Further Studies of a Lymphoutic Hemogram and Its Relation to Lymphocytopoiesis

Transcription

1 Further Studies of a Lymphoutic Hemogram and Its Relation to Lymphocytopoiesis III. The Mode of Reaction of the Blood Lymphocytes and of the Lymphatic Tissue to Ovalbumin in Young Adult Albino Rats By Hidetoshi Imamura Department of Anatomy, Yamaguchi Medical School, Ube (Director : Prof. B. Osogoe) In the previous reports from our laboratory, it has been shown that a marked elevation of the average number of mitochondria per lymphocyte in the blood may be correlated with an extensive new formation of F l e m m i n g's secondary nodules in the lymphoid organs (0sogoe et al., 1953; Awaya, 1956; and Imamur a, 1958 a, b). This indicates that a lymphocytic hemogram based on mitochondrial number of the blood lymphocytes is useful as a measure of reflecting the lymphocytopoietic activities of the lymphoid tissue, the F l e m m i n g's secondary nodules in particular. The purpose of this paper is to report further observations on the relationship between the average number of mitochondria in blood lymbhocytes and the lymphocytopoietic activities of the lymphoid organs in adult albino rats after single or repeated injections of ovalbumin which is known to produce lymphocytosis and hyperplasia of lymphoid tissue (W i s e m a n, 1931 ; Wiseman et al., 1936 ; and F u k a s e, 1949). The mode of reaction of the blood lymphocytes and of the lymphoid organs to ovalbumin in adult rabbits has already been reported by A w a y a (1956), who demonstrated a marked transient rise of the mitochondria] number in blood lymphocytes almost coincident with an extensive new formotion of F l e m m i n g's secondary nodules in the lymphoid organs after single or repeated * Supported in part by a grant to Professor Bunsuke Osogoe from the Ministry of Education (Grant-in-Aid for Fundamental Scientific Research, Cooperative). 305

2 306 Hidetoshi Imamura intravenous injections of ovalbumin. The present work is intended to follow up the experiments of A way a, using albino rats. Material and Methods The animals used in the present experiments were from the same albino rat colony of a subline of the W i s t a r strain as used in the preceding studies of this series. They were raised on a standard laboratory diet as described previously (I in am u r a, 1958b). Sixteen healthy male rats weighing around 200 g were divided into two groups. The first group consisted of 6 animals, among which 4 animals received a single intraperitoneal injection of 20 mg of ovalbumin (E. M e r k) dissolved in 2 ml of saline, and the remainder a single intraperitoneal injection of saline. The second group consisted of 10 animals, among which 5 animals received daily intraperitoneal injections of 20 mg of ovalbumin dissolved in 2 ml of saline, and the remainder daily intraperitoneal injections of 2 ml of saline. For staining mitochondria of lymphocytes, the same supravital technique with Janus green B and neutral red was used as in the earlier studies (0 so g o e et al., 1953 ; A way a, 1956 ; and Imamura, 1958a, b). The other procedures employed (i.e., calculation of average number of mitochondria per lymphocyte and of mitochondrial index, and other hematological and histological methods) were also the same as used in the preceding studies by the present author. In the present study a semi-apochromatic lens was used for counting mitochondria of lymphocytes instead of an achromatic lens employed in the earlier studies. This resulted in a considerable increase in the average number of mitochondria per lymphocyte as compared with the values obtained in the earlier studies,* probably because the semi-apochromatic lens is much superior to the achromatic lens in neutralizing chromatic abberration. Results The chief results of blood examinations are summerized in Tables 1 and 2. After a single intraperitoneal injection of ovalbumin, * The average number of mitochondria per lymphocyte in the blood of no rmal adult rats estimated in the present study ranged from to (Tables 1-2), whereas the corresponding figures obtained in the earlier studies were in the neighborhood of (I m a mura, 1958 a, b).

3 A Lymphocytic Hemogram and Lymphocytopoiesis Table 1. Changes in the lymphocyte count and the average number of mitochondria per lymphocyte in the peripheral blood of adult rats after a single intraperitoneal injection of 20 mg of ovalbumin. Table 2. Changes in the lymphocyte count and the average number of mitochondria per lymphocyte in the peripheral blood of adult rats after repeated intraperitoneal injections of 20 mg of ovalbumin.

4 308 Hidetoshi lmamura the average number of mitochondria per lymphocyte in the blood was significantly elevated already on the 3rd day (P<0.01) and thereafter tended to decline whilst the blood lymphocyte count showed no increase (Table 1). In the control animals that had received a single injection of saline alone, there were no significant changes either in the lymphocyte count or in the average number of mitochondria per lymphocyte. After daily intraperitoneal injections of ovalbumin, both the average number of mitochondria per lymphocyte and the lymphocyte count were significantly raised. It should be emphasized here that a marked elevation in mitochondrial number of blood lymphocytes was observed already on the 3rd day (P<0.01), whereas the number of these cells did not show any noticeable rise until the 7th day after the initial injection (Table 2). In the control animals that had received daily injections of saline alone, no significant changes were recognized either in the lymphocyte count or in the average number of mitochondria per lymphocyte. Histological examination of the mesenteric lymph nodes and spleen disclosed that, while after a single injection of ovalbumin there was no remarkable alteration in the lymphatic tissue, the F 1 e m m in g's secondary nodules of the cortex were greatly enlarged after 6 daily injections of ovalbumin. The majority of the enlarged F 1 e m m i n g's nodules had a large germinal center composed of densely packed, actively dividing medium-sized lymphocytes, and for this reason these nodules are called " active " forms according to our terminology. In addition to the changes of the pre-existing nodules, new formation of F l e m e m m i n g's nodules, though on a smaller scale, was also recognized (Figs. 1-3). In general, the cortical tissue became broader in width, extending toward the medulla, and its cellularity was markedly increased after repeated injections of ovalbumin. At the same time, mitotic figures became very numerous in the lymphatic tissue, especially in the F 1 e m m i n g's secondary nodules. It is therefore evident that, after repeated injections of ovalbumin, there occurs a hyperplasia of lymphatic tissue, the Flemmin g's nodules in particular, resulting in an increase in production and dilivery of lymphocytes from the lymphoid organs to the blood. That this is accompanied by a marked rise in mitochondrial number of blood lymphocytes, is the most important finding in the present study. In some instances, palsma cells which are normally present in

5 A Lymphocytic Hemogram and Lymphocytopoiesis III 309 variable numbers became very numerous in the medullarly cords of the mesenteric lymph node after injection of ovalbumin ; while in others a marked degree of sinus histiocytosis was observed without any remarkable increase in number of plasma cells in the medullary cords of the mesenteric node. The reason for this inconstancy in the mode of reaction of the medullary tissue to ovalbumin is unknown. Discussion In the earlier publications from our laboratory, it has been claimed that a marked rise in mitochondrial number of blood lymphocytes may be correlated with an extensive new formation of F I e m m i n g's secondary nodules in the lymphoid organs, which, in turn, indicates an increase in production and delivery of lymphocytes from these organs to the blood. This is based on the following observations : (1) In the normal course of the postnatal development of lymphoid organs in the rabbit and rat, there was observed a significant transient elevation of the average number of mitochondria per lymphocyte in the blood, almost coincident with an extensive new formatien of F 1 m m i n g's secondary nodules in the lymphatic tissue, during the period between 3 weeks and 2 months after birth. It was also disclosed' that the newly formed Fl e m m i n g's secondary nodules were initially centers of active proliferation of medium-sized lympocytes, and that only later they became " reaction centers " of Hellman (Osogoe et al., 1953; Awaya, 1956; and Imamura, 1958a). (2) In the earlier stages of the regenerative processes of the lymphatic tissue of young adult rats that had received 600r of total body X-irradiation, there also appeared a significant transient rise in mitochondrial nun'iber of blood lymphocytes and a simultaneous extensive new formation of F 1 e m m i n g's secondary nodules in the lymphoid organs (I ma mu r o, 1958b). (3) Single or repeated intravenous injection of ovalbumin into adult rabbits also produced a remarkable transient rise in mitochondrial content of blood lymphocytes, which was accompanied by an extensive new formation of F I e m m i n g's nodules in the lymphoid organs, during the period from 3 to 7 days after the initial injection. In addition to new formation of Flemmin g's nodules, the " active " forms having a large germinal center, which is composed of densely packed, actively dividing medium-sized lymphocytes, were markedly increased in percentage among the pre-existing Flemmin g's nodules (A w a y a,

6 310 Hidetoshi Imamura 1956). The chief findings of the present study generally agree with the earlier observations, particularly those of A way a (1956), in that a marked elevation of the average number of mitochondria in blood lymphocytes is associated with new formation of F 1 e m m i n g's nodules in the lymphoid organs. However, there are some differences between the present and the earlier observations. It should be pointed out, first, that in the present experiments with ovalbumin, enlargement of the pre-existing F l m m i n g's nodules predominated over new formation of these nodules and, secondly, that after a single injection of ovalbumin there occurred a significant rise in mitochondrial number of blood lymphocytes without any noticeable hyperplastic change in the lymphocytopoietic organs.' Of particular interest in this connection is the finding that, in response to daily injections of ovalbumin, the average number of mitochondria in blood lymphocytes was significantly elevated already on the 3rd day, whereas the number of these cells did not show any significant rise until the 7th day. This indicates that the average number of mitochondria in blood lymphocytes varies very promptly in response to stimuli. Theoretically, it is also conceivble that a rise in mitochondrial content of blood lymphocytes, primarily linked with an increase in mobilization and not in production of lymphocytes in the lymphoid organs, may take place under special circumstances. Our collaborator I t o is now making an extensive study on this possibility. The results so far obtained seem to prove that this possibility really takes place. In conclusion, it may be stated that a rise in mitochondrial number of blood lymphocytes takes place as a consequence of an increase in younger, mitochondria-rich lymphocytes in the circulating blood, and that such a condition is most commonly associated with a hyperplaia of the lymphatic tissue, the F 1 e m m i n g's secondary nodules in particular. Summary 1) In an attempt to gain further information concerning the relationship between the lymphocytic blood picture and the lymphocytopoietic activites of the lymphoid organs, variations in mitochondrial number in blood lymphocytes were pursued in relation to histological chages of the lympbatic tissue after single or repeated

7 A Lymphocytic Hemogram and Ly mphocytopoiesis III 311 injections of ovalbumin into young adult male albino rats weighing around 200g. 2) After a single intraperitoneal injection of 20 mg of ovalbumin, the average number of mitochondria per lymphocytes in the blood was significantly elevated already on the 3rd day and thereafter tended to decline ; whilst the absolute number of blood lymphecytes showed no increase. In the mesenteric lymph nodes and spleen, there was observed no remarkable alteration in the lymphatic tissue after a single injection. 3) After daily intraperitoneal injections of 20 mg of ovalbumin, both the average number of mitochondria and the absolute number of blood lymphocytes had increased considerably. It was noticed, however, that a marked elevation in mitochondrial number of blood lymphocytes occurred already on the 3rd day, whereas the number of these cells did not show any noticeable increase until the 7th day after the initial injection. In the mesenteric lymph nodes and spleen, there was a marked hyperplasia of the lymphatic tissue, the Fl e mm in g's secondary nodules in particular. Besides, new formation of these nodules, though on a smaller scale, was also recognized after repeated injections. 4) On the basis of these findings, it is concluded that a marked elevation of the average number of mitochondria in blood lymphocytes is associated with a hyperplasia of the lymyhatic tissue, and especially of the F l e m m i n g's secondary nodules. However the possibility is also suggested, at the same time, that a rise in mitochondrial content of blood lymphocytes may occur without any noticeable hyperplastic change of the lymphocytopoietic tissue. References Away a, K The mode of reaction of the lymphatic system to ovalbumin, with remarks on the role of secondary nodules in lymphocytopoiesis. Bull. Yamaguchi Med. Sch., 3: F u k a s e, M Experimental studies on the lymphocyte mitochondria. III. Variations in mitochondrial content of lymphocytes in the case of lymphocytosis. Acta haematol. jap., 12: I m a m u r a, H a Further studies of a lymphocytic hemogram and its 'bl relation to lymphocytopoiesis. I. Variations in mitochondrial content of ood lymphocytes in relation to the postnatal development of the lymphatic apparatus in the rat. Okajimas Fol. anat. jap., 32 (in press). I m a m u r a, H b Further studies of a lymphocytic hemogram and its relation to lymphocytopoiesis. II. Variations in mitochodrial content of blood lymphocytes in relation to the processes of regeneration of the Iym-

8 312 ilidetoshi Imamura phatic apparatus of rats after total body X-irradiation. Okajimas Fol. anat. jap., 32 (in press). It o, H. Unpublished observations. Os o go e, B., L. C. Chang, K. A w a y a and K. Karasawa 1953 Variation in mitochondrial content of lymphocytes in the peripheral blood in relation to postnatal development of lymphoid organs in the rabbit. Bull. Yamaguchi Med. Sch., 1: W is em a n, B. K The induction of lymphocytosis and lymphatic hyperplasia by means of parenterally administered protein. J. Exp. Med., 53: W i se ma n, B. K., C. A. Doan and L. A. E r f 1936 A fundamental, reciprocal relationship between myeloid and lymphoid tissues. J. A. M. A., 106 : Explanation of Plate-figures Plate I Fig. 1. Mesenteric lymph node of a control rat (Rat No. 28) that received 6 daily intraperitoneal injections of 2 ml of saline. x 40. Fig. 2. Mesenteric lymph node of Rat No. 35 that received 6 daily intraperitoneal injections of 20 mg of ovalbumin dissolved in 2 ml of saline. The F 1 e m m i n g's secondary nodules are markedly enlarged. x40. Fig. 3. Mesenteric lymph node of Rat No. 36 treated with ovalbumin in the same manner as in Rat No. 35 illustrated in Fig. 2. Notice several greatly enlarged, mature F I e m min g's secondary nodules and a few developing, immature nodules of the F 1 e m m i n g type. x 40.

9 Plate I H. Imamura