, at least in the stages mentioned, is not as prominent as in the subcutaneous tissue; and, correspondingly, the majority of the polyblasts in the tissue are of local origin--mobilized histiocytes (resting wandering cells or clasmatocytes. Figs. 9 and 10 Plb).
In the manner described for the subcutaneous tissue, the small carbon particles, first sticking to the inner surface of the endothelium, enter its protoplasm. After from ten to fifteen hours, many of them have passed through the thin endothelial membrane and have found their way into the protoplasm of the adjacent pericytes (Figs. 8-10 Per). This is especially manifest and is being conducted on a large scale where the large carbon emboli are located; in all probability at these points the vitality of the endothelium is reduced and the permeability of its protoplasm increased. In such places the cells surrounding the plugged section of the capillary are engorged with carbon particles (Fig. 10); some of the latter may even be found freely scattered in the tissue (Fig. 10 II). The pericytes, crowded full of carbon, probably under the influence of this stimulus, round off and transform themselves into large ameboid polyblasts (Fig. 10 Plb), moving away from the vessel into the tissue. Many of them, however, may keep their position on the outer surface of the endothelium, or, if they also move away, partly keep their connection with the outer surface of the capillary (Fig. 10 Per'). But in this case the rounding off of the cell is delayed, and it first assumes the character of a fixed histiocyte with dark protoplasm, a sharp, rugged outline and a darker, irregular nucleus. Later these histiocytes can also become transformed into round ameboid polyblasts (Fig. 10 Plb).
The fibroblasts--which in the serous membranes generally seem to be less differentiated than in the common loose connective tissue of the subcutis--near the capillaries may sometimes also contain a few carbon granules (Fig. 10 Fbl).
The number of emigrated granular leukocytes and of polyblasts of hematogenous origin, emigrated lymphocytes and monocytes, containing or not containing carbon particles, in the stages now under consideration, is smaller in the tissue of the omentum than in the subcutaneous tissue.
The most important observation on the mildly irritated omentum in animals that received injections with India ink is the complete passivity of the endothelium in the process of production of the ameboid mononuclear phagocytic cells, the polyblasts. The endothelial wall of the capillaries, even in the embolized sections, always remains continuous; its cells do not change their position; they never transform themselves into ameboid elements and never produce polyblasts. There is also no production of "endothelial leukocytes" in the lumen. The carbon particles enter the endothelial protoplasm, pass through it and thus find their way into the tissue, where they are, for the most part, at once taken up by the pericytes. These cells in their turn may either remain unchanged and keep their place, or, especially when they have engulfed large quantities of carbon, they may move away into the tissue and transform themselves into either common histiocytes (resting wandering cells, clasmatocytes) and then into polyblasts or directly into round, ameboid polyblasts.
In confirmation of the previous findings of Maximow,(2) the polyblasts, the mononuclear exudate cells in the inflamed tissue, arise partly through mobilization of the local histiocytes, the resting wandering cells of the loose connective tissue, and partly through rapid hypertrophy of the emigrated lymphocytes and monocytes. The hematogenous cells, which in the earliest stages of inflammation are much smaller than the polyblasts of local histiocytic origin, quickly become indistinguishable from the latter and join them in their further transformations. It may be pointed out in connection with these findings that Maximow(26) has recently succeeded in obtaining results with the method of tissue culture which strongly corroborate the concepts of the origin of the polyblasts as outlined in the present paper. If leukocytes of the blood of an adult rabbit are cultivated outside the body in a suitable nutritive medium, the lymphocytes as well as the monocytes in the course of a relatively short time are seen to develop into large, ameboid, phagocytic, carmine-storing polyblasts of the macrophage type; these cells also show a considerable capacity for mitotic proliferation.
M. Lewis(27) came to similar conclusions while incubating drops of blood of various animals in a moist chamber. However, she draws a sharp line of distinction between the monocytes and lymphocytes, and believes only the former to be capable of hypertrophy and macrophage formation.
That the lymphocytes and monocytes, on one hand, and the resting wandering cells or histiocytes, on the other hand, are closely related cell types, has been shown by the embryologic researches of Maximow(28) and confirmed recently by Alfejew.(29) Therefore, the double origin of the polyblasts both from local and hematogenous elements seems natural.
The endothelium of blood vessels in tissue cultures, as Maximow(26) has shown, does not give rise to ameboid elements. Long slender fusiform cells grow out of the severed ends of small arteries and gradually become transformed into strands of typical fibroblasts.
In our experiments, performed by the method of intravenous injections of India ink according to McJunkin and Foot, we failed to find any proof for the presumed active participation of the endothelium of blood vessels in the formation of polyblasts. The particles of India ink, after having entered the endothelium, pass through its protoplasm and are taken up by cells, surrounding the endothelial tube--the pericytes.
(*) From the Department of Anatomy, University of Chicago.
(*) Aided by a grant from the Douglas Smith Foundation for Medical Research of the University of Chicago.
(1.) Marchand, F.: Der Prozess der Wundheilung. Stuttgart, 1901; Beitr. z. path. Anat. u. z. allg. Path. 69:1, 1921.
(2.) Maximow, A.: Beitr. z. path. Anat. u. z. allg. Path., Suppl.5, 1902: 34: 153, 1903; 35:93, 1904; 38:301, 1905; 39:333, 1906; Verhandl. d. Internat. med. Congr. zu Budapest, 1909.
(3.) Rossle. R.: Verhandl. d. Deutsch. path. Ges. 19:18, 1923.
(4.) Foot, N.: J. Med. Res. 40:353, 1919; J. Exper. Med. 32:513, 533, 1920; 33:271,625, 192.1; 36:607. 1922; 37:139, 1923; Anat. Rec. 30:15, 1925.
(5.) Metchnikoff, E.: L'Immunite dans les maladies infectieuses. Paris, 1901.
(6.) Maximow, A.: Arch. f. mikr. Anat. 67:680, 1906.
(7.) Aschoff, L., and Kiyono, K.: Fol. haematol. Arch. 15:383, 1913.
(8.) Kiyono, K.: Die vitale Karminspeicherung. Jena, G. Fischer, 1914.
(9.) McJunkin, F.: Am. J. Anat. 25:27, 1919.
(10.) Herzog, G.: Klin Wchnschr. 11:684, 1923.
(11.) Marchand, F.: Haematologica 5:304, 1924.
(12.) Tschaschin, S.: Fol. haematol. Arch. 16:247, 1913.
(13.) I received the lecithin through the courtesy of Dr. F. Koch of the Department of Physiology.
(14.) Bergel. S.: Ztschr. f. exper. Pathol. u. Therap. 21:216, 1920. Die Lymphozytose, Berlin, J. Springer, 1921.
(15.) Maximow, A.: Ztschr. f. wissensch. Mikr. 26:177, 1909.
(16.) Wislocki, G.: Am. J. Anat. 32:423, 1924.
(17.) Herzog. F.: Ztschr. f. d. ges. exper. Med. 43:79. 1924.
(18.) Stilwell, F.: Fol. ham. Arch., to be published.
(19.) Simpson, M.: J. M. Res. 43:77, 1922.
(20.) Ferrata. A.: Haematologica 2:242, 1921.
(21.) Zimmermann, K.: Ztschr. f. Anat. u. Entwgesch. 68:29, 1923.
(22.) Ciaccio, C.: Anat. Anz. 35:17, 1910.
(23.) Marchand, F.: Verhandl. d. Deutsch. path. Gesellsch. 4:124, 1901.
(24.) Herzog, G.: Beitr. z. path. Anat. u. z. allg. Path. 61:377, 1916.
(25.) Maximow, A.: "Bindegewebe und blutbildende Gewebe," in von Mollendorff, W.: "Handbuch der mikroskopischen Anatome," Berlin, J. Springer. to be published.
(26.) Maximow, A.: Klin. Wchnschr. 4:1486, 1925.
(27.) Lewis, M.: Am. J. Path. 1:91, 1925.
(28.) Maximow, A.: Arch. f. mikr. Anat. 73:444, 1909; Fol. ham. 4:611, 1907.
(29.) Alfejew, S.: Fol. ham. Arch. 30:111, 1924.
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|Author:||LANG, F. J.|
|Publication:||Archives of Pathology & Laboratory Medicine|
|Date:||Jan 1, 2001|
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