Lymphoma models for B cell activation and tolerance. V. Anti-Ig mediated growth inhibition is reversed by phorbol myristate acetate but does not involve changes in cytosolic free calcium.
ABSTRACT B cell lymphomas which can be growth inhibited by crosslinking their surface IgM receptors by anti-Ig reagents provide models for normal B cell regulation and tolerance. WEHI-231 and CH31 are two independently derived lines that are exquisitely sensitive to negative signalling by antibodies specific for mu or kappa chains, but are unaffected by antibodies against MHC class 1 or 2 antigens. In order to determine the mechanism of this growth inhibition as a model for tolerance, we have examined the roles played by protein kinase C activation and calcium mobilization/influx during negative signalling in these cells. We found that growth inhibition caused by anti-mu crosslinking was reversed in the presence of either phorbol myristate acetate (PMA) or by lipopolysaccharide (LPS) from E. coli. The effect of PMA on negative signalling was a true reversal since phorbol esters could be added after anti-mu treatment, thus allowing nearly normal cellular progression into the S phase of the cell cycle. In contrast, pretreatment with PMA did not provide protection against the growth inhibition from anti-mu. Indeed, a "desensitization" protocol demonstrated that PMA pretreatment actually decreased reversal by both PMA and LPS of the effects of anti-mu on B lymphoma growth. These studies suggest that both LPS and PMA act via at least one common intermediate, which is assumed to involve activation and translocation of protein kinase C. Analysis of changes in calcium ion concentration after treatment with anti-Ig reagents showed both mobilization from internal stores and influx via calcium channels in WEHI-231, as has been reported for normal B cells. However, these changes did not correlate with negative signalling for the several reasons. Firstly, anti-mu inhibition of the growth of WEHI-231 could be induced in the relative absence of extracellular Ca++ or in quin-2 loaded (buffered) cells. Secondly, pretreatment with high concentrations of PMA ablated calcium mobilization, yet failed to modulate growth inhibition in WEHI-231 cells. Moreover, LPS provided protection from the effects of anti-mu yet did not alter cellular [Cai++]. In addition, PMA posttreatment (under conditions causing a reversal of the effects of anti-mu) can be applied as long as four hours after the initial exposure to anti-mu and the rapid measurable changes in calcium flux. Indeed, such changes in intracellular free calcium occurred in elutriated WEHI-231 lymphoma cells at all phases of the cell cycle, although we have previously identified early G1 as the only critical period in which negative signalling can be delivered.(ABSTRACT TRUNCATED AT 400 WORDS)
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ABSTRACT: Upon encountering the antigen (Ag), the immune system can either develop a specific immune response or enter a specific state of unresponsiveness, tolerance. The response of B cells to their specific Ag can be activation and proliferation, leading to the immune response, or anergy and activation-induced cell death (AICD), leading to tolerance. AICD in B lymphocytes is a highly regulated event initiated by crosslinking of the B cell receptor (BCR). BCR engagement initiates several signaling events such as activation of PLCgamma, Ras, and PI3K, which generally speaking, lead to survival. However, in the absence of survival signals (CD40 or IL-4R engagement), BCR crosslinking can also promote apoptotic signal transduction pathways such as activation of effector caspases, expression of pro-apoptotic genes, and inhibition of pro-survival genes. The complex interplay between survival and death signals determines the B cell fate and, consequently, the immune response.Cell Research 10/2000; 10(3):179-92. DOI:10.1038/sj.cr.7290047 · 11.98 Impact Factor
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ABSTRACT: Cross-linking surface immunoglobulin (Ig)M on the WEHI-231 B-cell lymphoma results in decreased cell size, G1/S growth arrest, and finally DNA cleavage into oligonucleosomal fragments that are the classical features of apoptotic cells. Treatment of WEHI-231 cells with anti-IgM in early G1 phase prevents phosphorylation of the retinoblastoma gene product (pRb) and inhibits entry into S phase. Using unsynchronized cells, we previously demonstrated that cyclin A-associated and Cdk2-dependent GST-pRb kinase activity were inhibited in WEHI-231 cells treated with anti-IgM. We now show that progression of elutriated early G1 phase WEHI-231 cells from early into late G1 phase is accompanied by an increase in the abundance of cyclin A protein and cyclin A-associated kinase activity. Treatment of early G1 cells with anti-IgM prevented this increase in cyclin A-associated kinase activity at late G1, despite minimal changes in the overall level of cyclin A and Cdk2 proteins. Late G1 cells, which already possess high cyclin A-associated kinase activity, were insensitive to anti-IgM treatment and were able to complete the cell cycle. We also found that anti-IgM-treated cells contained increased amounts of the Cdk inhibitor protein p27Kip1. Essentially all of the cyclin A in treated cells was associated with p27, a result which we propose explains the lack of cyclin A/Cdk2 kinase activity. Accumulation of p27 in cyclin A kinase complexes, however, did not decrease the amount of Cdk2 bound to cyclin A. Thus, cross-linking IgM on growth-inhibitable B-cell lymphomas affects cyclin A kinase activity by increasing the levels of p27 in this complex, thus preventing productive pRb phosphorylation and leading to cell cycle arrest and subsequent apoptosis. These results are discussed in terms of the cell cycle restriction points that regulate lymphocyte function, as well as the lineage-specific differences in cell cycle control.Molecular Biology of the Cell 05/1996; 7(4):553-64. DOI:10.1091/mbc.7.4.553 · 4.55 Impact Factor
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ABSTRACT: Summary The antigen receptors on T and B lymphocytes can transduce both agonist and antagonist sig- nals leading either to activation/survival or anergy/death. The outcome of B lymphocyte anti- gen receptor (BCk) triggering depends upon multiple parameters which include (a) antigen concentration and valency, (b) duration of BCP,. occupancy, (c) receptor affinity, and (d) B cell differentiation stages. Herein, using anti-immunoglobulin K and k light chain antibodies, we analyzed the response of human naive, germinal center (GC) or memory B cells to BCR cross- linking regardless of heavy chain Ig isotype or intrinsic BCR specificity. We show that after CD40-activation, anti-BCR.(K+k) can elicit an intracellular calcium flux on both GC and non-GC cells. However, prolonged BCP, cross-linking induces death of CD40-activated GC B cells but enhances proliferation of naive or memory cells. Anti-K antibody only kills K + GC B cells without affecting surrounding k + GC B cells, thus demonstrating that BCR-mediated killing of GC B lymphocytes is a direct effect that does not involve a paracrine mechanism. BCR-mediated killing of CD40-activated GC B cells could be partially antagonized by the ad- dition of IL-4. Moreover, in the presence of IL-4, prestimulation through CD40 could prevent subsequent anti-Ig-mediated cell death, suggesting a specific role of this combination in selec- tion of GC B cells. This report provides evidence that in human, susceptibility to BCP, killing is regulated along peripheral B cell differentiation pathway.