Inducible Fas-resistance mediated by IL-4 in activated B cells.
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Animals↗
Biomedical subjects
A Marshak-Rothstein
Publications and source records attributed to A Marshak-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↗
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.
Animals↗
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.
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↗
beta2-microglobulin dependence of the lupus-like autoimmune syndrome of MRL-lpr mice.
MRL-lpr/lpr mice develop a distinctive immunologic disease characterized by accumulation of unusually large numbers of T cells in the peripheral lymphoid organs. Most of the accumulating T cells express an alpha beta-TCR but are peculiar in that they express neither CD4 nor CD8 co-ligands. Concurrent with lymphoaccumulation of such double negative (DN) T cells, MRL-lpr/lpr mice develop a lethal systemic lupus erythematosus-like autoimmune syndrome. This study focuses on the role of MHC class I molecules in this latter pathologic process. Highly backcrossed class I molecule-deficient MRL and MRL-lpr mice carrying a functionally defective allele of the gene beta 2-microglobulin (B2m) were produced. Class I deficient MRL-lpr/lpr mice demonstrated a substantial reduction in DN T cells, confirming other reports indicating that most DN T cells arise from progenitors positively selected on MHC class I molecules. Significantly, class I-deficient MRL-lpr/lpr mice also demonstrated a diminution of every autoimmune disease indicator analyzed including hypergammaglobulinemia; autoantibodies including anti-DNA, anti-Smith antigen, and rheumatoid factor; and glomerulonephritis. The results indicate that class I-dependent T cells are crucial not only for the development of DN T cells, but for multiple features of the MRL-lpr/lpr systemic lupus erythematosus syndrome. Moreover, the pattern of hypergammaglobulinemia suggests that the requirement for MHC class I proteins is restricted temporally to later stages of the disease.
Animals↗
Restriction of the TCR repertoire inhibits the development of memory T cells and prevents autoimmunity in lpr mice.
The lpr mutation, a disruption of the fas gene, induces spontaneous autoimmunity characterized by high titers of autoantibodies, lymphadenopathy, autoreactive T cells, and early mortality. The mechanism of autoimmunity, however, remains unknown. The driving force for disease could result from the T cell recognition of autoantigen or, alternatively, an intrinsic T cell defect that promotes autoreactivity. We investigated the role of antigen-TCR interaction in the pathogenesis of lpr autoimmunity by transferring the DO-11.10 TCR beta-chain transgene (Vbeta8.2-Dbeta1.1-Jbeta1.1) to the MRL-lpr/lpr background producing the MRL-lprbeta strain. Our results show that the MRL-lpr beta transgenic strain has increased survival, lower titers of autoantibodies, and decreased lymphadenopathy compared with nontransgenic littermates. These beneficial effects were associated with decreased expansion of CD4+ T cells expressing memory phenotypes (CD44+, CD45RB-, and LECAM-) in the transgenic compared with nontransgenic strains. A role for impaired recognition of autoantigen by T cells expressing the TCR transgene was suggested by comparing the phenotypes of Vbeta8.2+ (transgene+) vs Vbeta8.2- (transgene-) CD4+ T cells within the transgenic mice. These experiments show that Vbeta8.2- T cells, which express endogenously rearranged TCR, are the major contributors to the expansion of memory T cells in the transgenic mice. In contrast, T cells with memory phenotypes expand similarly in both the Vbeta8.2+ and Vbeta8.2- subsets of nontransgenic mice. Based on these results, we hypothesize that TCR recognition of autoantigen is a major contributor to autoimmunity in lpr mice and that T cells expressing a memory phenotype are perpetrators of this process.
Age Factors↗
Regulation of T-cell death genes: selective inhibition of FasL- but not Fas-mediated function.
Activation-induced cell death (AICD) requires coexpression of Fas and FasL. Hybridoma T-cells express detectable FasL mRNA 3 to 5 hr after culture in anti-CD3-coated wells. High and steady expression of FasL mRNA was observed after 8-10 hr of activation. Expression of FasL cytotoxicity and AICD is consistent with the time-course of FasL mRNA induction. Fas-Ig was effective in inhibiting AICD when added no later than 5 hr after activation, but was ineffective when added after 8-10 hr of activation. These observations suggest that FasL gene activation is a critical step for AICD. By contrast, Fas was constitutively expressed and the time-course study did not support the idea that up-regulation of Fas is critical for AICD. The critical role of FasL gene activation for AICD was confirmed by studies using inhibitors of AICD. Dexamethasone (Dex) inhibited FasL induction and Fas up-regulation, but not basal Fas expression of hybridoma T-cells. All-trans retinoic acid (RA) inhibited FasL induction, but had little effect on Fas up-regulation. Both agents inhibited FasL cytotoxicity. The Fas-mediated death pathway distal to Fas/FasL interactions remained intact in the protected hybridoma T-cells. These results demonstrate that FasL gene activation, but not Fas up-regulation, is critical for AICD and that Dex and all-trans RA selectively inhibits FasL but not Fas function. The system may prove useful for the identification of critical factors regulating T-cell death genes. It may also serve as a useful system to study gene regulation in AICD-dependent phenomena.
Animals↗
Characterization of lpr-derived T cell hybridomas: Fas-deficient hybridomas are deathless, growth-arrested, and cytotoxic upon activation.
T cell hybridomas that are deathless upon TCR crosslinking were generated from lpr mice. The deathless hybridomas (1.4 and 5D5) expressed extremely low Fas even after anti-CD3 activation, whereas activation-induced cell death (AICD) was observed for Fas-expressing hybridomas. The deathless hybridomas were activated to produce FasL and IL-2, indicating that the intrinsic defect in Fas expression or up-regulation resulted in AICD blockade. The deathless hybridoma cells expressed longer and stronger FasL cytotoxicity than AICD-sensitive hybridomas. Although deathless, activated 5D5 cells were arrested at the G1/S border. Growth arrest lasted for at least 5 days, but some cells eventually recovered and proliferated. The deathless 5D5 cells were used to demonstrate that AICD includes a fratricidal mechanism that kills AICD-sensitive bystanders. The deathless T cell hybridomas are useful tools for studying T cell activation-dependent functions sensitive to AICD.
Animals↗
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↗
B cells in systemic autoimmune disease: recent insights from Fas-deficient mice and men.
In mice functionally deficient for either Fas or Fas ligand expression, the failure of Fas-ligand-expressing cytotoxic T cells to eliminate autoreactive B cells can result in excessive autoantibody production. Recent in vitro studies have shown that B cells activated by CD40 ligand become extremely sensitive to Fas-mediated apoptosis while IL-4 and/or surface IgM receptor engagement protects B cells from Fas ligand cytolysis. Potential in vivo sites for Fas ligand regulation of self-reactive B cells have been suggested and implications for human disease have been investigated.
Animals↗
The role of Fas/FasL interactions in the regulation of B cell function.
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.
Animals↗
Requirement for a beta 2-microglobulin-associated Fc receptor for acquisition of maternal IgG by fetal and neonatal mice.
There is considerable evidence to suggest that an FcR similar in structure to class I MHC Ags, neonatal Fc receptor (FcRn), transports IgG across the intestinal epithelium of suckling mice. However, this has not previously been shown definitively, nor has it been shown whether FcRn is the only, or even the major, IgG transporter in the neonatal mouse gut. We report here that neonatal mice homozygous for a targeted disruption of the beta 2microglobulin (beta 2m) gene, which encodes one subunit of FcRn, had reduced FcRn alpha-chain at the lumenal plasma membrane of intestinal cells. These mice had strikingly lower serum IgG levels during the first month after birth than littermates that possessed functional FcRn. Furthermore, we found by fostering mice on mothers with a different IgG allotype that all of the IgG in sera of beta 2m-/- mice was endogenous, and that none was obtained from milk. We conclude that FcRn is the only transporter of IgG from mother to young in the mouse. The onset of IgG synthesis in mice that received no milk IgG lagged behind that in siblings with normal IgG transport, suggesting that maternal IgG stimulates Ab production in the neonate. We noted no difference between the IgG concentrations in the milk of beta 2m-/- and beta 2m+/- mice, indicating that FcRn is not involved in the secretion of IgG into milk.
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Fas ligand-mediated cytotoxicity is directly responsible for apoptosis of normal CD4+ T cells responding to a bacterial superantigen.
Exposure of naive CD4+ T lymphocytes to superantigens such as staphylococcal enterotoxin B (SEB) induces a strong proliferative response. Prolonged exposure or subsequent restimulation of the responding T cell population with SEB leads to the apoptotic events of activation-induced cell death (AICD). However, T cells derived from either Fas-deficient lpr or Fas ligand-deficient gld autoimmune mouse strains, fail to undergo AICD under these conditions. Instead, these autoimmune T cells mount a vigorous proliferative response, suggesting a critical role for Fas/FasL interactions in this form of autoapoptosis. In the current study, we found that SEB-induced AICD was tied to the rapid induction of FasL expression in cells constitutively expressing high levels of Fas. Furthermore, the addition of soluble Fas-IgG fusion protein to the SEB-restimulated cultures blocked AICD and resulted in a 2 degrees proliferative response that was comparable in magnitude and kinetics to that of the lpr and gld T cells. The rapid onset of apoptosis in normal T cells subsequent to restimulation with SEB was in direct contrast to the proliferative response of the initial cultures, even though comparable levels of Fas and FasL RNA were found in T cells after 1 degree and 2 degrees challenge. The clonal expansion of the normal T cells responding to the initial SEB stimulation was, however, dramatically compromised when the normal cells were cocultured with an MRL-lpr responder population; addition of soluble Fas-IgG rescued the normal component of the response. Together, these data demonstrate first, that Fas/FasL interactions are intimately tied to superantigen-induced AICD, a form of autocrine cell death, and second, that FasL-mediated cytotoxicity is responsible for the disappearance of normal CD4+ T cells in lpr cocultures.
Animals↗
Protection against Fas-dependent Th1-mediated apoptosis by antigen receptor engagement in B cells.
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.
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Fas(CD95)/FasL interactions required for programmed cell death after T-cell activation.
Receptor crosslinking of T-cell hybridomas induces cell activation followed by apoptosis. This activation-induced cell death requires de novo synthesis of RNA and proteins, but the actual gene products that provide the death signal have not been identified. We show here that receptor crosslinking induces Fas ligand and upregulates Fas, and that the ensuing engagement of Fas by Fas ligand activates the cell-death programme. Cell death, but not activation, can be selectively prevented by a soluble Fas-immunoglobulin fusion protein. Thus, Fas and Fas ligand are the death-gene products, and their interaction accounts for the molecular mechanism of activation-induced T-cell death.
3T3 Cells↗
The effect of VH residues 6 and 23 on IgG3 cryoprecipitation and glomerular deposition.
MRL/lpr mice spontaneously develop a lupus-like autoimmune syndrome characterized by immunopathologic manifestations such as necrotizing vasculitis of the skin and glomerulonephritis. A feature of this autoimmune syndrome is the production of extremely large amounts of monoclonal IgG3 cryoglobulins. The structural basis of IgG3 cryoprecipitation is not well understood. Although the IgG3 isotype is necessary for cryoprecipitation, not all IgG3 antibodies cryoprecipitate. It has been postulated that electrostatic charge may be influential in cryoprecipitation. To investigate this problem, the VH and VL sequences of a panel of IgG3 cryoglobulins and non-cryoglobulins were compared, with particular attention to charged amino acid differences. At VH residues 6 and 23 the cryoglobulins were more positively charged than their non-cryoglobulin counterparts. To analyze further the effect of charge on cryoprecipitation, the sequence of an IgG3 monoclonal cryoprecipitating rheumatoid factor was modified by site-directed mutagenesis. The more positive residues at VH 6 and 23 present in some of the cryoglobulin antibodies were mutated to the more negative residues found in the non-cryoglobulins. The results show that VH residue 6 affects cryoprecipitation while residue 23 does not. When injected into normal BALB/c mice, the unmutated antibody produced glomerular immune deposits and focal glomerulonephritis, whereas loss of cryoprecipitability by mutating residue 6 completely abrogated glomerular immune deposition and glomerular injury. In contrast, the mutation at residue 23 which retains cryoprecipitability reduced glomerular immune deposition and prevented glomerular injury.
Amino Acid Sequence↗