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.     

Abnormal IgG galactosylation and arthritis in MRL-Fas(lpr) or MRL-FasL(gld) mice are under the control of the MRL genetic background.

MRL mice bearing the lpr (Fas) or gld (Fas ligand) mutation, MRL-Fas(lpr) or MRL-FasL(gld), respectively, develop arthritis similar to rheumatoid arthritis, but C3H and C57BL/6 mice bearing such mutations do not. In MRL-Fas(lpr) mice, agalactosylated oligosaccharides in serum IgG increase significantly in comparison to MRL-+/+ mice without arthritis. In this study, an increased level of agalactosylation in IgG, as compared to MRL-+/+, was found in both MRL-Fas(lpr) and MRL-FasL(gld) mice. In contrast, the incidence of IgG without galactose was comparable among C3H-Fas(lpr), C3H-FasL(gld), and C3H-+/+ mice as well as between C57BL/6-Fas(lpr) and C57BL/6-+/+ mice. These results suggest that the increase in agalactosylated IgG and the development of arthritis in MRL-Fas(lpr) and MRL-FasL(gld) mice are controlled by the MRL genetic background.     

Genetic diversity of murine rheumatoid factors

Anti-Ig autoantibodies (rheumatoid factors, RF) have been implicated in the pathogenesis of human and murine rheumatoid arthritis as well as in the regulation of normal immune responses. Their genetic origin, clonal diversity, and inducing agents, and the relatedness between RF associated with disease and those occurring under physiologic conditions are not well understood. In this study, the genetic and clonotypic origin of 34 monoclonal IgM RF-secreting hybridomas from arthritic MRL-lpr/lpr and nonarthritic MRL-+/+ and C57BL/6-lpr/lpr mice was examined by RNA hybridization. For this purpose, we used probes for 10 VH and 13 Vk gene families as well as all JH and Jk gene segments. The majority of hybridomas expressed distinct Ig gene segment patterns and, hence, were clonally unrelated. Overall, a variety of different V and J gene segments were expressed in the hybridoma panel, suggesting that a large number of distinct genetic elements participates in expression of RF-like activity. RF from arthritic mice expressed Vk messages from the overlapping Vk22 and Vk28 gene families more frequently than did those from nonarthritic mice. RF from autoimmune MRL mice, both arthritic MRL-lpr/lpr and nonarthritic MRL-+/+, showed skewed JH4 segment usage, whereas those from C57BL/6-lpr/lpr preferentially expressed JH2.     

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.     

Suppression of antigen-specific interleukin 2 production in the MRL/MpJ-lpr/lpr mouse

Several murine strains with spontaneously occurring systemic lupus erythematosus-like disease demonstrate defects in immunoregulation. The MRL/MpJ-lpr/lpr (MRL-1) strain develops a severe age-progressive defect in interleukin 2 (IL 2) production in response to mitogen or antigen. In this study, we demonstrate in vitro the presence of suppressor cells in the lymph nodes of naive mice of the MRL background. Suppression by MRL-1 lymph node cells was partially reversed by treatment with anti-Lyt-1.2 monoclonal antibody and complement and was moderately radiosensitive. Suppression by lymph node cells from the congenic MRL/MpJ-+/+ (MRL-+) mouse was somewhat more resistant to treatment with anti-Lyt-1.2 and complement, or radiation. Lymph node cells from the H-2-syngeneic mouse strain, C3H/HeJ, failed to suppress. Thus, lymph nodes from mice of the MRL background contain cells capable of suppressing in vitro IL 2 responses. We next performed cell transfers to determine whether suppressor cells contribute in vivo to the IL 2 defect. Lymph node cells, but not spleen cells, from MRL-1 mice by 5 to 6 mo of age suppressed antigen-specific IL 2, CTL, and DTH responses when transferred into young MRL-+ recipients. Transfer of identical numbers of lymph node cells from age-matched MRL-+ mice failed to suppress IL 2 production. Transfer of suppression was sensitive to treatment with monoclonal anti-Lyt-2.1 and complement, and to 250 rad of radiation. Thus, this study suggests a role for active suppression of IL 2 production in the establishment of the IL 2 defect in the MRL-1 mouse. Further, suppression may involve phenotypically distinct T lymphocyte subpopulations.     

High level expression of the plasma cell antigen PC.1 on the T-cell subset expanding in MRL/MpJ-lpr/lpr mice: detection with a xenogeneic monoclonal antibody and alloantisera

MRL/MpJ-lpr/lpr (MRL-lpr/lpr) mice spontaneously develop a systemic lupus erythematosus-like syndrome associated with the expansion of a T-cell subset exerting helper activity for autoantibody production. Several studies have demonstrated that these T cells have unusual phenotypic characteristics including the expression of the B220 B-cell marker. To further characterize the antigenic profiles of these T cells, we have generated monoclonal antibodies (MAb) by immunizing rats with MRL-lpr/lpr T cells. Using flow cytofluorometry analysis, one of these MAb (4G6), described here, was found to react strongly with T cells of MRL-lpr/lpr mice but weakly with T cells of congenic mice lacking the lpr mutation (MRL/MpJ-+/+ mice). This MAb also stained brightly T cells from C3H/Hej-lpr/lpr mice and dimly those from normal C3H/Hej mice. However, it failed to react with T cells from C57Bl/6-lpr/lpr mice or normal C57Bl/6 (B6) mice. Analysis of 4G6 reactivity (weak vs negative) of T cells in a series of inbred strains demonstrated a correlation with the Pca-1a genotype known to result in expression of the PC.1 antigen on plasma cells. Immunoprecipitation studies revealed that the 4G6 antigen has a mean apparent molecular weight of 115,000, under reducing conditions, similar to that of PC.1. Moreover, high expression of 4G6 was found on plasmacytoma lines and B blasts but not on T blasts. Identity of the 4G6 antigen with PC.1 was confirmed by the finding that conventional anti-PC.1 alloantisera could block the cell surface binding of the 4G6 MAb. Therefore, T cells from MRL-lpr/lpr (and C3H-lpr/lpr) mice aberrantly carry high levels of a plasma cell antigen, detected by the 4G6 MAb, which substantiates further that these T cells represent a unique subset with some surface properties of the B-cell lineage.     

C1q deficiency and autoimmunity: the effects of genetic background on disease expression

Gene-targeted C1q-deficient mice have been shown to develop a syndrome reminiscent of human systemic lupus erythematosus with antinuclear Abs and proliferative glomerulonephritis. Initial phenotypic analysis conducted in (129 x C57BL/6) hybrid mice showed that background genes were a significant factor for the full expression of the autoimmune disease. To assess the contribution of background genes in the expression of the autoimmune phenotype, the disrupted C1qa gene was backcrossed for seven generations onto C57BL/6 and MRL/Mp(+/+) strains. These were intercrossed with C57BL/6.lpr/lpr and MRL/Mp-lpr/lpr strains to generate C1q-deficient substrains. In C1q-deficient C57BL/6 mice, no evidence of an autoimmune phenotype was found, and C1q deficiency in both the C57BL/6.lpr/lpr and MRL/Mp-lpr/lpr strains did not modify the autoimmune phenotype observed in wild-type controls. However, in C1q-deficient MRL/Mp(+/+) animals an acceleration of both the onset and the severity of antinuclear Abs and glomerulonephritis was seen. Disease was particularly pronounced in females, which developed severe crescentic glomerulonephritis accompanied by heavy proteinuria. In addition, the C1q-deficient MRL/Mp(+/+) mice had an impairment in the phagocytic clearance of apoptotic cells in vivo. These data demonstrate that the expression of autoimmunity in C1q-deficient mice is strongly influenced by other background genes. The work also highlights the potential value of the C1q-deficient MRL/Mp(+/+) strain as a tool with which to dissect further the underlying mechanisms of the autoimmune syndrome associated with C1q deficiency.     

Specificity and molecular characteristics of monoclonal IgM rheumatoid factors from arthritic and non-arthritic mice

Two-hundred twenty-four hybridomas secreting monoclonal IgM rheumatoid factor (hIgMRF) derived from MRL-lpr/lpr, MRL-+/+ and C57BL/6-lpr/lpr autoimmune mice were analyzed with regard to IgG subclass and domain specificity, and some for VH gene expression patterns. Among these mice, only MRL-lpr/lpr develop arthritis. Clonotypes specific for each of the four mouse IgG subclasses and clonotypes reacting with more than one IgG subclass were identified. Although each panel contained several clonotypes, the predominant one differed in each strain (MRL-lpr/lpr, anti-IgG2a; MRL-+/+, combined anti-IgG2a and 2b; C57BL/6-lpr/lpr, anti-IgG1 or combined anti-IgG1, 2a, and 3). The IgG domains recognized by these monoclonals were defined with mutant Ig carrying IgG1 heavy chains that lacked either the CH1 or CH3 domains, variant Ig carrying hybrid IgG2b-2a heavy chains, and IgG fragments. Inhibition of hIgMRF binding to IgG substrates by protein A was also assessed. Most determinants were assigned to the CH3 domain, but determinants in the hinge region, CH2 domain, and in some instances, even in the Fab portion, could also be identified. Hybridization of cytoplasmic RNA from 35 classes of diverse IgG subclass specificity with VH gene probes representing seven of the approximately 10 VH families (7183, S107, Q52, J558, J606, 36-60, X24) indicated that approximately 90% of these clones expressed VH genes belonging to the large J558 gene family. The results indicate that murine IgMRF are extremely heterogeneous in IgG subclass and domain specificities; the genetic background influences RF specificity characteristics that may relate to pathogenicity; and considering the complexity of the J558 VH gene family and reported RF heavy chain assignments to additional VH gene families, it appears that VH genes encoding RF are diverse.     

CD4-CD8- thymocytes from MRL-lpr/lpr mice exhibit abnormal proportions of alpha beta- and gamma delta-TCR+ cells and demonstrate defective responsiveness when activated through the TCR.

MRL-lpr/lpr (lpr) mice develop profound lymphadenopathy resulting from the accumulation of CD4-CD8- (double-negative, DN) cells in the peripheral lymphoid organs. Earlier studies from our laboratory demonstrated an increased proportion of DN cells in the thymus of lpr mice with age. Inasmuch as the DN thymocytes constitute a heterogenous population of cells, in the present study, we investigated the TCR phenotype of DN thymocytes and their responsiveness to activation through the TCR. The DN thymocytes of young (1 month of age) lpr mice contained approximately 65% CD3+ cells of which approximately 60% were alpha beta-TCR+ and approximately 39% were gamma delta-TCR+ as detected by using pan anti-TCR mAbs. In old (4-6 months of age) or young MRL-(+/+) mice, similar proportions of CD3+, alpha beta- or gamma delta-TCR+ DN thymocytes were detected. Interestingly, however, in old (4-6 months of age) lpr mice, the CD3+ T cells increased to approximately 86% and the majority of these (approximately 81%) were alpha beta-TCR+ and only approximately 3% were gamma delta-TCR+. Also, in old lpr mice, there was a 10-fold increase in the absolute number of alpha beta-TCR+ DN cells in the thymus, whereas, the absolute number of gamma delta-TCR+ DN cells in the thymus did not alter significantly. Furthermore, a majority (approximately 84%) of the old lpr DN thymocytes expressed CD45R, similar to the peripheral DN T cells. In contrast, only a small number (approximately 1%) of DN thymocytes from young lpr or MRL-(+/+) mice expressed CD45R. The DN thymocytes from young lpr or MRL-(+/+) mice demonstrated strong and similar proliferative responsiveness to stimulation with PMA + calcium ionophore or PMA + IL-2, or to immobilized mAb directed against the TCRs (CD3, alpha beta and gamma delta). In contrast, the DN thymocytes and the DN peripheral T cells from old lpr mice demonstrated marked defect in responding to the above stimuli. The present study suggests that with the onset of lymphadenopathy, the DN cells in the thymus of old lpr mice are increasingly skewed toward the alpha beta-TCR repertoire, the majority of which express CD45R and respond poorly to mitogenic stimuli or when activated through the TCR. It is suggested that migration of such cells continuously to the periphery may result in severe lymphadenopathy seen in old MRL-lpr/lpr mice.     

Autoantibody in MRL/Mp-lpr/lpr mice. A monoclonal antibody specific for the abnormal T cells from mice bearing the lpr/lpr gene

We generated mAb from an unimmunized autoimmune MRL/Mp-lpr/lpr mouse. One of these mAb A108, reacted with cell surface Ag present on abnormal T cells from MRL/Mp-lpr/lpr, C3H-lpr/lpr, and C57BL/6-lpr/lpr mice. We failed to detect significant numbers of A108 bearing cells in the lymph nodes of MRL-Mp/+/+, normal C3H or normal C57BL/6 mice. Therefore, the expression of A108 correlates with the presence of the lpr/lpr gene. A108 binds to a variety of murine T cell tumor lines (e.g., EL4, BW5147, and YAC-1) and human T cell tumor lines (e.g., MOLT-3, Sup T1, and Jurkat). A108 does not bind to normal human PBL. Immunoprecipitation of surface iodinated EL-4 and BW5147 with A108 identified one major protein with a Mr of about 17.5-kDa. The significance of these findings with respect to the development of lymphoid proliferation and autoimmune disease in mice bearing the lpr/lpr gene will be discussed.     

T cells from autoimmune "IL 2-defective" MRL-lpr/lpr mice continue to grow in vitro and produce IL 2 constitutively

MRL-lpr/lpr mice and other autoimmune strains that bear the lpr gene develop a profound lymphadenopathy characterized by an expansion of a unique dull Lyt-1+2- T cell population. Because fresh splenic and lymph node T cells from such mice stimulated Con A in vitro are extremely defective in IL 2 production and proliferation, T cell lines derived from MRL-lpr/lpr spleens were established and maintained for several months, and were analyzed for their factor production to define their growth requirements. The results indicate that cultivation in vitro leads to constitutive production of IL 2 and the capacity to respond to growth factors, thereby facilitating the continuous proliferation of T cells bearing the dull Lyt-1+2- phenotype in vitro in the absence of exogenous antigen or mitogen. These studies indicate that MRL-lpr/lpr T cells have the ability to produce IL 2 and to respond to IL 2 with long-term proliferation. In addition, the impaired responsiveness to Con A of fresh MRL-lpr/lpr lymph node T cells was found to be quite transitory, because even short-term culture allowed MRL lpr/lpr T cells to respond normally.     

Studies of lymphoproliferation in MRL-lpr/lpr mice

MRL-lpr/lpr mice develop massive lymphoproliferation and an associated autoimmune process that includes anti-DNA formation, vasculitis, and glomerulonephritis. We have investigated the lymphoproliferation of MRL-lpr/lpr mice and have found that multiple factors are operative. Although neonatal thymectomy markedly retards the syndrome, chronic injection of poly rI.rC could substitute for the thymus. The resulting cells had the phenotype characteristic of the abnormal MRL-lpr/lpr T cells, Thy-1+, dull Ly-1+, Lyt-2-, 6B2+, Ig-. Splenectomy at 2 wk of age markedly retarded the development of this syndrome; however, splenectomy at birth did not. Some protection was afforded by splenectomy at 5 wk. Thus, there appears to be a critical period in the life of an MRL-lpr/lpr mouse when the spleen contributes importantly to the lymphoproliferation. A most remarkable observation was that an MRL-lpr/lpr spleen graft under the kidney capsule could induce lymphadenopathy characteristic of lpr/lpr mice in MRL +/+ recipients. Cells within the graft had to be able to proliferate for the adenopathy to occur because irradiation of the spleen with 800 R just before grafting abrogated the lymphadenopathy-inducing potential. No adenopathy was induced by +/+ spleen grafts placed into +/+ mice. Although MRL-lpr/lpr males develop disease slightly more slowly than female littermates, the differences are small. Manipulations that retard disease, such as splenectomy at 2 wk or neonatal thymectomy, magnified the sex differences. Male MRL-lpr/lpr mice that were thymectomized and splenectomized and given polyclonal immune activators failed to develop either anti-DNA or lymphadenopathy, whereas their female littermates expressed both abnormalities. We conclude from these studies that multiple factors serve to modulate the magnitude of the lymphoproliferation and autoimmune syndrome of MRL-lpr/lpr mice.     

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.     

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.     

Stimulation of murine T cells via the Ly-6C antigen: lack of proliferative response in aberrant T cells from lpr/lpr and gld/gld mice despite high Ly-6C antigen expression

Cross-linking of cell surface Ly-6C molecules with the 6C3 rat monoclonal antibody (MAb) followed by anti-rat immunoglobulin antibody acts in concert with phorbol myristate acetate (PMA) as a potent mitogenic stimulus for normal T cells. Specificity of this stimulation was demonstrated by its absence in T cells from NZB, NOD, or STb/J mice which lack the 6C3 determinant. In 6C3+ normal strains, the extent of 6C3-mediated stimulation varied, depending on the level of 6C3 antigen expression. Analysis of this stimulation in purified T cell subsets revealed that in Ly-6.1 strains (e.g., BALB/c, CBA/J), Lyt-2+ cells responded, but not L3T4+ cells, whereas in Ly-6.2 strains (e.g., C57BL/6, MRL-+/+), both subsets produced IL 2 and proliferated, although with different kinetics. Moreover, in adult MRL-+/+ mice, the minor Lyt-2-/L3T4- subset from the lymph nodes gave low responses to 6C3 cross-linking, whereas that from the thymus reacted strongly. Stimulation via Ly-6C therefore provides a pathway for differential activation of normal T cells. In contrast, the expanding population of Lyt-2-/L3T4- T cells from lpr/lpr or gld/gld mice did not proliferate in response to 6C3 antigen cross-linking plus PMA despite high levels of 6C3 antigen expression. Responsiveness of lpr/lpr T cells could not be restored with IL 1, IL 2, or both. These T cells also failed to be triggered by conjunction of PMA with either Thy-1 antigen cross-linking or concanavalin A. Moreover, they were not stimulated, in the presence of PMA, by doses of ionomycin that were optimal for normal T cells, but did respond to higher ionomycin concentrations (2 micrograms/ml), and this response was not altered by Ly-6C cross-linking. It is concluded that the Ly-6C pathway of T cell activation is not functional in the aberrant lpr/lpr (and gld/gld) T cells, and that this defect may reflect abnormalities of intracellular signaling.     

Metaphase-specific cell death in meiotic spermatocytes in mice

Apoptosis of male germ cells is a widespread but little-understood phenomenon in many animal species. The elucidation of its mechanisms could be useful in the understanding of male infertility. We have examined the distribution of dying cells with the terminal transferase-mediated nick-end labeling (TUNEL) method and by an electron-microscopic procedure in the testes of 10 mouse strains, viz., C57BL/10 (B10), SL/NiA (SL), C57BL/6 (B6), C3H/He (C3H), BALB/c (BALB), DBA2 (DBA), CBA/J (CBA), MRL/MpJ(-)+/+ (M+), MRL/MpJ-lpr/lpr (lpr), and wild-type NJL mice (Mus musculus musculus). In the testes of the B10, NJL, SL, B6, C3H, BALB, DBA, and CBA mice, very few TUNEL-positive cells are distributed in the seminiferous tubules, whereas in the testes of the M+ and lpr mice, many TUNEL-positive cells, which are restricted to stage XII seminiferous tubules, have been identified. The most important finding is that many metaphases of meiotic spermatocytes show a marked TUNEL-positive reaction. Some metaphases show apoptotic morphology electron-microscopically. These results suggest that the testes of MRL strains will provide a useful model for the study of the mechanism of metaphase-specific apoptosis in meiotic spermatocytes.     

Fas/Fas ligand deficiency results in altered localization of anti-double-stranded DNA B cells and dendritic cells

Autoantibodies directed against dsDNA are found in patients with systemic lupus erythematosus as well as in mice functionally deficient in either Fas or Fas ligand (FasL) (lpr/lpr or gld/gld mice). Previously, an IgH chain transgene has been used to track anti-dsDNA B cells in both nonautoimmune BALB/c mice, in which autoreactive B cells are held in check, and MRL-lpr/lpr mice, in which autoantibodies are produced. In this study, we have isolated the Fas/FasL mutations away from the autoimmune-prone MRL background, and we show that anti-dsDNA B cells in Fas/FasL-deficient BALB/c mice are no longer follicularly excluded, and they produce autoantibodies. Strikingly, this is accompanied by alterations in the frequency and localization of dendritic cells as well as a global increase in CD4 T cell activation. Notably, as opposed to MRL-lpr/lpr mice, BALB-lpr/lpr mice show no appreciable kidney pathology. Thus, while some aspects of autoimmune pathology (e.g., nephritis) rely on the interaction of the MRL background with the lpr mutation, mutations in Fas/FasL alone are sufficient to alter the fate of anti-dsDNA B cells, dendritic cells, and T cells.     

Idiotypic analysis of a monoclonal anti-Sm antibody. II. Strain distribution of a common idiotypic determinant and its relationship to anti-Sm expression

The idiotypes borne by Y2, a monoclonal anti-Sm antibody of MRL-lpr/lpr mouse strain origin, were investigated to elucidate genetic mechanisms in this autoantibody response. An anti-Y2 anti-idiotypic antiserum was raised in a rabbit and was rendered specific for idiotype by extensive absorption with globulins of the B6 and BALB/c strains as well as the BALB/c myeloma UPC 10. By using a sensitive assay for idiotype by inhibition ELISA, the Y2 determinant was found to be commonly expressed in sera of MRL-lpr/lpr and MRL-+/+ mice. Moreover, sera of several normal strain mice also bore the idiotype and, in mice bearing the lpr gene, idiotype levels were increased 1.5 to fivefold, even in the absence of a serum anti-Sm response. The relationship of this idiotype to anti-Sm expression was further assessed by determining the idiotype content of affinity-purified anti-Sm antibodies from MRL-lpr/lpr mice. Anti-Sm from serum pools or individual animals showed no significant enrichment of the Y2 idiotype in comparison to unselected MRL-lpr/lpr IgG. These results suggest that the Y2 idiotype defines only a minor component of the anti-Sm autoantibody response, and that most antibodies with this determinant express other antigenic specificities.