Anti-IgM but not anti-IgD antibodies inhibit cell division of normal human mature B cells.

Insolubilized anti-IgD antibody markedly increased DNA synthesis in and cell division of normal peripheral blood B cells (PBL-B) when used in combination with IL-4. Anti-IgM antibodies also induced DNA synthesis of PBL-B, but their ability to induce cell division was less than that of anti-IgD antibodies even when used in combination with IL-4. Moreover, anti-IgM antibodies inhibited cell division of PBL-B stimulated with insolubilized anti-IgD antibody plus IL-4 without affecting DNA synthesis. Anti-IgM antibodies also inhibited Staphylococcus aureus Cowan I-induced cell division of PBL-B without affecting DNA synthesis. These results indicate that cross-linkage of surface IgM (sIgM) in mature B cells generates negative signals to inhibit cell division of mature B cells. Because anti-IgD antibodies did not inhibit cell division at all, the role of sIgD in the regulation of cell division of mature B cells may be quite different from that of sIgM. IFN-alpha/beta promoted cell division of PBL-B stimulated with insolubilized anti-IgD antibody plus IL-4. They also counteracted the inhibitory effect of anti-IgM antibody on cell division of PBL-B.     

Growth regulation of a human mature B cell line, B104, by anti-IgM and anti-IgD antibodies

An EBNA- human B lymphoma cell line, B104, was established. B104 cells express IgD as well as IgM on their surface, which is thought to be a basic characteristic of mature B cells. The growth of B104 cells was inhibited by treatment with a panel of anti-IgM antibodies. Cell cycle analyses revealed that the transition of B104 cells from the G2/M to the G0/G1 phase of the cell cycle was markedly inhibited by treatment with anti-IgM antibodies. Progression of B104 cells to the M phase of the cell cycle was found to be suppressed in the presence of anti-IgM antibodies. In contrast, both the entrance of G0/G1 phase cells into the S phase and the progression of S phase cells to the G2/M phase of the cell cycle did not seem to be inhibited significantly by treatment with anti-IgM antibodies. These results indicate that the mechanism of the inhibition of growth of B104 cells by anti-IgM antibodies is blockage of the transition from the G2 to the M phase of the cell cycle. In contrast to anti-IgM antibodies, anti-IgD antibodies could not cause growth inhibition of B104 cells at all. B cell growth factors such as IL-4 and IL-6 had no effect on the inhibition of growth of B104 cells by anti-IgM antibody. IFN-alpha and -beta, which have no B cell growth factor activity, did increase the number of cells that survived the treatment with anti-IgM antibodies. B104 is an excellent experimental model for the study of the mechanism of signal transduction through sIg as well as the functional difference between sIgM and sIgD.     

Anti-IgM antibody-induced cell death in a human B lymphoma cell line, B104, represents a novel programmed cell death.

We investigated the mechanisms of anti-IgM antibody-induced cell death in a recently established human surface IgM+ IgD+ B lymphoma cell line, B104, the growth of which is irreversibly inhibited by anti-IgM antibody but not by anti-IgD antibody, and compared it with the cell death of T cells via TCR/CD3 complex and with the cell death of a murine anti-IgM antibody-sensitive B lymphoma cell line, WEHI-231. The rapid time course of B104 cell death and its requirements for de novo macromolecular synthesis and Ca2+ influx suggest that anti-IgM antibody-induced B104 cell death is an active Ca(2+)-dependent programmed cell death. Moreover, cyclosporin A rescued B104 cells from this lethal signal, via surface IgM, suggesting that the intracellular mechanisms involved are quite similar to those of T cell death. DNA fragmentation, which has been reported in TCR/CD3 complex-mediated T cell death, apoptosis, was not involved in the B104 cell death process, but the possible involvement of DNA single-strand breaks was suggested. Observations under light microscopy and transmission electron microscopy indicated that the morphologic features of dying B104 cells resembled necrosis rather than apoptosis. B104 cell death was shown to be quite distinct from that of WEHI-231 in cell death kinetics, the mode of cell death, and the response to cyclosporin A. These data collectively indicate that the death of B104 cells resulting from surface IgM cross-linking represents a hitherto undefined mode of programmed cell death.     

Inhibition of human B cell responsiveness by prostaglandin E2

The capacity of prostaglandin E2 (PCE2) to modulate human peripheral blood B cell proliferation and the generation of immunoglobulin-secreting cells (ISC) stimulated by Cowan 1 strain Staphylococcus aureus and mitogen-stimulated T cell supernatant was examined. PGE2 significantly inhibited both responses, whereas PGF2 alpha had no inhibitory effect. Responses of highly purified B cells obtained from spleen, lymph node, and tonsil were also inhibited. In addition PGE2 suppressed B cell responses supported by recombinant interleukin 2 rather than T cell supernatant. PGE2-mediated inhibition was mimicked by forskolin, a direct activator of adenylate cyclase. Kinetic studies indicated that PGE2 inhibited B cell responses by a progressively greater increment as cultures were prolonged. Evaluation by flow cytometry after staining with acridine orange or mithramycin indicated that PGE2 had no effect on initial B cell entry into the G1 phase of the cell cycle, passage through G1, and entry into S, G2, and M. Rather, PGE2 inhibited responses of postdivisional daughter cells. PGE2 inhibited responses in cultures stimulated by the calcium ionophore ionomycin and T cell supernatant but had minimal effects in cultures stimulated by the combination of ionomycin and phorbol myristate acetate. Moreover, phorbol myristate acetate reversed PGE2-mediated inhibition of proliferation stimulated by S. aureus or S. aureus + T cell supernatant. These results indicate that PGE2 suppresses the continued growth and differentiation of human B cells, although it has no effect on initial B cell activation and suggest that PGE2 may play a role in regulating human B cell responses in vivo.     

Molecular signals in B cell activation. II. IL-2-mediated signals are required in late G1 for transition to S phase after ionomycin and PMA treatment

We report that sustained increase of intracellular calcium ion concentration and protein kinase C (PKC) activation maintained throughout the G1 phase of cell cycle do not provide sufficient signals to cause S-phase entry in rabbit B cells, and that additional signals transduced by IL-2 and IL-2 receptor interaction are essential for G1 to S transition. We have shown earlier that rabbit B cells can be activated to produce IL-2 and express functional IL-2 receptors after treatment with ionomycin and PMA. Herein we have compared the response of rabbit PBLs, which contain about 50% T cells, with those of purified B cells. After activation with ionomycin or PMA, comparable numbers of PBLs and B cells entered the cell cycle; but DNA synthesis by the PBL cultures was three to four times higher than that of cultures of purified B cells. Interestingly, IL-2 production by the PBL cultures was also three to four times higher than in B cell cultures, suggesting an involvement of IL-2 in inducing DNA synthesis in these cells. The hypothesis that IL-2, which is produced in early G1, acts in late G1 and is required for G1 to S transition in B cells was supported by the following observations: (i) IL-2 production by B cells was detected as early as 6 hr after activation and preceded DNA synthesis by at least 24 hr. (ii) B cell blasts in G1 (produced by treatment of resting B cells with ionomycin and PMA) showed DNA synthesis in response to IL-2, but showed very little DNA synthesis in response to restimulation with ionomycin and PMA. (iii) A polyclonal rabbit anti-human IL-2 antibody caused nearly complete inhibition of DNA synthesis by B cells activated by ionomycin and PMA. (iv) A PKC inhibitor, K252b, inhibited DNA synthesis in ionomycin and PMA-stimulated cells if added at the beginning of culture but was not inhibitory if added 16 hr later. We conclude that increased [Ca2+]i and PKC activation are not sufficient signals for G1 to S transition in B cells; entry into S is signaled by IL-2, and IL-2-mediated signal transduction probably does not involve increased [Ca2+]i or PKC activation.     

Anti-CD45 inhibition of human B cell proliferation depends on the nature of activation signals and the state of B cell activation. A study with anti-IgM and anti-CDw40 antibodies

The proliferation of human peripheral and tonsillar B cells stimulated with the anti-CDw40 mAb 626.1 and/or anti-IgM antibody (Ab) in the presence of anti-CD45 mAb A.1.1 was investigated. The anti-CD45 mAb suppressed the anti-CDw40-stimulated proliferation of peripheral blood B cells but had no effect on the proliferation of unfractionated tonsillar B cells stimulated similarly. When tonsillar B cells were separated according to their sizes, the anti-CDw40-induced proliferation of small tonsillar B cells was inhibited by the anti-CD45 mAb, whereas large tonsillar B cells were resistant. In contrast, anti-IgM-induced proliferation of human B cells was always significantly inhibited by the anti-CD45 mAb regardless of cell size and tissue origin. The anti-CD45 mAb also inhibited the anti-IgM-induced initial rise in intracellular [Ca2+] and the G0-G1 cell cycle transition of small tonsillar B cells. However, co-stimulation with anti-IgM/anti-CDw40 Ab resulted in the resistance to the anti-CD45 inhibitory effect on proliferation of peripheral blood B cells and the majority of tonsillar B cells. In contrast, B cell proliferation co-stimulated with anti-IgM Ab/and B cell growth factors were always suppressed by the anti-CD45 mAb. These results demonstrate that certain activational signal mechanisms utilized by anti-CDw40/anti-IgM Ab and anti-IgM Ab/B cell growth factors are different in that B cells stimulated with these agents differ in their sensitivity to the anti-CD45 mAb. Moreover, both the activational state of human B cells and the nature of activation signals given determine their response to the inhibitory signals delivered by the anti-CD45 mAb.     

Redistribution of protein kinase C during mitogenesis of human B lymphocytes

G0 human tonsillar B-lymphocytes were stimulated to divide by the polyclonal mitogen Staphylococcus Aureus Cowan strain 1 (SAC) and by the combined use of 12-O-tetradecanoyl phorbol-13-acetate (TPA) and the calcium ionophore ionomycin. The activities of protein kinase C, which requires Ca++ and phospholipid as co-factors, and a proteolytically cleaved form of this enzyme (protein kinase M), which is independent of calcium and phospholipid control, were determined in soluble and particulate fractions obtained from activated B cells. Treatment of G0 B cells with SAC or TPA together with ionomycin caused redistribution of protein kinase C from the soluble to the particulate fraction where the 80,000-Dalton protein kinase C was cleaved to give rise to a 50,000-Dalton form of the kinase which was also found in the cytoplasm. These data suggest that redistribution and proteolytic cleavage of protein kinase C are key signal transduction events in B cell mitogenesis.     

Microfilament assembly is required for antigen-receptor-mediated activation of human B lymphocytes

The mechanisms responsible for initiating the conversion of globular to filamentous actin (assembly) after stimulation of B lymphocytes and the role of these cytoskeletal changes in cell activation are incompletely understood. We investigated the molecular basis of the signals leading to actin polymerization and concentrated on the involvement of guanosine triphosphate (GTP)-binding regulatory proteins, and protein kinase C (PKC). In addition, we related these early events to later events in B-cell activation, including cell proliferation. Cross-linking the Ag receptor with Staphylococcus aureus Cowan I (SAC) or anti-IgM antibodies, or stimulation of PKC with phorbol ester induced a time- and concentration-dependent increase in the filamentous actin content of B cells. Inhibition or depletion of PKC resulted in decreased actin assembly induced by anti-IgM, SAC, and PMA, suggesting that the signal for polymerization is generated distally to PKC activation. Pertussis toxin pretreatment inhibited the responses to anti-IgM and SAC but not PMA, and direct stimulation of permeabilized cells with GTP gamma S induced microfilament assembly, indicating the involvement of a GTP-binding protein for receptor-mediated events. Disruption of actin polymerization with botulinum C2 toxin or cytochalasin D inhibited the assembly of actin and [3H]TdR incorporation induced by all stimuli. We conclude that human B cell activation by receptor-mediated stimuli results in actin polymerization by signaling pathways coupled to GTP-binding proteins. These changes in the cytoskeleton may be involved in the transduction of messages leading to responses such as proliferation in B lymphocytes.     

Role of phosphoinositide-derived second messengers in mediating anti-IgM-induced growth arrest of WEHI-231 B lymphoma cells

Anti-IgM irreversibly inhibits the growth of WEHI-231 B lymphoma cells and induces phosphoinositide hydrolysis--producing diacylglycerol, which activates protein kinase C, inositol 1,4,5-trisphosphate, which induces the release of calcium from intracellular storage sites into the cytoplasm, and other inositol polyphosphates. The roles of two of the possible second messengers, cytoplasmic free calcium and diacylglycerol, in mediating the action of anti-IgM on WEHI-231 cells were assessed by elevating [Ca2+]i with ionomycin and by activating protein kinase C with phorbol 12,13-dibutyrate (PdBu). The combination of 250 nM ionomycin and 4 to 7 nM PdBu was found to cause growth arrest and cell volume decrease responses in WEHI-231 cells which were similar to those caused by anti-IgM, although clearly slower. Both anti-IgM and the combination of mimicking reagents induced growth arrest of WEHI-231 cells in the G1 phase of the cell cycle. In both cases, this growth arrest was mitigated by addition of bacterial LPS. Moreover, 250 nM ionomycin plus 4 to 7 nM PdBu did not inhibit the growth of two other murine B lymphoma cell lines, each of which did exhibit increased phosphoinositide hydrolysis but not growth arrest in response to anti-Ig. Taken together, these results suggest that ionomycin and PdBu, at the concentrations used, did not inhibit WEHI-231 growth by general toxicity, but rather by mimicking the effects of the natural second messengers generated from Ag receptor cross-linking. Thus, the phosphoinositide-derived second messengers Ca2+i and diacylglycerol are capable of playing important roles in mediating the action of anti-IgM on WEHI-231 B lymphoma cells. However, the response of WEHI-231 cells to anti-IgM could not be fully reproduced with ionomycin and phorbol diester. These results suggest that another second messenger induced by anti-IgM may also play an important role in mediating the growth arrest of these cells.     

Molecular signals in B cell activation. I. Differential refractory effects of incomplete signaling by ionomycin or PMA relate to autocrine IL-2 production and IL-2R expression

The molecular signals required by resting (G0) B cells for the induction of cell cycle entry, IL-2 production, and high-affinity IL-2 receptor (IL-2R) expression were defined and the effects of incomplete activation signals on the subsequent response to complete signals were examined. Highly enriched rabbit peripheral blood B cells were activated with a calcium ionophore, ionomycin, and a protein kinase C (PKC) activating phorbol ester, phorbol myristate acetate (PMA). It was observed that cell cycle entry to early G1 was induced by either reagent acting alone, but both reagents were required to stimulate IL-2 production, IL-2R expression, and DNA synthesis. These effects of ionomycin and PMA were shown to be mediated by increased intracellular calcium ion concentration [Ca2+]i and PKC activation, respectively. Although, increased [Ca2+]i or PKC activation each led to cell cycle entry, the subsequent response of these preactivated cells to complete activation with both signals was different: Cells pretreated with PMA alone for up to 24 hr could progress further to DNA synthesis after the addition of ionomycin. In contrast, cells activated with ionomycin alone, or those cultured without any stimulus, progressively lost the ability to show DNA synthesis after complete activation. The failure to progress to DNA synthesis in these two cases was, however, differentially regulated by the ability of these cells to produce IL-2 and to express IL-2R. Ionomycin-pretreated cells retained the ability to produce IL-2 but showed about 70% reduction in the numbers of IL-2R; whereas cells cultured without any stimulus lost the ability to produce IL-2 after subsequent complete activation, but showed lesser reduction in IL-2R expression.     

Persistent intracellular calcium pool depletion by thapsigargin and its influence on cell growth.

The intracellular Ca2+ pump inhibitor, thapsigargin, added to DDT1MF-2 smooth muscle cells in culture, irreversibly inhibited accumulation of Ca2+ within cells, permanently emptied the inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pool, and simultaneously induced profound alteration of cell growth. After only a brief (30-min) treatment of cultured cells with 3 microM thapsigargin followed by extensive washing, the total releasable InsP3-sensitive Ca2+ pool remained entirely empty, even after 7 days of culture without thapsigargin. After thapsigargin treatment, cells retained viability, usual morphology, and normal mitochondrial function. Despite the otherwise normal appearance and function of thapsigargin-treated cells, cell division was completely blocked by thapsigargin. DNA synthesis was completely inhibited when thapsigargin was added immediately after passaging, but was suppressed only slowly (4-6 h) when added to rapidly synthesizing cells (24 h after passaging). Protein synthesis was reduced by approximately 70% in thapsigargin-treated cells. The sensitivity of thapsigargin-mediated inhibition of cell division, DNA synthesis, protein synthesis, and Ca(2+)-pumping activity were all similar with the EC50 values for thapsigargin in each case being close to 10 nM. Upon application to DDT1MF-2 cells, thapsigargin transiently increased resting cytosolic Ca2+ (0.15 microM) to a peak of 0.3 microM within 50 s; thereafter, free Ca2+ declined to 0.2 microM by 150 s and continued to slowly decline toward resting levels. Cells treated with thapsigargin for 1-72 h in culture displayed normal resting cytosolic Ca2+ levels. However, application of thapsigargin or epinephrine to such cells resulted in no change in the intracellular Ca2+, indicating that the internal Ca2+ pool remained completely empty. These results suggest that emptying of Ca2+ from intracellular thapsigargin-sensitive Ca(2+)-pumping pools induces profound alteration of cell proliferation.     

Glycoprotein biosynthesis in B lymphocytes: induction of protein N-glycosylation, RNA synthesis, and DNA synthesis by phorbol ester plus ionomycin is blocked by protein kinase inhibitors

The combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin produces a dramatic increase in the incorporation of [2-3H]mannose into Glc3Man9GlcNAc2-P-P-dolichol and glycoprotein, and the induction of RNA and DNA synthesis in murine splenic B lymphocytes (B cells). The kinetics of the induction processes and the concentrations of PMA and ionomycin required for the optimal response have been defined. While the levels of induction of RNA and DNA synthesis by PMA + ionomycin were similar to the mitogenic response to bacterial lipopolysaccharide, activation by PMA and the calcium ionophore resulted in a threefold higher stimulation in dolichol-linked oligosaccharide biosynthesis and protein N-glycosylation. These results indicate that all signalling mechanisms that trigger RNA and DNA synthesis may not be sufficient to produce maximal induction of the N-glycosylation apparatus. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7), a potent protein kinase C inhibitor, prevented the induction of protein N-glycosylation activity (IC50 = 11 microM), as well as RNA (IC50 = 18 microM) and DNA synthesis (IC50 = 12 microM), two common indices of B cell activation. N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) also inhibited the induction of oligosaccharide-lipid intermediate, glycoprotein, RNA, and DNA synthesis, but required higher concentrations than H-7 for 50% inhibition. N-(2-Guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a potent inhibitor of cyclic nucleotide-dependent protein kinases, had little effect on the activation of the B cell metabolic processes. The H-7-sensitive reactions involved in the induction of RNA and DNA synthesis occurred within 4 h, but induction of lipid intermediate and glycoprotein biosynthesis remained sensitive to H-7 for 10 h after exposure to PMA and ionomycin. Direct in vitro assays in the presence of 0.6% Brij 58 reveal that a cytosolic, phospholipid-dependent protein kinase activity is translocated to a membrane site(s) after treatment with PMA and ionomycin, and the translocated protein kinase is sensitive to H-7. The relative order of potency of the protein kinase inhibitors on the metabolic processes strongly supports the hypothesis that protein kinase C, acting synergistically with Ca2+ mobilization, plays a key regulatory role in the early stages of B cell activation. The synthesis of oligosaccharide-lipid intermediates and protein N-glycosylation are also shown to be induced in B cells activated by PMA + ionomycin.     

Muscarinic receptor-linked elevation of cAMP in SH-SY5Y neuroblastoma cells is mediated by Ca2+ and protein kinase C.

The mechanisms of muscarinic receptor-linked increase in cAMP accumulation in SH-SY5Y human neuroblastoma cells has been investigated. The dose-response relations of carbachol-induced cAMP synthesis and carbachol-induced rise in intracellular free Ca2+ were similar. The stimulated cAMP synthesis was inhibited by about 50% when cells were entrapped with the Ca2+ chelator BAPTA or in the presence of the protein kinase C (PKC) inhibitor staurosporine. Production of cAMP could be induced also by the Ca2+ ionophore, ionomycin and by TPA, an activator of PKC. When added together TPA and ionomycin had a synergistic effect. When cAMP synthesis was activated with cholera toxin, PGE1 or PGE1 + pertussis toxin carbachol stimulated cAMP production to the same extent as in control cells. Ca2+ and protein kinase C thus seem to be the mediators of muscarinic-receptor linked cAMP synthesis by a direct action on adenylate cyclase.     

Inhibition of DNA synthesis by protein kinase C-activating phorbol esters in NIH/3T3 cells

Fibroblast growth factor (FGF) plus insulin induced DNA synthesis in and proliferation of NIH/3T3 cells. The protein kinase C-activating phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), inhibited both the DNA synthesis and cell proliferation induced by FGF plus insulin. The concentration of TPA required for 50% inhibition of the DNA synthesis was about 5 nM. Phorbol-12,13-dibutyrate, another protein kinase C-activating phorbol ester, also inhibited the DNA synthesis but 4 alpha-phorbol-12,13-didecanoate, known to be inactive for this enzyme, was ineffective. DNA synthesis started at about 12 h after the addition of FGF plus insulin. The inhibitory action of TPA on the DNA synthesis was observed when it was added within 12 h after the addition of FGF plus insulin. These results suggest that phorbol esters exhibit an antiproliferative action through protein kinase C activation in NIH/3T3 cells, and that this action of phorbol esters is due to inhibition of the progression from the late G1 to the S phase of the cell cycle.     

Antiproliferative action of protein kinase C in cultured rabbit aortic smooth muscle cells

In rabbit aortic smooth muscle cells (SMC), protein kinase C-activating 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited the whole blood serum (WBS)-induced DNA synthesis. The inhibitory action of TPA was mimicked by another protein kinase C-activating phorbol ester, phorbol-12,13-dibutyrate (PDBu), but not by 4 alpha-phorbol-12,13- didecanoate known to be inactive for this enzyme. Prolonged treatment of the cells with PDBu caused the down-regulation of protein kinase C. In these cells, WBS still induced DNA synthesis but the inhibitory action of TPA was abolished. DNA synthesis started at 18 h and reached a maximal level 24 h after the addition of WBS. TPA inhibited the WBS-induced DNA synthesis even when added 12 h after the addition of WBS. These results suggest that protein kinase C has an antiproliferative action in rabbit aortic SMC and that this action is attributed to the inhibition of the progression from the late G1 into S phase of the cell cycle. TPA also inhibited the phospholipase C-mediated hydrolysis of phosphoinositides which was induced by WBS within several minutes, but the relevance of this effect on the antiproliferative action of TPA is uncertain.     

Inhibition of protein kinase C-dependent cellular proliferation by interaction of endogenous ganglioside GM1 with the B subunit of cholera toxin

In quiescent Swiss 3T3 fibroblasts, the B subunit of cholera toxin, a protein which binds specifically to ganglioside GM1 on the cell surface, stimulates DNA synthesis and potentiates the effects of several other growth factors such as insulin, epidermal growth factor, bombesin, and even unfractionated serum. In contrast to its synergistic effect with other known growth factors, the B subunit markedly inhibited DNA synthesis induced by the phorbol ester, 12-O-tetradecanoyl-phorbol 13-acetate (TPA). The inhibitory effect of the B subunit was observed even in the presence of insulin, which greatly potentiates the mitogenic response to TPA or the B subunit. In contrast to the effect of the B subunit, calcium ionophores and cholera toxin stimulated DNA synthesis induced by TPA. The antagonism between the B subunit and TPA is not simply due to their abilities to modify their mutual binding sites or known effector systems. TPA did not block the early rise in cytosolic free calcium in response to the B subunit, and conversely, the B subunit did not modify the ability of TPA to activate protein kinase C. However, in protein kinase C-deficient cells, the antagonistic effect between TPA and the B subunit was abolished. In addition, there was no indication for the involvement of a pertussis toxin-sensitive G protein in the antagonism. Maximum inhibition was found when the B subunit was added 2 h after the addition of TPA. Significant inhibition was still evident when the time of addition of the B subunit was delayed until 6 h after the addition of TPA. This suggests that the cross-talk between signal transduction induced through endogenous gangliosides and protein kinase C is a late step in mitogenesis.     

Tyrosine phosphorylation is an essential event in the stimulation of B lymphocytes by Staphylococcus aureus Cowan I.

Staphylococcus aureus Cowan I (SAC) is a potent mitogen for purified human B cells. By using Western blotting with antiphosphotyrosine antibodies, we demonstrated that the mitogenic effect of SAC is associated with rapid tyrosine phosphorylation of proteins of 45, 68, 75, 97, and 145 kDa. This tyrosine phosphorylation was detected within 30 s of the addition of SAC; it reached a maximum within 10 min, after which it declined gradually. In contrast to SAC, most soluble anti-IgM antibodies do not induce proliferation of isolated human B cells. As indicated by Western blotting, soluble anti-IgM antibodies induced a similar pattern of tyrosine phosphorylation, with the exception of the 68-kDa protein, which was the most heavily phosphorylated protein in SAC-treated cells. A similar but less intense 68-kDa band was also induced by mitogenic anti-IgM bound to beads. This suggested that tyrosine phosphorylation, especially of p68, may play an important role in B cell mitogenesis. To test this hypothesis, we determined the effect of specific tyrosine kinase inhibitors (tyrphostins) on SAC-induced tyrosine phosphorylation, oncogene expression, and B cell proliferation. The concentration dependencies of inhibition of these processes suggested that they were linked. Nonspecific toxic effects of the tyrphostins were ruled out by the demonstration that the tyrphostins did not alter cell viability and did not inhibit B cell proliferation induced by phorbol esters, which do not induce tyrosine phosphorylation. For maximal inhibition of SAC-induced cell proliferation, the tyrophostins needed to be added before or shortly after addition of SAC. Taken together, these data indicate that tyrosine phosphorylation is an obligatory early signal in B cell proliferation.     

Selective suppressive effects of glucocorticoids on the early events in the human B cell activation process

The effect of hydrocortisone on the induction of human B cell activation and proliferation has been described. Hydrocortisone prevents the anti-mu-induced cell enlargement of small tonsillar B cells, blocks expression of the activation markers 4F2 and 5E9 induced by anti-mu, inhibits RNA synthesis of small B cells stimulated by anti-mu with or without BCGF, and suppresses B cell proliferation in response to anti-mu and BCGF or to Staphylococcus aureus Cowan I. In contrast, hydrocortisone does not affect the proliferative response of in vitro or in vivo preactivated B cells. Therefore, hydrocortisone has a selective inhibitory effect on early events in the human B cell cycle that subsequently leads to inhibition of total RNA and DNA synthesis. Possible mechanisms of this action are discussed. These studies further define the nature of glucocorticoid-induced modulation of human B cell activation and proliferation.     

A specific defect of prosolin phosphorylation in T cell leukemic lymphoblasts is associated with impaired down-regulation of DNA synthesis

Prosolin is a major cytosolic phosphoprotein of proliferating normal PBL. Treatment of growing PBL with phorbol ester (12-O-tetradecanoylphorbol-13-acetate (TPA)) or calcium ionophore (A23187) for 1 h caused phosphorylation of prosolin with the production of up to four prominent phosphorylated forms differing in degree of phosphorylation and/or two-dimensional electrophoretic mobility (peptides B to E). Formation of these phosphopeptides coincided with rapid down-regulation of DNA synthesis. A23187 was particularly effective in inducing phosphorylation of the more highly phosphorylated peptides D and E, suggesting the existence of a (Ca2+)-activated mechanism in their phosphorylation. The T cell leukemia cell lines Jurkat, HuT-78, CCRF-CEM, and Molt-4 showed reduced to absent ability to phosphorylate prosolin peptides rapidly in response to A23187 and also showed diminished down-regulation of DNA synthesis. In leukemic cells treated with both TPA and A23187, peptides B and C were rapidly phosphorylated, but the phosphorylation of peptides D and E seen in normal PBL remained deficient. The T cell leukemic cells appear to have intact a TPA-activated mechanism for phosphorylating prosolin peptides B and C, but share an impairment of a specific Ca2(+)-activated mechanism, possibly a Ca2(+)-dependent protein kinase, required for phosphorylation of prosolin phosphopeptides D and E. The degree of rapid down-regulation of DNA synthesis was correlated with degree of phosphorylation of peptide E in PBL and in three of four T cell leukemic cell lines. Thus, rapid phosphorylation of prosolin may mediate responses to TPA and A23187 in normal proliferating PBL, including down-regulation of DNA synthesis. A deficiency of this pathway in leukemic T cells may impede their response to physiologic growth regulatory signals utilizing this pathway and contribute to unrestrained cell growth.