Restoration of surface IgM-mediated apoptosis in an anti-IgM-resistant variant of WEHI-231 lymphoma cells by HS1, a protein-tyrosine kinase substrate.

The HS1 protein is one of the major substrates of non-receptor-type protein-tyrosine kinases and is phosphorylated immediately after crosslinking of the surface IgM on B cells. The mouse B-lymphoma cell line WEHI-231 is known to undergo apoptosis upon crosslinking of surface IgM by anti-IgM antibodies. Variants of WEHI-231 that were resistant to anti-IgM-induced apoptosis expressed dramatically reduced levels of HS1 protein. Expression of the human HS1 protein from an expression vector introduced into one of the variant cell lines restored the sensitivity of the cells to apoptosis induced by surface IgM crosslinking. These results suggest that HS1 protein plays a crucial role in the B-cell antigen receptor-mediated signal transduction pathway that leads to apoptosis.

Signaling properties of anti-immunoglobulin--resistant variants of WEHI-231 B lymphoma cells.

Stimulation of the B cell antigen receptor (BCR) of the murine immature WEHI-231 B lymphoma with anti-immunoglobulin antibodies leads to irreversible growth arrest and apoptosis. As in normal B cells, membrane immunoglobulin (mIg) ligation in WEHI-231 cells triggers a series of signaling cascades from the BCR to intracellular compartments. In order to address the role of early signals in mediating the growth arrest of WEHI-231 cells, we have generated two variants resistant to the anti-Ig-mediated inhibitory effect. Some of the properties of these variants have been recently described in terms of bcl-2 and c-myc gene regulation. We report here that these variants can be further distinguished from the wild type on the basis of significant alterations in the early biochemical events which follow mIg ligation. Both Ca2+ signals and patterns of protein tyrosine phosphorylation were affected in these variants, suggesting that alterations in the early signal transduction machinery may have profound effects on the fate of B cells. In addition, we found that expression of the p75HS1 substrate of p53/56lyn was strikingly reduced in both variants as compared to the wild type. These findings support the view that p75HS1 may play a critical role in BCR-dependent signaling cascades.

Signaling of programmed cell death induction in WEHI-231 B lymphoma cells.

The murine WEHI-231 B lymphoma is highly sensitive to membrane immunoglobulin ligation which leads to programmed cell death (PCD) in this cell line. To study the molecular pathways involved in PCD induction in these cells, we derived two variants of WEHI-231 resistant to anti-Ig treatment. The level of bcl-2 mRNA was identical in the wild type and the variants, either untreated or anti-Ig treated, suggesting that PCD is not under the control of bcl-2 in WEHI-231 cells. In contrast, c-myc gene expression was markedly different in the wild type and the variants, both in the unstimulated and anti-Ig-stimulated state. Our findings are interpreted in the context of the dual capacity of c-myc to promote cell growth or cell death, in conjunction with other growth regulatory signals.

Expression and biological activity of interleukin-1 receptors in mouse gamma/delta thymocytes during ontogeny.

We have recently shown that the response of mouse thymocytes to interleukin (IL)-1 + IL-2 was maximal at birth and that the responding cells displayed a CD4-CD8- T cell receptor (TcR) gamma/delta + phenotype. Unexpectedly, despite their high proportion of gamma/delta + cells, fetal thymocyte populations responded only weakly to IL-1 + IL-2. In this report, we demonstrate that the discrepancy between the day 17.5 fetal and newborn sensitivities to the combined action of IL-1 and IL-2 is a consequence of the different patterns of high-affinity IL-1 receptor (IL-1R) expression displayed by these two cell subsets. Actually, high- and low-affinity IL-1R are found in TcR gamma/delta + newborn cells and, in contrast, only low-affinity IL-1R are detectable in day 17.5 fetal cells. Our binding and functional studies strongly support the hypothesis that high-affinity IL-1R on the one hand, and low-affinity ones on the other hand, are involved in the response to IL-1 + IL-2 of newborn and day 17.5 fetal thymocytes, respectively. In addition, the high-affinity IL-1R appear to be far more efficient than the low-affinity receptors in promoting IL-2 responsiveness of thymocytes.

Anti-immunoglobulins induce death by apoptosis in WEHI-231 B lymphoma cells.

Anti-membrane immunoglobulin (anti-mIg) antibodies exert inhibitory effects in immature B lymphocytes such as WEHI-231 cells. We show here that anti-mIg treatment causes DNA fragmentation (apoptosis) in these cells. We also report that co-treatment with the protein kinase C activator phorbol 12-myristate 13-acetate prevents apoptosis induced by anti-mIg. These results are in agreement with our initial proposal that sensitivity to the toxic effects of anti-mIg reflects, at least partially, altered signal transduction in immature B lymphocytes. Variations in signal transduction pathways during B lymphocyte ontogeny may, therefore, play a critical role in determining whether B cells should be activated or inhibited via their mIg.

Early membrane potential and cytoplasmic calcium changes during mitogenic stimulation of WEHI 231 cell line by polyene antibiotics, lipopolysaccharide and anti-immunoglobulin.

Changes in free cytosolic calcium concentration and in membrane voltage are thought to be important initiating events in lymphocyte activation. The antifungal agent amphotericin B (AmB) holds interesting immunomodulating properties and its N-thiopropionyl derivative (AmBSH) is a potent polyclonal B-cell activator. These molecules may then exert their stimulating activity through the production of early ionic signals similar to those delivered by the classical activators lipopolysaccharide (LPS) and anti-immunoglobulin (anti-Ig). We addressed this question in a B-cell line (WEHI 231) which has previously been shown to exhibit characteristic response to LPS and anti-Ig. AmBSH protected these cells against anti-Ig-induced cell growth inhibition, providing a LPS-like response. In contrast, the parental compound AmB did not. The two polyene antibiotics did not modify the resting Ca2+i level of the cells, neither did LPS, whereas anti-Ig induced a rapid increase in the cytosolic calcium concentration. On the other hand, polyene antibiotics and LPS promoted membrane depolarization, whereas membrane voltage remained unchanged after anti-Ig treatment. Polyene antibiotics-induced depolarization originated from the increase of membrane permeability to Na+ ions and occurred independently of Ca2+i changes. The relationship between membrane potential and Ca2+i changes in lymphocyte activation are discussed on the basis of these results. Our conclusion was that constitutive Ca2+(-)dependent K+ channels are absent in the WEHI 231 cell line.

Membrane effects of the polyene antibiotic amphotericin B and of some of its derivatives on lymphocytes.

Amphotericin B (AmB) exhibits immunomodulating properties in mice. In vitro studies on lymphocytes, in relation with these properties, are reported here with AmB and two of its derivatives: the N-Fructosyl (N-Fru AmB) and the N-thiopropionyl (AmBSH) derivatives. Interactions of these molecules with thymocytes, a sensitive cell type, demonstrated that the extent of binding is not a toxicity parameter. In contrast, membrane fluidity changes have been observed and appeared to be related to toxicity. Experiments performed with normal B lymphocytes have shown that Amphotericin B derivatives were more potent polyclonal B cell activators than the parent compound. To go further in the understanding of these events, we have investigated in a B cell line WEHI 231, the changes in intracellular Ca2+ and membrane potential induced by AmB and AmBSH. The two polyenes were shown to induce membrane depolarization but no intracellular Ca2+ increase.

Membrane IgM cross-linking is not coupled to protein kinase C translocation in WEHI-231 B lymphoma cells.

The early molecular events involved in the process of signal transduction via membrane immunoglobulins (mIg) include phosphatidyl inositol metabolism, intracellular Ca2+ mobilization, and protein kinase C (PKC) activation. Anti-mIg antibodies exert either stimulating or inhibitory effects depending on the activation state and/or the differentiation stage of B cells. WEHI-231 is a murine B lymphoma that becomes inactivated upon anti-mIg treatment. This lymphoma has an immature B cell phenotype and is considered as a model for tolerance induction in B lymphocytes. In this study, we have investigated the relationship between mIg triggering, Ca2+ elevation, PKC translocation, and growth inhibition in WEHI-231 cells. Monoclonal antibodies to mu and kappa chains of the mIgM receptor promoted a rapid increase in intracytoplasmic Ca2+ and were potent inhibitors of cell growth. Ca2+ elevation and PKC translocation have been previously shown to be associated in B lymphocytes. To study the subcellular distribution of PKC in WEHI-231 cells, we used enzymatic assays and immunodetection methods. Although phorbol 12-myristate 13-acetate induced a rapid and almost complete redistribution of cytosolic PKC to the membrane fraction, anti-mIg treatment failed to modify the compartmentalization of PKC. These findings extend recent observations suggesting that B cell triggering through mIg receptors may involve additional pathways independent from PKC activation. PKC activation in normal B cells is also believed to provide a regulatory signal which limits the magnitude of the early signals produced by anti-mIg. Such a regulatory control is unlikely in WEHI-231 cells, due to the dissociation between Ca2+ mobilization and PKC translocation. Our findings therefore suggest that the sensitivity of immature B cells such as WEHI-231 to the inhibitory effects of anti-mIg antibodies may result in part from alterations of the phosphoinositide signal transduction pathway.

Phorbol ester induces class II gene expression in pre-B cell lines.

The effect of phorbol myristate acetate on the induction of major histocompatibility complex class II gene expression was studied in four Abelson murine leukemia virus-transformed pre-B cell lines. In three cell lines, low concentrations of PMA (0.1-10 ng/ml) induced the expression of high levels of surface Ia molecules, and this effect was mediated at the transcriptional level. PMA induced a program of coordinated transcription of all four genes involved in the biosynthesis of Ia molecules. A spontaneous Ia-positive variant pre-B cell line was derived from an Ia-negative parental cell line. This variant was highly sensitive to the toxic effects of PMA, and highly responsive to the Ia-inductive effects of phorbol ester. Our findings suggest that in pre-B cells the appearance of Ia molecules and the regulation of their expression are controlled, at least partially, by variations in the activity of protein kinase C, which is the cellular receptor for phorbol esters.

Cyclic AMP-modulated potassium channels in murine B cells and their precursors.

A voltage-dependent potassium current (the delayed rectifier) has been found in murine B cells and their precursors with the whole-cell patch-clamp technique. The type of channel involved in the generation of this current appears to be present throughout all stages of pre-B-cell differentiation, since it is detected in pre-B cell lines infected with Abelson murine leukemia virus; these cell lines represent various phases of B-cell development. Thus, the presence of this channel is not obviously correlated with B-cell differentiation. Although blocked by Co2+, the channel, or channels, does not appear to be activated by Ca2+ entry. It is, however, inactivated by high intracellular Ca2+ concentrations. In addition, elevation of intracellular adenosine 3', 5'-monophosphate induces at all potentials a rapid decrease in the peak potassium conductance and increased rates of activation and inactivation. Therefore, potassium channels can be physiologically modulated by second messengers in lymphocytes.

B cell triggering properties of a nontoxic derivative of amphotericin B.

The immunomodulating properties of amphotericin B (AMB), an antifungal polyene antibiotic, have been reported in multiple studies. However, many findings on the subject are conflicting, and the precise mechanism of AMB action on the immune system is yet unknown. Because toxicity and limited solubility of AMB are likely to be responsible for these discrepancies, we synthesized a nontoxic derivative of AMB (AMBSH), and we investigated its immune modulating effects on murine B cells. Our results show that AMBSH induces a strong proliferative response under conditions where AMB is weakly efficient or toxic, and that AMBSH supports maturation to Ig secretion. When suboptimal doses of LPS (or BCGF) are present together with AMBSH, a synergistic effect on B cell proliferation occurs. Frequency analyses reveal that, although only a limited number of B cells respond to AMBSH alone, a large population of B cells will respond to subthreshold doses of LPS in the presence of this polyene. Finally, we show that incubation of spleen cells with AMBSH results in an increase in Ia expression. These results are discussed in terms of the membrane disorganizing properties of polyene antibiotics.

Transport and hydrolysis of peptides in Saccharomyces cerevisiae.

The transport and hydrolysis of several radioactive di- and tripeptides in Saccharomyces cerevisiae was studied. A peptide-transport-deficient mutant isolated on the basis of its resistance to nikkomycin Z lost most of its capacity to take up di- and tripeptides. The transport kinetics of [14C]methionylglycine, [14C]methionylsarcosine and [3H]nikkomycin Z indicated that peptide transport is not dependent on intracellular hydrolysis. Intact cells had some peptidase activity towards methionylsarcosine but not towards nikkomycin Z. The relationship between this activity and peptide transport is discussed.

Sensitivity to nikkomycin Z in Candida albicans: role of peptide permeases.

The uptake of tritiated nikkomycin Z, a potent inhibitor of chitin synthetase, is mediated by a peptide transport system in Candida albicans. Kinetic transport assays with radioactive di- and tripeptides and competition studies suggest that two distinct systems operate in this yeast. Nikkomycin Z was transported through one of these systems, common to di- and tripeptides. A peptide transport-deficient mutant was isolated on the basis of its resistance to nikkomycin Z. The mutant lost most of its capacity to take up dipeptides but simultaneously increased its ability to transport tripeptides. These results indicate that C. albicans handles peptides through multiple transport systems and adjusts their expression to environmental conditions.

Photoaffinity inhibition of peptide transport in yeast.

A photoaffinity label, 4-azidobenzoyltrimethionine has been synthesized. It competitively inhibits trimethionine uptake in the yeast C. albicans. Upon UV irradiation it irreversibly and specifically blocks oligopeptide uptake. These results give the first example of photoinhibition of peptide uptake in yeast.