Immunology Today, vol. 5, No. 10, 1984
286
M]
)
More pieces of the jigsaw puzzle SIR, Roland Scollay ~discusses intrathymic events in the differentiation o f T cells and begs a few more pieces of the jigsaw puzzle to make the thymus story fit together. I would like to contribute a few pieces, and would suggest that the jigsaw puzzle appears to have missing pieces because we have not got the correct picture on the front of the box, and we are trying to make one jigsaw from two or more sets of pieces. Firstly, considering pyknotic cells in the thymus gland as evidence for lymphocyte cell death, Scollay's 1 excellent data allows the conclusion that most thymocytes die in situ. This does not accord with histological studies. Cortical tymphocytes in mice have a cell cycle time of about nine hours 2and usually the mitotic figures are visible for less than 25 % of the duration of a cell cycle; thus there should be morphological evidence of mitotis for two to three hours of its cycle. It is not known how long it takes for a cell to become pyknotic and be completely degraded, but from studies ofthymic involution after steroids 3, pyknotic cells and celt debris in macrophages are very commonly seen in the thymus for about four days after treatment. Examination by LM or EM, therefore, of any fixed normal thymus gland should show a vast number ofpyknotic cells and only
a few mitotic figures, even allowing for a number of cell generations from each prothymocyte. In practice there are usually more, or as many, mitotic figures as pyknotic cells. Thus, the morphological picture does not fit the conclusions from Scollay's fluorescent cell marker studies. Second, is pyknosis always cell death of thymocytes? Pyknosis is common in the thymus glands of young mice (as used by Metcalf and Wiadrowski 4 in studies on cell death), in fetal animals of many species, in adult animals in late pregnancy and during lactation 5, and in many birds, especially during the moult cycle 6. To begin to understand the thymus, one must study these events separately. Certainly thymocyte cell death (overt pyknosis) occurs during breeding, according welt with the effect of eorticosteroids on the thymus. However, pyknosis is not always caused by steroids, nor is it always thymocytes that become pyknotic, especially in young and fetal animals. In studies on wild animals and birds 7"-9,thymic changes are clearer and more dramatic than in laboratory reared animals. Our extensive studies on Quelea quetea, which appear to be typical of most small birds in the wild, show that when quelea are in poor condition (judged by whole body weight loss) anaemia often results in, and is measurable by, low packed celt volumes (PCVs) and increased reticulocyte counts in peripheral blood ~°. Then the thymus becomes greatly reduced in size (each of the 14 lobes being < 1 mm tong). If better environmental conditions occur (e.g. more food, cessation of restriction to the
Fig. 1. The cortex of the thymus of Quelea quelea with nucleated erythrocytes (R) developing between epithelial reticular ceils (E) and thymocytes (T). Azur II, 1 [a thick section, x 1600 © 1984, Elsevier Science PublJshem B.V., Amsterdam 0167 - 4919/841502.00
nest by parents), both mate and female adult birds frequently show a rapid and huge increase in thymus weight (each lobe becomes up to 5 mm long). As the thymus lobes increase in size, there is extensive mitosis and the production of some lymphocytes, but at the same time vast numbers of developing erythocytes can be seen (Fig.l). So extensive is erythropoiesis that parts of the thymus may have no lymphocytes present at all, only epithelial reticular cells (ER cells) being readily identified. This occurs over about two to four days, and when RBCs leave the gland it collapses to a small mass of cells which can regenerate via the new differentiation of medulla and cortex to form an apparently normal lymphoid thymus again. During mouk, when there is a drain on the birds' nutritional balance, the thymus gland is also erythropoletie and normoblasts are common in the gland. However, perhaps because moulting birds do not reach the dangerously low body weights that breeding birds reach, erythropoiesis is often ineffective (at least in part of the glands) and vast erythrocyte pyknosis occurs in the gland (Fig. 2). Herein, I believe, lies the key to the phenomenal pyknosis sometimes seen in young and fetal animals. The thymus of all young animals studied (including man) can support erythropoeisis. Indeed the thymus of man can in adult life too H. Thus it is more likely that pyknotic cells in young animals derive from erythrocytes, not lymphocytes. The next piece of jigsaw which is not placed in position concerns the lymphatics of the thymus which have been misinterpreted or ignored. Despite some
Fig. 2. The cortex of the thymus of a moulting Quelea quelea. Note the pyknotic nuclei (P) amongst the thymocytes (T) and epithelial reticular cells haemotoxytin and eosin (E) x 1600
Immunology Today, voL 5, No. 10, 1984
287 active throughout life (cells and hormones), is necessary for healthy life, the better 15. Let us forget this nonsense that the thymus is only of value in early childhood and seek instead better ways of understanding and observing the thymus in adult life. [] MARION D. KENDALL
Department of Anatomy, St. Thomas's Hospital Medical School, Lambeth Palace Road, London, SE1 7EH, UK. References
Fig. 3. The cortex of the thymus of a mouse after being subjected to hypoxic conditions (simulation of 15 000 ft), Arrow indicates lymphatics filled with thymocytes. Azur II, 1 #m. x 252 negations, the thymus does contain good well developed lymphatics 1z-14 in the thymus capsule and in the septal extensions of capsular connective tissue that carry blood vessels deep into the gland. EMs of the thymus reveal a rich and varied assortment of cells (thymocytes, probable B lymphocytes, plasma cells, eosinophils, mast cells, fibroblasts, adipocytes and macrophages) around most blood vessels. Superficially, that is including L M examination, these ceils appear to be within the gland but they are not within the interstices of the E R cells that ramify throughout the thymus. Usually all of the above ceil types (except young immature mast cells and of course thymocytes) reside in perivascular spaces bounded on the thymus side by type 1 E R cells. Confirmation of this requires, at the E M level, examination of the arrangement of E R cells and their basal laminae, and of connective tissue fibres (e.g. collagen) in perivascular spaces. I suspect that much of what we consider to he intrathymic is not technically so, but is intralymphatic or inside the perivascular spaces. L M studies are particularly prone to these misinterpretations. Most perivascular spaces contain fine lymphatics, some being so wide and full of thymocytes that their identity is easily lost, and one can mistake them for regions of thymic cortex. Thus the perivascular space may be considered as a main highway 1~-15. I hope that any scheme for the thymus will take the above points into consideration as the thymus is not just a home for uneducated T cells: it is a complex organ, in some ways like bone
marrow with its finely controlled environment. The thymus is essential to life in the young, and its continued thymic hormone output seems to be inextricably bound up with our ability to withstand certain illnesses, particularly some of those of old age such as cancer. AIDS and severe combined immunodeficiency patients, the chronically ill and the aged all have one thing in common - little or no thymus. The sooner we recognize that a thymus,
Surface carbohydrate recognition determinants for phagocytosis SIR, While the review article by Nathan Sharon (Immunol. Today, 1984 5, 143147) was very pertinent and timely, it may be appropriate to indicate some important points that he did not discuss. Firstly, Sharon stated that sialidasetreated erythrocytes are removed from the circulation through exposed surface galactose residues via binding to cellular lectins of the macrophages. Although this has some support 1-3, we recently have reported both in vitro 4 and in vivo 5 that exposure of senescent antigen which is followed by binding of an autoantibody (directed to senescent antigen on erythrocytes) is responsible for their phagocytosis by autologous monocytes and/or macrophages. We have found that after sialidase treatment of erythrocytes, at least three determinants will be exposed: M N antigen, Arachis hypagdea receptor (peanut lectin) and senescent antigen (unpublished observation); others have also reported a similar
1 ScoUay, g . (1983) Immunol. Today 4, 282 2 Zaitoun, A. M., Lander, I. and Aherene, W. A. (1979) Cell Tiss. Kinet. 12, 191 3 Lundin, M. and Schelin, U. (1966) PathoL Eur. 1, 15 4 Metcalf, D. and Wiadrowski, M. (1966) Cancer Res. 26, 483 5 McLean, J. M., Mosley, J. G. and Gibbs, A. C. C. (1974)J. Anat. 118, 223 6 Bacchus, S. and Kendall, M. D. (1975) Philos. R. Soc. London Ser. B 273, 65 7 Kendall, M. D. and Ward, P. (1974) Nature (London) 249, 366 8 Kendall, M. D. (ed.) (1981) The thymus gland, Academic Press, London 9 Kendall, M. D. (1980)Dev. Comp. Immuno. 4, 191 10 Jones, P. J. (1983).]. Zool. 210, 217 11 Kendall, M. D. and Singh,J. (1980),]. Anat. 130, 183 12 Smith, C. (1955) VIII Intrathymic lymphatic vessels. Anat. Rec. 122(2) 173-179 13 Goldstein, G. and MacKay, I. R. (1969) The Human Thymus, Heinemann Medical, London 14 Bearman, R. M., Levine, G. D. and Bensch, K. G. (1978)Anat. Soc. 190, 755 15 Kendall, M. D. Experientia (in press)
binding 6. Furthermore, we reported that elimination of either aging red blood ceils or sialidase-treated erythrocytes is dependent on the presence of the Fc portion of the serum autoantibody, by which monocytes or macrophages can recognize these cells via Fc receptors on their surface 7. However, whether these Fc receptors have affinity to bind lectin deserves further investigation. In addition, if elimination of sialidase-treated erythrocytes from the circulation was through lectin binding, the peripheral blood monocytes (which in our hands do not bind to any lectin studied thus far) should not be able to bind and phagocytose the sialidase-treated RBCs. The second important point is that, as stated in Sharon's article, tissue, as well as peritoneal macrophages, do bind some lectins; namely, Soya bean lectin, peanut lectin and so forth. In contrast, peripheral blood monocytes bind to none of 10 lectins studied (peanut lectin Ricinu~" communis lectin, Dolichosbiflorus lectin, Lotus tetragonatobus lectin, Pisum satirum lectin, Aleura aurantia lectin, Lima~ flavus lectin, Vicea villosa lectin, Soya bean lectin, Bandeiral Simplificifolia lectin); therefore,
© 1984.ElsevierSciencePublishersB.V.,Amsterdam 0167- 4919/84/$02.00
286
M]
)
More pieces of the jigsaw puzzle SIR, Roland Scollay ~discusses intrathymic events in the differentiation o f T cells and begs a few more pieces of the jigsaw puzzle to make the thymus story fit together. I would like to contribute a few pieces, and would suggest that the jigsaw puzzle appears to have missing pieces because we have not got the correct picture on the front of the box, and we are trying to make one jigsaw from two or more sets of pieces. Firstly, considering pyknotic cells in the thymus gland as evidence for lymphocyte cell death, Scollay's 1 excellent data allows the conclusion that most thymocytes die in situ. This does not accord with histological studies. Cortical tymphocytes in mice have a cell cycle time of about nine hours 2and usually the mitotic figures are visible for less than 25 % of the duration of a cell cycle; thus there should be morphological evidence of mitotis for two to three hours of its cycle. It is not known how long it takes for a cell to become pyknotic and be completely degraded, but from studies ofthymic involution after steroids 3, pyknotic cells and celt debris in macrophages are very commonly seen in the thymus for about four days after treatment. Examination by LM or EM, therefore, of any fixed normal thymus gland should show a vast number ofpyknotic cells and only
a few mitotic figures, even allowing for a number of cell generations from each prothymocyte. In practice there are usually more, or as many, mitotic figures as pyknotic cells. Thus, the morphological picture does not fit the conclusions from Scollay's fluorescent cell marker studies. Second, is pyknosis always cell death of thymocytes? Pyknosis is common in the thymus glands of young mice (as used by Metcalf and Wiadrowski 4 in studies on cell death), in fetal animals of many species, in adult animals in late pregnancy and during lactation 5, and in many birds, especially during the moult cycle 6. To begin to understand the thymus, one must study these events separately. Certainly thymocyte cell death (overt pyknosis) occurs during breeding, according welt with the effect of eorticosteroids on the thymus. However, pyknosis is not always caused by steroids, nor is it always thymocytes that become pyknotic, especially in young and fetal animals. In studies on wild animals and birds 7"-9,thymic changes are clearer and more dramatic than in laboratory reared animals. Our extensive studies on Quelea quetea, which appear to be typical of most small birds in the wild, show that when quelea are in poor condition (judged by whole body weight loss) anaemia often results in, and is measurable by, low packed celt volumes (PCVs) and increased reticulocyte counts in peripheral blood ~°. Then the thymus becomes greatly reduced in size (each of the 14 lobes being < 1 mm tong). If better environmental conditions occur (e.g. more food, cessation of restriction to the
Fig. 1. The cortex of the thymus of Quelea quelea with nucleated erythrocytes (R) developing between epithelial reticular ceils (E) and thymocytes (T). Azur II, 1 [a thick section, x 1600 © 1984, Elsevier Science PublJshem B.V., Amsterdam 0167 - 4919/841502.00
nest by parents), both mate and female adult birds frequently show a rapid and huge increase in thymus weight (each lobe becomes up to 5 mm long). As the thymus lobes increase in size, there is extensive mitosis and the production of some lymphocytes, but at the same time vast numbers of developing erythocytes can be seen (Fig.l). So extensive is erythropoiesis that parts of the thymus may have no lymphocytes present at all, only epithelial reticular cells (ER cells) being readily identified. This occurs over about two to four days, and when RBCs leave the gland it collapses to a small mass of cells which can regenerate via the new differentiation of medulla and cortex to form an apparently normal lymphoid thymus again. During mouk, when there is a drain on the birds' nutritional balance, the thymus gland is also erythropoletie and normoblasts are common in the gland. However, perhaps because moulting birds do not reach the dangerously low body weights that breeding birds reach, erythropoiesis is often ineffective (at least in part of the glands) and vast erythrocyte pyknosis occurs in the gland (Fig. 2). Herein, I believe, lies the key to the phenomenal pyknosis sometimes seen in young and fetal animals. The thymus of all young animals studied (including man) can support erythropoeisis. Indeed the thymus of man can in adult life too H. Thus it is more likely that pyknotic cells in young animals derive from erythrocytes, not lymphocytes. The next piece of jigsaw which is not placed in position concerns the lymphatics of the thymus which have been misinterpreted or ignored. Despite some
Fig. 2. The cortex of the thymus of a moulting Quelea quelea. Note the pyknotic nuclei (P) amongst the thymocytes (T) and epithelial reticular cells haemotoxytin and eosin (E) x 1600
Immunology Today, voL 5, No. 10, 1984
287 active throughout life (cells and hormones), is necessary for healthy life, the better 15. Let us forget this nonsense that the thymus is only of value in early childhood and seek instead better ways of understanding and observing the thymus in adult life. [] MARION D. KENDALL
Department of Anatomy, St. Thomas's Hospital Medical School, Lambeth Palace Road, London, SE1 7EH, UK. References
Fig. 3. The cortex of the thymus of a mouse after being subjected to hypoxic conditions (simulation of 15 000 ft), Arrow indicates lymphatics filled with thymocytes. Azur II, 1 #m. x 252 negations, the thymus does contain good well developed lymphatics 1z-14 in the thymus capsule and in the septal extensions of capsular connective tissue that carry blood vessels deep into the gland. EMs of the thymus reveal a rich and varied assortment of cells (thymocytes, probable B lymphocytes, plasma cells, eosinophils, mast cells, fibroblasts, adipocytes and macrophages) around most blood vessels. Superficially, that is including L M examination, these ceils appear to be within the gland but they are not within the interstices of the E R cells that ramify throughout the thymus. Usually all of the above ceil types (except young immature mast cells and of course thymocytes) reside in perivascular spaces bounded on the thymus side by type 1 E R cells. Confirmation of this requires, at the E M level, examination of the arrangement of E R cells and their basal laminae, and of connective tissue fibres (e.g. collagen) in perivascular spaces. I suspect that much of what we consider to he intrathymic is not technically so, but is intralymphatic or inside the perivascular spaces. L M studies are particularly prone to these misinterpretations. Most perivascular spaces contain fine lymphatics, some being so wide and full of thymocytes that their identity is easily lost, and one can mistake them for regions of thymic cortex. Thus the perivascular space may be considered as a main highway 1~-15. I hope that any scheme for the thymus will take the above points into consideration as the thymus is not just a home for uneducated T cells: it is a complex organ, in some ways like bone
marrow with its finely controlled environment. The thymus is essential to life in the young, and its continued thymic hormone output seems to be inextricably bound up with our ability to withstand certain illnesses, particularly some of those of old age such as cancer. AIDS and severe combined immunodeficiency patients, the chronically ill and the aged all have one thing in common - little or no thymus. The sooner we recognize that a thymus,
Surface carbohydrate recognition determinants for phagocytosis SIR, While the review article by Nathan Sharon (Immunol. Today, 1984 5, 143147) was very pertinent and timely, it may be appropriate to indicate some important points that he did not discuss. Firstly, Sharon stated that sialidasetreated erythrocytes are removed from the circulation through exposed surface galactose residues via binding to cellular lectins of the macrophages. Although this has some support 1-3, we recently have reported both in vitro 4 and in vivo 5 that exposure of senescent antigen which is followed by binding of an autoantibody (directed to senescent antigen on erythrocytes) is responsible for their phagocytosis by autologous monocytes and/or macrophages. We have found that after sialidase treatment of erythrocytes, at least three determinants will be exposed: M N antigen, Arachis hypagdea receptor (peanut lectin) and senescent antigen (unpublished observation); others have also reported a similar
1 ScoUay, g . (1983) Immunol. Today 4, 282 2 Zaitoun, A. M., Lander, I. and Aherene, W. A. (1979) Cell Tiss. Kinet. 12, 191 3 Lundin, M. and Schelin, U. (1966) PathoL Eur. 1, 15 4 Metcalf, D. and Wiadrowski, M. (1966) Cancer Res. 26, 483 5 McLean, J. M., Mosley, J. G. and Gibbs, A. C. C. (1974)J. Anat. 118, 223 6 Bacchus, S. and Kendall, M. D. (1975) Philos. R. Soc. London Ser. B 273, 65 7 Kendall, M. D. and Ward, P. (1974) Nature (London) 249, 366 8 Kendall, M. D. (ed.) (1981) The thymus gland, Academic Press, London 9 Kendall, M. D. (1980)Dev. Comp. Immuno. 4, 191 10 Jones, P. J. (1983).]. Zool. 210, 217 11 Kendall, M. D. and Singh,J. (1980),]. Anat. 130, 183 12 Smith, C. (1955) VIII Intrathymic lymphatic vessels. Anat. Rec. 122(2) 173-179 13 Goldstein, G. and MacKay, I. R. (1969) The Human Thymus, Heinemann Medical, London 14 Bearman, R. M., Levine, G. D. and Bensch, K. G. (1978)Anat. Soc. 190, 755 15 Kendall, M. D. Experientia (in press)
binding 6. Furthermore, we reported that elimination of either aging red blood ceils or sialidase-treated erythrocytes is dependent on the presence of the Fc portion of the serum autoantibody, by which monocytes or macrophages can recognize these cells via Fc receptors on their surface 7. However, whether these Fc receptors have affinity to bind lectin deserves further investigation. In addition, if elimination of sialidase-treated erythrocytes from the circulation was through lectin binding, the peripheral blood monocytes (which in our hands do not bind to any lectin studied thus far) should not be able to bind and phagocytose the sialidase-treated RBCs. The second important point is that, as stated in Sharon's article, tissue, as well as peritoneal macrophages, do bind some lectins; namely, Soya bean lectin, peanut lectin and so forth. In contrast, peripheral blood monocytes bind to none of 10 lectins studied (peanut lectin Ricinu~" communis lectin, Dolichosbiflorus lectin, Lotus tetragonatobus lectin, Pisum satirum lectin, Aleura aurantia lectin, Lima~ flavus lectin, Vicea villosa lectin, Soya bean lectin, Bandeiral Simplificifolia lectin); therefore,
© 1984.ElsevierSciencePublishersB.V.,Amsterdam 0167- 4919/84/$02.00
