The potassium ionophore nigericin induces cell death and promotes the maturation and release of IL-1b in lipopolysaccharide (LPS)-primed monocytes and macrophages, the latter depending on caspase-1 activation by an unknown mechanism. Here, we investigate the pathway that triggers cell death and activates caspase-1. We show that without LPS priming, nigericin alone triggered caspase-1 activation and IL-18 generation in THP-1 monocytic cells. Simultaneously, nigericin induced caspase-1-independent necrotic cell death, which was blocked by the cathepsin B inhibitor CA-074-Me and other cathepsin inhibitors. Cathepsin B activation after nigericin treatment was determined biochemically and corroborated by rapid lysosomal leakage and translocation of cathepsin B to the cytoplasm. IL-18 maturation was prevented by both caspase-1 and cathepsin B inhibitors in THP-1 cells, primary mouse macrophages and human blood monocytes. Moreover, IL-18 generation was reduced in THP-1 cells stably transformed either with cystatin A (an endogenous cathepsin inhibitor) or antisense cathepsin B cDNA. Collectively, our study establishes a critical role for cathepsin B in nigericin-induced caspase-1-dependent IL-18 maturation and caspase-1-independent necrosis.

Based on high sequence homology, there are six members in the caspase-1 subfamily: caspases 1, 4, 5, and 13 in humans and caspases 1, 11, and 12 in mice. Only caspase-1 is known to activate interleukin-1␤ and interleukin-18, and caspase-11 activates pro-caspase-1 in vivo. Almost nothing is known about caspases 4, 5, and 13. Here we report a sensitive and specific polymerase chain reaction system to analyze closely related genes.We employed this system to analyze the gene expression and regulation of human caspases 1, 4, 5, and 13, demonstrating that they have different expression patterns in normal tissues and cell lines. Interferon-␥ strongly induced CASP1 and CASP5 but not CASP4 or CASP13 gene expression in HT-29 colon carcinoma cells. In contrast to the mRNA, interferon-␥ up-regulated caspase-1 but not caspase-5 protein. In the monocytic cell line THP-1, CASP1 mRNA and caspase-1 protein are expressed constitutively, and their levels were not increased by lipopolysaccharide, whereas both CASP5 mRNA and caspase-5 protein were induced by lipopolysaccharide. Caspase-1 subfamily members displayed different in vitro activities toward pro-caspases 1 and 3 and pro-interleukin-1␤. Our results demonstrate that caspase-1 and caspase-5 levels are modulated by interferon-␥ and lipopolysaccharide, respectively, and suggest that caspase-1 subfamily members are differentially regulated and may have distinct functions.

The phenotypically immature B cell lymphoma WEHI-231 undergoes apoptotic cell death when cultured with anti-immunoglobulin (Ig) antibodies, via a bcl-2-independent mechanism. We have therefore studied the role of the bcl-2-related protein bcl-x in controlling cell death in WEHI-231. We find that overexpression of the long form of bcl-x (bcl-XL) renders these cells refractory to anti-Ig-induced cell death. Stimulation of WEHI-231 via CD40 has similar protective effects. We show here that ligation of CD40 rapidly induces the appearance of the bcl-XL protein in WEHI-231, while stimulation via sIgM, sIgD, CD5 or CD45 receptors, or with phorbol esters plus ionomycin does not. WEHI-231 cells also rapidly undergo massive apoptosis following culture with thapsigargin, a specific inhibitor of the Ca(2+)-ATPase of the endoplasmic reticulum: this is also reversed by anti-CD40, or by overexpression of bcl-XL. We, therefore, conclude that bcl-XL plays a key role in the regulation of antigen receptor-mediated apoptosis via CD40 in WEHI-231. In addition, the fact that this protein is not induced in WEHI-231 in response to phorbol dibutyrate plus ionomycin points to a fundamental signaling defect in these cells, which could conceivably be a reflection of their immature, apoptosis-susceptible phenotype.

No abstract

The role of arachidonic acid in the regulation of steroidogenesis in rat Leydig cells was studied. A dose- and time-dependent biphasic effect on maximal and submaximal LH- and dibutyryl-cAMP-stimulated testosterone production was found. The locus of the inhibition, which occurred during 3 h incubation, was prior to the side chain cleavage of cholesterol and after cAMP production. The same inhibitory effect was found with the protein kinase C (PKC) activators, phorbol-12-myristate, 13-acetate (PMA) and oleic acid, also with no change in LH-stimulated cAMP production. Arachidonic acid, PMA, and diolein, all stimulated PKC activity in a dose-dependent fashion in partially purified Leydig cell homogenates. When the cells were incubated for 5 h, arachidonic acid potentiated LH- and dibutyryl-cAMP-stimulated testosterone production. Similarly, incubation with PMA for 5 h, potentiated subsequent basal and dibutyryl-cAMP-stimulated testosterone production. PKC was down-regulated over 5 h (but not during 3 h) by pretreating Leydig cells with PMA or arachidonic acid in the presence of LH. Lipoxygenase and cyclooxygenase inhibitors did not alter the stimulatory effects of arachidonic acid. We conclude that the short-term inhibitory effect of arachidonic acid (and PMA) is via activation of PKC, but when protein kinase C (PKC) is down-regulated by these ligands, steroidogenesis is enhanced. These results suggest that steroidogenesis is normally under tonic inhibitory control by PKC.

Stimulation of the phenotypically immature B cell lymphoma WEHI-231 with anti-IgM induces G 1 arrest followed by apoptotic cell death, which can be reversed by stimulation via the CD40 receptor. Here, we show that cells expressing bcl-x L (WEHI-bcl-x L ) arrest at G 0 /G 1 following culture with anti-IgM but do not undergo apoptosis. These arrested cells can be induced to reenter the cell cycle by ligation of CD40. We have therefore used these cells as a model to study the regulation of the transcription factor E2F, which is critically involved in transit through the cell cycle. We found that anti-IgM treatment induces the appearance of an inhibitory DNA binding complex containing the pRB-related pocket protein p130 together with E2F and a concomitant decrease in "free" E2F, consisting of E2F1 and its partner DP1; these effects were reversed following stimulation via CD40. These changes in free E2F levels were regulated by changes in E2F1 gene transcription, which is at least partly a result of control of E2F1 promoter activity through its E2F binding sites. A key mechanism for the control of autoreactivity within the immune system is the purging of self-reactive clones during the early stages of T and B lymphocyte development (1-3). In B lymphocytes, such negative selection occurs in immature B cells within the bone marrow or in recent bone marrow emigrants. Studies with a variety of transgenic strains of mice have clearly shown that encounter of immature B lymphocytes with self antigen can lead to growth arrest followed by apoptotic cell death (clonal deletion) or to functional silencing (clonal anergy), depending on the degree of antigen receptor cross-linking (4 -8). Currently, little is known about the intracellular events that lead to deletion or anergy or, indeed, how negative selection can be modulated by co-stimulatory (e.g. T cell-derived) signals.A well established in vitro model for studying clonal deletion of B cells is the WEHI-231 B cell lymphoma. These phenotypically immature (sIgM hi sIgD lo ) cells arrest in the G 1 phase of the cell cycle following culture with anti-IgM antibodies and subsequently die by apoptosis (9 -11). Significantly, these events can be abrogated by concurrent stimulation via the CD40 receptor (12). CD40 is now known to play a central role in the initiation of T cell-dependent antibody responses, via its interaction with a counterreceptor, the CD40-ligand, which is expressed on activated CD4 T helper cells. CD40-mediated signals in normal B cells promote cellular proliferation, immunoglobulin isotype switching, germinal center formation, and the development of B memory cells (13,14). In addition, CD40 appears to be the most important receptor on B cells for promoting cell survival and in reversing sIg-induced apoptotic signals (15-17).We and others have found that CD40 stimulation rapidly induces the appearance of the anti-apoptotic protein bcl-x L in WEHI-231 cells (17)(18)(19)(20). However, although constitutive expression of bcl-x L protects these cells from anti-IgM-in...

The possible role of chloride channels in luteinizing hormone (LH) action on steroidogenesis in rat Leydig cells had been investigated. A chloride channel blocker, SITS (4-acetamido-4'-isothiocyanatostilbene-2,~-disulphonic acid), inhibited LH-stimulated steroidogenesis at low (6; 1 ng/ml), but not at high (100 ng/ml) LH concentrations. In addition, dibutyryl cyclic AMP-and forskolin-stimula~ steroidogenesis was unaffected by SITS. The removal of extra~ll~ar chloride ~tentiat~ steroidogen~is stim~at~ by subm~mal but not maximal doxes of LH. These results suggest that at low levels of LH, steroidogenesis depends on chloride channels whereas with high levels, cyclic AMP is the mediator of LH action.

We have studied the expression of the novel anti-apoptotic protein bcl-x during mouse B cell differentiation and activation. We find that bcl-x is expressed throughout all stages of B cell differentiation in the bone marrow, and is only down-regulated in mature (sIgD+) B cells. Immature peripheral B cells express low levels of bcl-x even in adult animals, whereas mature resting B cells do not. Mature B cells re-express the protein following activation, achieving maximal levels after 36-48 h. The highest levels of bcl-x are observed with potent mitogenic stimuli (such as anti-CD40 + anti-Ig): B cells first express bcl-x in the G1 phase of the cell cycle and contain maximal levels in S phase. In addition, B cells from CBA/N mice, which do not proliferate when stimulated with anti-Ig, anti-CD40 or both, exhibited only low levels of the protein following culture with these stimuli. To investigate the functional significance of bcl-x in activated B cells, we tested their sensitivity to apoptosis induced by the Ca2+ ATPase inhibitor thapsigargin: B cell blasts activated with anti-CD40 and anti-Ig were resistant to this agent. The available data therefore suggest that bcl-x fulfils two roles in B cells: it promotes survival of immature B cells (which lack bcl-2) and secondly, it apparently plays an additional role in protecting activated mature B cells (perhaps those in germinal centers) from apoptotic stimuli.