Abnormal thymocyte development and production of autoreactive T cells in T cell receptor transgenic autoimmune mice.

Development of a C57BL/6-+/+ TCR transgenic mouse containing the rearranged TCR alpha- and beta-chain specific for the Db + HY male Ag results in production of a nearly monoclonal population of early thymocytes expressing the Db + HY reactive TCR. These thymocytes are autoreactive in H-2Db male mice and undergo clonal deletion and down-regulation of CD8. To study the effect of the lpr gene on development of autoreactive T cells, these transgenic mice were backcrossed with C57BL/6-lpr/lpr mice. T cell populations in the thymus and spleen were analyzed by three-color flow cytometry for expression of CD4, CD8, and TCR. The thymus of TCR transgenic H-2b/b lpr/lpr male mice had an increase in percent and absolute number of CD8dull thymocytes compared to TCR transgenic H-2b/b +/+ male mice. However, there was not a complete defect in clonal deletion, because clonal deletion and down-regulation of CD8 was apparent in both +/+ and lpr/lpr H-2Db HY+ male mice compared to H-2Db HY- female mice. The phenotype of splenic T cells was almost identical in TCR transgenic +/+ and lpr/lpr males with about 50% CD4-CD8- T cells and 50% CD8+ T cells. However, there was a dramatic increase in the SMLR proliferative response of splenic T cells from TCR transgenic lpr/lpr males compared to TCR transgenic +/+ males. To determine the specificity of this response, spleen cells from TCR transgenic lpr/lpr and +/+ mice were cultured with irradiated H-2b/b and H-2k/k male and female spleen cells. T cells from TCR transgenic C57BL/6-lpr/lpr male mice had an increased proliferative response to H-2b/b male spleen cells compared to T cells from TCR transgenic C57BL/6(-)+/+ male mice, but both lpr/lpr and +/+ mice had a minimal response to irradiated H-2b/b female or H-2k/k male or female stimulator cells. The splenic T cells from TCR transgenic lpr/lpr mice also had an increased specific cytotoxic activity against H-2b/b male target cells compared to TCR transgenic +/+ mice. These results demonstrate that there is a defect in negative selection of self-reactive T cells in the thymus of lpr/lpr mice and a defect in induction or maintenance of clonal anergy of self-reactive T cells in the periphery of lpr/lpr mice.     

Accelerated programmed cell death of MRL-lpr/lpr T lymphocytes.

MRL-lpr/lpr (lpr) mice develop a polyclonal accumulation of abnormal peripheral T lymphocytes, which bear surface alpha beta TCR, CD3, and the B220 isoform of CD45, but lack CD4, CD8, and CD2. These T cells have a constitutively phosphorylated CD3 zeta chain and manifest a defect in signal transduction that results in a lack of IL-2 production and proliferation. We investigated whether this signaling abnormality might contribute to their accumulation via a defect in T cell elimination in the periphery. T cell deletion occurs through a process of programmed cell death with DNA degradation, or apoptosis. Viable lymphocytes from lpr mice were found to undergo rapid programmed cell death in culture within 4 h without additional activation, which was not observed in lymphocytes from normal MRL-+/+ or C57BL/6-+/+ mice. Both nonmature B220+ and mature B220- T lymphocytes from lpr mice display this accelerated programmed cell death, indicating that this is a defect affecting all peripheral T lymphocytes in lpr mice. In vitro apoptosis of lpr T cells could be inhibited with PMA, a stimulator of protein kinase C. Thus, the massive accumulation of T lymphocytes in the lymphoid tissue of lpr mice is not due to a defect in their ability to undergo programmed cell death in vitro. The activation state of lpr T cells may contribute to their rapid degradation of DNA in vitro.     

Autoreactive T cells in MRL/Mpr-lpr/lpr mice. Characterization of the lymphokines produced and analysis of antigen-presenting cells required

Lymph node cells from 4-wk-old MRL/Mp-lpr/lpr mice, but not from MRL/Mp-+/+ mice, when cultured in vitro for 5 to 7 days, will spontaneously proliferate and produce IL-2. We examined the expression of several cell surface Ag on lymph node cells from MRL/Mp-lpr/lpr mice before and after in vitro culture. There is an increase in the expression of Thy-1, L3T4, IL-2R, T cell activating protein, T cell receptor, and T3 complex on the surface of cultured cells. Cultured cells produced IL-3, IFN-gamma, and small but detectable amounts of IL-1 in addition to IL-2. Gamma irradiation of APC from young MRL/Mp-lpr/lpr mice or treatment of APC with a mAb (J11D) and C, completely abrogated their stimulatory capacity. These experiments suggest that B cells are the predominant APC responsible in the activation of autoreactive T cells in MRL/Mp-lpr/lpr mice. Lymph node cells from C57BL/6-lpr/lpr or C3H-lpr/lpr mice were unable to spontaneously proliferate or produce IL-2. Lymph node cells from (MRL/Mp-lpr/lpr x C57BL/6-lpr/lpr) F1 mice or (C3H-lpr/lpr x MRL/Mp-lpr/lpr) F1 mice did proliferate and produced IL-2 after in vitro culture. Using T cells from these F1 animals and APC from each parental haplotype, we found that APC from MRL/Mp-lpr/lpr mice induced more proliferation and greater amounts of IL-2, when compared to APC from F1 animals. APC from C57BL6-lpr/lpr mice or C3H-lpr/lpr were unable to induce spontaneous proliferation and IL-2 production. Therefore, B cells from MRL/Mp-lpr/lpr mice appear to possess unique features that enable them to activate autoreactive T cells more effectively than B cells from other mice bearing the lpr/lpr gene.     

Autoimmune CD4+ T cells interfere with immune tolerance to a thymic-independent antigen

The ability of autoimmune T cell subsets to interfere with tolerization of B cells can be studied by using thymic-independent Ag. We have defined an abnormality within the CD4+ T cell compartment in young NZB and MRL-lpr/lpr mice by studying tolerance of spleen and B cells to the thymic independent Ag, fluorescein-Brucella abortus. Tolerization of spleen cells is defective in MRL-lpr/lpr mice, but not MRL-+/+ or C3H.lpr mice, suggesting that the defect requires both the autosomal MRL background and the lpr gene to be present. T enriched cells from NZB mice and from MRL-lpr/lpr mice (but not MRL-+/+ or C3H.lpr mice) reverse tolerance in spleen cells from [NZB X DBA/2]F1 and C3H/HeJ mice, respectively. This interference is removed by treatment with anti-CD4 antibody and C. Supernatants from cultured T cells of NZB and MRL-lpr/lpr mice also prevent tolerance in spleen cells of [NZB X DBA/2]F1 and MRL-+/+ mice, respectively, unless CD4+ cells are removed prior to T cell culture. Removal of T cells from NZB and MRL-lpr/lpr spleen cells allows normal tolerization of B cells, which is abrogated by the addition of syngeneic T cells or cultured T cell supernatants. This effect also depends on the presence of CD4+ T cells. These studies show that in MRL-lpr/lpr mice, through interaction of the lpr and MRL background genes in a T cell subset, and in NZB mice, CD4+ T cells interfere with B cell tolerance to a thymic-independent Ag.     

Neutrophil apoptosis in autoimmune Fas-defective MRL lpr/lpr mice

The apoptosis-defective lpr (fas) mutation in MRL mice causes the early onset of a lupus-like autoimmune disease with concomitant inflammation. In order to analyse the consequences of the impaired Fas-dependent apoptosis on inflammation, the susceptibility to apoptosis of polymorphonuclear leukocytes (PMN), obtained from MRL lpr/lpr mice, has been studied. Peritoneal PMN from lpr/lpr and control (+/+) mice were recruited with a mild inflammatory stimulus. The number of cells collected from the peritoneal cavity of young lpr/lpr mice was comparable to that obtained from age-matched control mice, indicating that PMN homeostasis is maintained regardless of the loss-of-function Fas mutation. Recruited neutrophils were exposed in culture to apoptosis-inducing stimuli. Treatment with agonist anti-Fas antibody increased apoptosis of +/+ PMN, but did not affect lpr/lpr PMN which do not express Fas on their surface. However, lpr/lpr PMN could undergo both spontaneous and stimulus-induced apoptosis in a fashion comparable to or higher than that of control +/+ mice. Analysis of mRNA expression revealed that lpr/lpr PMN have reduced expression of IL-18, whereas IL-1beta, IFNgamma, caspase 1 and caspase 3 are expressed at levels comparable to those of +/+ cells. However, caspase-3-like activity was higher in PMN from lpr/lpr mice than in +/+ cells, and correlated with enhanced apoptosis. It could be concluded that in young, uncompromised lpr/lpr mice, PMN homeostasis is still fully regulated through the involvement of Fas-independent, compensatory, apoptotic mechanisms. This could include an increased participation of caspase 3 in the apoptotic pathway, consequent to enhanced activation of the enzyme and to the decreased production of IL-18, which acts as a competitive caspase 3 substrate.     

Rapid T cell receptor modulation accompanies lack of in vitro mitogenic responsiveness of double negative T cells to anti-CD3 monoclonal antibody in MRL/Mp-lpr mice

The role of the CD8-, CD4- (double negative) (DN) T cells accumulating in MRL/Mp-lpr/lpr (lpr) mice is unclear. Although they bear the TCR/CD3, the lpr DN cells do not respond to Ag, and the specificity of TCR/CD3 on these cells is unknown. With the aid of monoclonal anti-murine CD3 epsilon (145-2C11), we have investigated the function of the CD3 molecule on the DN cells. 145-2C11 was not mitogenic for lpr DN lymph node cells (LNC), even in the presence of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, whereas MRL/Mp-+/+ (+/+) LNC responded strongly. Surprisingly, CD3 modulation induced by 145-2C11 was much more rapid for lpr DN than for +/+ LNC. For example, the modulation observed after 10 min in lpr DN LNC required at least 2 h in +/+ cells. This was not due solely to a property of the 145-2C11 antibody, because both TPA and the F23.1 anti-TCR mAb also provoked a faster modulation of the TCR in lpr DN LNC. Double-staining experiments showed that co-culturing +/+ and lpr DN LNC did not alter their respective rates of modulation, which suggests an intrinsic defect in the lpr DN cells. Moreover, in LNC from 6-wk-old lpr mice (before the appearance of DN cells), as well as in normal phenotype-bearing T cells (CD8+ or CD4+) from 6-mo-old lpr mice, the CD3 modulation was similar to that of +/+ LNC. After modulation, the CD3 molecule was reexpressed at the surface of both +/+ and lpr DN cells during subsequent incubation of the cells without 145-2C11. In addition, spontaneous recycling of CD3 was similar in +/+ and lpr DN LNC. The rapid modulation of the lpr DN TCR/CD3 is presumably related to the anergy of this cell population.     

Autoreactivity accelerates the development of autoimmunity and lymphoproliferation in MRL/Mp-lpr/lpr mice

Lymph node T cells from autoimmune MRL/Mp-lpr/lpr mice, but not from congeneic MRL/Mp-+/+ mice, spontaneously proliferate and produce IL 2 when cultured in vitro for 5 to 7 days. This autologous activation depends critically on the length of in vitro culture and the initial culture density, indicating that cell to cell interaction may be essential. Phenotypic characterization of cultured cells suggests that both L3T4+ and Lyt-2+ T cells proliferate. However, only L3T4+ T cells produce IL 2. Mixing experiments reveal that the inability of freshly isolated lymph node cells from MRL/Mp-lpr/lpr mice to proliferate is not due to the presence of suppressor cells. Supernatant from 7-day cultures failed to induce freshly isolated cells to proliferate. Thus, the failure of freshly isolated cells to spontaneously proliferate and secrete IL 2 is not due to the inability of the cells to produce soluble mediators. Similar to the inactivation of normal T lymphocytes, in vitro addition of monoclonal anti-L3T4 or anti-IL 2 receptor antibody significantly inhibits the activation of these cultured lymphocytes. Spontaneous proliferation and IL 2 production can be blocked by the addition of monoclonal anti-I-Ak but not by monoclonal anti-I-Ad. Spontaneous proliferation and IL 2 production can be detected in young (4-wk-old) MRL/Mp-lpr/lpr mice at a time when their lymphocyte composition and physiology appear to be normal. More interestingly, spontaneous proliferation and IL 2 production cannot be detected in C57BL/6J mice bearing the lpr/lpr gene. These experiments support the notion that aberrant syngeneic autoreactivity may act as an accelerating factor in the pathogenesis of lymphoproliferation and autoimmunity in MRL/Mp-lpr/lpr mice.     

Fas modulates both positive and negative selection of thymocytes.

We studied the functional role of Fas (CD95) in thymic T cell development using the TCR transgenic mice homozygous for the lpr mutation, DO10 lpr/lpr mice. In DO10 lpr/lpr mice, the differentiation of CD4(+)CD8(+) double-positive (DP) thymocytes to CD4(+) single-positive (SP) thymocytes was markedly impaired, as indicated by decreased generation of CD4(+) SP thymocytes and reduced ratio of CD4(+) SP thymocytes to DP thymocytes in lpr/lpr mice compared with those of +/+ mice. Activation of DP thymocytes in the process of positive selection was also significantly inhibited in DO10 lpr/lpr mice, as shown by the lower levels of CD69 expression on DP thymocytes in lpr/lpr mice compared to +/+ mice. Furthermore, the deletion of DP thymocytes induced by in vivo administration of OVA peptide (up to 150 micrograms) and anti-TCR clonotype mAb did not occur in DO10 lpr/lpr mice, whereas these treatments significantly decreased DP thymocytes in DO10 +/+ mice. On the other hand, no significant difference in DO10 transgenic TCR expression on DP thymocytes was found between DO10 lpr/lpr and +/+ mice. Together, these results indicate that Fas is importantly involved in both positive and negative selection of thymocytes.     

Altered expression of self-reactive T cell receptor V beta regions in autoimmune mice.

Intrathymic tolerance results in elimination of T cells bearing self-reactive TCR V beta regions in mice expressing certain combinations of I-E and minor lymphocyte stimulatory (Mls) phenotypes. To determine if autoimmune strains of mice have a defect in intrathymic deletion of self-reactive TCR V beta regions, expression of V beta 3, V beta 6, V beta 8.1, and V beta 11 were examined in lpr/lpr and +/+ strains of mice; MRL/MpJ(H-2K, I-E+, Mlsb,), C57BL/6J(H-2b, I-E-, Mlsb,), C3H/HeJ(H-2k, I-E+, Mlsc), AKR/J(H-2k, I-E+, Mlsa); and in autoimmune NZB/N(H-2d, I-E+, Mlsa) and BXSB(H-2b, I-E-, Mlsb) mice. The results suggest that, during intrathymic development, self-reactive T cells are deleted in autoimmune strains of mice as found in normal control strains of mice. However, the TCR V beta repertoire is skewed in autoimmune strains compared to normal strains of mice. For example, MRL-lpr/lpr mice, but not other lpr/lpr strains, had increased expression of V beta 6 relative to expression in control MRL(-)+/+ mice, which is associated with collagen-induced arthritis. These data are consistent with a model of normal affinity for negative selection of self-reactive T cells in the thymus of autoimmune strains of mice followed by expansion of autoreactive T cell clones in the peripheral lymphoid organs. The peripheral lymphoid organs of lpr/lpr mice contain an expanded population of abnormal CD4-, CD8-, 6B2+ T cells. Elimination of self-reactive peripheral T cells suggests that these abnormal cells are derived from a CD4+ subpopulation in the thymus. Flow cytometry analysis of peripheral lymph node T cells from MRL-lpr/lpr mice reveal three populations of CD4+ T cells expressing low, intermediate and high intensity of B220 (6B2). This supports the hypothesis that in lpr/lpr mice, self-reactive CD4+ T cells are eliminated in the thymus, and that these cells lose expression of CD4 and acquire expression of 6B2 in the periphery.     

STAT1 affects lymphocyte survival and proliferation partially independent of its role downstream of IFN-gamma

Lymphocytes derived from mice deficient in STAT1 showed reduced apoptosis and enhanced proliferation in vitro. To understand the involvement of STAT1 in the observed reduction in apoptosis, we examined the levels of caspase and bcl-2 family genes that are involved in cell survival and/or apoptosis. The levels of caspase 1 and 11, two enzymes involved in both cytokine protein processing and induction of apoptosis, were reduced in STAT1-/- cells compared with wild-type. However, the levels of bcl-2 genes were comparable in both mice. STAT1-/- cells also displayed an enhanced proliferation following TCR stimulation. This hyperproliferation could not be ascribed completely to the loss of IFN-gamma-mediated antiproliferation. First, similar phenotypes were also observed in fibroblasts and pre-B cells derived from STAT1-/- mice, which do not produce IFN-gamma. Second, comparisons with cells lacking the gene for IFN-gamma or with cells treated with neutralizing Abs to IFN-gamma only partially mimicked the STAT1-/- phenotype. Interestingly, the kinetics of degradation of p27kip1, a CDK inhibitor, following TCR ligation were faster, and, concomitantly, the up-regulation of CDK2 kinase activity and protein levels were increased in stimulated T cells of STAT1-/- mice relative to those of wild-type mice. Furthermore, STAT1-/- animals were more susceptible to carcinogen-induced thymic tumors, a possible consequence of altered T cell growth and/or survival. These results demonstrate an essential role for STAT1 for lymphocyte survival and proliferation that is only partially dependent on IFN-gamma signaling.     

T cell receptor ligation triggers novel nonapoptotic cell death pathways that are Fas-independent or Fas-dependent

Short-term culture of activated T cells with IL-2 renders them highly susceptible to apoptotic death triggered by TCR cross-linking. Activation-induced apoptosis is contingent upon caspase activation and this is mediated primarily by Fas/Fas ligand (FasL) interactions that, in turn, are optimized by p38 mitogen-activated protein kinase (MAPK)-regulated signals. Although T cells from mice bearing mutations in Fas (lpr) or FasL (gld) are more resistant to activation-induced cell death (AICD) than normal T cells, a significant proportion of CD8(+) T cells and to a lesser extent CD4(+) T cells from mutant mice die after TCR religation. Little is known about this Fas-independent death process. In this study, we demonstrate that AICD in lpr and gld CD4(+) and CD8(+) T cells occurs predominantly by a novel mechanism that is TNF-alpha-, caspase-, and p38 MAPK-independent and has morphologic features more consistent with oncosis/primary necrosis than apoptosis. A related Fas- and caspase-independent, nonapoptotic death process is revealed in wild-type (WT) CD8(+) T cell blasts following TCR ligation and treatment with caspase inhibitors, the p38 MAPK inhibitor, SB203580, or neutralizing anti-FasL mAb. In parallel studies with WT CD4(+) T cells, two minor pathways leading to nonapoptotic, caspase-independent AICD were identified, one contingent upon Fas ligation and p38 MAPK activation and the other Fas- and p38 MAPK-independent. These data indicate that TCR ligation can activate nonapoptotic death programs in WT CD8(+) and CD8(+) T blasts that normally are masked by Fas-mediated caspase activation. Selective use of potentially proinflammatory oncotic death programs by activated lpr and gld T cells may be an etiologic factor in autosensitization.     

Primed T cells are more resistant to Fas-mediated activation-induced cell death than naive T cells.

Memory T cells respond in several functionally different ways from naive T cells and thus function as efficient effector cells. In this study we showed that primed T cells were more resistant to Fas-mediated activation-induced cell death (AICD) than naive T cells using OVA-specific TCR transgenic DO10 mice and Fas-deficient DO10 lpr/lpr mice. We found that apoptosis was efficiently induced in activated naive T cells at 48 and 72 h after Ag restimulation (OVA peptide; 0.3 and 3 microM), whereas apoptosis was not significantly increased in activated primed T cells at 24-72 h after Ag restimulation. We further showed that the resistance to AICD in primed T cells was due to the decreased sensitivity to apoptosis induced by Fas-mediated signals, but TCR-mediated signaling equally activated both naive and primed T cells to induce Fas and Fas ligand expressions. Furthermore, we demonstrated that primed T cells expressed higher levels of Fas-associated death domain-like IL-1beta-converting enzyme inhibitory protein (FLIP), an inhibitor of Fas-mediated apoptosis, at 24-48 h after Ag restimulation than naive T cells. In addition, Bcl-2 expression was equally observed between activated naive and primed T cells after Ag restimulation. Thus, these results indicate that naive T cells are sensitive to Fas-mediated AICD and are easily deleted by Ag restimulation, while primed/memory T cells express higher levels of FLIP after Ag restimulation, are resistant to Fas-mediated AICD, and thus function as efficient effector cells for a longer period.     

Cytokine secretion by C3H-lpr and -gld T cells. Hypersecretion of IFN-gamma and tumor necrosis factor-alpha by stimulated CD4+ T cells.

Mice homozygous for lpr and gld develop profound lymphadenopathy characterized by the expansion of two unusual T cell subsets, a predominant Ly-5(B220)+ CD4- CD8- double negative (DN) population and a minor CD4 dull+ Ly-5(B220)+ population. The mechanisms promoting lymphoproliferation are unknown, but one possibility is a abnormality in the production of cytokines that regulate T cell growth. In the present report, unfractionated LN cells and sorted T cell subsets from C3H-lpr, -gld, and -+/+ mice were compared for spontaneous and induced secretion of a spectrum of lymphokines. In addition, CD4+, CD4 dull+ Ly-5(B220)+, and DN T cells were examined for expression of CD3 epsilon, TCR-alpha/beta heterodimers, Ly-6C, and CD44 and for proliferative responses to immobilized anti-TCR mAb and cofactors. These studies revealed that sorted DN T cells did not secrete IL-3, IL-4, IL-5, IL-6, GM-CSF, TNF-alpha, or IFN-gamma spontaneously or after TCR-alpha/beta cross-linking. In contrast, stimulated unfractionated lpr and gld LN cells proliferated strongly and secreted high levels of IFN-gamma and TNF-alpha and low levels of IL-3, IL-4, and IL-6. Despite a 5- to 10-fold deficit in the frequency of CD4+ and CD8+ T cells, cytokine secretion by lpr and gld LN generally exceeded that of +/+ LN. Comparisons of cytokine secretion by stimulated CD4+ T cells revealed that +/+, lpr, and gld CD4+ Ly-5(B220)- T cells proliferated strongly, but only lpr and gld cells produced significant levels of IFN-gamma. The lpr and gld CD4+ T cells also produced higher levels of TNF-alpha and IL-2 than +/+ cells. In contrast to normal CD4+ T cells, lpr and gld CD4+ Ly-5(B220)+ T cells proliferated weakly and did not secrete TNF-alpha, IL-2, or, in most experiments, IFN-gamma after stimulation. Phenotypic studies of T cell subsets revealed that unstimulated lpr and gld CD4+ Ly-5(B220)- T cells express significantly higher levels of CD44 than +/+ CD4+ T cells. In addition, CD4 dull+ Ly-5(B220)+ cells closely resembled DN T cells in size and expression of TCR-alpha/beta, CD3epsilon, CD44, and Ly-6C. Since elevated CD44 expression is generally associated with T cell activation and only previously activated normal CD4+ T cells produce high levels of IFN-gamma in vitro, our data suggest that lpr and gld CD4+ Ly-5(B220)- T cells contain a higher than normal proportion of primed or memory T cells and thus may be polyclonally activated in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)     

Accelerated Progression of a Murine Retrovirus-Induced Immunodeficiency Syndrome In Fas Mutant C57BL/6/lpr/lpr Mice

We reported previously that CD4⁺ T cells and B cells in mice with retrovirus-induced murine acquired immunodeficiency syndrome (MAIDS) caused by LP-BM5 murine leukemia virus (MuLV) mixtures increased the expression of Fas antigen (Fas) during progression of the disease. However, the contribution of the Fas/Fas ligand (Fas L) system to the pathogenesis of MAIDS remained unknown. Here, we examined the susceptibility of C57BL/6 (B6) lpr/lpr mice, which has been reported to be defective for the expression of Fas, to MAIDS. We found that the Thy 1.2^? CD4 T cells and IgK dull B220⁺ cells, which are characteristic of MAIDS, increased after the inoculation of LP-BM5 MuLV in B6 lpr/lpr mice. B22⁺ TCR αβ T cells, unique to lupus prone mice, also increased in the B6 lpr/lpr mice after infection. CD4⁺ B220⁺ TCR αβ T cells increased profoundly among the B220⁺ TCR αβ T cells from LP-BM5 MuLV-infected B6 lpr/lpr mice, while the B220⁺ TCR αβ T cells observed in non-infected B6 lpr/lpr mice were largely of the CD4^? CD8^? phenotype. A DNA PCR analysis of the LP-BM5 MuLV-infected B6 lpr/lpr mice revealed the genome integration of defective LP-BM5 virus, further confirming that MAIDS is inducible to B6 lpr/lpr mice. LP-BM5 MuLV-infected lpr/lpr mice died within 3 months, while MAIDS-infected B6 +/+ mice usually died within 5 to 6 months, and B6 lpr/lpr mice not infected with LP-BM5 MuLV lived more than 6 months. Taken together, these results suggest that MAIDS is inducible independently with functional Fas expression and the possibility of accelerated progression of murine AIDS and lpr-associated autoimmune disease in B6 lpr/lpr mice infected with LP-BM5 MuLV.     

Lymphocytes from autoimmune MRL lpr/lpr mice are hyperresponsive to IL-18 and overexpress the IL-18 receptor accessory chain

MRL lpr/lpr mice spontaneously develop a severe autoimmune lupus syndrome characterized by strong autoantibody production and massive lymphoproliferation, in which IFN-gamma plays a major pathogenic effect. The role of the IFN-gamma-inducing cytokine IL-18 in the autoimmune syndrome of lpr/lpr mice has been investigated. In response to IL-18, lymph node cells of lpr/lpr mice produce significant amounts of IFN-gamma and proliferate more potently as compared with cells from +/+ mice. Cells likely responsible for such hyperresponsiveness to IL-18 include NK cells and the CD4(+)/CD8(+) self-reactive T lymphocytes characteristically present in lymph nodes of lpr/lpr mice. Analysis of the expression of IL-18R complex revealed that mRNA for the IL-18R alpha-chain is constitutively expressed at similar level both in +/+ and lpr/lpr lymphocytes. In contrast, the expression of the accessory receptor chain IL-18R beta is low in unstimulated +/+ cells but significantly high in lpr/lpr cells. Thus, the abnormally high expression of the IL-18R chain IL-18R beta could be one of the causes of the hyperresponsiveness of lpr/lpr cells to IL-18 at the basis of consequent enhancement of IFN-gamma production and development of IFN-gamma-dependent autoimmune pathology.     

Overexpressed Bcl-x(L) prevents bacterial superantigen-induced apoptosis of thymocytes in vitro

bcl-x, a homologous gene of bcl-2, has an anti-apoptotic function and appears to play a critical role in the development of lymphoid systems. To investigate the effect of overexpressed Bcl-x(L) on the development of T lymphocytes, we established two lines of transgenic mice by using Emu-chicken bcl-x(L) (cbcl-x(L)) transgene, where the cBcl-x(L) protein was expressed mainly in lymphoid cells. Although thymocytes and splenocytes from cbcl-x(L) transgenic mice are resistant to apoptosis in vitro, clonal deletion of thymocytes, recognizing endogenous self-superantigens in the thymus, still normally proceeded and no self-reactive T cells were found in the spleen of the transgenic mice. To dissect clonal deletion, we utilized two in vitro models, thymocytes/antigen presenting cells co-culture system and fetal thymus organ culture system. In both, bacterial superantigen staphylococcus aureus enterotoxin B (SEB) induces apoptosis of T cells with Vbeta8+ T cell receptor (TCR) reacting to SEB, which mimics clonal deletion of self-reactive thymocytes in vivo. SEB-induced depletion of Vbeta8+ T cells from thymocytes when taken from the transgenic mice was effectively inhibited. The data might raise the possibility that cell death process involved in clonal deletion in the thymus is a form of apoptosis inhibited by Bcl-x(L).     

CD21/CD19 coreceptor signaling promotes B cell survival during primary immune responses

The adaptive immune response is tightly regulated to limit responding cells in an Ag-specific manner. On B cells, coreceptors CD21/CD19 modulate the strength of BCR signals, potentially influencing cell fate. The importance of the CD95 pathway was examined in response of B cells to moderate affinity Ag using an adoptive transfer model of lysozyme-specific Ig transgenic (HEL immunoglobulin transgene (MD4) strain) B cells. Although adoptively transferred Cr2+/+ MD4 B cells are activated and persist within splenic follicles of duck egg lysozyme-immunized mice, Cr2-/- MD4 B cells do not. In contrast, Cr2-/- MD4 lpr B cells persist after transfer, suggesting that lack of CD21/CD35 signaling results in CD95-mediated elimination. Cr2 deficiency did not affect CD95 levels, but cellular FLIP (c-FLIP) protein and mRNA levels were reduced 2-fold compared with levels in Cr2+/+ MD4 B cells. In vitro culture with Cr2+/+ MD4 B cells demonstrated that equimolar amounts of rHEL-C3d3 were more effective than hen egg lysozyme alone in up-regulating c-FLIP levels and for protection against CD95-mediated apoptosis. Collectively, this study implies a mechanism for regulating B cell survival in vivo whereby the strength of BCR signaling (including coreceptor) determines c-FLIP levels and protection from CD95-induced death.     

Expression of a bone marrow-associated Ly-6 determinant on the T cell population expanding in the lymph nodes of the autoimmune mouse strain MRL/Mp-lpr/lpr

In this report we describe the reactivity patterns of two monoclonal anti-Ly-6.2 antibodies toward lymph node (LN) cells of the autoimmune MRL/Mp-lpr/lpr (lpr) and the normal congenic MRL/Mp-+/+ (+/+) mouse strains. The monoclonal antibody 34-11-3, which has previously been found to detect a LN-associated Ly-6.2 antigen, reacted with both lpr and +/+ LN cells. Another monoclonal antibody, 34-10-7, which has been demonstrated to detect a bone marrow- (BM) associated determinant, was found to react with a small proportion of +/+ LN cells and unexpectedly with a high percentage of lpr LN cells. The expression of this BM determinant found in the LN of lpr mice was age dependent, and its presence on Thy-1.2+ cells increased with the expansion of a subset of Lyt-1+2- cells. In contrast, dual labeling experiments showed that in +/+ and young lpr mice a small subset of Lyt-1+2+ cells express this BM-associated Ly6.2 determinant. Therefore these findings demonstrate that the lpr gene-dependent T cell expansion in the LN results in both aberrant expression and association of lymphocyte cell surface markers.     

Attenuation of retinal endothelial cell migration and capillary morphogenesis in the absence of bcl-2

Apoptosis plays a critical role during development and in the maintenance of the vascular system. B-cell leukemia lymphoma 2 (bcl-2) protects endothelial cells (EC) from apoptosis in response to a variety of stimuli. Previous work from this laboratory demonstrated attenuation of postnatal retinal vascular development and retinal neovascularization during oxygen-induced ischemic retinopathy in bcl-2-deficient (bcl-2-/-) mice. To gain further insight into the function of bcl-2 in the endothelium, we isolated retinal EC from bcl-2+/+ and bcl-2-/- mice. Retinal EC lacking bcl-2 demonstrated reduced cell migration, tenascin-C expression, and adhesion to vitronectin and fibronectin. The bcl-2-/- retinal EC also failed to undergo capillary morphogenesis in Matrigel. In addition, using an ex vivo angiogenesis assay, we observed reduced sprouting from aortic rings grown in culture from bcl-2-/- mice compared with bcl-2+/+ mice. Furthermore, reexpression of bcl-2 was sufficient to restore migration and capillary morphogenesis defects observed in bcl-2-/- retinal EC. Mechanistically, bcl-2-/- cells expressed significantly less endothelial nitric oxide synthase, an important downstream effecter of proangiogenic signaling. This may be attributed to increased oxidative stress in the absence of bcl-2. In fact, incubation of retinal EC or aortic rings from bcl-2-/- mice with the antioxidant N-acetylcysteine rescued their capillary morphogenesis and sprouting defects. Thus, bcl-2-mediated cellular functions play important roles not only in survival but also in proangiogenic phenotype of EC with a significant impact on vascular development and angiogenesis.