Inducible Fas-resistance mediated by IL-4 in activated B cells.
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Animals↗
Biomedical subjects
T L Rothstein
Publications and source records attributed to T L Rothstein.
Unique site of autoantibody production in Fas-deficient mice.
The inability of B and T lymphocytes from mice expressing the lpr mutation to express functional Fas on their cell surface leads to an immunoregulatory defect associated with excessive autoantibody production. Nevertheless, T-dependent antibody response to foreign antigens in these mice appears relatively normal. To better understand exactly how Fas/FasL interactions control autoantibody production, studies were undertaken to determine (1) what kind(s) of B cells are sensitive to Fas-mediated apoptosis and (2) where the autoantibody-producing cells in lpr mice are located. We found that B cells activated by CD40L are extremely sensitive as targets in assays of Th1 CMC. However, B cells that receive a complete signal through their sIgM antigen receptor acquire a FasL-resistant phenotype. In situ analysis of splenic sections from lpr mice demonstrated that autoantibody-producing cells were uniquely localized to the T cell-rich inner PALS. A similar distribution pattern of IgG AFC was found in mice with chronic GVH disease. These data are consistent with the premise that the inner PALS, and not the germinal center, is the major site of FasL regulation of B cell activity and that, as a result of genetic or inducible loss of sensitivity to Fas-mediated apoptosis, autoreactive B cells may survive and differentiate in this location to cause serological autoimmunity.
Animals↗
Signal transducer and activator of transcription-3 (STAT3) is constitutively activated in normal, self-renewing B-1 cells but only inducibly expressed in conventional B lymphocytes.
Cytokine and growth factor receptor engagement leads to the rapid phosphorylation and activation of latent, cytosolic signal transducers and activators of transcription (STAT) proteins, which then translocate to the nucleus where they regulate transcriptional events from specific promoter sequences. STAT3 expression in particular has been associated with Abl, Src, and HTLV-1 transformation of normal cells. B-1 lymphocytes are self-renewing, CD5+ B cells that display a propensity for malignant transformation and are the normal counterpart to human chronic lymphocytic leukemias. Further, B-1 cells are characterized by aberrant intracellular signaling, including hyperresponsiveness to phorbol ester PKC agonists. Here we demonstrate that B-1 lymphocytes constitutively express nuclear activated STAT3, which is not expressed by unmanipulated conventional (B-2) lymphocytes. In contrast, STAT3 activation is induced in B-2 cells after antigen receptor engagement in a delayed fashion (after 3 h). Induction of STAT3 is inhibited by both the serine/threonine protein kinase inhibitor H-7 and the immunosuppressive drug rapamycin and requires de novo protein synthesis, demonstrating novel coupling between sIg and STAT proteins that differs from the classical paradigm for STAT induction by cytokine receptors. The inability of prolonged stimulation of conventional B-2 cells with anti-Ig, a treatment sufficient to induce CD5 expression, to result in sustained STAT3 activation suggests that STAT3 is a specific nuclear marker for B-1 cells. Thus, STAT3 may play a role in B cell antigen-specific signaling responses, and its constitutive activation is associated with a normal cell population exhibiting intrinsic proliferative behavior.
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The role of evoked potentials in coma.
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Brain↗
Receptor-specific regulation of NF-kappa B, c-Myc and Fas-mediated apoptosis in primary B cells.
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Animals↗
Unique site of IgG2a and rheumatoid factor production in MRL/lpr mice.
MRL/lpr (Fas-deficient) mice develop an autoimmune syndrome associated with excessive production of autoantibodies. A significant portion of these autoantibodies are IgG2a molecules specific for many of the autoantigens recognized by the sera of patients with systemic lupus erythematosus. In addition, MRL/lpr mice make exceedingly high titers of IgG or IgA rheumatoid factors (RF) specific for autologous IgG2a. The microenvironment of the IgG2a-producing B cells as well as the prototypic RF autoantibodies was determined by a combination of immunohistochemical and in situ hybridization techniques. In contrast to the antibody-producing cells present in mice responding to conventional foreign antigens, both IgG2a+ and RF+ B cells were found to be densely clustered in the T-cell-rich inner periarteriolar lymphatic sheath of the spleen. These results suggest that conventional antibody and autoantibody production in MRL/lpr mice may be mechanistically distinct processes.
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Fas expression in pulmonary alveolar type II cells.
Fas, a type I membrane receptor protein, transduces a signal culminating in apoptosis after binding to the Fas ligand. Information regarding the expression of Fas in nonlymphoid tissues, although limited, suggests a role for Fas in epithelial progenitor cell populations. In this paper, we provide several lines of evidence indicating that the progenitor cell of the alveolus, the type II cell, displays restricted expression of Fas. We found 1) Fas gene expression in RNA derived from fresh isolates of primary rat type II cells; 2) restriction of Fas expression to a subset of alveolar type II cells by in situ hybridization and immunohistochemistry of the normal mouse lung; 3) induction of apoptosis in a mouse lung type II epithelial cell line (MLE) after activation of Fas; and 4) induction of apoptosis in a subpopulation of type II cells after the intratracheal instillation of an activating anti-Fas antibody in mice. These findings suggest that Fas-dependent apoptosis is involved in regulating turnover of the alveolar epithelium.
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Isolation and characterization of murine fra-1: induction mediated by CD40 and surface Ig is protein kinase C dependent.
The murine fra-1 gene, encoding Fos-related Ag 1, was isolated from a splenic cDNA library and sequenced. Murine fra-1 was highly homologous to rat and human fra-1. Oligonucleotide primers based on the murine sequence were used to construct a quantitative reverse transcription-PCR assay for gene expression. B lymphocyte stimulation via both CD40 and surface Ig (sIg) receptors substantially induced fra-1 expression, and for both receptors, induction was protein kinase C (PKC) dependent. This contrasts with induction of c-fos by both CD40 and sIg, which is PKC independent and indicates that CD40 is capable of signaling through PKC or a closely related kinase. Induction of fra-1 following engagement of CD40 did not require protein synthesis, suggesting that the PKC-dependent linkage between CD40 and fra-1 is direct. CD40-mediated fra-1 induction did require tyrosine kinase activity. These results demonstrate that CD40, like sIg, may employ PKC in producing select outcomes, that individual B cell receptors may signal downstream events via both PKC-dependent and PKC-independent pathways, and that multiple signal transduction pathways may be used to activate the expression of closely related genes.
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IL-4 induces Fas resistance in B cells.
Activated B cells express Fas (CD95) and are targets for apoptosis induced by CD4+ Th1 effector cells that kill in a Fas-dependent fashion. We report here that IL-4 reverses the susceptibility to Fas-mediated apoptosis that characterizes CD40-stimulated primary B cells. IL-4-induced Fas resistance is not associated with an alteration in the elevated level of Fas expression produced by CD40 ligand and does not depend on additional receptor-mediated signals. However, IL-4-induced resistance to Th1 cell-mediated cytotoxicity (Th1-CMC) develops more slowly than resistance mediated by surface Ig and is not affected by protein kinase C depletion, unlike anti-Ig-induced Fas resistance. By these two criteria, IL-4-and anti-Ig-induced resistance to Th1-CMC appear to be driven through distinct mechanisms; in keeping with this, suboptimal doses of IL-4 and anti-Ig act in synergy to induce marked protection against Th1-CMC. An important role for IL-4-induced Fas resistance is suggested by the observation that sera from IL-4-overexpressing transgenic mice contain autoreactive Abs.
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Delayed tyrosine phosphorylation and nuclear expression of STAT1 following antigen receptor stimulation of B lymphocytes.
The regulation of the STAT1 alpha transcription factor was assessed during B cell activation induced by cross-linking the surface IgM Ag receptor. Surface Ig ligation or pharmacologic stimulation with PMA and ionomycin resulted in the delayed (2-3 h after stimulation) nuclear appearance of tyrosine-phosphorylated STAT1 alpha, in contrast to the rapid induction that follows cytokine treatment. Nuclear expression of phosphorylated STAT1 alpha was abrogated by co-incubation of anti-Ig-treated B cells with the protein synthesis inhibitor cycloheximide (CHX), with the protein kinase inhibitor H-7, or with the immunosuppressive drug rapamycin. Tyrosine-phosphorylated STAT1 alpha was found to be recruited to the STAT binding site of the IFN regulatory factor-1 (IRF-1) gene promoter only after 2 to 3 h, and this association was also inhibitable by CHX and rapamycin. The arrival of STAT1 alpha coincided with attenuation of anti-Ig-induced STAT-binding activity specific for the IRF-1 promoter site, and both rapamycin and CHX treatment counteracted the loss of this activity. Furthermore, basal transcription of the endogenous IRF-1 gene was decreased as a result of anti-Ig treatment, and this effect of anti-Ig was blocked by co-incubation with rapamycin. Thus, STAT1 alpha plays a dynamic role in the composition of IRF-1 promoter-specific DNA binding complexes stimulated by B cell Ag receptor ligation, and nuclear expression of phosphorylated STAT1 alpha is regulated in a unique fashion by Ag receptor engagement. In addition, surface Ig cross-linking imparts negative regulatory control of IRF-1 gene expression, possibly through activation of STAT1 alpha.
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Inhibition of NF-kappaB/Rel induces apoptosis of murine B cells.
Apoptosis of the WEHI 231 immature B cell lymphoma line following membrane interaction with an antibody against the surface IgM chains (anti-IgM) is preceded by dramatic changes in Nuclear Factor-kappaB (NF-kappaB)/ Rel binding activities. An early transient increase in NF-kappaB/Rel binding is followed by a significant decrease in intensity below basal levels. Here we have explored the role of these changes in Rel-related factors in B cell apoptosis. Treatment of WEH1 231 cells with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), a protease inhibitor which prevents degradation of the inhibitor of NF-kappaB (IkappaB)-alpha, or with low doses of pyrrolidinedithiocarbamate (PDTC) selectively inhibited NF-kappaB/Rel factor binding and induced apoptosis. Bcl-XL expression protected WEHI 231 cells from apoptosis induced by these agents. Microinjection of WEHI 231 cells with either IkappaB-alpha-GST protein or a c-Rel affinity-purified antibody induced apoptosis. Ectopic c-Rel expression ablated apoptosis induced by TPCK or anti-IgM. Treatment of BALENLM 17 and A20 B lymphoma cells or normal murine splenic B lymphocytes with either TPCK or PDTC also resulted in apoptosis. These findings indicate that the drop in NF-kappaB/Rel binding following anti-IgM treatment activates apoptosis of WEHI 231 cells; furthermore, they implicate the NF-kappaB/Rel family in control of apoptosis of normal and transformed B cells.
Animals↗
Intracellular signaling for inducible antigen receptor-mediated Fas resistance in B cells.
CD40 ligand-activated B cells are sensitive targets for CD4+ Th1 effector cells that kill in a Fas-dependent fashion. Susceptibility to apoptosis is counteracted by Ag receptor binding that produces a state of resistance to Fas engagement in otherwise sensitive targets. In the present study, protection from Th1-mediated apoptosis was found to be induced by protein kinase C and calcium signals, which in combination mimicked the level of Fas resistance produced by surface Ig engagement. Signaling for Fas resistance did not alter Fas expression. Furthermore, B cells that were protected against Th1-mediated apoptosis were also resistant to apoptosis mediated by soluble, rFas ligand. Taken together, these results indicate that signaling for protection against Fas-mediated apoptosis does not depend on alteration of the interaction between B cell target and Th1 effector populations. Instead, surface IgM-derived protein kinase C and calcium signals appear to produce an intracellular change in the Fas signaling pathway that develops over a period of hours and interferes with the apoptotic process through a mechanism that depends on protein synthesis.
Animals↗
Regulation of the B cell response to T-dependent antigens by classical pathway complement.
Mice deficient in complement components C3 (C3 -/-) and C4 (C4 -/-) were found to have a profound defect in their Ab response to a T-dependent Ag (bacteriophage (phi X174). Characterization of the deficient mice demonstrated a diminished level of peanut agglutinin+ germinal centers and a failure in isotype switching despite normal B cell signaling in vitro. The nature of the defect was found to lie at the B cell level, as the T cells were primed in C3- and C4-deficient mice as well as those in wild-type mice. These results, and the finding that the defect could be partly reversed by a 10-fold increase in Ag dose, support the hypothesis that covalent attachment of complement ligands, i.e., C3b and C3d to the Ag-Ab complex, increases its immunogenicity.
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Antigen-receptor engagement in B cells induces nuclear expression of STAT5 and STAT6 proteins that bind and transactivate an IFN-gamma activation site.
The signal transducer and activator of transcription (STAT) family of transcription factors is triggered by cytokine and growth factor receptors in a number of cell types, and binds to a consensus sequence defined in part by the IFN-gamma activation site (GAS). It is not known whether these transcription factors respond to other kinds of growth stimuli, and, with particular relevance to lymphocytes, it is not known whether STAT proteins participate in Ag-specific responses. To determine the role of STAT proteins, coupling between Ag-receptor cross-linking and nuclear expression of DNA-binding protein complexes that recognize GAS sequences was evaluated. Ag-receptor triggering in primary B lymphocytes stimulated nuclear expression of a complex that specifically binds the IFN response factor-1 (IRF-1) GAS sequence, and is distinguished by electrophoretic mobility and GAS preference from IRF-1 GAS-binding complexes induced by IFN-gamma. Activation of nuclear IRF-1 GAS-binding activity by sIg was inhibited by the tyrosine kinase inhibitor, herbimycin A, and binding activity was eliminated by tyrosine phosphatase treatment. Activation of IRF-1 GAS-binding activity was blocked by depletion of protein kinase C. The IRF-1 GAS-binding activity induced by sIg engagement in B cells was transcriptionally active, and was found to consist of immunoreactive STAT5 and STAT6 proteins. This work demonstrates that the STAT signaling pathway previously associated with cytokine signaling is triggered in B lymphocytes through Ag-receptor engagement in a protein kinase C-dependent fashion. This heretofore described cytokine signaling pathway may play a role in bringing about Ag-specific proliferative and differentiative responses.
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Maintenance of nuclear factor-kappa B/Rel and c-myc expression during CD40 ligand rescue of WEHI 231 early B cells from receptor-mediated apoptosis through modulation of I kappa B proteins.
Engagement of surface IgM (sIgM) on WEHI 231 murine B lymphoma cells leads to abortive activation and apoptosis, suggesting that this cell line may represent a model for tolerance. Loss of viability in these cells is preceded by an early increase in c-myc RNA levels followed by a decline below control values, implicating c-myc in the control of apoptosis. Costimulation with CD40 ligand (CD40L) has been shown to rescue WEHI 231 cells from anti-sIgM-induced apoptosis, and therefore, the effects of CD40L on c-myc RNA and protein levels were measured. Treatment of these cells with the combination of CD40L and anti-sIgM led to induction and maintenance of elevated levels of c-myc RNA and protein. Since transcriptional regulation of c-myc is mediated through two nuclear factor-kappa B (NF-kappa B) sites in WEHI 231, the effects of CD40L on DNA binding by this family of transcription factors were evaluated. CD40L induced and sustained the levels of NF-kappa B binding to both of these sites, paralleling the changes in c-myc RNA levels. Elevated levels of NF-kappa B were partially achieved through a sustained decrease in the steady state amount of the NF-kappa B/Rel-specific inhibitory protein, I kappa B alpha, and a transient decrease in I kappa B beta. These data lend support to the hypothesis that anti-sIgM-induced apoptosis is caused by a drop in c-myc expression and that an appropriate second signal, such as that provided by CD40L, is able to rescue these cells by inducing NF-kappa B and thereby maintaining c-myc RNA levels.
Animals↗
Signaling pathways for antigen receptor-mediated induction of transcription factor CREB in B lymphocytes.
We previously reported that cross-linking surface immunoglobulin (sIg) leads to induction of the transcription factor CREB in B lymphocytes through phosphorylation at Ser133, despite the lack of an increase in cAMP. Further, cAMP-raising agents fail to induce CREB Ser133 phosphorylation and CRE-dependent gene expression in these cells, which differs sharply from the situation in PC12 rat pheochromocytoma cells where CREB responds to elevation of cAMP through the activity of protein kinase A. In this study, we characterized the signal transduction pathways leading from sIg engagement to CREB activation. By using specific inhibitors for protein kinase C (PKC), Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), and protein kinase A (PKA), we found that anti-Ig-induced CREB Ser133 phosphorylation depends on PKC, but does not require activation of PKA or CaM kinase II. The differential responsiveness of CREB to forskolin in PC12 cells and BAL-17 B cells may relate to the more marked elevation of cAMP in the former as opposed to the latter; however, high concentrations of dbcAMP which should readily enter B cells and artificially increase cAMP levels still failed to induce CREB Ser133 phosphorylation, even in conjunction with a phosphodiesterase inhibitor. Taken together, the cAMP/PKA pathway does not appear to be as active a contributor to CREB phosphorylation in B lymphocytes as in PC12 cells, and does not appear to be involved in sIg-induced, PKC-dependent, CREB activation.
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Fas (CD95)/Fas ligand interactions regulate antigen-specific, major histocompatibility complex-restricted T/B cell proliferative responses.
The effect of Fas ligand (FasL) cytotoxicity on T/B collaboration was examined in vitro using cloned T helper 1 cells and antigen-pulsed, activated B cells. We compared antigen-pulsed B cells that had been activated through different membrane receptors (IgM, CD14 and CD40) for their ability to induce T cell proliferation and to respond to T cell help. We also used a Fas-Ig fusion protein, an inhibitor of FasL-mediated cytotoxicity, to determine the effect of FasL cytotoxicity on the T and B cell proliferative responses. The data show that the extent of both T and B cell proliferative responses correlate with the relative resistance of activated B cell populations to FasL cytotoxicity. Moreover, both T and B cell proliferation could be enhanced by Fas-Ig. Our results demonstrate that FasL cytotoxicity is a negative regulatory mechanism for both T and B cell proliferative responses and that Fas-Ig can be an immunopotentiating agent for both T and B cell immunity.
Animals↗
Inducible resistance to Fas-mediated apoptosis in primary B lymphocytes.
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Animals↗