Kirstine A. Knox

Inhibition by glucocorticoid and staurosporine of IL-4-dependent CD23 production in B lymphocytes is reversed on engaging CD40.

IL‐4 synergizes with signals delivered through CD40 both for the induction of CD23/FcRH expression and for IgE synthesis. Moreover, engagement of CD40 on the B cell surface by MoAb overcomes the ability of intcrferons. transforming growth factor‐beta, or anti‐CD 19 to inhibit IL‐4‐dependent change. We now report that occupancy of CD40 relieves potent suppression of IL‐4‐induced CD23 production by glucocorticoid or the relatively broad‐acting kinase inhibitor staurosporine. Interruption of the IL‐4 signal was observed with concentrations of staurosporine considered to be selective for protein kinase C (PKC) inhibition (IC50= 10 nm) but not with genistein or tyrphostins. effective inhibitors of tyrosine kinase activity. On ligation of CD40, staurosporine no longer inhibited the IL‐4 signal: at concentrations of between I and 20 nM. staurosporine actually increased by as much as 100% the rate of CD23 production stimulated on simultaneous activation through CD40 and 1L‐4R. Such augmentation was not observed when the more specific PKC inhibitor RO‐31‐8220 was used; indeed. CD40 engagement was unable to overcome the ability of this inhibitor to block IL‐4‐promoted CD23 induction (IC50= 10 μm). Occupancy of CD40 did, however, thwart completely the usual ability of prednisolone to inhibit the IL‐4 signal leading to CD23 induction. Activation through CD40 left inhibition of phorbol ester‐induced CD23 expression by staurosporine, RO‐31‐8220, or glucocorticoid unchecked. These findings further highlight the intimate level of cross‐talk existing between CD40 and IL‐4R on resting B lymphocytes to promote CD23 expression, a phenotypic change which preludes IgE synthesis.

Stimulation of human B lymphocytes by phorbol esters reported to be selective in the protein kinase C isoforms they activate

It is reported here that B cells can be stimulated by two phorbol esters which, in cell free substrate phosphorylation assays, are selective in the PKC isoforms they activate: thymeleatoxin (Thy) stimulates all of the classical (c) or Group A PKCs (alpha, beta 1, beta 2 and gamma) but not PKC delta and epsilon which belong to the novel (n) or Group B PKCs, while 12-deoxyphorbol-13-O-phenylacetate-20-acetate (dPPA) is a specific activator of PKC beta 1. By itself, phorbol 12-myristate, 13-acetate (PMA)--which activates all cPKC and nPKC--was, on a molar basis, some 40-times more potent than either Thy or dPPA which were themselves equipotent at promoting DNA synthesis in resting B cells: the peak response achieved with Thy and dPPA was higher (1.4 x) than that obtained with PMA. In the presence of calcium ionophore, PMA, Thy and dPPA all stimulated a higher (and equivalent) peak response which was achieved at a lower phorbol ester concentration in each case: however, whereas Thy now approached PMA in potency, dPPA remained some 40-times less potent.

Stimulation of tyrosine phosphorylation without inositol lipid hydrolysis in human B lymphocytes on engaging CD72.

Occupancy of CD72 on resting tonsillar B cells by monoclonal antibodies (mAb) promotes entry into the G1 phase of the cell cycle with an accompanying increase in MHC Class II expression and provides a co‐stimulus to immobilized anti‐μ for driving DNA synthesis. We now report that engagement of CD72 by mAb stimulates tyrosine phosphorylation in B cells with a peak of activity seen at 5–10 min. Two major substrates of 29 and 57 kDa showed a basal level of phosphorylation which increased with time, while a 40 kDa protein and several other minor components were phosphorylated de novo on the addition of mAb to CD72. Inositol lipid hydrolysis was found to be unperturbed, although a shallow rise in the basal level of intracellular free Ca2+ was provoked on engaging CD72. Receptor cross‐linking was not a requirement for signaling human B cells through CD72: simple occupancy by univalent antibody was sufficient both to trigger the rise in basal [Ca2+]i and to promote DNA synthesis.