The Wandering Cells in the Loose Connective Tissue of the Bird And

THE WANDERING CELLS IN THE LOOSE CONNECTIVE TISSUE OF THE BIRD AND THEIR ORIGIN VERA DAKCHAKOFF Rockefeller Institute, New York Cloose connective tissue of the hen.

1.' MORPHOLOGICAL DATA

As may be gathered from the title of this communication, the loose connective tissue contains permanently a certain amount of wandering cells. This is especially true for the embryonic connective tissue. Just as the circulating blood contains many kinds of differentiated blood cells, so the loose connective tissue of an adult hen includes, beside the fibroblasts, many kinds of amoeboid cells. Among these are included small lymphocytes, wandering histiotopic cells, granular wandering cells, Mast cells and occasionally plasma cells and true polymorphonuclear special leucocytes. All of these cells are found in the adult organism, but the majority of them appear in the embryo. They are numerous in the connective tissue of the adult hen. The free amoeboid cells are sometimes arranged in the form of character- 483 484 VERA DANCHAKOFF istic cords and groups. Thus in some regions the free amoeboid cells form complete layers which appear in sections as uninter- rupted lines. They are densely arranged in some of the thin connective septa between small fat accumulations. The morphological structure of different kinds of cells encountered in the connective tissue of the adult hen were described by Dr. Solucha in the year 1908. In 1908 I published the result of my studies on the embryogenesis of these elements in the chick embryo. Later this problem was worked out for the reptiles and the account of this work became a part of the work about the general haematopoiesis of the reptiles. The cells mentioned a,bove as well as the tissue of other haematopoietic organs, all develop in the last instance from a common, undifferentiated mesenchymal anlage. The mode of development of the mesenchymal cells into amoeboid cells is easily deducible. The long irregular processes so characteristic of the mesenchy.ma1 cells are withdrawn and the cell becomes free, potentially spherical, but very mobile. Some- times innumerable short and irregular pseudopods cover the whole surface of the cell. Such a cell may remain as a wandering cell of the connective tissue, undoubtedly capable of further differentiation. In other cases it may immediately undergo further differentiation and assume the morphological structure of the large lymphocyte. This latter must be a very active cell, for its appearance is usually followed by a further differentiation, this depending on the environmental conditions. The environmental conditions for the differentiation of an erythro-granulopoietic organ are more or less definite; a large amount of nutritive material, a region isolated from mechanical disturbance favor an intensive development of haemocytoblasts. The localization within or outside the vessels determines the further differentiation of the haeniatocytoblasts. A distension of the capillary net and a subsequent retarding of the blood current, offer again favorable conditions for multiplication and differentiation of the haematocytoblasts. It is much more difficult to define the specific environmental conditions, which direct the differentiation of the various cell WANDERING CELLS IN TISSUE OF THE BIRD 485 groups found scattered in the connective tissue. However, some indications show the existence of intimate relations between definite environmental conditions and differentiation. A striking example is presented by the differentiation of the Mast cells along the vessels of the omentum; these follow the vessels in whole ranges and envelop them by their long processes, which, as well as the whole cytoplasm, are filled up by metachromatic granules. An analogous intensive development of the Mast cells is seen under the epithelium of the intestine. Another instance of the interdependence of cell differentiation on the environmental conditions is offered by the development of the granulocytoblasts in the spleen of the reptiles and birds, this being chiefly confined to the pulpa in the normal embryos, while the small lymphocytes are principally developed in the external layers of the organ and in the splenic follicles. The embryogenesis of the diffuse granulo-lymphatic tissue is easily established. The question of its reproduction in the adult organism is however obscure. Are the numerous granular and lymphatic cells in the connective tissue the direct offspring of analogous cells split off from the mesenchyme during embryonic life, securing their existence through their own reproduction? Or do these cells represent a renewal by cells brought by the blood current, or finally is the mode of their re6roduction analogous to the one, observed in the embryonic development and may they not differentiate in the adult organism as well as in the embryo from the same source, namely, from young mesenchymal cells? A definite answer to these questions would greatly facili- tate the solution of the problems stated at the beginning of this paper. . The peculiar structure of the granular cells of the connective tissue, which are not found in the circulating blood, speaks with great certitude for their local differentiation. The presence of mi- tosis in the non-granular amoeboid cells confirms the possibility of an independent reproduction of these cells in the connective tissue. In studying the granulo-lymphatic cells in the connective tissue of the adult hen, I observed numerous small agglomerations of true lymphadenoid tissue. The center of such an ag- 486 VERA DANCHAKOFF glomeratioii is usually occupied by a mesenchymal syncytium, as described by Mall and Sabin in the case of the lymph glands of mammals. This syncytium. must be considered as a young, undifferentiated tissue. Mall derived the connective reticulum from this tissue. Another line of differentiation is characteristic of the syncytium in the lymphatic nodes scattered in the loose connective tissue of the hen. 'This seems to consist in the development of free amoeboid cells from the mesenchymal syncytium cells. The syncytium of the small agglomerations of the lymphadenoid tissue consists of ramified cells, connected with their neighbors by rather short processes. The whole aspect of this syncytium strongly reminds one of such regions of the embryo body where the mesenchyme multiplies intensely and therefore becomes denser. Both in the embryo and in the adult organism the mesenchymal syncytium appears to be the source of the free amoeboid cells. Only the adult organism fails to show intense differentiation of free cells at the expense of the mesenchymal syncytium in the lymphnodes, as is so easily observed in the Ioose mesenchyme in the embryo. The multiplication of the granular and lymph cells in the connective tissue proceeds in the adult chiefly in a more homoplastic way, at the expense of intermediate stages of differentiation. It is known that most of the birds and hens particularly do not possess special lymph glands and it is permissible to assume a homology between the numerous small agglomerations of lym- phatjc tissue and the granular and lymphatic cells, diffusely scattered in the connective tissue of birds on the one hand and the precisely localized lymph glands of the mammals on the other hand. The conception of the haematopoietic organ, as established in recent years, requires not only the presence in it of proliferation and differentiation of various blood cells, but also the presence of ancestral mother cells. At the expense of such young cells in tke lymphnodes the regeneration of various amoeboid cells takes place normally in the connective tissue of birds, and most intensely after loss of blood elements. Accordingly the loose connective tissue should be considered as a haematopoietic organ both in the adult organism and in the embryo. WANDERING CELLS IN TISSUE OF THE BIRD 487

The data referred to above are obtained as a result of the study of fixed material by the use of technical methods giving minute morphological details. The existence of numerous granular and lymph cells diffusely scattered in the connective tissue of the hen, as well as of numerous localized centers of mesenchymal syncytium, must be ad- mitted as uncontested facts. However, the whole line of development and differentiation is merely the result of interpretation, which may give a true understanding of the development, sup- posing no links in the series of observation to be omitted. But the omitting of certain data may give rise to the wildest specula- tions. One may recall only the possibility of admitting the small lymphocytes found in accumulations in the bone-marrow of birds, as being the common stem-cells for granular leucocytes and erythrocytes (Venalaff). The validity of the interpretation may be accepted if they are confirmed by experimental tests. 2. EXPERIMENTAL DATA Observations have shown the presence in the loose connective tissue of the adult hen of swarms of amoeboid cells. Small . sgglomerations of mesenchymal syncytium are also irregularly scattered about in the connective tissue. Around these agglomerations the amoeboid cells appear in greater numbers and the peripheral layers of the mesenchymal syncytium contain numerous free cells in the form of large lymphocytes, which apparently differentiate at the expense of the cells of the syncytium and partly reproduce themselves by mikosis. The following-questions, if possible, should be tested experimentally. a) Are the different amoeboid cells of the connective tissue the product of the development and differentiation of the mesenchymal nodes? If so, the mesenchymal nodes would appear to be stocks of undifferentiated cells, preserved in the adult organism from the time of the embryonic development and retain- ing their faculty of further differentiation. If by some means it were possible to destroy the differentiated cells without destroy- ing the stem cells and if such destruction were followed by a new regeneration of differentiated cells, it would be easy to define the 488 VERA DANCHAKOFF

source and the mode of regeneration in the adult organism. Such a destruction of lymphoid tissue was obtained by the use of the X-rays, which given in definite doses destroy chiefly the lymphoid elements, leaving the younger istern cells uninjured. Anintense regeneration follows the loss of the lymphoid elements and chiefly proceeds at thc expense of the cellular syncytium in the lymphnodes. b) The second question to be tested experimentally is: What are the relations to other haemopoietic organs of the lymphoid elements found in the connective tissue? In other words, is the anlage for the amoeboid cells of the connective tissue and for the blood cells identical? This latter question may be answered affirmatively on the basis of experiment only in case, for example, we succeed in changing the actual line of differentiation of the loose inesenchyrrie into another one-if by means of certain in- tervention we may force the loose mesenchyme of the embryonic body which normally in certain regions gives rise only to lym-

, phatic cells, to differentiate, lei us say, into granular leucocytes. Such a inetaplasis of the mesenchyme in the embryo body is obtainable, as will be shortly related. Whether the adult organism preserves a stock of young mesenchymal cells is difficult to say at present. However, this assump- tion is true for the embryo and may be based not only on morphological observations but also on experiment. In studying the factors of resistance to heteroplastic tissue- grafting, Dr. Murphy of the ]Rockefeller Institute made a very interesting observation. The tumor grafts give intense growth on the allantois of a chick embryo. The spleen graft of an adult chick made simultaneously with a cancer graft inhibits the cancer growth. At the same time t’he spleen of the embryo becomes much larger. As already mentioned in my other papers, the spleen and other agglomerations of lymphatic tissue may be considered as localized hypertrophic centers of diffuse analogous processes, present in all parts of the loose connective tissue, which is especially true for. the embryo. If so, we should certainly expect, after grafting, the same changes throughout the whole mesenchyme, as are noticed in the WANDERING CELLS IN TISSUE OF THE BIRD 489 spleen. The changes in the spleen were not studied, but they obviously existed, for the organ became many times larger. In order to obtain uncomplicated results I made in my experiments single spleen grafts to 7-10 day embryos. If after grafting, a hypertrophy of amoeboid cells, or whatever changes they may be, were confined both to the spleen and to other mesenchymal haematopoietic anlages and if these changes were identical in all regions, we could deduce herefrom that the mesenchyme of the whole embryo body and the mesenchyme of the spleen is equivalent. The single grafts of adult spleen on the allantois of a chick embryo were also followed by enlargement of the host spleen. On the other hand, the study of the loose mesenchyme of the embryo body and of other haematopoietic anlages showed intense and widespread changes. These changes consisted in the splitting off from the mesenchyme of haematocytoblasts in the form of the morphological unity of a large lymphocyte. It would be too long to mention all the regions of the embryo body where such an intense splitting takes place. I will merely state that no place exists in the embryo body where the mesenchyme does not show this process. Innumerable haemocytoblasts lie along the vessels in the form of thick cords, they are diffusely scattered in places where the mesenchyme is looser, the haemocytoblasts are crowded in groups in every skin papilla, strands of haemocytoblasts separate the smaller and larger muscle bundles and muscle fibers; even the mesenchyme in the centers of developing feathers shows an analogous splitting. The splitting off of haemocytoblasts may be normally observed in the mesenchyme. The grafts may generalize, accelerate and intensify the same development through all the regions, where mesenchyme is found in the embryo body. The extension of identical reactions to the whole loose mesenchyme and the mesenchymal haematopoietic organs amply confirm the value of the equivalence of the mesenchyme in the spleen, the thymus and in other parts of the embryo body, where an identical reaction takes place. The intense splitting off of the haemocytoblasts in the loose mesenchyme of the embryo body, of the spleen and of the 490 VERA DA:NCHAKOFF

thymus is accompanied by differentiation of these cells, analogous in all these regions. The study of this differentiation gives decisive data about the equivalence of the anlage of the lymphoid elements in the connective tissue and the anlages of other haematopoietic organs. For the differentiation of the mesenchyme now consists in a development of granulocytoblasts and granular leucocytes, even between muscle fibers. I cannot here analyze the causes which lead to this wonderful metaplasis of the mesenchyme. I would merely like to point out the value of the results obtained for the clear understanding of the r81e of the embryonic loose mesenchyme as of an undiff er- entiated haematopoietic anlage. Under normal conditions the mesenchyme gives rise to a certain number of amoeboid cells, the direct offsprings of which remain as a part of the wandering cells of the loose connective tissue in the adult organism. This process undergoes intense stimulation which here evidently depends upon resorption and circulation of certain enzymes, furnished by the grafted spleen cells. At the same time a differentiation takes place in the lymphoid cells, which in many places is normally absent. Not only the diffuse mesenchyme of the body, but the spleen and the thymus become at certain periods after grafting centers of a most intensive true granulo-leukopoiesjs (giving rise tospecial leucocytes). What under these circunistances becomes of the specificity of the lympho- and leukoblasts? The same cells, which under ordi- nary conditions give lymphatic non-granular cells as products of their differentiation, now becoiine tthe source of the most intensive leukopoiesis. Thus, the embryonic meseiich y?nr appecrrs to bc (I diffuse uttluye for bcth lymphopoiesis mid yranu2oieukopaieuis. The faculty of polyvalent development for the embryonic mesenchyme is proved by the study of the changes jn the mesenchyme, resulting after the grafting of spleen from an adult, chick on the allantois of the embryo. Other important results arc: obtained by a closer study of the changes in the spleen occurring after grafting. These result,s be- WANDERING CELLS IN TISSUE OF THE BIRD 491 come an additional proof of other polyvalent differentiation possibilities of the mesenchyme. They demonstrate once more, that only environmental conditions direct the differentiation of the haematocytoblasts derived from the spleen mesenchyme, for, numerous haemocytoblasts may under definite conditions differentiate in the spleen into erythroblasts. The spleen of the chick embryo is normally a lympho- and granuloleuko-poietic organ. In the earliest stages of development it appears in the form of a dense, scarcely vascularized mesenchymal anlage. The splitting off of lymphoid haemocytoblasts is gradual; the latter remain outside the vessels and many of them differentiate into granulocytoblasts. The developing vessels contain mostly differentiated erythrocytes. The development in a spleen after grafts proceeds very intensely. The splitting off of lymphoid haemocytoblasts is most active. At the time when the spleen anlage becomes vascuolarized, numerous lymphoid haemocytoblasts pass into the lumen of the vessels. The blood current in the spleen being at this time very slow, many of them remain here and differentiate inside the vessels into erythroblasts and erythrocytes. This differentiation of the haemocytoblasts (that is, of the large lymphocytes), into erythroblasts inside the vessels with a very slow current again shows that the haemocytoblasts are polyvalent cells, of which the differentiation is directed by environmental conditions. The results of the experiments confirm fully the deductions made with regard to the observations of the embryogenesis of the different blood cells. The study of the changes in the embryo, obtained by grafting have shown that the loose niesenchyme of the embryo at certain stages is equivalent in all the regions of its localization. The mesenchyme is moreover polyvalent in its potencies of development. It may give typical connective tissue cells as well as all the different kinds of blood cells. The differentiation of the mesenchymal cells is directed by the force of external conditions, whether these conditions consist in the presence or absence of different food material, or in the special physico-chemical milieu

THE ANATOMICAL RECORD, VOL. 10, NO. 7 492 VERA DANCHAKOFF of the vessels. Their proliferation seems to depend upon specific stimuli, which may consist in enzyme-like material. These experimental results give strong support to the mono- phyletic views regarding the olrigin of different blood cells, and seem to stand in contradiction to the polyphyletic origin of the various blood cells. The completely different cells (the red and the different kind of white blood cells) are so completely different only because tJheypresent the final steps of various directions of differentiation. These cells, as such, are certainly not capable of interchanging, but, as the above described experiments have shown, the various lines of differentiation of their. common stem cells may be inter- changed.