Dept. of Anat., Yamaguchi Med. School, Ube (Director: Prof. B. OSOGOE and Assist. Prof. K. AWAYA).

Transcription

1 Arch. hist. jap. Vol. 18, n. 4 (January 1960). P Dept. of Anat., Yamaguchi Med. School, Ube (Director: Prof. B. OSOGOE and Assist. Prof. K. AWAYA). A Quantitative Study of the Plasma Cell Population in Lymphoid Organs of Young Adult Albino Rats.* It is generally believed that plasma cells are the major site of antibody production and proliferate in response to the stimulus exerted by the antigens like heterologous protein. However, little is known about the mechanism of the proliferation of these cells. Recently AWAYA et al. have pointed out that the propagation of the plasma cell is intimately related to the destruction of lymphocytes (AWAYA et al. 1955, 1959; IMAMURA 1959). A consideration of the relation between the lymphocyte and the plasmocytic reaction would require a knowledge about the appearance of plasma cells in normal lymphoid organs, for fairly numerous plasma cells are to be found in the red pulp of the spleen and the medullary cords of the lymph nodes of normal animals. I. Material and Methods. WISTAR strain male albino rats weighing about 200g at the age of 4-5 months were used. After sacrificing the animals by stunning, 8 lymph nodes including superficial and deep cervical, superficial and deep axillary, popliteal, superficial inguinal, iliac, and mesenteric lymph nodes and 3 lymphoid organs including spleen, thymus and PEYER's patch were excised and their imprint and smear preparations were prepared; after drying and fixing, these preparations were stained with MAY- GIEMSA. The method of preparing smear preparations on slides was a modification of that described by MC NEIL (1948). In this method a piece of the lymphoid organs was placed on a slide glass and the tissue was disintegrated in 1-2 drops of human plasma by the aid of the tip of a needle, to mix the cellular material with the plasma homogeneously. They were then smeared towards the other end of the slide glass as in the routine method. Such a procedure was employed in order that various cellular elements occurring in different parts of the lymphoid organs, such as the cortex and medullary cords of the lymph node or the cortex and medulla of the thymns, were mixed homogeneously, which will ensure an accurate estimation of the percentual ratio of different cellular elements. It should be pointed out here that the imprint method hitherto employed would only allow the observation of * Supported in part by a grant to Professor Bunsuke Osogoe from the Ministry of Education (Grant-in-Aid for Fundamental Scientific Research, Cooperative). 479

2 480 H. FUJII: Table 1. Differential cell counts from the mesenteric lymph nodes of young mature obtained by the smear The cerresponding smears and imprints were made from the same materials. * Smear method: By crushing and mixing small portions of fresh tissues of lymphoid for morphological and quantitative studies. cellular population at a certain section of the tissue only. The results obtained with such a smear method and with the imprint method hitherto employed were compared in the mesenteric lymph node of one and the same animal (Table 1). As to the percentage of the plasma cells, a slightly higher value was obtained by the imprint than by the smear, but the difference between them in 18 animals ranged between 0.1 and 10 percent by the present methed and between 0 and 21% by the imprint method. Therefore a broader range of variation was obtained by the imprint method, but both methods gave the same variation coefficient. Such results suggest that our method may be superir to the routine imprint method. In this study observations on individual lymphatic organs were based on 5-10 animals, and the classification of the cells were conducted by counting 2,000 cells per organ. In addition, the methyl green-pyronine stain was also employed for the identification of cells. We included in plasma cells not only cells of typical MARSCHALKO type (mature form) but also what appeared to be these of relatively young stage. The latter were distinguished from the typical cells by their larger nuclei, but the size of the cytoplasm, the presence of a perinuclear clear zone and the absence of nucleolus were common to both types. Lymphocytes were classified, as usual, into large, medium and small forms. Besides, granulocytes such as neutrophilic, acidophilic, or basophilic leukocytes, reticular cells, and monocytes were classified also as usual. Sometimes, unclassifiable or disintegrated cells were also encountered. The latter tended to appear in larger numbers in smears than in imprint preparations. Further, paraffin sections were prepared of each lymphoid organs after fixing in ZENKER-formol to examine the appearance of plasma cells; results of such observations will be reported elsewhere.

3 A Quantitative Study of the Plasma Cell Population in Lymphoid Organs etc. 481 male rats weighing around 200g (average of 18 rats). A comparison of the results and imprint methods. organs with plasma on a glass slide, satisfactory smears were obtained which can be used imprint method. II. Results. Principal results are summarized in Table 2. First of all, it should be noted that plasma cells occur consistently in all the lymphoid organs examined. However, there was a fairly considerable variation in the percentage of plasma cells in all nucleated cells (to be called simply 'percentage' below) among individual lymphoid organs. The organs in the order of decreasing percentage are: 2.51% Mesenteric lymph node % Superficial and deep cervical and iliac lymph nodes % Popliteal lymph nodes and spleen % Superficial axillary lymph nodes % Superficial inguinal lymph nodes, PEYER's patch and thymus. Divided, for convenience' sake, into higher and lower percentage groups above and below a borderline of 0.41%, the higher percentage group comprised the spleen and all the lymph nodes except superficial inguinal nodes, which, together with PEYER's patch and thymus, belonged to the lower percentage group. So-called primary and secondary lymph nodes are distinguished among the lymph nodes. Out of the organs examined here, superfical cervical, superficial axillary, popliteal and superficial inguinal lymph nodes belonged to the primary node, while the deep cervical, deep axillary, iliac and mesenteric lymph nodes were to be included in the secondary one. Compared between the primary and secondary lymph nodes in the same region, the percentage of plasma cells was higher in the secondary the latter group. In this way, plasma cells occurred in a higher percentage in the secondary lymph node than in the primary lymph nodes in this region (the differ- teric lymph node belonging to the secondary lymph node also gave a high percentage

4 482 H. FUJII: Table 2. Plasma cell counts (%) from the various lymphoid Two thousand cells were counted in each organ. * Others: This column contains monocytes and unclassified cells. Designations are the same as used in Table 1. Table 3. Plasma cell counts (%) from the mesenteric lymph Group A: Average of 12 rats in which plasma cell counts are less than 3.7%. Group B: Average of 6 rats in which plasma cell counts are more than 7.5%. The designations are the same as in Table 1.

5 A Quantitative Study of the Plasma Cell Population in Lymphoid Organs etc. 483 organs of young mature male rats weighing around 200g. nodes of young mature male rats weighing around 200g. 0.16%) and of the deep cervical lymph nodes pertaining to the secondary lymph

6 484 H. FUJII: Next, it must be stressed that, as shown in Table 2, the percentage of plasma cells show, even in the same organ, an appreciable individual variation. The lymph nodes with relatively small variation coefficients (below 50per cent) are the superficial cervical, iliac and mesenteric lymph nodes and the spleen, those of the other organs being above 50per cent. Especially in the superficial and deep axillary, superficial inguinal and popliteal lymph nodes, the variation coefficient was higher than 80per cent. Table 3 lists the variation of the percentage of plasma cells in the mesenteric lymph node of 18 albino rats. It contains 12 instances of relatively low percentages ( per cent) and 6 instances of relatively high ones ( per cent). And each animal of the latter group tended to show a higher percentage of neutrophilic and easinophilic leukocytes than the former group. III. Recapitulation and Discussion. The principal findings of the present study are that all the lymphatic organs of the normal adult albino rats always contain plasma cells, the percentage of which in the total nucleated cells is higher in the lymph nodes or spleen and lower in the thymus or PEYER's patch, and that there is a considerable regional or individual variation of the percentage of plasma cells in lymph nodes. Although the percentage of plasma cells is high in all the lymph nodes except the superficial inguinal lymph nodes and spleen and low in the thymus and PEYER's patch, the microscopic inspection of the sections revealed the occurrence of plasma cells in the medullary cord of the lymph node and the red pulp of the spleen without exception. In most cases they are clustered to form cellular foci, and a small number of mitotic figures were also observed. In contrast, thymus and PEYER's patch show a very few plasma cells which do not form any cellular foci. CARLSON et al. (1958) have recently pointed out that in young guinea pigs the medullary cord of the lymph nodes and the red pulp of the spleen are the important sites of the plasma cell production, but they are scarcely encountered in the thymus and PEYER's patch, an observation fully in accord with our own findings. The occurrence of plasma cells in the thymus has already been pointed out by SHIRAKI (1934) and KANOH (1956). It should be noted that plasma cells always occur, although in a small number, in the thymus (and also in the PEYER's patch). Concerning the regional difference of the percentage of plasma cells in the lymph node, there are very few reports of systematic studies on the occurrence of plasma cells in lymphatic organs. We only know the work of KANOH et al. (1954) who classified cells in various lymph nodes of albino rats by the imprint method. Although plasma cells were not their main subject of study, they noticed a regional difference in the percentage of the plasma cell. According to their study, the highest percentage of the plasma cell was found in the mesenteric lymph node (2.98per cent) and the lowest in the superficial axillary lymph nodes (0.53per cent), which agree fairly well with the results of the present study.

7 A Quantitative Study of the Plasma Cell Population in Lymphoid Organs etc. 485 The reason why such a regional difference appears is not clear at the moment. As mentioned above, a trend of higher percentage of plasma cells was noticed in the secondary lymph nodes than in the primary one in the lower half of the animal body, but such was not recognized in the lymph nodes of the upper half of the body. We can not, therefore, explain the observed regional difference by that between the primary and secondary lymph nodes alone. Further, much individual variation can be observed, as noted above, in the percentage of plasma cells among different lymphoid organs; at the same time, it was observed that the higher percentage of plasma cells was accompanied by a higher percentage of the granulocytes (Table 3). Since the presence of a slight inflammation is inferred in such cases, the main reason for the regional and individual differences may well be explained by the occurrence, or the extent, of inflammation. Finally, mention should be made of the relationship between the plasma cells and other cellular species. JORDAN et al. (1927, 1929, 1954) repeatedly claimed that plasma cells found in lymph nodes of rabbits and albino rats are an incomplete form of erythroblasts transformed from lymphocytes. Also KELSALL et al. (1958) maintained, in agreement with JORDAN, that plasma cells in lymph nodes or spleen were originated from lymphocytes. SHIRAKI (1934) also stated that plasma cells in the thymus were produced by a transformation of small lymphocytes in the cortex of the thymus. However, the observation of the present author has demonstrated that plasma cells in any organs examined could be clearly distinguished from other cellular species without any discernible intermediary forms between themselves and lymphocytes or erythroblasts. The AMANO's theory on the transformation of adventitial cells into plasma cells UNNO 1953) will be discussed separa- (AMANO et al. 1948, 1956; AMANO 1958; tely. IV. Summary. 1. The percentage of plasma cells in total nucleated cells was determined with smear and imprint preparations of various lymph nodes, spleen, thymus, and PE- YER's patches of normal albino rats. Plasma cells were found consistently in all the lymphoid organs examined, but their percentage was relatively high in all the lymph nodes except the superficial inguinal lymph nodes and also in the spleen, and remarkably low in the thymus or PEYER's patch, The organs mentioned in the 2. A fairly remarkable regional difference was noticed in the percentage of the plasma cells in lymph nodes. Among lymph nodes of the same region, secondary lymph nodes tended to show higher percentage of plasma cells than primary ones in the lower half of the body, but such a trend could not be found in the upper half

8 486 H. FUJII: of the body. 3. There was a fairly considerable individual variation in the percentage of the plasma cells in lymphoid organs, the variation coefficient of the organs except for the superficial, iliac, and mesenteric lymph nodes as well as the spleen being more than 50per cent. 4. There was a trend of higher percentage of plasma cells being generally accompanied by a higher percentage of the granulocytes. References. Amano, S.: Studies on plasma cells-cytogenesis, defensive function and ultracytophysiology. A review of our original studies since Ann. Report. Inst. Virus Research. Kyoto Univ. Series A. 1 (1958). P Amano, S., G. Unno and M. Hanaoka: Studies on the differentation of lymphocytes and plasma cells. An advocation of 'lymphogonia' theory. Acta haem. jap. 14 (1954). P Amano, S. and H. Tanaka: Further observation of the plasma cell generation from the vascular adventitial cells through metamorphosis by ultrathin sections under the electron microscope. Acta haem. jap. 19 (1956). P Awaya, K., T. Ikeda and N. Tagawa: Nucleic acid and plasmacytic reaction. 8 (1955). P Awaya, K., H. Fujii and A. Inada: On the plasma cell reaction in lymphatic tissue. I. The relationship between lymphatic tissue degeneration and plasma cell reaction. Kaibo. Z. 34 (1959). P Carlson, B. and L. Gyllensten: Plasma cells in the growing lymphatic system of young guinea pigs. A quantitative investigation. Acta path. et microbiol. scandinav. 43 (1958). P Imamura, H.: Further studies of a lymphatic hemogram and its relation to lymphocytopoiesis. II. Variations in mitochondrial content of blood lymphocytes in relation to the processes of regeneration

9 A Quantitative Study of the Plasma Cell Population in Lymphoid Organs etc. 487 of the lymphatic apparatus of rats after total body x-irradiation. Okajimas Fol. anat. jap. 32 (1959) P Jordan, H. E. and J. B. Looper: The comparative histology of the lymph nodes of the rabbit. Amer. J. Anat. 39 (1927). P Jordan, H. E.: On the genetic relation between so-called plasma cells and erythroblasts in certain lymph nodes. Anat. Rec. 42 (1929). P Jordan, H. E.: The origin and fate of plasmacytes. A comparative histologic study of plasmacytes of normal lymph nodes and tumors of multiple myeloma. Anat. Rec. 119 (1954). P Kanoh, S., M. Nagata and H. Nakamura: Studies on the cells in the lymph and the lymph nodes of albino rat and myocaster coypus (molina). Collect. Pap. II. Div. Anat. Inst. Kyoto Univ. 2 (1954). -Kanoh, S.: Studies on the thymus. I. Age change, especially cytological, of the thymus. Collect. Pap. II. Div. Anat. Inst. Kyoto Univ. 4 (1956). P Kelsall, M. A. and E. D. Crabb: Lymphocytes and plasmacytes in nucleoprotein metabolism. Ann. New York Acad. Sci. 72, Art 9 (1958). P McNeil, C.: Cellular changes in rabbits during antibody formation. 1. Response to Eberthella thyphosa. Amer. J. Path. 24 (1948). P. 127I Shiraki, Y.: Uber die hamatopoietische Funktion des Thymus der Ratte. Kaibo. Z. 7 (1934). P Unno, G.: On the lymphogonia. Acta haem. jap. 16 (1953). P Addendum. In the present paper, the lymph nodes receiving 'peripheral lymph', which has not yet passed through a lymph node or come into contact with any lymphoid tissue, were called the 'primary lymph nodes'; whereas the lymph nodes receiving 'intermediate lymph', which has already passed through at least a lymph node or has been in contact with some lymphoid tissue, were referred to as the 'secondary lymph nodes'.