This year we celebrate the first centennial of the discovery of germinal centers by Flemming in 1884. The present paper reviews and adds new data to the functional anatomy of a germinal center. Emphasizing its reactive nature, we first describe a germinal center reaction and then deal with its infrastructural aspects and constituent cell populations, both lymphoid and nonlymphoid. Elements involved in the de novo formation of a germinal center, like antigen, T cells, and the mysterious germinal-center-precursor cell, are discussed. Next, attention is paid to the requirements for lymphoid cells to migrate into germinal centers, and novel features of germinal-center-seeking cells are presented. Subsequently, we discuss kinetic aspects of the high proliferative activity in a germinal center; and finally a description of the functional capacities of germinal-center-derived cells, such as B memory cells and IgM-antibody-forming cell precursors, completes this picture of present-day knowledge of the germinal center, a structure which has yet to reveal its last secrets.

Haemopoietic cells carry a variety of cell-surface molecules, some of which are known to have allotypic variation. In rats, the RT7 alloantigenic system has been well documented using alloantisera. We have produced the first mouse hybridoma cell line secreting an antibody, HIS41, which binds to leucocytes of rat strains carrying the RT7.2 but not the RT7.1 determinant. An IgG2b isotype switch variant (HIS41.2b) of the original HIS41 (IgG1 isotype) was also made. HIS41 showed a clear and discrete binding in immunofluorescent and histological experiments and has already been used in several studies on haemopoietic cell turnover and differentiation employing PVG rats congenic for RT7. The present study addresses the question of whether the RT7 gene products are members of the L-CA family, which has been a matter of controversy over the last decade. When using HIS41 for the analysis of tissue distribution and molecular weight of RT7 gene products, a strong similarity was evident with the data reported for the L-CA detected by MRC OX-1 and MRC OX-30. These two MoAb have been reported to bind to all members of the L-CA family. All haemopoietic cells, excluding erythrocytes and the more mature stages of erythropoiesis, stained with HIS41. The molecular weights of HIS41 binding molecules on thymocytes and peripheral T cells were comparable to the L-CA precipitated by MRC OX-1. Capping and sequential immunoprecipitation studies indicated that HIS41 and MRC OX-30-binding molecules were identical. MRC OX-1, however, appeared to bind only a subset of these molecules. Thus, our study confirms the identity of RT7.2 gene products and L-CA. It also revealed a difference between MRC OX-1 and MRC OX-30 not noticed previously.

Occlusal abnormality may contribute to osteoarthrosis of the temporomandibular joint (TMJ). Since mechanical force may induce changes in the extracellular matrix, we tested the hypothesis that unilateral removal of teeth and the resulting unilateral mastication change the content of glycosaminoglycans (GAGs) in the TMJ condyle and disc. Lower-right-side teeth were extracted from 12 adult male rabbits, which were killed 3 or 6 weeks later. Three normal rabbits served as controls. Sections were analyzed for morphological changes and levels of sulfated GAGs in the condyle and disc. Unilateral removal of teeth led to thickening of the condylar cartilage, alterations in the morphology of chondrocyte nuclei in the condylar cartilage and disc, and increases in levels of negatively charged ions in the hypertrophic layer of condylar cartilage. Small differences were observed, after unilateral removal of teeth, between the functional and non-functional sides of the TMJ. The results suggest that in response to mechanical stress, chondrocytes alter sulfated glycosaminoglycan (GAGs) synthesis and degradation rates, resulting in an elevated level of sulfated GAGs in the condylar cartilage.

Three mouse monoclonal antibodies (mAb) directed against rat B lineage antigens were produced. The mAb, designated HIS14 (IgG1), HIS22 (IgM) and HIS24 (IgG2b), were characterized for binding to lymphoid and nonlymphoid tissues by immunoperoxidase staining of frozen sections and by (double-) immunofluorescence staining of single cell suspensions from lymphoid organs. HIS14 recognized a pan B cell determinant: it reacted with virtually all cells of each anatomic B cell compartment and with about 95% of surface (s)Ig+ cells in thoracic duct lymph and in suspensions of spleen and lymph nodes. HIS22 and HIS24 detected B lineage-associated antigens expressed by major subpopulations of B cells. HIS22 predominantly stained the lymphocyte corona, but not (or weakly) the germinal centers and splenic marginal zones, whereas HIS24 reacted with both corona and germinal center and not (or weakly) with marginal zone. In accordance with this, substantial proportions of sIg+ cells in spleen cell suspensions did not express HIS22 or HIS24 determinants (20% and 27%, respectively). In bone marrow the vast majority of cytomplasmic mu+ pre-B cells were HIS14+ and HIS24+, and up to one third also HIS22+, indicating an appearance of the determinants early in B lymphocytopoiesis. The antigens recognized by HIS14, HIS22 and HIS24 are lost during the final stage of B cell differentiation: none of the mAb bound to plasma cells. As far as detectable, neither cells of myeloid and erythroid lineages in bone marrow nor thymocytes were stained by HIS14, HIS22, or HIS24. In suspensions of peripheral lymphoid organs (spleen and lymph nodes) but not in thoracic duct lymph, HIS14 and HIS24 labeled a small proportion (12% and 14%, respectively) of Ig- cells. HIS22 did not bind to Ig- peripheral lymphocytes. Reactivity of HIS14, HIS22 and HIS24 with nonlymphoid tissues was virtually absent; HIS22 stained the high endothelial venules in lymph nodes and Peyer's patches. As determined by immunoblotting, the antigenic determinants on lymph node cells recognized by HIS14, HIS22 and HIS24 were present on molecules with an apparent molecular mass of 205 kDa, 210 (and 175) kDa and 205 kDa, respectively, which is similar to the molecular mass of the B cell form of the rat leukocyte common antigen. In addition, the antigens recognized by HIS14, HIS22 and HIS24 co-capped with the leukocyte common antigen. This suggests that each of the three mAb recognize determinants present on the B cell form of the leukocyte common antigen.

Occlusal abnormality may contribute to osteoarthrosis of the temporomandibular joint (TMJ). Since mechanical force may induce changes in the extracellular matrix, we tested the hypothesis that unilateral removal of teeth and the resulting unilateral mastication change the content of glycosaminoglycans (GAGs) in the TMJ condyle and disc. Lower-right-side teeth were extracted from 12 adult male rabbits, which were killed 3 or 6 weeks later. Three normal rabbits served as controls. Sections were analyzed for morphological changes and levels of sulfated GAGs in the condyle and disc. Unilateral removal of teeth led to thickening of the condylar cartilage, alterations in the morphology of chondrocyte nuclei in the condylar cartilage and disc, and increases in levels of negatively charged ions in the hypertrophic layer of condylar cartilage. Small differences were observed, after unilateral removal of teeth, between the functional and non-functional sides of the TMJ. The results suggest that in response to mechanical stress, chondrocytes alter sulfated glycosaminoglycan (GAGs) synthesis and degradation rates, resulting in an elevated level of sulfated GAGs in the condylar cartilage.

To identify factors influencing the in vivo proliferate activity of bone marrow pre-B cells, the metaphase-blocking drug, vincristine sulfate, was injected into (a) mice depleted of B lymphocytes by treatment with anti-mouse IgM antibodies from birth; (b) hematologically deficient W/Wv and Sl/Sld mutants, and (c) mice injected with a foreign agent, sheep red blood cells (SRBC). Subsequently, a quantitative measure of pre-B cell proliferation was provided by examining marrow cells by immunofluorescence labeling for the absolute number of pre-B cells, identified by the presence of cytoplasmic mu chains (c mu) without surface mu (s mu), which had been arrested in metaphase. In anti-IgM-treated mice, some changes were observed in the size of the large pre-B cell population and in the incidence of mitotic cells after vincristine administration, but the overall production rate of pre-B cells did not differ from that in controls given normal rabbit serum. Pre-B cell kinetics in W/Wv and Sl/Sld mice also generally resembled those in homozygous controls. In contrast, after SRBC injection, there was an increase in the rate at which large pre-B cells entered mitosis. Thus, the proliferation of c mu + s mu- bone marrow pre-B cells shows no evidence of feedback control from the mature B lymphocyte pool, as indicated by lack of stimulation of pre-B cell production in anti-IgM-treated mice, and is independent of the hemopoietic defects of W/Wv or Sl/Sld mutants. On the other hand, the increased bone marrow pre-B cell proliferation after SRBC injection demonstrates that the magnitude of B cell genesis in the bone marrow can be influenced by extrinsic agents and thus may be influenced by environmental stimuli.

The cycling B precursor cells in rat bone marrow (BM) that carry the B220 antigen and no surface Ig daily produce 780 million new cells. The pool of recirculating B lymphocytes in the rat, however, renew at a rate of only about 40 million cells/day. To analyze at which stages in B lymphocyte genesis the cell loss occurs, we identified post-mitotic cells in the rat BM B lineage, and determined their renewal rates. We used 5-bromo-deoxyuridine (BrdUrd) to label DNA-synthesizing cells, identifying incorporated BrdUrd with the mouse monoclonal antibody BU-1. B lineage cell subsets were identified by the markers HIS24 antigen (rat B220), terminal deoxynucleotidyl transferase (TdT), Ig mu heavy chain, and complete Ig. By use of double and triple immunocytology, we determined the extent of BrdUrd incorporation in the various B lineage compartments [HIS24+TdT-Ig-, TdT+, cytoplasmic mu chain (c mu)+ surface (s) IgM- pre-B, sIgM+ B]. Both sIgM+ B lymphocytes and all B precursors with cell diameters less than 11-12 microns were virtually devoid of DNA synthesis, as indicated by S-phase indices below 2%. In contrast, S-phase indices of large B precursors ranged between 43%-66%. We established the renewal rates of nondividing BM B lineage cells by placing osmotic minipumps containing BrdUrd subcutaneously in the flank of rats. The nondividing BM B lineage cells all renewed rapidly at rates between 2.4% and 5.6%/h, representing average half-lives of 29 to 12 h. In absolute numbers, the renewal/day/whole body BM was 165 X 10(6) for sIgM+ B lymphocytes, 422 X 10(6) for small c mu+ sIgM- pre-B cells, 89 X 10(6) for small TdT+ cells and 35 X 10(6) for small HIS24+TdT-Ig- cells. Assuming that recirculating B lymphocytes in the periphery are the descendants of BM sIgM+ B lymphocytes, which in their turn are the progeny of small pre-B cells, the renewal data indicate the following. Of the 165 million potentially available BM B lymphocytes, only 40 million cells become incorporated in the pool of recirculating B lymphocytes, representing a loss of 75%. BM B lymphocytes, in turn, use only (165/422 X 100% = ) 40% of the potential output from their immediate precursors. The 60% loss that occurs here may reflect the extent of aberrant Ig light chain gene rearrangement in normal B lymphocyte genesis.(ABSTRACT TRUNCATED AT 400 WORDS)

SummaryTo gain insight into the clonal organization of lymphoid organs, we studied the distribution in situ of donor-derived cells in near-physiological chimeras. We introduced RT7 b fetal liver cells into nonirradiated congenic RT7 a neonatal rats. The chimerism 6-20 wk after injection ranged from 0.3 to 20%. The numbers of cell clones simultaneously contributing to cell generation in a particular histological feature were deduced from the variance in donor cell distribution. In bone marrow and thymus, donor-derived lymphoid cells were found scattered among host cells, indicating a high mobility of cells. In bone marrow, donor cells were evenly distributed over the entire marrow, even at low chimerism. This indicates that leukopoiesis is maintained by the proliferation of many clones. In the thymus, the various lobules showed different quantities of donor-derived lymphoid cells. Mathematical analysis of these differences indicated that 17-18 cell division cycles occur in the cortex. In spleen, the distribution of donor-derived cells over the germinal centers indicated that 5 d after antigenic stimulation, germinal centers develop oligoclonaUy. The main conclusions of this work are that (a) bone marrow and thymus are highly polyclonal; (b) 17-18 divisions occur between prothymocyte and mature T cell; and (c) lymphoid cells disperse rapidly while proliferating and differentiating.YhProliferation and differentiation from pluripotent emopoietic stem cells to lymphocytes, cell populations with highly diverse antigen-receptor specificities are generated and maintained. To understand this generation of the antigen-receptor repertoire, a better insight into the donal organization of the lymphoid system is required. Particularly relevant issues are the numbers orb and T lymphoid cell clones (a clone is the progeny of a single ancestor cell) that simultaneously contribute to the generation of lymphocytes, the size and antigen receptor composition of individual clones, and the nature of the interactions of the progeny cells with the environment. To study the number of clones simultaneously engaged in production of hemo/lymphopoietic cells, chimeric animals are used since genotypic fluctuations in these animals are an indication for progenitor cell numbers.