SummaryCD45 expression is essential for immunoglobulin (Ig)-mediated B cell activation. Treatments with either anti-Ig or anti-CD45 suggest that CD45 may facilitate early signaling events such as calcium mobilization, and phosphoinositide hydrolyis as well as later events leading to transcription of genes such as c-myc. To examine the role of CD45 more extensively, CD45-deficient mice were generated by disruption of exon 6. Although normal numbers of B cells were found in peripheral lymphoid tissues, CD45-deficient cells failed to proliferate upon IgM crosslinking. In the present study, we demonstrate that the fraction of high buoyant density B cells is reduced while low buoyant density cells are increased. Moreover, there is a significant decline in the number of splenic B cells of the mature lgD hi, IgM I~ phenotype. Although both the basal and anti-Ig-induced levels of phosphorylation of Ig-0t and phospholipase C',/2 are indistinguishable from that observed in CD45 + control B cells, a major distinction was found in Ca 2+ mobilization. While anti-Ig-induced mobilization of intracellular Ca 2+ stores was normal, influx from extracellular sources was abrogated. This finding reveals a novel pathway of regulating B cell responses mediated by CD45. Evidence from a variety of sources clearly demonstrates that the expression of CD45 is essential for activation of B lymphocytes after cross-linking of the B cell receptor (BCR) (1-6). In vitro activation studies using primary cells treated with either anti-Ig or anti-CD45 suggest that CD45 facilitates early signaling events such as calcium mobilization (6), and phosphoinositide hydrolyis as well as later events leading to transcription of genes such as c-myc (7). Cell lines have also been used to examine the role of CD45. In some cases conflicting results have emerged from such studies. For example, it was demonstrated that the plasmacytoma J558LIxm3, which lacks CD45, failed to mobilize Ca 2+ after BCR cross-linking. This effect was restored in J558LF~m3 cells by transfection with a eDNA encoding CD45 (1). In contrast, CD45-variants of the CD45 + immature cell line WEHI231, had a slightly delayed but increased and prolonged Ca 2+ response upon BCR cross-linking (8). Whether this discrepancy accurately reflects the different maturation states of these cell lines, or is due to abnormalities peculiar to transformed cells, remains to be determined.To examine the role of CD45 more extensively, CD45-deficient mice were generated by disruption of exon 6 (9). Mice homozygous for the CD45 exon 6 mutation lacked CD45 expression on B cells and on the majority of thymocytes and peripheral T cells. A block in T cell development occurred at the transition from immature CD4+CD8 + double-positive to mature single-positive thymocytes resulting in a significant reduction in peripheral T cells. Residual peripheral T cells exhibited impaired anti-CD3 and anti-TCR signaling. In contrast to the paucity of peripheral T cells, approximately normal numbers of B cells were found in peripheral ...

Cell preparations in cytochemistry are conventionally analyzed with transmitted light after fixation and reaction with agents such as azo-coupling dyes. With cell suspensions stained with fluorescent cytochemical dyes, cells can also be analyzed and sorted by flow cytometry. We have exploited the intense red fluorescence of Fast Red Violet LB generated in cytochemical reactions to perform flow cytometric analyses of alkaline phosphatase (AP) expression in rat bone marrow stromal cells. By modifying staining protocols of single-cell suspensions, we demonstrate that in comparison to staining with Fast Red TR, the method is specific, can distinguish among various levels of enzyme expression within the whole population, and permits enzyme kinetic studies of heterogeneous cell populations. The method was applied to study the effect of the glucocorticoid dexamethasone (Dx) on cell proliferation and AP expression. In low AP-expressing cells, Dx treatment at 10(-8) M increased the [3H]-thymidine labeling index from 3.85% to 5.24% (p less than 0.01). In contrast, high AP-expressing cells were unlabeled by [3H]-thymidine. The staining and analytical methods reported here facilitate the detection, isolation, and quantification of subpopulations of bone marrow stromal cells that express alkaline phosphatase activity. These experiments demonstrate the value of flow cytometry as an adjunct to conventional cytochemical methods.

Bone marrow stromal cells are a mixed population that contribute to the formation of the hematopoietic microenvironment. The osteogenic lineage includes populations of cells that, in culture, form discrete nodules of mineralized tissue when grown in the presence of ascorbic acid and sodium beta-glycerophosphate. We have used nodule formation to assay for the self-renewal capacity of osteoprogenitor cells in chick bone marrow cultures. To examine the regulatory influence of dexamethasone (Dx), first subcultures were grown continuously or split 1:1 at repeated subculture. Cells in continuous culture exhibited less than two population doublings, while cellular proliferation and alkaline phosphatase area were inhibited by 10(-8) mol/L Dx. Cells in split (redistributed) cultures exhibited up to 14 population doublings and cellular proliferation was also inhibited by Dx. In contrast with continuous cultures, redistributed cultures treated with Dx had increased alkaline phosphatase area and 15-fold larger amounts of mineralized tissue formation than controls. Osteogenesis was sustained for up to four subcultures and the ratio of mineralized tissue area to alkaline phosphotase positive cell area was at most 0.55. These data indicate that the osteogenic lineage of bone marrow stromal cells contains self-renewing progenitors that are recruited by Dx in culture and that at a maximum, only 55% of the alkaline phosphatase-positive cell population contributes to osteogenesis.

Glucocorticoids and sex-steroids can modulate osteogenesis in vivo and in vitro. Although the effects of glucocorticoids on bone cells in vitro have been described in detail, the role of sex-steroids is not as well defined. We examined whether sex-steroids influence bone metabolism indirectly by regulating glucocorticoid effects on bone. Interactions of the sex-steroid progesterone or its analog RU38486 with the glucocorticoid dexamethasone (dex) were studied in functional assays of osteogenesis. Three osteoblastic models were evaluated: (1) the rat bone marrow stromal cell (RBMC) nodule system; (2) the chick periosteal osteogenesis (CPO) model; and (3) ROS 17/2.8 cells. RU38486, progesterone, and unlabelled dex competitively inhibited 3H-dex uptake by ROS 17/2.8 cells as well as its (3H-dex) binding to cytosol preps. Both RU38486 and progesterone inhibited dex-induced increases in alkaline phosphatase in CPO cultures, in RBMC cultures, and in ROS 17/2.8 cells. Dex-induced decreases in cell proliferation in ROS 17/2.8 cells were reversed by RU38486 but dex-induced increases in proliferation in the CPO model were not affected. In CPO cultures, dex-induced increases in collagen synthesis were inhibited completely by RU38486 and progesterone. Dex-dependent nodule formation in the RBMC was blocked by RU38486. Both RU38486 and dex mediated reduction of calcium uptake in the CPO model but did not affect mineralized tissue area. The data indicate that RU38486 and progesterone competitively inhibit dex-mediated stimulation of osteogenesis in vitro; this inhibition is exerted on early but not late stage differentiation events of osteoprogenitor cells.