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Morphology of antibody-forming cells in young and aged experimental animals
Mechanisms of Ageing and Development
Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
M O R P H O L O G Y OF A N T I B O D Y - F O R M I N G A G E D E X P E R I M E N T A L ANIMALS
CELLS IN Y O U N G
AND
EDIT BEREGI Research Department of Gerontology, Medical University of Budapest, Budapest (Hungary)
(Received January 24th, 1972) (Revised manuscript received May 8th, 1972)
SUMMARY The morphology of the primary immunological reaction was studied in the popliteal lymph nodes of rats 6 days after immunisation with bovine serum albumin. The lymph-node indices of young animals significantly increased, and light-microscopic examination showed the activity of the germinal centers of the follicles following immunisation. These changes were not observed in old animals. Analysis of the cells of the germinal centers of the follicles by light and electron microscopy, showed immunoblasts and transitory cells in both young and old animals, but the number of cytoplasmic organelles proved to be decreased in the old animals; degenerative changes could be also observed in the cytoplasmic organelles. Mature and immature plasma cells could be found both in the young and old animals, but the number of these cells proved to be much less in the old rats than in the young ones. Earlier immunopathologic investigations of the author had also shown a decreased number of antibody-containing cells in old animals. On the basis of her experimental results, the author hypothesised inhibition of immunoblast transformation in old animals, which may be related to the degenerative changes described; this inhibited transformation may explain the decreased number of antibody-producing cells in aged animals.
INTRODUCTION In most publications dealing with antibody formation, antibody formation in young individuals is dealt with. It can be stated on the basis of data in the literature that immunocapacity rapidly increases during neonatal and juvenile life and reaches its peak in the young-adult stage; it remains constant for a while, then decreases during aging of the individual 1. Mech. Age. Dev., 1 (1972) 233-244
233
Three changes in connection with aging should be mentioned here: (i) The quantity of natural gamma globulins (i.e. without antigen stimulus) increases with aging2 5; (ii) Immune reactions to extrinsic antigens (of bacterial and other origin) decrease6-10; (iii) Antibody production to endogenous antigens increases with aging (rheumatoid factor, antinuclear antibody etc.)L In my earlier investigations I have studied the immunological reactions to extrinsic antigens in young and old animals. In these investigations I compared some morphological manifestations of early and delayed allergic reactions, of experimental glomerulonephritis in autoimmune diseases and of antibody formation (primary reaction) in young and old animals. A very marked morphological difference could be seen in all these reactive alterations. By histologic and electronmicroscopic examination a decreased cellular reaction and morphologic signs of decreased immunological activity could be observed in the aged animals it la. After immunisation, the draining popliteal lymph-node index proved to be significantly higher in young animals than in old ones. By immunofluorescence investigations a larger number of antibody-producing cells could be demonstrated in the lymph nodes of the young animals than in those of the old ones la. The results of these experiments raised the question of whether the decrease in antibody production during old age could have been caused by the finer structural changes of the immunocompetent cells, i.e. by changes in the cytoplasmic organelles inhibiting the transformation of immunocompetent cells into plasma cells. MATERIALS AND METHODS Ninety Wistar rats were used for the experiments. Forty of the 90 rats served as controls. Twenty of the 40 control rats were 6-months old and 20 were 25-months old. To twenty 6-month-old and thirty 25-month-old rats, bovine serum albumin (10 mg/g body weight) in 0.3 ml Freund's adjuvant was injected into the footpad. The primary immune reaction in the draining popliteal lymph node was studied 6 days after the immunisation. The lymph-node index [(weight of lymph node (mg) × 100) -- body weight (kg)] was determined in both the immunised and control animals. Small parts of the cortical regions of the lymph nodes freshly dissected out were fixed in osmium tetroxide after short fixation in cold glutaraldehyde, then embedded in Araldite. Sections were cut using a Reichert ultramicrotome, stained with lead citrate and then studied in a Hitachi Hu 10 electron microscope. The remaining parts of the lymph nodes were prepared for light-microscopic and immunofluorescence investigations by the method of Sainte-Marie 14. The slides were incubated with anti-rat gamma-globulin conjugated to ftuorescein isothiocyanate (Sylvana Chemical Co., New Jersey, U.S.A.) and with bovine serum albumin conjugated to fluorescein isothiocyanate to demonstrate the antibody-producing cells. Light microscopicexaminations were carried out with an Opton microscope; an HBO 200W lamp was used for the immunofluorescent studies.
234
Mech. Age. Dev., 1 (1972) 233-244
TABLE I LYMPH-NODE INDICES OF GROUPS I-IV Statistical analysis: Groups I-H, not significant; Groups 1-III, P < 0.001; Groups ll-IV, not significant; Groups Ill-IV, 0.02 :> P ~ 0.01.
Age (months) No. of animals Treatment Average value of lymph-node indices
Group I
Group 11
Group 1H
Group IV
6 20 --
25 20 --
6 20 BSA*
25 20 BSA
19.5
10.4
7.2
7.2
* Sensitization with bovine serum albumin.
EXPERIMENTAL RESULTS Table I shows the l y m p h - n o d e indices o f the different groups. It can be seen that there is no significant difference b e t w e e n the indices o f the y o u n g and old rats o f the c o n t r o l group. T h e r e is, however, a significant difference between the indices o f the y o u n g rats o f the i m m u n i s e d and c o n t r o l g r o u p s : the l y m p h - n o d e indices o f the i m m u n i s e d y o u n g rats were m a r k e d l y increased. T h e r e is no significant difference
Fig. 1. A number of large cells in the lymph node of a young rat showing fluorescence in the cytoplasm.
Mech. Age. Dev., I (1972) 233-244
235
Fig. 2. Electronmicrograph of an immunoblast in the follicle of a young rat, after immunisation. In the cytoplasm of the immunoblast some swollen mitochondria (Mi) are visible as well as a number of ribosomes (Ri) and polysomes (Po). N, nucleus.
again between the indices of the old rats of the immunised and control groups. By comparing the indices of the young and old rats of the immunised group, however, a significantly higher value for the young rats could be observed. Under histologic examination large germinal centers could be seen in the follicles of the lymph nodes of the immunised young rats; on the contrary, such germinal centers could hardly be seen in the follicles of the lymph nodes of the old animals. In the germinal centers of the lymph nodes of the young rats a marked polymorphism could be observed; besides lymphoblasts and macrophages many large immature plasma cells with eccentric nuclei could be seen. In the germinal centers of the follicles of the old rats only a few lymphoblasts and immature plasma cells could be seen besides the lymphocytes. In the follicles of the young animals a number 236
Mech. Age. Dev., 1 (1972) 233 244
l
Fig. 3. Electronmicrograph showing only a few small mitochondria (Mi), with dense matrix and undeveloped cristae, in the cytoplasm of an immunoblast in the follicle of an old rat. N, nucleus; Ri, ribosome; Po, polysome.
of cells with a broad cytoplasm showing specific fluorescence by immunofluorescence investigation could be seen (Fig. 1). Under electronmicroscopic investigation a number of plasma ceils of different maturity could be demonstrated in the germinal centers of the lymphoid follicles of the young rats besides cytoplasmic fragments and degrading cell organelles, immunoblasts and lymphoblasts. In the old animals plasma cells could scarcely be seen, only lymphocytes and a few immunoblasts and lymphoblasts. F r o m analysis of the cells and cell organelles at high magnification the following data could be obtained. Immunoblasts of young animals have large nuclei and in the cytoplasm a number of mitochondria can be seen besides many ribosomes and polysomes (Fig. 2). Immunoblasts could also be seen in the follicles of the old animals; their nuclei were similar to those of young rats. However, we could see in the follicles Mech. Age. Dev., 1 (1972) 233-244
237
Fig. 4. lmmunoblast of an old rat, showing swollen mitochondria (Mi) in the cytoplasm. Degenerative changes in the mitochondria such as the formation of myelin-like structures (M) and disintegration of the cristae are visible. Other abbreviations as in previous figures.
Of the old rats immunoblasts with cytoplasmic invaginations to the nuclei and with myelin-like structures in the invaginated cytoplasmic particles. In the cytoplasm, ribosomes and polysomes could be observed also in the cells, but we gained the impression that in some cases the immunoblasts o f the old animals contained less mitochondria, and some of these had undeveloped cristae and a dense matrix (Fig. 3). The mitochondria of some o f these cells were swollen; myelin-like structures and disappearance o f the cristae were also noted (Fig. 4). Similar degenerative changes could not be observed in the y o u n g animals. Cells transitory between lymphocytes and large blastocytes 1~ could be seen in both y o u n g and old animals. In these cells of" the y o u n g rats, many cytoplasmic organelles with normal structure and nucleoli showing marked activity could be seen (Fig. 5). In the cells of the old animals, the n u m b e r o f cytoplasmic organelles was less. 238
Mech. Age. Dev., 1 (1972) 233-244
Fig. 5. Lymphoblast of a young rat. The nucleolus (Nu) shows a high activity; a developed Golgi apparatus (Go) and some endoplasmic reticulum can be seen in the cytoplasm beside a number of ribosomes and polysomes. Abbreviations as in previous figures.
There were cytoplasmic invaginations in the nuclei, and degenerative changes in the cytoplasm could also be seen (Figs. 6 and 7). We found cytolysosomes and emperipolesis in some reticular cells in the old rats. A large number of plasma cells of different maturity could be demonstrated in the young animals; on the contrary, in old animals only a few such cells could be seen. A comparison of the immature plasma cells of young and old animals showed that the cells of the young animals had a marked activity. Signs of micropyknocytosis16 were apparent in the cytoplasm, and a large number of cytoplasmic organelles with intact structure could be also seen. Mature plasma cells of young animals have endoplasmic reticula forming broad cisternae and containing granular material (Fig. 8). Only a few immature plasma cells could be observed in the follicles of the old rats and in these cells degenerative changes, lipid drops in the cytoplasm and myelin-like Mech. Age. Dev., 1 (1972) 233-244
239
Fig. 6. Lymphoblast of an old rat. Note the cytoplasmic invagination (Cy) in the nucleus. The number of cytoplasmic organelles is low, but a lipid drop (L) can be seen. End, endoplasmic reticulum. Other abbreviations as in previous figures.
structures could be frequently demonstrated (Fig. 9). Similar changes were seen in the plasma cells too. Plasma cells with broad cisternae could not be detected in the old animals. In this paper we do not deal with the histologic and electronmicroscopic structure o f normal, control animals, but refer the reader to other publications17,18. DISCUSSION AND CONCLUSIONS M a k i n o d a n and Peterson 6 described that after immunisation the antibodyproducing capacity o f spleen cells o f old mice proved to be only one-quarter that of 8-month-old mice. They explained this observation by the diminishing o f the potential antibody-producing cells in relation to aging. In addition, it was stated 240
Mech. Age. Dev., 1 (1972) 233-244
Fig. 7. Cytoplasmic invagination into the nucleus of a lymphoblast of an old rat. In the cytoplasm a swollen and cleared-up mitochondrion, and a myelin-like structure can be seen besides ribosomes and polysomes. Abbreviations as in previous figures.
that the number of 7S immunglobulin molecules decreased to a greater extent than that of the 19S molecules, i.e. immunological potency returned to the embryonic and neonatal level, which is also characterized by the synthesis of 19S molecules. H a n n a et al. 19 showed that the decrease of immunological potency is not only caused by the decrease of immunocompetent cells in aging, but that changes in the microenvironment, and in the reticular elements of the spleen, thymus, lymph nodes and bone marrow also play a major role in this phenomenon. A study of the morphology of these elements is planned for the future. White 20 showed that immunocompetent cells are situated in the cortex of the lymph nodes and spleen, mostly in the germinal centers. Antigen-containing cells appear also in this region, according to many authors. On the basis of these observations a structural and functional relation between antigen-containing cells and immunocompetent cells was hypothesised by White 2°. Mech. Age. Dev., I (1972) 233-244
241
Fig. 8. Developed rough endoplasmic reticulum forming broad cisternae and containing a granular material (G)in the cytoplasm (C)of the plasma cell of a young rat. Other abbreviations as in previous figures,
The cause of the diminished number of antibody-producing cells of old animals can be explained by the known fact that the capacity of adaptation markedly decreases in old age and therefore reacts only weakly to the immunising stimulus. On the other hand, the proliferative capacity of the cells also decreases in old age as shown by Letterer 21, and Beneke, Finger and Emmerling z2. In our earlier inves242
Mech. Age. Dev., 1 (1972) 233-244
Fig. 9. Rough endoplasmic reticulum in the cytoplasm of the plasma cell of an old rat and myelinlike structure formation. tigations we also described a reduction of allergic inflammations in old animalsaL These data from the literature explain the results of the experiment described in this paper, that the lymph-node indices of old animals did not significantly change and the germinal centers of the follicles did not prove to be active after primary immunisation. On the contrary, however, the lymph-node indices of young rats increased significantly and the germinal centers of the follicles became active after primary immunisation. We can state from the electronmicroscopic investigations that fewer mitochondria can be observed in the immunoblasts and transitory cells of old animals than in young ones, and that the mitochondria of old animals show morphologic signs of degeneration. Nuclear invagination and degenerative changes were seen in the cytoplasm of the cells of the old animals. Similar changes have been described in the livers of aged mice 2a. These observations and the experimental result that Mech. Age. Dev., 1 (1972) 233-244
243
mature and immature plasma cells of old animals were found only in a small number 6 days after the primary immunisation, can be explained by the inhibited transformation of the immunoblasts, which can be related to the degenerative changes described. Our observations help to understand the decrease in number of antibodyproducing cells in old animals. REFERENCES 1 J. Sri Ram, Aging and immunological phenomena - - a review, J. Gerontol., 22 (1967) 92. 2 J. L. Karel, Y. M. Wilder and M. Beber, Electrophoretic serum patterns in the aged, J. Am. Geriatr. Soc., 4 (1956) 667. 3 I. Haferkamp, D. Schlettwein-Gsell, H. G. Schwick and K. St6riko, Serum protein in an aging population with particular reference to evaluation of immune globulins and antibodies, Gerontologia, Basel, 12 (1966) 30. 4 B. C. Das and S. K. Bhattacharya, Change in human serum protein fractions with age and weight, Can. J. Biochem. Physiol., 39 (1961) 569. 5 Ph. Goullet and H. Kaufmann, Aging and antibody production in the rat, Experientia, 21 (1965) 46. 6 T. Makinodan and W. J. Peterson, Further studies on the secondary antibody-forming potential of juvenile, young adult and aged mice, Dev. Biol., 14 (1966) 112. 7 J. F. Albright and T. Makinodan, Growth and senescence of antibody forming cells, J. Cell. Comp. Physiol., (Suppl. 1) 67 (1966) 185. 8 D. Metcalf, R. Moulds and B. Pike, Influence of the spleen and thymus on immune responses in aging mice, Clin. Exp. lmmunol., 2 (1966) 109. 9 M. J. Rowley, H. Buchanan and [. R. Mackay, Reciprocal changes with age in antibody to extrinsic and intrinsic antigens, l ancet, 11 (1968) 24. 10 S. Kishimoto, 1. Tsuyuguchi and Yuichi Yamamura, Immune response in aged mice, Clht. Exp. lmmunol., 5 (1969) 525. 11 E. Beregi, J. Simon and 1. F61des, Anaphylaxis of rabbits and rats of different ages, Gerontologia, Basel, 10 (1964-65) 183. 12 E. Beregi, Vergleichende immunmorpbologische Untersuchungen bei Arthus-Reaction in verschiedenen Alter, Gerontologia, Basel, 15 (1969) 256. 13 E. Beregi, Comparative morphological studies of the allergic reactions of young and old animals, Gerontologia, Basel, 16 (1970) 141. 14 G. Sainte-Marie, A paraffin embedding technique for studies employing immunofluorescence, J. Histochem. Cytochem., 10 (1962)250. 15 H. H. Movat and N. V. P. Fernando, The fine structure of the lymphoid tissue during antibody formation, Exp. Mol. Pathol., 4 (1965) 155. 16 W. Bernhard and N. Grandboulan, Ultrastructure of immunologically competent cells, in G. E. W. Wolstenholme and Maeve O'Connor (Eds.), Ciba Foundation Symposium on Celhdar Aspects c~flmmunity, Churchill, London, 1960, p. 92. 17 G. Y. V. Nossal and G. L. Ada, Antigens, Lymphoid Cells" and the Immune Response, Academic Press, New York and London, 1971, p. 69. 18 E. Grundmann, Cytologische Untersuchungen fiber Formen und Orte der kymphocytenreifung bei der Ratte, Verh. Dtseh. Ges. Pathol., 41 (1958) 261. 19 M. G. Hanna, Jr., P. Nettesheim and L. C. Peters, Evidence of functional microenvironments in lymphatic tissue response to antigen, Nature New Biol., 232 (1971) 204. 20 R. G. White, Functional recognition of immunologically competent cells by means of the fluorescent antibody technique, in G. E. W, Wolstenholme and J. Knight (Eds.), The hnmunologieally Competent Cell. Its Nature and Origin, Ciba Foundation Study Group No. 16., Churchill, London, 1963, p. 6. 21 E. Letterer, A[tern und Krankheit, Dtsch. Med. Woehensehr., 79 (1954) 1473. 22 G. Beneke, H. Finger and P. Emmerling, Einfluss von Bordetella pertussis auf das lymphatische Gewebe von M~usen, Z. Med. Microbiol. lmmunol., 154 (1968) 179. 23 W. Andrew, An electronmicroscope study of age changes in the liver of mouse, Anat. Ree., 139 (1961) 203.
244
Mech. Age. Dev., 1 (1972) 233-244
Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
M O R P H O L O G Y OF A N T I B O D Y - F O R M I N G A G E D E X P E R I M E N T A L ANIMALS
CELLS IN Y O U N G
AND
EDIT BEREGI Research Department of Gerontology, Medical University of Budapest, Budapest (Hungary)
(Received January 24th, 1972) (Revised manuscript received May 8th, 1972)
SUMMARY The morphology of the primary immunological reaction was studied in the popliteal lymph nodes of rats 6 days after immunisation with bovine serum albumin. The lymph-node indices of young animals significantly increased, and light-microscopic examination showed the activity of the germinal centers of the follicles following immunisation. These changes were not observed in old animals. Analysis of the cells of the germinal centers of the follicles by light and electron microscopy, showed immunoblasts and transitory cells in both young and old animals, but the number of cytoplasmic organelles proved to be decreased in the old animals; degenerative changes could be also observed in the cytoplasmic organelles. Mature and immature plasma cells could be found both in the young and old animals, but the number of these cells proved to be much less in the old rats than in the young ones. Earlier immunopathologic investigations of the author had also shown a decreased number of antibody-containing cells in old animals. On the basis of her experimental results, the author hypothesised inhibition of immunoblast transformation in old animals, which may be related to the degenerative changes described; this inhibited transformation may explain the decreased number of antibody-producing cells in aged animals.
INTRODUCTION In most publications dealing with antibody formation, antibody formation in young individuals is dealt with. It can be stated on the basis of data in the literature that immunocapacity rapidly increases during neonatal and juvenile life and reaches its peak in the young-adult stage; it remains constant for a while, then decreases during aging of the individual 1. Mech. Age. Dev., 1 (1972) 233-244
233
Three changes in connection with aging should be mentioned here: (i) The quantity of natural gamma globulins (i.e. without antigen stimulus) increases with aging2 5; (ii) Immune reactions to extrinsic antigens (of bacterial and other origin) decrease6-10; (iii) Antibody production to endogenous antigens increases with aging (rheumatoid factor, antinuclear antibody etc.)L In my earlier investigations I have studied the immunological reactions to extrinsic antigens in young and old animals. In these investigations I compared some morphological manifestations of early and delayed allergic reactions, of experimental glomerulonephritis in autoimmune diseases and of antibody formation (primary reaction) in young and old animals. A very marked morphological difference could be seen in all these reactive alterations. By histologic and electronmicroscopic examination a decreased cellular reaction and morphologic signs of decreased immunological activity could be observed in the aged animals it la. After immunisation, the draining popliteal lymph-node index proved to be significantly higher in young animals than in old ones. By immunofluorescence investigations a larger number of antibody-producing cells could be demonstrated in the lymph nodes of the young animals than in those of the old ones la. The results of these experiments raised the question of whether the decrease in antibody production during old age could have been caused by the finer structural changes of the immunocompetent cells, i.e. by changes in the cytoplasmic organelles inhibiting the transformation of immunocompetent cells into plasma cells. MATERIALS AND METHODS Ninety Wistar rats were used for the experiments. Forty of the 90 rats served as controls. Twenty of the 40 control rats were 6-months old and 20 were 25-months old. To twenty 6-month-old and thirty 25-month-old rats, bovine serum albumin (10 mg/g body weight) in 0.3 ml Freund's adjuvant was injected into the footpad. The primary immune reaction in the draining popliteal lymph node was studied 6 days after the immunisation. The lymph-node index [(weight of lymph node (mg) × 100) -- body weight (kg)] was determined in both the immunised and control animals. Small parts of the cortical regions of the lymph nodes freshly dissected out were fixed in osmium tetroxide after short fixation in cold glutaraldehyde, then embedded in Araldite. Sections were cut using a Reichert ultramicrotome, stained with lead citrate and then studied in a Hitachi Hu 10 electron microscope. The remaining parts of the lymph nodes were prepared for light-microscopic and immunofluorescence investigations by the method of Sainte-Marie 14. The slides were incubated with anti-rat gamma-globulin conjugated to ftuorescein isothiocyanate (Sylvana Chemical Co., New Jersey, U.S.A.) and with bovine serum albumin conjugated to fluorescein isothiocyanate to demonstrate the antibody-producing cells. Light microscopicexaminations were carried out with an Opton microscope; an HBO 200W lamp was used for the immunofluorescent studies.
234
Mech. Age. Dev., 1 (1972) 233-244
TABLE I LYMPH-NODE INDICES OF GROUPS I-IV Statistical analysis: Groups I-H, not significant; Groups 1-III, P < 0.001; Groups ll-IV, not significant; Groups Ill-IV, 0.02 :> P ~ 0.01.
Age (months) No. of animals Treatment Average value of lymph-node indices
Group I
Group 11
Group 1H
Group IV
6 20 --
25 20 --
6 20 BSA*
25 20 BSA
19.5
10.4
7.2
7.2
* Sensitization with bovine serum albumin.
EXPERIMENTAL RESULTS Table I shows the l y m p h - n o d e indices o f the different groups. It can be seen that there is no significant difference b e t w e e n the indices o f the y o u n g and old rats o f the c o n t r o l group. T h e r e is, however, a significant difference between the indices o f the y o u n g rats o f the i m m u n i s e d and c o n t r o l g r o u p s : the l y m p h - n o d e indices o f the i m m u n i s e d y o u n g rats were m a r k e d l y increased. T h e r e is no significant difference
Fig. 1. A number of large cells in the lymph node of a young rat showing fluorescence in the cytoplasm.
Mech. Age. Dev., I (1972) 233-244
235
Fig. 2. Electronmicrograph of an immunoblast in the follicle of a young rat, after immunisation. In the cytoplasm of the immunoblast some swollen mitochondria (Mi) are visible as well as a number of ribosomes (Ri) and polysomes (Po). N, nucleus.
again between the indices of the old rats of the immunised and control groups. By comparing the indices of the young and old rats of the immunised group, however, a significantly higher value for the young rats could be observed. Under histologic examination large germinal centers could be seen in the follicles of the lymph nodes of the immunised young rats; on the contrary, such germinal centers could hardly be seen in the follicles of the lymph nodes of the old animals. In the germinal centers of the lymph nodes of the young rats a marked polymorphism could be observed; besides lymphoblasts and macrophages many large immature plasma cells with eccentric nuclei could be seen. In the germinal centers of the follicles of the old rats only a few lymphoblasts and immature plasma cells could be seen besides the lymphocytes. In the follicles of the young animals a number 236
Mech. Age. Dev., 1 (1972) 233 244
l
Fig. 3. Electronmicrograph showing only a few small mitochondria (Mi), with dense matrix and undeveloped cristae, in the cytoplasm of an immunoblast in the follicle of an old rat. N, nucleus; Ri, ribosome; Po, polysome.
of cells with a broad cytoplasm showing specific fluorescence by immunofluorescence investigation could be seen (Fig. 1). Under electronmicroscopic investigation a number of plasma ceils of different maturity could be demonstrated in the germinal centers of the lymphoid follicles of the young rats besides cytoplasmic fragments and degrading cell organelles, immunoblasts and lymphoblasts. In the old animals plasma cells could scarcely be seen, only lymphocytes and a few immunoblasts and lymphoblasts. F r o m analysis of the cells and cell organelles at high magnification the following data could be obtained. Immunoblasts of young animals have large nuclei and in the cytoplasm a number of mitochondria can be seen besides many ribosomes and polysomes (Fig. 2). Immunoblasts could also be seen in the follicles of the old animals; their nuclei were similar to those of young rats. However, we could see in the follicles Mech. Age. Dev., 1 (1972) 233-244
237
Fig. 4. lmmunoblast of an old rat, showing swollen mitochondria (Mi) in the cytoplasm. Degenerative changes in the mitochondria such as the formation of myelin-like structures (M) and disintegration of the cristae are visible. Other abbreviations as in previous figures.
Of the old rats immunoblasts with cytoplasmic invaginations to the nuclei and with myelin-like structures in the invaginated cytoplasmic particles. In the cytoplasm, ribosomes and polysomes could be observed also in the cells, but we gained the impression that in some cases the immunoblasts o f the old animals contained less mitochondria, and some of these had undeveloped cristae and a dense matrix (Fig. 3). The mitochondria of some o f these cells were swollen; myelin-like structures and disappearance o f the cristae were also noted (Fig. 4). Similar degenerative changes could not be observed in the y o u n g animals. Cells transitory between lymphocytes and large blastocytes 1~ could be seen in both y o u n g and old animals. In these cells of" the y o u n g rats, many cytoplasmic organelles with normal structure and nucleoli showing marked activity could be seen (Fig. 5). In the cells of the old animals, the n u m b e r o f cytoplasmic organelles was less. 238
Mech. Age. Dev., 1 (1972) 233-244
Fig. 5. Lymphoblast of a young rat. The nucleolus (Nu) shows a high activity; a developed Golgi apparatus (Go) and some endoplasmic reticulum can be seen in the cytoplasm beside a number of ribosomes and polysomes. Abbreviations as in previous figures.
There were cytoplasmic invaginations in the nuclei, and degenerative changes in the cytoplasm could also be seen (Figs. 6 and 7). We found cytolysosomes and emperipolesis in some reticular cells in the old rats. A large number of plasma cells of different maturity could be demonstrated in the young animals; on the contrary, in old animals only a few such cells could be seen. A comparison of the immature plasma cells of young and old animals showed that the cells of the young animals had a marked activity. Signs of micropyknocytosis16 were apparent in the cytoplasm, and a large number of cytoplasmic organelles with intact structure could be also seen. Mature plasma cells of young animals have endoplasmic reticula forming broad cisternae and containing granular material (Fig. 8). Only a few immature plasma cells could be observed in the follicles of the old rats and in these cells degenerative changes, lipid drops in the cytoplasm and myelin-like Mech. Age. Dev., 1 (1972) 233-244
239
Fig. 6. Lymphoblast of an old rat. Note the cytoplasmic invagination (Cy) in the nucleus. The number of cytoplasmic organelles is low, but a lipid drop (L) can be seen. End, endoplasmic reticulum. Other abbreviations as in previous figures.
structures could be frequently demonstrated (Fig. 9). Similar changes were seen in the plasma cells too. Plasma cells with broad cisternae could not be detected in the old animals. In this paper we do not deal with the histologic and electronmicroscopic structure o f normal, control animals, but refer the reader to other publications17,18. DISCUSSION AND CONCLUSIONS M a k i n o d a n and Peterson 6 described that after immunisation the antibodyproducing capacity o f spleen cells o f old mice proved to be only one-quarter that of 8-month-old mice. They explained this observation by the diminishing o f the potential antibody-producing cells in relation to aging. In addition, it was stated 240
Mech. Age. Dev., 1 (1972) 233-244
Fig. 7. Cytoplasmic invagination into the nucleus of a lymphoblast of an old rat. In the cytoplasm a swollen and cleared-up mitochondrion, and a myelin-like structure can be seen besides ribosomes and polysomes. Abbreviations as in previous figures.
that the number of 7S immunglobulin molecules decreased to a greater extent than that of the 19S molecules, i.e. immunological potency returned to the embryonic and neonatal level, which is also characterized by the synthesis of 19S molecules. H a n n a et al. 19 showed that the decrease of immunological potency is not only caused by the decrease of immunocompetent cells in aging, but that changes in the microenvironment, and in the reticular elements of the spleen, thymus, lymph nodes and bone marrow also play a major role in this phenomenon. A study of the morphology of these elements is planned for the future. White 20 showed that immunocompetent cells are situated in the cortex of the lymph nodes and spleen, mostly in the germinal centers. Antigen-containing cells appear also in this region, according to many authors. On the basis of these observations a structural and functional relation between antigen-containing cells and immunocompetent cells was hypothesised by White 2°. Mech. Age. Dev., I (1972) 233-244
241
Fig. 8. Developed rough endoplasmic reticulum forming broad cisternae and containing a granular material (G)in the cytoplasm (C)of the plasma cell of a young rat. Other abbreviations as in previous figures,
The cause of the diminished number of antibody-producing cells of old animals can be explained by the known fact that the capacity of adaptation markedly decreases in old age and therefore reacts only weakly to the immunising stimulus. On the other hand, the proliferative capacity of the cells also decreases in old age as shown by Letterer 21, and Beneke, Finger and Emmerling z2. In our earlier inves242
Mech. Age. Dev., 1 (1972) 233-244
Fig. 9. Rough endoplasmic reticulum in the cytoplasm of the plasma cell of an old rat and myelinlike structure formation. tigations we also described a reduction of allergic inflammations in old animalsaL These data from the literature explain the results of the experiment described in this paper, that the lymph-node indices of old animals did not significantly change and the germinal centers of the follicles did not prove to be active after primary immunisation. On the contrary, however, the lymph-node indices of young rats increased significantly and the germinal centers of the follicles became active after primary immunisation. We can state from the electronmicroscopic investigations that fewer mitochondria can be observed in the immunoblasts and transitory cells of old animals than in young ones, and that the mitochondria of old animals show morphologic signs of degeneration. Nuclear invagination and degenerative changes were seen in the cytoplasm of the cells of the old animals. Similar changes have been described in the livers of aged mice 2a. These observations and the experimental result that Mech. Age. Dev., 1 (1972) 233-244
243
mature and immature plasma cells of old animals were found only in a small number 6 days after the primary immunisation, can be explained by the inhibited transformation of the immunoblasts, which can be related to the degenerative changes described. Our observations help to understand the decrease in number of antibodyproducing cells in old animals. REFERENCES 1 J. Sri Ram, Aging and immunological phenomena - - a review, J. Gerontol., 22 (1967) 92. 2 J. L. Karel, Y. M. Wilder and M. Beber, Electrophoretic serum patterns in the aged, J. Am. Geriatr. Soc., 4 (1956) 667. 3 I. Haferkamp, D. Schlettwein-Gsell, H. G. Schwick and K. St6riko, Serum protein in an aging population with particular reference to evaluation of immune globulins and antibodies, Gerontologia, Basel, 12 (1966) 30. 4 B. C. Das and S. K. Bhattacharya, Change in human serum protein fractions with age and weight, Can. J. Biochem. Physiol., 39 (1961) 569. 5 Ph. Goullet and H. Kaufmann, Aging and antibody production in the rat, Experientia, 21 (1965) 46. 6 T. Makinodan and W. J. Peterson, Further studies on the secondary antibody-forming potential of juvenile, young adult and aged mice, Dev. Biol., 14 (1966) 112. 7 J. F. Albright and T. Makinodan, Growth and senescence of antibody forming cells, J. Cell. Comp. Physiol., (Suppl. 1) 67 (1966) 185. 8 D. Metcalf, R. Moulds and B. Pike, Influence of the spleen and thymus on immune responses in aging mice, Clin. Exp. lmmunol., 2 (1966) 109. 9 M. J. Rowley, H. Buchanan and [. R. Mackay, Reciprocal changes with age in antibody to extrinsic and intrinsic antigens, l ancet, 11 (1968) 24. 10 S. Kishimoto, 1. Tsuyuguchi and Yuichi Yamamura, Immune response in aged mice, Clht. Exp. lmmunol., 5 (1969) 525. 11 E. Beregi, J. Simon and 1. F61des, Anaphylaxis of rabbits and rats of different ages, Gerontologia, Basel, 10 (1964-65) 183. 12 E. Beregi, Vergleichende immunmorpbologische Untersuchungen bei Arthus-Reaction in verschiedenen Alter, Gerontologia, Basel, 15 (1969) 256. 13 E. Beregi, Comparative morphological studies of the allergic reactions of young and old animals, Gerontologia, Basel, 16 (1970) 141. 14 G. Sainte-Marie, A paraffin embedding technique for studies employing immunofluorescence, J. Histochem. Cytochem., 10 (1962)250. 15 H. H. Movat and N. V. P. Fernando, The fine structure of the lymphoid tissue during antibody formation, Exp. Mol. Pathol., 4 (1965) 155. 16 W. Bernhard and N. Grandboulan, Ultrastructure of immunologically competent cells, in G. E. W. Wolstenholme and Maeve O'Connor (Eds.), Ciba Foundation Symposium on Celhdar Aspects c~flmmunity, Churchill, London, 1960, p. 92. 17 G. Y. V. Nossal and G. L. Ada, Antigens, Lymphoid Cells" and the Immune Response, Academic Press, New York and London, 1971, p. 69. 18 E. Grundmann, Cytologische Untersuchungen fiber Formen und Orte der kymphocytenreifung bei der Ratte, Verh. Dtseh. Ges. Pathol., 41 (1958) 261. 19 M. G. Hanna, Jr., P. Nettesheim and L. C. Peters, Evidence of functional microenvironments in lymphatic tissue response to antigen, Nature New Biol., 232 (1971) 204. 20 R. G. White, Functional recognition of immunologically competent cells by means of the fluorescent antibody technique, in G. E. W, Wolstenholme and J. Knight (Eds.), The hnmunologieally Competent Cell. Its Nature and Origin, Ciba Foundation Study Group No. 16., Churchill, London, 1963, p. 6. 21 E. Letterer, A[tern und Krankheit, Dtsch. Med. Woehensehr., 79 (1954) 1473. 22 G. Beneke, H. Finger and P. Emmerling, Einfluss von Bordetella pertussis auf das lymphatische Gewebe von M~usen, Z. Med. Microbiol. lmmunol., 154 (1968) 179. 23 W. Andrew, An electronmicroscope study of age changes in the liver of mouse, Anat. Ree., 139 (1961) 203.
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