Search

Anti-CD45RA antibody defined by anti-2H4 monoclonal antibody has been reported to split CD4+T cells into two distinct subpopulations. CD45RA antigen is present on the surface of virtually more than 95% B lymphocytes in the purified tonsillar B-cell preparations. We examined the role of CD45RA antigen on human B-cell function using this antibody. The addition to anti-2H4 to tonsillar B cells inhibited the proliferative response induced by Staphylococcus aureus Cowan strain I(SAC) in a dose-dependent manner. Kinetic analysis indicated that anti-2H4 exerted its inhibitory effect when added within the first 24 h of culture initiation during a 72-h culture period. Anti-2H4 inhibited the transferrin receptor expression without interfering with the expression of the IL-2 receptor on SAC-stimulated B cells in a short-term culture. Anti-2H4 blocked the progress of SAC-stimulated B cells from the G1 to S phase of the cell cycle. These events suggested that anti-CD45RA MoAb inhibited the proliferative response by directly acting on B cells in the G1 phase. In addition, anti-CD45RA antibody also had a suppressive effect on early phase of B-cell differentiation. This effect appeared to be independent of its suppressive effect on proliferation, because anti-CD45RA did not inhibit the proliferative response of preactivated B cells with lymphokines. These studies suggested that the restricted epitope recognized by anti-2H4 antibody may be directly involved in regulatory function on B cells.

The role of prostaglandin E2 (PGE2) in the regulation of human B cell proliferation in vitro was examined. Initial studies demonstrated that monocyte (M phi)-mediated suppression of B cell proliferation in Staphylococcus aureus (SA)-stimulated cultures was diminished by indomethacin, and thus it was suggested that cyclooxygenase pathway products of arachidonic acid played a role in the regulation of B cell activation. The possibility that PGE2, one of the major products of this pathway generated by M phi, affected human B cell responses was therefore investigated. PGE2 was found to suppress B cell DNA synthesis and proliferation stimulated by SA in the presence or absence of B cell growth factor (BCGF)-containing T cell supernatants. Suppression was concentration-dependent; significant inhibition was observed in indomethacin-blocked cultures with 10(-10) to 10(-9) M PGE2. In contrast to the effect of PGE2 on SA-induced B cell proliferation, it had minimal effect on pokeweed mitogen (PWM)-stimulated B cell or T cell DNA synthesis. The effect of PGE2 on the generation of BCGF activity from mitogen-stimulated T cells was also examined. PGE2 (10(-7) M) did not inhibit the production of BCGF activity from PWM-stimulated T cells, or from T cells stimulated with the combination of phytohemagglutinin and phorbol myristate acetate. Thus, PGE2 inhibits B cell proliferation in response to SA but not PWM, and has little effect on the production of BCGF by T cells. These results indicate that PGE2 at physiologically relevant concentrations exerts selective regulatory effects on human B cell responses.

The responses of resting human B lymphocytes to a variety of activation signals were studied. Human tonsillar B lymphocytes were separated according to size by countercurrent elutriation. The small B lymphocytes were then stimulated in vitro with various concentrations of anti-mu antibody in the presence or absence of B cell growth factor (BCGF) or with Staphylococcus aureus Cowan strain I (SAC). Cellular volume changes and RNA synthesis were measured over the first 24 h of stimulation and were similar with either 15 micrograms/ml of anti-mu, 100 micrograms/ml of anti-mu, or SAC. In the subsequent 24 h, however, substantial increases occurred in the amount of RNA synthesis and cell enlargement only in those cultures stimulated with 100 micrograms/ml of anti-mu or SAC, but not in the cultures stimulated with 15 micrograms/ml of anti-mu. The addition of BCGF to those cultures stimulated with 15 micrograms/ml of anti-mu did not alter the increases in cellular volume and RNA synthesis found 24 h after stimulation with anti-mu alone. However, over the subsequent 24 h, the presence of BCGF in culture enhanced both B cell volume changes and RNA synthesis, when compared to cultures stimulated with 15 micrograms/ml of anti-mu alone. In addition, BCGF enhanced DNA synthesis in cultures stimulated with low and high concentrations of anti-mu. DNA content changes following stimulation with anti-mu, anti-mu plus BCGF, and SAC were also measured using propidium iodide staining and flow cytometry. Optimal concentrations of anti-mu induced 20% of the resting B cells to enter S phase, while optimal concentrations of anti-mu plus BCGF or SAC induced approximately 40%. Finally, prestimulation of resting B cells for 24 h with a low concentration of anti-mu, sufficient for cell enlargement but not S phase progression, allowed for rapid entrance of the prestimulated B cells into S phase when a high concentration of anti-mu or SAC was added. These findings suggest the existence of a control point in the progression of human B cells through the cell cycle. This control point is located in the G1 phase of the cycle and is reached 24 to 36 h after a surface immunoglobulin-mediated stimulus.

In certain human IgM and IgG cell lines, immunoglobulin (Ig) secretion is highly stimulated by a B cell inducing factor (BIF) that is free of interleukin 2 (IL 2). BIF also induces Ig secretion in purified peripheral blood B cell populations that have been mitogenically stimulated by Staphylococcus aureus bacteria. Low concentrations of IL 2 (less than 20 U/ml) are not active in these systems. We now show that IL 2 at concentrations above 100 U/ml can induce Ig secretion in these blood B cells and B cell lines. Both conventional IL 2, purified from the human JURKAT and gibbon MLA-144 cell lines, and recombinant IL 2 are active. Very high concentrations approaching 10(4) U/ml are optimal for Ig secretion. Antibody to the T cell IL 2 receptor, anti-Tac, did not inhibit stimulation of the IgM cell line SKW6.4 by IL 2, and no Tac antigen was detected on the cells. The 9B11 monoclonal anti-IL 2 antibody that neutralizes T cell growth activity also abrogates stimulation of Ig secretion by conventional and recombinant IL 2 in the SKW6.4 cell line. However, the 1H11 monoclonal anti-(conventional thr3-glycosylated IL 2), which does not neutralize T cell growth activity, does inhibit induction of Ig secretion by the corresponding IL 2 in the B cell line. These results suggest that IL 2 stimulates B cells via a low-affinity interaction with a receptor different from the Tac receptor identified on T cells, and that the active site on the IL 2 molecule for B cells differs from that for T cell targets. If IL 2 promotes Ig secretion by binding with a low affinity to the B cell BIF receptor, IL 2 and BIF could be homologous proteins.

Transferrin receptors are expressed on proliferating cells and are required for their growth. Transferrin receptors can be detected after, but not before, mitogenic stimulation of normal peripheral blood T and B cells. T cells demonstrate a functional requirement for transferrin receptors in the activation process. These receptors, in turn, are induced to appear by T cell growth factor (interleukin 2). In the experiments reported here, we examined the regulation of transferrin receptor expression on activated human B cells and whether these receptors are necessary for activation to occur. Activation was assessed by studying both proliferation and immunoglobulin secretion. We determined that transferrin receptor expression on B cells is regulated by a factor contained in supernatants of mitogen-stimulated T cells (probably B cell growth factor). This expression is required for proliferation to occur, because antibody to transferrin receptor (42/6) blocks B cell proliferation. Induction of immunoglobulin secretion, however, although dependent on phytohemagglutinin-treated T cell supernatant, is not dependent on transferrin receptor expression and can occur in mitogen-stimulated cells whose proliferation has been blocked by anti-transferrin receptor antibody. These findings support a model for B cell activation in which mitogen (or antigen) delivers two concurrent but distinct signals to B cells: one, dependent on B cell growth factor and transferrin receptor expression, for proliferation; and a second, dependent on T cell-derived factors and not requiring transferrin receptors, which leads to immunoglobulin secretion.

The role of IL-4 in human B cell activation, proliferation, and differentiation was examined. rIL-2, but not rIL-4, was able to promote maximum proliferation and generation of Ig-secreting cells in cultures of highly purified B cells stimulated with Cowan I Staphylococcus aureus (SA). Addition of rIL-4 to rIL-2-supported cultures of SA-stimulated peripheral blood, spleen, or lymph node B cells dramatically suppressed both proliferation and differentiation. Results from experiments in which rIL-4 was added to culture at progressively later times indicated a requirement for rIL-4 to be present during the first 2 days of a 5-day incubation to cause inhibition of responsiveness. When a two-stage culture system was utilized, rIL-4 was found to support proliferation or differentiation of B cells initially activated with SA for 2 days only minimally. However, rIL-4 did not inhibit responses of SA preactivated B cells supported by IL-2. The presence of rIL-4 during the initial 48-h activation of B cells with SA and rIL-2 resulted in a profound inhibition of the ability of the activated B cells to respond subsequently to rIL-2 or lymphokine-rich T cell supernatants. A similar 48-h incubation with rIL-4 alone without SA had no effect on subsequent B cell responsiveness. The presence of rIFN-gamma during B cell activation decreased the inhibitory effect of IL-4. Other cytokines including IFN-alpha, IL-1, and commercially available low m.w. B cell growth factor also diminished the inhibitory effect of IL-4. These results indicate that IL-4 inhibits the capacity of human B cells to be activated maximally by SA and rIL-2 and therefore suggest a new immunomodulatory role for this cytokine.