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Histologic changes in mouse lymph nodes after treatment with antitheta-globulin
Exp. Path., Bd. 14, S. 171-179 (1977) Central Laboratory of the Departments of Pathology, University of Helsinki, Finland
Histologic changes in mouse lymph nodes after treatment with antitheta-globulin By K. J. SYRJANEN With 7 figures (Received April 7, 1977) Key-words: lymph node; theta-globulin; anti-theta-globulin; gamma-globulin; pyroninophilic cells; plasma cells; lymphocyte; theta-isoantigen; cell surface isoantigen; anti-thy mus-globulin ; thymus cells; antibody; mouse
Summary The DBAj2 mice were treated with 4 successive subcutaneous injections of rabbit anti-thetagammaglobulin and the histology of the axillary lymph nodes was studied 1, 2, 4, 7, 14 and 21 days after the last dose. The histology of the lymph nodes was assessed according to the standardized reporting system of COTTIER et al. (1973). The histologic findings were compared with those found in mice treated with anti-thymus-gammaglobulin, normal rabbit gammaglobulin and those in untreated DBAj2 mice. Anti-theta-gammaglobulin was found to be a potent suppressor of the small lymphocytes in the lymph node paracortical areas. At th same time, it elicited a strong humoral immune response in the nodes manifested as proliferation of large pyroninophilic cells and plasma cells in the medulla and germinal centers. The standardized reporting system of C01'lIER et al. was found to be extremely suitable for assessing the histology of the experimental animals' lymph nodes and its acceptance into general use is strongly advocated.
Increasing evidence has accumulated suggesting that two distinct populations of lymphocytes are present in the spleen and lymph nodes. One population is thymus-dependent (T-Iymphocytes), the other is thymus-independent (B-Iymphocytes) (PARROTT et al. 1966, RAFF 1969, RAFF et al. 1970). Light-microscopically these two populations are indistinguishable, but they are readily identifiable by scanning electron microscopy (POLLIACK et al. 1973). Recently, it was suggested that theta isoantigen (0) could serve as a marker for the T-Iymphocytes (RAFF 1969, RAFF et al. 1970, SCHLESINGER 1970). Theta isoantigen is determined by a single locus with two alleles; theta AKR found in AKR and RF mice and theta CaR present in most other inbred strains of mice (REIF et al. 1964, REIF et al. 1966, RAFF 1969, RAFF et al. 1970). Theta is a cell surface isoantigen mainly found in the cells of thymus and brain (RAFF 1969, RAFF et al. 1970, SCHLESINGER 1970). The mice treated with heterologous anti-lymphocyte serum or globulin prepared against thymus cells showed a marked paracortical depletion in the lymph nodes and a reduction, in number of the small lymphocytes in the spleen periarteriolar regions (LEVEY et al. 1966, GRAY et al. 1966, MARTIN et al. 1967, TURK et al. 1967, TAuB et al. 1968). Anti-theta-serum, too, was found to have a cytotoxic effect on the T-Iymphocyte population in vivo and in vitro (RAFF 1969, RAFF et al. 1970, SCHLESINGER 1970). Recently, a proposal has been made for a standardized system of reporting human lymph node morphology in relation to the immunological function (COTTIER et al. 1973). The present work is an attempt to describe the morphology of mouse lymph nodes, after anti-thetaglobulin treatment, according to the standardized reporting system of COTTIER et al.
Material and methods Animals White rabbits weighing 2.0 to 3.0 kg were used to prepare the antiglobulins. Inbred mice of DBAj2 strain, reared in our laboratory by strict brother and sister matings, were used at the age of two three months.
171
Preparation of anti-theta-globulin Cells from the brain of DBAj2 mice were rendered into washed single celled suspensions in MEM with 10 per cent fetal calf serum at +4 °C. From 300 to 650 million cells were injected into rabbits intravenously at 14 day intervals six times. The viability of the injected cells was tested by trypan blue staining and was found to be more than 90 per cent. The rabbits were bled 7 days after the injection, starting from the 2nd injection. The blood was allowed to clot at room temperature and then to stand overnight at +4°C. The serum was separated, pooled and stored at -20°C after addition of Merthiolate (1: 10000). Prior to use the antiserum was decomplemented at +56 °C for 30 minutes. The gammaglobulin was extracted by ammonium sulphate precipitation according to the routine procedures. The protein concentration was then adjusted to 10 mg per ml. Preparation of anti-thy mus-globulin The method of preparation was equal to that above except that this serum was absorbed with mouse erythrocytes as previously described (GRAY et al. 1966). From 60 to 300 million thymus cells were injected into rabbits which were bled 7 days after each injection starting from the 2nd day. Preparation of normal rabbit gammaglobulin The normal rabbit gammaglobulin was prepared according to the routine procedures described above. The protein concentration was adjusted to 10 mg per ml. Cytotoxicity tests The efficacy of the prepared antiglobulins was tested by a cytotoxicitiy test for thymus cells of the mice (DBAj2). Two drops of normal saline were placed in a well on a glass slide. Mouse thymus cells in suspension were added in that well followed by two drops of the globulin to be tested in dilutions 1: 1, 1: 2, 1: 4, 1: 8 and so on down to 1: 128. Two drops of fresh rabbit complement prepared by agar absorption (COHEN et al. 1971) were added in the well which was then incubated at 37°C for 30 minutes. The cytolysis was analysed after adding a drop of freshly prepared trypan blue (0.20 'Yo). Two other sets of wells were tested in the same way. In each of them either antiglobulin or complement was lacking. In one set of wells only thymus cells and saline were present. All the test were made as triplicates. The cytotoxicity of the antiglobulins increased along with the times of rabbit immunizations. In general, a level was reached where anti-theta and anti-thy mus-globulins had a 75 per cent cytotoxicity in dilutions 1: 8, and more than 90 per cent in dilutions 1: 4. The normal rabbit gamma was only weakly cytotoxic, less than 10 per cent in 1: 1 dilution. Treatment of the mice with gammaglobulins Four series of mice were tested, each comprising 15 animals of 2 to 4 months of age. The 1st series comprised mice without any kind of treatment, i.e. they were normal mice used as controls. The 2nd series was treated with anti-theta-globulin, 5mg subcutaneously on back at 2 days intervals four times. The 3rd series got anti-thy mus-globulin according to the same schedule. The 4th series was treated with normal rabbit gamma-globulin in the same way. Animals in each series were sacrificed 1, 2, 4, 7, 14 and 21 days after the last globulin injection. Axillary lymph nodes were dissected out and processed into 5 micron sections according to the routine procedures. The microscopic sections were stained with hematoxylin-eosine and methyl greenpyronine stains. Lymph node histology The morphology of the lymph nodes was analysed according to the expanded protocol as previously described (COTTIER et al. 1973). Thus, the morphology of the nodal cortex, paracortex, medulla and sinuses was analysed separately as suggested by COTTIER et al. 1973.
Results Histology of the node in control series The morphologic features of the lymph nodes in control series are summarized in table l. Lymph nodes were small in size compared with those in the other series. Cortical areas were a rim band of densely packed lymphocytes inside the lymph node capsule. In this series only a single node was found that contained lymphocyte follicles in the cortex. In this case the follicles also had features fulfilling the criteria of active germinal centers. Paracortical areas were inconspicuous and showed a few postcapillary venules with cuboidal endothelium. The main type of cell in paracortex was a small lymphocyte, loosely arranged among the reticulum cells which formed the framework of this area. The number of medium-sized lymphocytes was small and large lymphoid cells were extremely rare,
172
Table 1.
Lymph node histology in the different series Grading
Lymph node*) size architecture altered diffusely altered focally Cortical area size lymphocyte content lymphocyte follicles number size germinal centers number size content of large lymphoid cells relative number of mitotic figures relative number of macrophages Paracortical area size content of small lymphocytes content of medium-sized lymphocytes content of large lymphoid cells content of histiocytes mitotic activity Medulla size medullary cords width content of large lymphoid cells content of plasma cells content of lymphocytes Sinuses width content of lymphocytes content of large lymphoid cells content of histiocytes
-N+ 1
+++
2,3
4
2,3
1
2, 3
++
4
1
1,2,3
4
4
1 1
2, 3
2, 3
4
1 1 1 1 1
2,3 2,3 2,3 2,3 2,3
4 4 4 4 4
2 2 1 1
3 3 2,3
1,4 1,4 1,4
1,2
3, 4
4
2,3,4 2,3,4
1
2,34
1 1 1
2,34 2,34 2,3,4 4
1,2,3 1, 2, 1, 2, 1,2, 1,2,
Explanation of the symbols: N, normal; one, two or three moderate or marked deviation above or below the normal range; 2: lymph nodes of the anti-theta series; 3: lymph nodes of the of the normal rabbit globulin series. *) The table has been complied by taking into account the globulin dose.
3, 3, 3, 3,
4 4 4 4
plus or minus signs indicate slight, 1: lymph nodes of the control series; anti-thymus series; 4: lymph nodes nodes of the 4th day after the last
as were the mitotic figures. Due to the small size of cortex and paracortex, the medulla comprised the major area of these small nodes (fig. 1). Mostly, medullary cords were narrow and contained a few large lymphoid cells, mature plasma cells and lymphocytes. Sinuses were inconspicuous in general and contained scattered lymphocytes and histiocytes. A florid sinus histiocytosis was found in one node only. Histology of the node after anti-theta-globulin treatment The size of the nodes was greatly increased from the first day on after the last globulin injection. The lymph node size was more than double that of the untreated controls as evaluated from the surface area of the node section. This enlargement seemed to persist at least two weeks following the last injection. The architecture was altered diffusely as evident from table l. The cortical area appeared normal at first (fig. 4) but later the increased hypertrophy of the medulla tended to obscure the cortical area (fig. 6 and 7). Inspite of the increasing number of large pyroniophilic cells the number of small lymphocytes in the cortex remained
173
1 Fig. 1. Lymph node of an untreated mouse. Cortical areas are inconspicuous, lymphocyte follicles are absent like germinal centers, paracortical areas are of average size and medulla is prominent here. A few sinuses with sparse cellular contents are visible in the left part of the node. HE, x 50.
Fig. 2. Lymph node of the first day after the last dose of normal rabbit gammaglobulin. Large lymphocyte follicles are present in the cortex, paracortical area is unchanged and medullary elements are prominent. HE, X 50.
174
Fig. 3. The same node as in fig. 2 with greater magnification. Note the prominent lymphocyte follicles (If), normal paracortical area (pc) and hyperplastic medullary elements (m). HE, x 12S.
Fig. 4. Lymph node of the first day after the last dose of anti-theta-globulin. Medullary elements (m) are prominent by that time and expand to the paracortical areas (pc) and cortex (c). An almost empty sinus (s) is also present. HE, x 50.
175
Fig. 5. The same node as in fig. 4 with greater magnification. Paracortical area (pc) is masked by the expanded medullary elements. Cortical areas (c) seem unchanged in this node. HE, X 200.
Fig. 6. Lymph node of the fourth day after the last dose of anti-theta-globulin. Medullary hyperplasia (m) is extreme, paracortical areas (pc) are totally obliterated by the medullary elements. Cortex (c) is a rim band of lymphocytes beneath the node capsule. HE, x50.
176
Fig. 7. The sa,me node as in fig. 6 with greater power. The extreme hyperplasia of the meduilary cells (m) obscures the paracortical area (pc). Cortical area (c) is devoid of medullary cells. HE, X 128.
unaltered as revealed by the methyl green-pyronine staining. Lymphocyte follicles in the cortex were prominent on the 4th day following the last globulin injection and persisted, although with lesser intensity, all the three weeks of observation. The number of these follicles rarely exceeded 10 per one node. These follicles were large in size and contained active germinal centers with large lymphoid cells, actively phagocytozing macrophages and many mitotic figures. The paracortical area was totally altered in the nodes of this series. The number of the small lymphocytes was greatly reduced and the framework of reticulum cells appeared prominent in the nodes of the first day (fig. 4 and 5). The number of large pyroninophilic lymphoid cells was small first, but their count greatly increased in the nodes of the second and fourth day (fig. 6 and 7). Indeed, their number was so great that the cellular density of the paracortex appeared quite normal in Hand E stained sections (fig. 6 and 7). The truth was revealed in methyl green-pyronine stained sections where most of the paracortical cells were large pyroninophilic lymphoid cells and only a few small lymphocytes were seen among these and the reticular framework. These pyroninophilic cells were the precursors of mature plasma cells and invaded the paracortex from the medulla. The mitotic activity remained high between days 2 and 7. The number of the small lymphocytes was low up to 7 days, normal counts being found in the nodes of the 14th day. By that time the large pyroninophilic cells were few, postcapiIIary venules had gained back their cuboidal endothelium and recirculating small lymphocytes were seen in their lumen. The medulla also underwent profound alterations during the days 1 and 14 as seen in table 1. The medullary elements, especially the large pyroninophilic cells and mature plasma cells increased in number and massive medullary hyperplasia was evident on the 2nd day (fig. 6 and 7). Medullary cords were prominent extending into the paracortical areas and even into the cortical regions masking the true extent of these regions. It was difficult to assess accurately the count of small lymphocytes in the medullary cords due to the hyperplasia of the above mentioned elements but their number seemed to be fairly constant. The hyperplasia of the medullary elements had returned to normal state by the 14th day of observation. The sinuses were flat, their lining endothelial cells were close together and the cellular content was sparse. 12 Exp. Path. Bd. 14
177
Histology of the node after anti-thymus-globulin treatment The architecture of the nodes in this series was altered diffusely as seen in table 1. The table shows that the alterations in morphology were similar to those in the anti-theta series, in general, but a few differences must be emphasized. First, it should be pointed out that large lymphocyte follicles with active germinal centers were seen on the 1st day in contrast to the 2nd and 4th day in the anti-theta series. Secondly, the depletion of the smalllymphocytes in the paracortical areas was not so prominent in this series compared with the antitheta series. Table 1 shows that the two series do not differ as to the morphologic alterations in the medulla and in the sinuses. Histology of the node after normal rabbit globulin treatment Table 1 shows the morphologic alterations following the treatment with normal rabbit gammaglobulin. These alterations were localized in the cortex and medulla leaving the other regions intact. The cortical area was greatly expanded due to the app'earance of large lymphocyte follicles with active germinal centers (fig. 2 and 3). The number of those follicles varied from 4 to 12 per node and exeeded the number found in the other series. No depletion in the paracortical constituents was found and the morphology of this area was similar to that in the control series. All the medullary elements were hyperplastic. Medullary cords were wide containing numerous large pyroninophilic cells and mature plasma cells. The mitotic activity was high. Medullary cords expanded into paracortex and even to the cortical areas (fig. 2 and 3). Sinuses were inconspicuous and their cellular content was sparse.
Discussion The histologic changes in the lymphoid organs after treatment with anti-thymus-serum have been studied (MARTIN et al. 1967, TURK et al. 1967, TAUB et al. 1968). These studies have confirmed that anti-thymus-serum specifically depletes the small lymphocytes in the paracortical areas of the lymph node. Similar results have been reported by other workers with anti-theta-serum thus confirming the thymus-dependence of the theta-bearing small lymphocytes (RAFF 1969, RAFF et al. 1970, SCHLESINGER 1970). Some controversy exists, however, concerning the changes found in the medulla and lymphocyte follicles. An intraperitoneal injection of normal rabbit serum in mice caused lymphoid hyperplasia in paracortex and germinal centers whereas these changes were not observed after anti-thymus serum (TURK et al. 1967). On the other hand, the histologic features of immunization with pronounced medullary hyperplasia and germinal center formation were found in mice after normal rabbit serum and anti-thymus serum subcutaneously (TAUB et al. 1968). The results of the present study confirm that anti-theta as well as anti-thymus globulins act as suppressive agents for the small lymphocytes of the lymph node paracortex. Evidence of this suppressive action was found in the nodes taken one day after the last globulin injection and the changes in both series persisted for 7 or 14 days. As to the quantitative differences between the two globulins, it was evident that the effects of anti-theta-globulin were more pronounced. This could support the view that there are at least two populations of lymphocytes in the thymus; the one bearing theta-antigen, the other not. Thus an antiglobulin prepared against these cells would not be as potent and selective as that prepared against the theta-isoantigen proper. The results also support the view that thymus-dependent small lymphocytes with theta-isoantigen are localized in the paracortical areas of the lymph nodes. The strong signs of immunization including the medullary hyperplasia and the appearance of lymphocyte follicles with active germinal centers were observed in the nodes after the treatment with all these globulins. In this respect, however, differences were observed that need further discussion. The signs of intense humoral immune response appeared in the nodes taken one day after the last dose of anti-thymus and normal rabbit globulins. On the
178
other hand, there was a delay of 2 to 4 days in the appearance of such humoral response in the anti-theta-globulin treated animals. The signs of humoral response were most pronounced in the animals treated with normal rabbit globulin, a finding contradictory to the earlier observations (TAuB et al. 1968). These differences in the initiation and strength of the humoral immune response could be explained on the basis of the T- and B-cell interaction. It has been suggested previously that an interaction between T- and B-lymphocytes is important in humoral immune response (MILLER et al. 1971). The exact mechanism of this interaction, however, remained obscure (MILLER et al. 1971). The results of the present study could suggest that anti-theta-globulin bound to its theta-isoantigen on T-cell surface would create a hindrance for the access of B-lymphocytes to the vicinity thus making the T-B interaction impossible. This interaction could be possible after some two to four days after the new circulating T-lymphocytes had horned in the paracortex via the postcapillary venules. Indeed, small lymphocytes were evident in the lumen and in the vicinity of paracortical postcapillary venules by that time. In the nodes of the animals treated with normal rabbit globulin no such disturbance in T-B interaction happened and consequently no delay in humoral response was observed. The fact that no delay in antibody response was observed in cases treated with antithymus-globulin suggests differences in potency between this and anti-theta-globulin, as stated earlier in this discussion. It seems evident that anti-theta-globulin is the more potent and more selective T-cell suppressor of these two. The present work indicates that the standardized reporting system of COTTIER et al. is a very applicable method in assessing the immunological state of the lymph node even in experimental animals. The findings are easy to summarize in tabular form and thus ready for mutual comparisons. These facts advocate the acceptance of this reporting system into general use.
Literature COHEN, A., and M. SCHLESINGER, Absorption of guinea pig serum with agar. Transplantation 10, 130-132 (1971). COTTIER, H., J. TURK and L. SOBIN, A proposal for a standardized system of reporting human lymph node morphology in relation to immunological function. J. Clin. Path. 26, 317-331 (1973). GRAY, J. G., A. P. MONACO, M. L. WOOD and P. S. RUSSELL, Studies on heterologous anti-lymphocyte serum in mice. I. In vitro and in vivo properties. J. Immunol. 96, 217-228 (1966). LEVEY, R. H., and P. B. MEDAWAR, Some experiments on the action of anti-lymphoid antisera. Ann. N. Y. Acad. Sci. 129, 164-177 (1966). MARTIN, W. J., and J. F. MILLER, Site of action of antilymphocyte globulin. Lancet 1,1285-1287 (19S7). MILLER, J. F., A. BAS'fEN, J. SPENT and C. CHEERS, Interaction between lymphocytes in immune responses. Cell. Immunol. 2, 469-495 (1971). PARROTT, D. M. V., M. A. B. DE SOUSA and J. EAST, Thymus-dependent areas in the lymphoid organs of neonatally thymectomized mice. J. Exp. Med. 123, 191-204 (1966). POLLIACK, A., N. LAMPEN, B. D. CLARKSON and E. DE HARVEN, Identification of human Band T lymphocytes by scanning electron microscopy. J. Exp. Med. 138, 607-616 (1973). RAFF, M. C., Theta isoantigen as a marker of thymus-derived lymphocytes in mice. Nature 224, 378-379 (1969). - and H. H. WORTIS, Thymus dependence of E)-bearing cells in the peripheral lymphoid tissues of mice. Immunology 18, 931-942 (1970). REIF, A. K, and J. M. V. ALLEN, The AKR thymic antigen and its distribution in leukemias and nervous tissue. J. Exp. Med. 120, 413-421 (1964). - - Mouse thymic iso-antigens. Nature 209, 521-522 (1966). SCHLESINGER, M., Anti-E)-antibodies for detecting thymus-dependent lymphocytes in the immune response of mice to SRBC. Nature 226, 1254-1255 (1970). TAUB, R. N., and K M. LANCE, Histopathological effects in mice of heterologous antilymphocyte serum. J. Exp. Med. 128, 1281-1307 (1968). TURK, J. L., and D. A. WILLOUGHBY, Central and peripheral effects of anti-lymphocyte sera. Lancet I, 249-251 (1967). Author's address: K. J. SYRJANEN, MD, Department of Pat,hology, Jorvi Hospital, SF -02740 Espoo 74 (Finland). 12*
179
Histologic changes in mouse lymph nodes after treatment with antitheta-globulin By K. J. SYRJANEN With 7 figures (Received April 7, 1977) Key-words: lymph node; theta-globulin; anti-theta-globulin; gamma-globulin; pyroninophilic cells; plasma cells; lymphocyte; theta-isoantigen; cell surface isoantigen; anti-thy mus-globulin ; thymus cells; antibody; mouse
Summary The DBAj2 mice were treated with 4 successive subcutaneous injections of rabbit anti-thetagammaglobulin and the histology of the axillary lymph nodes was studied 1, 2, 4, 7, 14 and 21 days after the last dose. The histology of the lymph nodes was assessed according to the standardized reporting system of COTTIER et al. (1973). The histologic findings were compared with those found in mice treated with anti-thymus-gammaglobulin, normal rabbit gammaglobulin and those in untreated DBAj2 mice. Anti-theta-gammaglobulin was found to be a potent suppressor of the small lymphocytes in the lymph node paracortical areas. At th same time, it elicited a strong humoral immune response in the nodes manifested as proliferation of large pyroninophilic cells and plasma cells in the medulla and germinal centers. The standardized reporting system of C01'lIER et al. was found to be extremely suitable for assessing the histology of the experimental animals' lymph nodes and its acceptance into general use is strongly advocated.
Increasing evidence has accumulated suggesting that two distinct populations of lymphocytes are present in the spleen and lymph nodes. One population is thymus-dependent (T-Iymphocytes), the other is thymus-independent (B-Iymphocytes) (PARROTT et al. 1966, RAFF 1969, RAFF et al. 1970). Light-microscopically these two populations are indistinguishable, but they are readily identifiable by scanning electron microscopy (POLLIACK et al. 1973). Recently, it was suggested that theta isoantigen (0) could serve as a marker for the T-Iymphocytes (RAFF 1969, RAFF et al. 1970, SCHLESINGER 1970). Theta isoantigen is determined by a single locus with two alleles; theta AKR found in AKR and RF mice and theta CaR present in most other inbred strains of mice (REIF et al. 1964, REIF et al. 1966, RAFF 1969, RAFF et al. 1970). Theta is a cell surface isoantigen mainly found in the cells of thymus and brain (RAFF 1969, RAFF et al. 1970, SCHLESINGER 1970). The mice treated with heterologous anti-lymphocyte serum or globulin prepared against thymus cells showed a marked paracortical depletion in the lymph nodes and a reduction, in number of the small lymphocytes in the spleen periarteriolar regions (LEVEY et al. 1966, GRAY et al. 1966, MARTIN et al. 1967, TURK et al. 1967, TAuB et al. 1968). Anti-theta-serum, too, was found to have a cytotoxic effect on the T-Iymphocyte population in vivo and in vitro (RAFF 1969, RAFF et al. 1970, SCHLESINGER 1970). Recently, a proposal has been made for a standardized system of reporting human lymph node morphology in relation to the immunological function (COTTIER et al. 1973). The present work is an attempt to describe the morphology of mouse lymph nodes, after anti-thetaglobulin treatment, according to the standardized reporting system of COTTIER et al.
Material and methods Animals White rabbits weighing 2.0 to 3.0 kg were used to prepare the antiglobulins. Inbred mice of DBAj2 strain, reared in our laboratory by strict brother and sister matings, were used at the age of two three months.
171
Preparation of anti-theta-globulin Cells from the brain of DBAj2 mice were rendered into washed single celled suspensions in MEM with 10 per cent fetal calf serum at +4 °C. From 300 to 650 million cells were injected into rabbits intravenously at 14 day intervals six times. The viability of the injected cells was tested by trypan blue staining and was found to be more than 90 per cent. The rabbits were bled 7 days after the injection, starting from the 2nd injection. The blood was allowed to clot at room temperature and then to stand overnight at +4°C. The serum was separated, pooled and stored at -20°C after addition of Merthiolate (1: 10000). Prior to use the antiserum was decomplemented at +56 °C for 30 minutes. The gammaglobulin was extracted by ammonium sulphate precipitation according to the routine procedures. The protein concentration was then adjusted to 10 mg per ml. Preparation of anti-thy mus-globulin The method of preparation was equal to that above except that this serum was absorbed with mouse erythrocytes as previously described (GRAY et al. 1966). From 60 to 300 million thymus cells were injected into rabbits which were bled 7 days after each injection starting from the 2nd day. Preparation of normal rabbit gammaglobulin The normal rabbit gammaglobulin was prepared according to the routine procedures described above. The protein concentration was adjusted to 10 mg per ml. Cytotoxicity tests The efficacy of the prepared antiglobulins was tested by a cytotoxicitiy test for thymus cells of the mice (DBAj2). Two drops of normal saline were placed in a well on a glass slide. Mouse thymus cells in suspension were added in that well followed by two drops of the globulin to be tested in dilutions 1: 1, 1: 2, 1: 4, 1: 8 and so on down to 1: 128. Two drops of fresh rabbit complement prepared by agar absorption (COHEN et al. 1971) were added in the well which was then incubated at 37°C for 30 minutes. The cytolysis was analysed after adding a drop of freshly prepared trypan blue (0.20 'Yo). Two other sets of wells were tested in the same way. In each of them either antiglobulin or complement was lacking. In one set of wells only thymus cells and saline were present. All the test were made as triplicates. The cytotoxicity of the antiglobulins increased along with the times of rabbit immunizations. In general, a level was reached where anti-theta and anti-thy mus-globulins had a 75 per cent cytotoxicity in dilutions 1: 8, and more than 90 per cent in dilutions 1: 4. The normal rabbit gamma was only weakly cytotoxic, less than 10 per cent in 1: 1 dilution. Treatment of the mice with gammaglobulins Four series of mice were tested, each comprising 15 animals of 2 to 4 months of age. The 1st series comprised mice without any kind of treatment, i.e. they were normal mice used as controls. The 2nd series was treated with anti-theta-globulin, 5mg subcutaneously on back at 2 days intervals four times. The 3rd series got anti-thy mus-globulin according to the same schedule. The 4th series was treated with normal rabbit gamma-globulin in the same way. Animals in each series were sacrificed 1, 2, 4, 7, 14 and 21 days after the last globulin injection. Axillary lymph nodes were dissected out and processed into 5 micron sections according to the routine procedures. The microscopic sections were stained with hematoxylin-eosine and methyl greenpyronine stains. Lymph node histology The morphology of the lymph nodes was analysed according to the expanded protocol as previously described (COTTIER et al. 1973). Thus, the morphology of the nodal cortex, paracortex, medulla and sinuses was analysed separately as suggested by COTTIER et al. 1973.
Results Histology of the node in control series The morphologic features of the lymph nodes in control series are summarized in table l. Lymph nodes were small in size compared with those in the other series. Cortical areas were a rim band of densely packed lymphocytes inside the lymph node capsule. In this series only a single node was found that contained lymphocyte follicles in the cortex. In this case the follicles also had features fulfilling the criteria of active germinal centers. Paracortical areas were inconspicuous and showed a few postcapillary venules with cuboidal endothelium. The main type of cell in paracortex was a small lymphocyte, loosely arranged among the reticulum cells which formed the framework of this area. The number of medium-sized lymphocytes was small and large lymphoid cells were extremely rare,
172
Table 1.
Lymph node histology in the different series Grading
Lymph node*) size architecture altered diffusely altered focally Cortical area size lymphocyte content lymphocyte follicles number size germinal centers number size content of large lymphoid cells relative number of mitotic figures relative number of macrophages Paracortical area size content of small lymphocytes content of medium-sized lymphocytes content of large lymphoid cells content of histiocytes mitotic activity Medulla size medullary cords width content of large lymphoid cells content of plasma cells content of lymphocytes Sinuses width content of lymphocytes content of large lymphoid cells content of histiocytes
-N+ 1
+++
2,3
4
2,3
1
2, 3
++
4
1
1,2,3
4
4
1 1
2, 3
2, 3
4
1 1 1 1 1
2,3 2,3 2,3 2,3 2,3
4 4 4 4 4
2 2 1 1
3 3 2,3
1,4 1,4 1,4
1,2
3, 4
4
2,3,4 2,3,4
1
2,34
1 1 1
2,34 2,34 2,3,4 4
1,2,3 1, 2, 1, 2, 1,2, 1,2,
Explanation of the symbols: N, normal; one, two or three moderate or marked deviation above or below the normal range; 2: lymph nodes of the anti-theta series; 3: lymph nodes of the of the normal rabbit globulin series. *) The table has been complied by taking into account the globulin dose.
3, 3, 3, 3,
4 4 4 4
plus or minus signs indicate slight, 1: lymph nodes of the control series; anti-thymus series; 4: lymph nodes nodes of the 4th day after the last
as were the mitotic figures. Due to the small size of cortex and paracortex, the medulla comprised the major area of these small nodes (fig. 1). Mostly, medullary cords were narrow and contained a few large lymphoid cells, mature plasma cells and lymphocytes. Sinuses were inconspicuous in general and contained scattered lymphocytes and histiocytes. A florid sinus histiocytosis was found in one node only. Histology of the node after anti-theta-globulin treatment The size of the nodes was greatly increased from the first day on after the last globulin injection. The lymph node size was more than double that of the untreated controls as evaluated from the surface area of the node section. This enlargement seemed to persist at least two weeks following the last injection. The architecture was altered diffusely as evident from table l. The cortical area appeared normal at first (fig. 4) but later the increased hypertrophy of the medulla tended to obscure the cortical area (fig. 6 and 7). Inspite of the increasing number of large pyroniophilic cells the number of small lymphocytes in the cortex remained
173
1 Fig. 1. Lymph node of an untreated mouse. Cortical areas are inconspicuous, lymphocyte follicles are absent like germinal centers, paracortical areas are of average size and medulla is prominent here. A few sinuses with sparse cellular contents are visible in the left part of the node. HE, x 50.
Fig. 2. Lymph node of the first day after the last dose of normal rabbit gammaglobulin. Large lymphocyte follicles are present in the cortex, paracortical area is unchanged and medullary elements are prominent. HE, X 50.
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Fig. 3. The same node as in fig. 2 with greater magnification. Note the prominent lymphocyte follicles (If), normal paracortical area (pc) and hyperplastic medullary elements (m). HE, x 12S.
Fig. 4. Lymph node of the first day after the last dose of anti-theta-globulin. Medullary elements (m) are prominent by that time and expand to the paracortical areas (pc) and cortex (c). An almost empty sinus (s) is also present. HE, x 50.
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Fig. 5. The same node as in fig. 4 with greater magnification. Paracortical area (pc) is masked by the expanded medullary elements. Cortical areas (c) seem unchanged in this node. HE, X 200.
Fig. 6. Lymph node of the fourth day after the last dose of anti-theta-globulin. Medullary hyperplasia (m) is extreme, paracortical areas (pc) are totally obliterated by the medullary elements. Cortex (c) is a rim band of lymphocytes beneath the node capsule. HE, x50.
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Fig. 7. The sa,me node as in fig. 6 with greater power. The extreme hyperplasia of the meduilary cells (m) obscures the paracortical area (pc). Cortical area (c) is devoid of medullary cells. HE, X 128.
unaltered as revealed by the methyl green-pyronine staining. Lymphocyte follicles in the cortex were prominent on the 4th day following the last globulin injection and persisted, although with lesser intensity, all the three weeks of observation. The number of these follicles rarely exceeded 10 per one node. These follicles were large in size and contained active germinal centers with large lymphoid cells, actively phagocytozing macrophages and many mitotic figures. The paracortical area was totally altered in the nodes of this series. The number of the small lymphocytes was greatly reduced and the framework of reticulum cells appeared prominent in the nodes of the first day (fig. 4 and 5). The number of large pyroninophilic lymphoid cells was small first, but their count greatly increased in the nodes of the second and fourth day (fig. 6 and 7). Indeed, their number was so great that the cellular density of the paracortex appeared quite normal in Hand E stained sections (fig. 6 and 7). The truth was revealed in methyl green-pyronine stained sections where most of the paracortical cells were large pyroninophilic lymphoid cells and only a few small lymphocytes were seen among these and the reticular framework. These pyroninophilic cells were the precursors of mature plasma cells and invaded the paracortex from the medulla. The mitotic activity remained high between days 2 and 7. The number of the small lymphocytes was low up to 7 days, normal counts being found in the nodes of the 14th day. By that time the large pyroninophilic cells were few, postcapiIIary venules had gained back their cuboidal endothelium and recirculating small lymphocytes were seen in their lumen. The medulla also underwent profound alterations during the days 1 and 14 as seen in table 1. The medullary elements, especially the large pyroninophilic cells and mature plasma cells increased in number and massive medullary hyperplasia was evident on the 2nd day (fig. 6 and 7). Medullary cords were prominent extending into the paracortical areas and even into the cortical regions masking the true extent of these regions. It was difficult to assess accurately the count of small lymphocytes in the medullary cords due to the hyperplasia of the above mentioned elements but their number seemed to be fairly constant. The hyperplasia of the medullary elements had returned to normal state by the 14th day of observation. The sinuses were flat, their lining endothelial cells were close together and the cellular content was sparse. 12 Exp. Path. Bd. 14
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Histology of the node after anti-thymus-globulin treatment The architecture of the nodes in this series was altered diffusely as seen in table 1. The table shows that the alterations in morphology were similar to those in the anti-theta series, in general, but a few differences must be emphasized. First, it should be pointed out that large lymphocyte follicles with active germinal centers were seen on the 1st day in contrast to the 2nd and 4th day in the anti-theta series. Secondly, the depletion of the smalllymphocytes in the paracortical areas was not so prominent in this series compared with the antitheta series. Table 1 shows that the two series do not differ as to the morphologic alterations in the medulla and in the sinuses. Histology of the node after normal rabbit globulin treatment Table 1 shows the morphologic alterations following the treatment with normal rabbit gammaglobulin. These alterations were localized in the cortex and medulla leaving the other regions intact. The cortical area was greatly expanded due to the app'earance of large lymphocyte follicles with active germinal centers (fig. 2 and 3). The number of those follicles varied from 4 to 12 per node and exeeded the number found in the other series. No depletion in the paracortical constituents was found and the morphology of this area was similar to that in the control series. All the medullary elements were hyperplastic. Medullary cords were wide containing numerous large pyroninophilic cells and mature plasma cells. The mitotic activity was high. Medullary cords expanded into paracortex and even to the cortical areas (fig. 2 and 3). Sinuses were inconspicuous and their cellular content was sparse.
Discussion The histologic changes in the lymphoid organs after treatment with anti-thymus-serum have been studied (MARTIN et al. 1967, TURK et al. 1967, TAUB et al. 1968). These studies have confirmed that anti-thymus-serum specifically depletes the small lymphocytes in the paracortical areas of the lymph node. Similar results have been reported by other workers with anti-theta-serum thus confirming the thymus-dependence of the theta-bearing small lymphocytes (RAFF 1969, RAFF et al. 1970, SCHLESINGER 1970). Some controversy exists, however, concerning the changes found in the medulla and lymphocyte follicles. An intraperitoneal injection of normal rabbit serum in mice caused lymphoid hyperplasia in paracortex and germinal centers whereas these changes were not observed after anti-thymus serum (TURK et al. 1967). On the other hand, the histologic features of immunization with pronounced medullary hyperplasia and germinal center formation were found in mice after normal rabbit serum and anti-thymus serum subcutaneously (TAUB et al. 1968). The results of the present study confirm that anti-theta as well as anti-thymus globulins act as suppressive agents for the small lymphocytes of the lymph node paracortex. Evidence of this suppressive action was found in the nodes taken one day after the last globulin injection and the changes in both series persisted for 7 or 14 days. As to the quantitative differences between the two globulins, it was evident that the effects of anti-theta-globulin were more pronounced. This could support the view that there are at least two populations of lymphocytes in the thymus; the one bearing theta-antigen, the other not. Thus an antiglobulin prepared against these cells would not be as potent and selective as that prepared against the theta-isoantigen proper. The results also support the view that thymus-dependent small lymphocytes with theta-isoantigen are localized in the paracortical areas of the lymph nodes. The strong signs of immunization including the medullary hyperplasia and the appearance of lymphocyte follicles with active germinal centers were observed in the nodes after the treatment with all these globulins. In this respect, however, differences were observed that need further discussion. The signs of intense humoral immune response appeared in the nodes taken one day after the last dose of anti-thymus and normal rabbit globulins. On the
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other hand, there was a delay of 2 to 4 days in the appearance of such humoral response in the anti-theta-globulin treated animals. The signs of humoral response were most pronounced in the animals treated with normal rabbit globulin, a finding contradictory to the earlier observations (TAuB et al. 1968). These differences in the initiation and strength of the humoral immune response could be explained on the basis of the T- and B-cell interaction. It has been suggested previously that an interaction between T- and B-lymphocytes is important in humoral immune response (MILLER et al. 1971). The exact mechanism of this interaction, however, remained obscure (MILLER et al. 1971). The results of the present study could suggest that anti-theta-globulin bound to its theta-isoantigen on T-cell surface would create a hindrance for the access of B-lymphocytes to the vicinity thus making the T-B interaction impossible. This interaction could be possible after some two to four days after the new circulating T-lymphocytes had horned in the paracortex via the postcapillary venules. Indeed, small lymphocytes were evident in the lumen and in the vicinity of paracortical postcapillary venules by that time. In the nodes of the animals treated with normal rabbit globulin no such disturbance in T-B interaction happened and consequently no delay in humoral response was observed. The fact that no delay in antibody response was observed in cases treated with antithymus-globulin suggests differences in potency between this and anti-theta-globulin, as stated earlier in this discussion. It seems evident that anti-theta-globulin is the more potent and more selective T-cell suppressor of these two. The present work indicates that the standardized reporting system of COTTIER et al. is a very applicable method in assessing the immunological state of the lymph node even in experimental animals. The findings are easy to summarize in tabular form and thus ready for mutual comparisons. These facts advocate the acceptance of this reporting system into general use.
Literature COHEN, A., and M. SCHLESINGER, Absorption of guinea pig serum with agar. Transplantation 10, 130-132 (1971). COTTIER, H., J. TURK and L. SOBIN, A proposal for a standardized system of reporting human lymph node morphology in relation to immunological function. J. Clin. Path. 26, 317-331 (1973). GRAY, J. G., A. P. MONACO, M. L. WOOD and P. S. RUSSELL, Studies on heterologous anti-lymphocyte serum in mice. I. In vitro and in vivo properties. J. Immunol. 96, 217-228 (1966). LEVEY, R. H., and P. B. MEDAWAR, Some experiments on the action of anti-lymphoid antisera. Ann. N. Y. Acad. Sci. 129, 164-177 (1966). MARTIN, W. J., and J. F. MILLER, Site of action of antilymphocyte globulin. Lancet 1,1285-1287 (19S7). MILLER, J. F., A. BAS'fEN, J. SPENT and C. CHEERS, Interaction between lymphocytes in immune responses. Cell. Immunol. 2, 469-495 (1971). PARROTT, D. M. V., M. A. B. DE SOUSA and J. EAST, Thymus-dependent areas in the lymphoid organs of neonatally thymectomized mice. J. Exp. Med. 123, 191-204 (1966). POLLIACK, A., N. LAMPEN, B. D. CLARKSON and E. DE HARVEN, Identification of human Band T lymphocytes by scanning electron microscopy. J. Exp. Med. 138, 607-616 (1973). RAFF, M. C., Theta isoantigen as a marker of thymus-derived lymphocytes in mice. Nature 224, 378-379 (1969). - and H. H. WORTIS, Thymus dependence of E)-bearing cells in the peripheral lymphoid tissues of mice. Immunology 18, 931-942 (1970). REIF, A. K, and J. M. V. ALLEN, The AKR thymic antigen and its distribution in leukemias and nervous tissue. J. Exp. Med. 120, 413-421 (1964). - - Mouse thymic iso-antigens. Nature 209, 521-522 (1966). SCHLESINGER, M., Anti-E)-antibodies for detecting thymus-dependent lymphocytes in the immune response of mice to SRBC. Nature 226, 1254-1255 (1970). TAUB, R. N., and K M. LANCE, Histopathological effects in mice of heterologous antilymphocyte serum. J. Exp. Med. 128, 1281-1307 (1968). TURK, J. L., and D. A. WILLOUGHBY, Central and peripheral effects of anti-lymphocyte sera. Lancet I, 249-251 (1967). Author's address: K. J. SYRJANEN, MD, Department of Pat,hology, Jorvi Hospital, SF -02740 Espoo 74 (Finland). 12*
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