Biomedical subjects
Publications and source records attributed to T L Rothstein.
Programmed death in B cells is a highly regulated process. During the past year it has become increasingly apparent that specific receptor signals influence B cell apoptosis in distinct ways as a function of developmental stage and/or apoptotic trigger. Studies making use of opposing signals for programmed death have begun to reveal molecular correlates of sensitivity and resistance to apoptosis.
Covalent attachment of activated products of the third component of complement to antigen enhances its immunogenicity, but the mechanism is not clear. This effect is mediated by specific receptors, mCR1 (CD35) and mCR2 (CD21), expressed primarily on B cells and follicular dendritic cells in mice. To dissect the role of mCR1 and mCR2 in the humoral response, we have disrupted the Cr2 locus to generate mice deficient in both receptors. The deficient mice (Cr2-/-) were found to have a reduction in the CD5+ population of peritoneal B-1 cells, although their serum IgM levels were within the range of normal mice. Moreover, Cr2-/- mice had a severe defect in their humoral response to T-dependent antigens that was characterized by a reduction in serum antibody titers and in the number and size of germinal centers within splenic follicles. Reconstitution of the deficient mice with bone marrow from MHC-matched Cr2+/+ donors corrected the defect, demonstrating that the defect was due to B cells themselves. These results indicate an obligatory role of B cell complement receptors in responses of the B cells to protein antigens.
In mice deficient in the Fas/FasL apoptotic signaling pathways, T-dependent antibody responses to conventional environmental antigens are mechanistically distinct from the antibody responses to self-antigens. The latter appear to be initiated in the T cell rich inner PALS of the spleen, where the majority of antibody producing cells are located early in the disease process. The data are consistent with the premise that the inner PALS, and not germinal centers, is the major site of FasL regulation of B cell activity.
CD40L, a membrane protein of activated T cells, interacts with the B cell receptor CD40. This interaction has been implicated in the rescue of germinal center B cells from apoptosis and in the rescue of WEHI-231 B lymphoma cells from sIg-induced apoptosis. In this report, we have demonstrated that the signal mediated by CD40L acts upon bcl-x, a bcl-2 homologue. bcl-x expression is strongly enhanced by CD40 receptor engagement, while there is little or no induction by sIg cross-linking. The expression of bax and bcl-2 is not significantly affected by either CD40L or sIg cross-linking. Antisense but not sense phosphorothioate oligonucleotide for bcl-x can partially block this CD40-mediated apoptotic rescue. This result suggests that the up-regulation of bcl-x by CD40L plays an important role in CD40-mediated apoptotic rescue in murine B cells.
The inducible nuclear transcription factor complex, AP-1, typically consists of heterodimers between Jun and Fos proteins. Although components are drawn from families of related molecules, little is known about the physiologic regulation of jun- and fos-related gene products. In particular, it is not known whether expression of individual family members is stimulus-specific or whether the same signaling pathways are responsible for induction of all subunits. To clarify these issues, AP-1 components were examined following activation of primary B lymphocytes through two separate receptors, the surface Ig Ag receptor, and the CD40 receptor for T cell influences. Both forms of stimulation led to expression of JunB and JunD mRNA and protein; however, induction of JunB mediated by anti-Ig Ab was protein kinase C (PKC)-dependent, whereas induction mediated by CD40 ligand was resistant to PKC depletion. The two forms of stimulation diverged even further with respect to Fos expression. Although both stimuli induced c-Fos, expression of FosB was stimulus specific at both the mRNA and protein levels, in that this component was induced by anti-Ig but not by CD40 ligand. Stimulated expression of c-Fos and FosB was in all cases PKC-independent. These results provide evidence for receptor-specific differences in the expression of AP-1 components, primarily with respect to FosB. They also indicate that separate intracellular pathways may be used for induction of jun and fos gene products and that the same transcription factor (junB) may be triggered by two surface receptors through separate pathways that differ in PKC dependence.
Cytotoxic CD4+ Th1-cells induce cell death by triggering a Fas-dependent apoptotic pathway. Potential targets include activated B cells, but it is not known whether the mode of B-cell stimulation influences susceptibility to Th1-mediated cytotoxicity. Here we report that CD40-ligand-stimulated B cells were extremely sensitive, whereas anti-IgM-stimulated B cells were resistant, to Fas-mediated apoptosis. B cells stimulated by both CD40L and anti-IgM were not susceptible to cytolysis, demonstrating that anti-IgM-mediated protection is an active, dominant process. Resistance to Th1-mediated cytotoxicity was similarly observed in CD40L-stimulated 3-83 (anti-H-2Kk,b) transgenic B cells co-cultured with H-2Kk or H-2Kb (but not H-2Kd) splenocytes. These results indicate that B cells can participate in regulating their own destruction. Protection against Fas-dependent apoptosis afforded by immunoglobulin-receptor engagement may constitute a fail-safe mechanism that eliminates bystander B cells activated by CD40L-expressing T cells, but ensures survival of antigen-specific B cells.
The cAMP response element-binding protein (CREB) is generally considered to be responsive to elevation of cAMP through the activity of protein kinase A (PKA). Although it is well known that cAMP-raising agents can strongly influence B cell stimulation, the regulation of CREB has been little studied. Recently, cross-linking of surface Ig (sIg) was shown to result in trans-activation of a cAMP response element (CRE)-dependent promoter to which bound B cell CREB. In this study, we explored the mechanism underlying this unexpected linkage between sIg and CREB. We found that sIg cross-linking results in phosphorylation of CREB at Ser133. Although this phosphorylation step is mediated by PKA in pheochromocytoma cells, it depends on protein kinase C (PKC) in B lymphocytes. This conclusion is based on abrogation of sIg-induced CREB Ser133 phosphorylation by long-term phorbol-ester treatment to deplete PKC, and mimicking of sIg-induced CREB phosphorylation and CRE-dependent gene expression by short-term PKC agonism. Furthermore, CD40 ligand (CD40L) and LPS, two PKC-independent forms of B cell stimulation, failed to induce phosphorylation of CREB Ser133. These results suggest that CREB responds to specific surface-receptor signals in B cells and that this response is mediated by PKC. Interestingly, forskolin failed to induce phosphorylation of CREB Ser133 in B cells, although it did so in PC12 pheochromocytoma cells. Taken together with PKC mediation of CREB Ser133 phosphorylation in B cells, these results suggest that the dominant mode of CREB regulation is cell-type specific.
CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type-specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.
B-1 lymphocytes constitute a mature B cell population that differs from conventional B cells in signaling requirements for cell cycle progression, being unusually responsive to PKC agonism but refractory to antigen receptor stimulation. Further, B-1 cells are self-renewing and derive from surface immunoglobulin-positive precursors. A previous report on clonally derived B-1 cells suggested that increased levels of expression of c-myc might underlie these unusual growth characteristics. This issue has now been reexamined using primary B-1 cells. The possible role of egr-1, in addition to c-myc, in specifying the responsiveness of B-1 cells was evaluated by determining levels of gene expression at baseline and after mitogenic treatment. Expression of the two genes was similar in B-1 and B-2 cells prior to stimulation. Subsequently, B-1 cells responded with higher levels of gene expression than B-2 cells regardless of ultimate cell cycle progression, with the exception of stimulation by anti-Ig. Overall, there was no apparent direct correlation between stimulated levels of expression of egr-1 or c-myc and mitogen-induced S phase entry, nor were B-1 cells characterized by constitutively elevated levels of either proto-oncogene that might explain their capacity for self-renewal.
To address elements that might uniquely characterize CD40 mediated signaling, the nuclear expression of three transcription factors was evaluated following B cell stimulation by CD40L and by anti-Ig antibody. Cross-linked CD40L was found to induce nuclear expression of NF-kappa B, AP-1 and NF-AT with a time course and intensity similar to that produced by anti-Ig. Examination of NF-kappa B in more detail demonstrated that the CD40 mediated expression of DNA binding complexes correlated with induction of trans-activating activity which again attained similar levels following cross-linking of CD40 and slg. Despite the marked similarity in transcription factor induction triggered through CD40 and slg, differences in the intracellular signaling pathways utilized were apparent in that protein kinase C (PKC) depletion did not affect CD40 mediated induction of NF-kappa B even as induction by anti-Ig was abolished. These results suggest that a 'final common pathway' or convergence of transcription factor induction may exist for two distinct receptors, each of which is individually capable of triggering cell cycle progression, despite the use of separate intracellular signaling pathways that differ at the level of PKC. Although transcription factor induction was similar for CD40L and anti-Ig early on, subtle differences in expressed NF-kappa B and AP-1 nucleoprotein complexes were apparent at 24 h. Such differences may play a role in determining the variant effects on B cells of stimulation through these two receptors.
We demonstrate herein that resting primary B lymphocytes do not contain detectable levels of AP-1 (TRE)-binding activity. Upon cross-linking of surface immunoglobulin (sIg) receptors, TRE-binding activity is induced within 2 hr and its appearance requires de novo protein synthesis. Antisera to Jun-B inhibits the vast majority of TRE-binding activity, indicating that Jun-B is a primary component of B cell TRE-binding complexes. In keeping with this, Jun-B protein is not detectable in cytosol or nuclear extracts from resting B lymphocytes, as determined by immunoblotting with Jun-B antisera. However, the nuclear expression of Jun-B is induced within 2 hr following sIg cross-linking and is completely blocked by cycloheximide. 35S-labeling studies suggest that the increase in Jun-B expression results from de novo protein synthesis. Moreover, Jun-B migrates in SDS-polyacrylamide gels as two distinct electrophoretic proteins that correspond to a 41-kDa species and a phosphorylated 47-kDa form. These results suggest that the induction of AP-1-binding activity in primary B lymphocytes following sIg cross-linking does not result from post-translational phosphorylation of a preexisting cellular pool of Jun-B protein, but rather is coupled to the stimulation of de novo Jun-B synthesis. Thus Jun-B synthesis represents an integral event in the production of receptor-mediated AP-1 in B cells. The significance of these results with respect to the role of Jun-B in controlling gene expression during the activation of primary B cells is discussed.
We report a DNA sequence encoding the nuclear factor NF-kappa B p50 precursor from primary murine B-lymphocytes that differs from that previously published for the murine 22D6 B-cell line. The variation is located in one of the ankyrin-like repeats at the C terminus of the molecule.
Primary B cells are induced to proliferate by cross-linking surface immunoglobulin or by its pharmacological equivalent, phorbol ester and calcium ionophore. However, nuclear responses that have been studied in activated B cells are typically inducible with phorbol esters alone. We show that a factor, indistinguishable from the nuclear factor of activated T cells (NF-AT), is induced in B cells in response to anti-immunoglobulin signals or the combined action of phorbol ester and ionomycin, but not in response to either reagent alone. The signals necessary for NF-AT induction in B cells, therefore, closely parallel those required to induce B cell proliferation. Transfection analysis shows that B cell NF-AT is a transcriptional activator. Furthermore, NF-AT induction in splenic cells is suppressed by cyclosporin A, suggesting a mechanism by which immunosuppressive agents act on the B cell compartment. We propose that NF-AT should be considered more generally as a nuclear factor of activated lymphoid cells.
Intracellular signaling triggered by the antigen receptor of primary B-1 (CD5+ B) lymphocytes is insufficient to bring about cell cycle progression to S phase; this appears to result from a block in signal propagation prior to the activation of protein kinase C. The present studies were undertaken to evaluate specific elements in the proximal portion of the intracellular signaling cascade to determine the basis for aberrant signal transduction in B-1 cells. There was no evidence for an alteration in src kinase composition or for diminished receptor mediated tyrosine phosphorylation on the part of B-1 cells. Further, phospholipase C gamma 2 was inducibly tyrosine phosphorylated following surface Ig ligation. However, the receptor mediated increase in enzymatic activity of immunoprecipitated phospholipase C gamma 2 was markedly diminished in B-1 as compared with B-2 cells. These results indicate that phospholipase C gamma 2 largely fails to become activated following surface Ig receptor interaction in B-1 cells and suggest that this enzyme may be directly involved in controlling the propagation of progression signals.
The regulation and function of CREB was examined in B cells to begin to elucidate the role of cAMP-derived signals in B cell activation. CRE-binding activity detected by the electrophoretic mobility shift assay was found to be constitutively expressed in nuclear extracts of primary murine splenic B cells and was unchanged in nuclear extracts obtained from B cells stimulated in a variety of ways. This activity was shown to be specific by competition analysis and to represent CREB or a closely related molecule on the basis of a "supershift" in the mobility of the nucleoprotein complex induced by anti-CREB antiserum. The function of B cell CREB was assessed by transient transfection of the murine B lymphoma cell line, BAL-17, with a CRE-dependent chloramphenicol acetyl-transferase (CAT) construct that contains a portion of the somatostatin promoter. Cross-linking of the surface Ig receptors of transfected BAL-17 B cells produced a threefold induction of CAT activity. Forskolin, which markedly induced CAT expression in PC12 cells transfected with the CRE-dependent construct, failed to stimulate CAT activity in transfected BAL-17 B cells despite an increase in cAMP. However, anti-Ig was found to act in synergy with forskolin to produce enhanced CAT activity. A phosphoprotein of appropriate molecular size for CREB was immunoprecipitated from anti-Ig plus forskolin treated BAL-17 B cells. These results suggest that CREB is present in primary B cells and that CRE-dependent gene expression is regulated by surface Ig either alone or in synergy with cAMP; the latter implies cross-talk between intracellular signaling pathways acting at the level of CREB.
In comparison to B cell stimulation mediated by surface immunoglobulin (Ig) antigen receptor ligation, little is known about the intracellular events associated with T cell-dependent B cell responses. A model for the efferent phase of T cell-B cell interaction was used to examine the capacity of activated T cells to trigger nuclear expression of the trans-acting transcription factor, NF-kappa B, in B cells. Fixed, activated, but not fixed, resting Th2 cells were found to induce increased binding activity for a kappa B site-containing oligonucleotide in a time-dependent manner. This induction of NF-kappa B was eliminated by an antibody directed against a 39-kD cell interaction protein on activated T cells as well as by a soluble form of B cell CD40. Of particular relevance to intracellular signaling, NF-kappa B induction was not diminished by prior depletion of B cell protein kinase C (PKC) with phorbol myristate acetate. These results strongly suggest that T cell-dependent B cell stimulation is associated with NF-kappa B induction via p39-CD40 interaction and that this is brought about by non-PKC dependent signaling, in marked contrast to the previously documented requirement for PKC in sIg receptor-mediated stimulation. This suggest that NF-kappa B responds to more than one receptor-mediated intracellular signaling pathway in B cells and may be part of a "final common pathway" for B cell stimulation.
Populations of murine peritoneal B-1 and splenic B-2 cells, highly purified by negative selection techniques, were used to demonstrate that B-1 cells completely fail to enter cell cycle in response to surface immunoglobulin M (sIgM) crosslinking without any decrease in cell number or viability. This failure of B-1 cell responsiveness appears to represent a specific defect in sIgM-derived signaling inasmuch as stimulation to enter S phase occurs normally in response to activated and fixed T cells, and to lipopolysaccharide (LPS). The level at which sIgM signaling fails was determined by evaluating the nuclear expression of the transcription factor complex, NF-kappa B, whose sIgM-mediated induction in B-2 cells is dependent on protein kinase C (PKC) activation but is independent of protein synthesis. There was no induction of nuclear NF-kappa B in B-1 cells stimulated by sIgM crosslinking, although NF-kappa B was stimulated by phorbol myristate acetate and by LPS. In contrast, NF-kappa B was induced in B-2 cells by all three stimuli. Thus, in B-1 cells, the sIgM-mediated induction of a transcription factor that is substantially stimulated by anti-IgM in B-2 cells is blocked. However, all sIgM-derived signaling in B-1 cells was not impaired inasmuch as anti-IgM increased I-A antigen expression. These results strongly suggest that sIgM receptor-mediated signaling in B-1 cells is interrupted early in the signal transduction pathway, at a point proximal to the activation of PKC. These results further demonstrate that transcription factor induction can be used to analyze the level at which receptor-mediated signaling is blocked.