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.     

Fas death receptor signaling represses monocyte numbers and macrophage activation in vivo

Over 1 billion monocytes are produced daily, with a small percentage differentiating into macrophages, suggesting that excess monocytes are deleted through a tightly regulated process. Although the in vivo mechanism governing monocyte/macrophage homeostasis is unknown, deletion of monocytes in culture is mediated by the Fas death pathway and is blocked by M-CSF. To determine the in vivo significance of Fas in monocyte development, mice lacking Fas (lpr/lpr) and mice deficient in Fas and M-CSF were examined. Compared with congenic control C57BL/6 (B6) mice, lpr/lpr mice displayed increased numbers of circulating monocytes. The lack of Fas in M-CSF-deficient mice resulted in an enhanced percentage, but not total numbers, of monocytes. Fas deficiency led to an increase in myeloid bone marrow progenitor potential only in M-CSF-intact mice. Although lpr/lpr and B6 mice had similar numbers of tissue macrophages, the loss of Fas in M-CSF-deficient mice was sufficient to increase the number of macrophages in a subset of tissues. Additionally, after stimulation with thioglycolate, lpr/lpr and B6 mice showed equivalent numbers of peritoneal macrophages. However, Fas-deficient peritoneal macrophages displayed a marked increase in spontaneous and LPS-induced proinflammatory molecule production. Moreover, Fas-deficient mice showed enhanced systemic inflammatory arthritis associated with up-regulation of IL-1beta and CCL2 secretion, elevated numbers of inflammatory monocytes, and increased numbers of tissue macrophages. Collectively, these data suggest that Fas may be required for maintaining circulating monocytes and for suppressing macrophage activation and recruitment that are stimulus dependent.     

Negative role of colony-stimulating factor-1 in macrophage, T cell, and B cell mediated autoimmune disease in MRL-Fas(lpr) mice

Inflammation in the kidney and other tissues (lung, and salivary and lacrimal glands) is characteristic of MRL-Fas(lpr) mice with features of lupus. Macrophages (Mphi) are prominent in these tissues. Given that 1) Mphi survival, recruitment, proliferation, and activation during inflammation is dependent on CSF-1, 2) Mphi mediate renal resident cell apoptosis, and 3) CSF-1 is up-regulated in MRL-Fas(lpr) mice before, and during nephritis, we hypothesized that CSF-1-deficient MRL-Fas(lpr) mice would be protected from Mphi-mediated nephritis, and the systemic illness. To test this hypothesis, we compared CSF-1-deficient MRL-Fas(lpr) with wild-type strains. Renal pathology is suppressed and function improved in CSF-1-deficient MRL-Fas(lpr) mice. There are far fewer intrarenal Mphi and T cells in CSF-1-deficient MRL-Fas(lpr) vs wild-type kidneys. This leukocytic reduction results from suppressed infiltration, and intrarenal proliferation, but not enhanced apoptosis. The CSF-1-deficient MRL-Fas(lpr) kidneys remain preserved as indicated by greatly reduced indices of injury (nephritogenic cytokines, tubular apoptosis, and proliferation). The renal protective mechanism in CSF-1-deficient mice is not limited to reduced intrarenal leukocytes; circulating Igs and autoantibodies, and renal Ig deposits are decreased. This may result from enhanced B cell apoptosis and fewer B cells in CSF-1-deficient MRL-Fas(lpr) mice. Furthermore, the systemic illness including, skin, lung, and lacrimal and salivary glands pathology, lymphadenopathy, and splenomegaly are dramatically suppressed in CSF-1-deficient MRL-Fas(lpr) as compared with wild-type mice. These results indicate that CSF-1 is an attractive therapeutic target to combat Mphi-, T cell-, and B cell-mediated autoimmune lupus.     

Molecular basis of elevated c-myb expression in the abnormal L3T4-, Lyt-2- T lymphocytes of autoimmune mice

The presence of the lpr/lpr genotype on a number of murine genetic backgrounds results in a systemic lupus erythematosus-like disease and lymphadenopathy. The T lymphocytes of these mice exhibit a variety of abnormalities; most pertinent to the present report is an abnormally high level of c-myb proto-oncogene mRNA. Since the c-myb protein is presumably the effector molecule that affects cellular functions, it is important to determine whether increased levels of this c-myb protein are produced. With the use of immunoprecipitation with an anti-v-myb reagent, we found high levels of c-myb protein in the lymph nodes of lpr mice. Detailed analysis showed that the c-myb protein is primarily expressed by an abnormal T lymphocyte population that does not express the mature T cell markers, L3T4 and Lyt-2. Analysis by two-dimensional gel electrophoresis showed that the c-myb proteins from normal thymocytes and from these L3T4-, Lyt-2-T cells are indistinguishable. DNA analysis with Southern hybridizations showed the lack of amplification, insertions, deletions, and rearrangements, which is in accord with results from the protein studies. Most interestingly, the c-myb gene in lpr L3T4-, Lyt-2- T cells is hypomethylated compared with normal controls. This suggests that a regulatory mechanism, rather than the structural alteration of the gene, is responsible for elevated expression of c-myb in these L3T4-, Lyt-2- cells.     

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.     

IgG3 production in MRL/lpr mice is responsible for development of lupus nephritis

MRL/Mp-lpr/lpr (MRL/lpr) mice spontaneously develop lethal glomerulonephritis (GN) similar to human lupus nephritis, associated with the expression of lymphoproliferation gene lpr. To examine whether a particular IgG subclass is responsible for development of GN in these mice, first quantitative analysis of IgG subclasses in serum and in kidney eluates was performed. Although IgG2a was the dominant subclass in serum throughout the lifespan of mice, the IgG3 level in kidney eluates was three times higher than that of IgG2a at the 16 wk of age, which is the time of onset of development of severe GN. In sera of the 12-wk-old mice, half of the IgG3 was in immune complex form, whereas IgG2a in this form was only 17% of the total amount. Second, cyclosporin A, which ameliorates GN in MRL/lpr mice despite autoantibody production, was found to reduce serum IgG3 and mRNA levels, associated with the revision of cationic shift of the serum IgG3 spectrotype seen in isoelectric focusing. Third, among the hybrid mice with non-autoimmune-prone C3H/HeJ-lpr/lpr (C3H/lpr) mice, MRL/lpr x (MRL/lpr x C3H/lpr) F1, in which the genetic background for GN is likely segregated, the mRNA level for IgG3 correlated well with the degree of glomerular lesion. These findings indicate that production of IgG3 in MRL/lpr mice is one of the major factors responsible for development of GN in these mice, and that this is due to the genetic background of the MRL strain.     

Effect of xid on autoimmune C3H-gld/gld mice

The xid gene was introduced into C3H-gld/gld mice to determine its effects on the development of autoimmune disease. C3H-gld/gld.xid mice were compared with C3H-gld/gld mice for the development of lymphadenopathy, surface phenotype of lymph node (LN) cells, c-myb oncogene RNA production, serum immunoglobulin (Ig) levels, and autoantibody production. In addition, C3H-gld/gld and C3H-lpr/lpr mice were examined for serum Ig and autoantibody levels. The results showed that the xid gene had no effect on either the development of the severe lymphadenopathy characteristic of C3H-gld/gld mice or the phenotype of the Ly-2-, L3T4-, Ly-5(B220)+ T-cell subset that is expanded in the LN and spleens of these mice. Similarly, xid did not affect the high levels of c-myb oncogene RNA expression by C3H-gld/gld LN and spleen cells. By contrast, the xid gene caused a significant reduction in serum IgM but not IgA levels and almost completely ablated the generation of both IgM and IgG anti-ssDNA antibodies and anti-dsDNA antibodies. These data suggest that the xid gene can dramatically decrease the B-cell manifestations of autoimmunity in gld homozygotes without affecting their abnormal T-cell expansion. Comparisons of age-matched C3H-gld/gld and C3H-lpr/lpr mice showed that they had similarly elevated serum IgM and IgA levels and anti-ssDNA and anti-dsDNA antibody levels providing further evidence that gld and lpr produce parallel defects in C3H mice.     

Anti-Sm B cell differentiation in Ig transgenic MRL/Mp-lpr/lpr mice: altered differentiation and an accelerated response

To determine the regulation of B cells specific for the ribonucleoprotein Sm, a target of the immune system in human and mouse lupus, we have generated mice carrying an anti-Sm H chain transgene (2-12H). Anti-Sm B cells in nonautoimmune 2-12H-transgenic (Tg) mice are functional, but, in the absence of immunization, circulating anti-Sm Ab levels are not different from those of non-Tg mice. In this report, we compare the regulation of anti-Sm B cells in nonautoimmune and autoimmune MRL/Mp-lpr/lpr (MRL/lpr) and bcl-2-22-Tg mice. Activation markers are elevated on splenic and peritoneal anti-Sm B cells of both nonautoimmune and autoimmune genetic backgrounds indicating Ag encounter. Although tolerance to Sm is maintained in 2-12H/bcl-2-22-Tg mice, it is lost in 2-12H-Tg MRL/lpr mice, as the transgene accelerates and increases the prevalence of the anti-Sm response. The 2-12H-Tg MRL/lpr mice have transitional anti-Sm B cells in the spleen similar to nonautoimmune mice. However, in contrast to nonautoimmune mice, there are few if any peritoneal anti-Sm B-1 cells. These data suggest that a defect in B-1 differentiation may be a factor in the loss of tolerance to Sm and provide insight into the low prevalence of the anti-Sm response in lupus.     

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.     

Mice with mutations in Fas and Fas ligand demonstrate increased herpetic stromal keratitis following corneal infection with HSV-1

HSV-1 infection of the cornea leads to a potentially blinding immunoinflammatory lesion of the cornea, termed herpetic stromal keratitis. It has also been shown that one of the factors limiting inflammation of the cornea is the presence of Fas ligand (FasL) on corneal epithelium and endothelium. In this study, the role played by FasL expression in the cornea following acute infection with HSV-1 was determined. Both BALB/c and C57BL/6 (B6) mice with HSV-1 infection were compared with their lpr and gld counterparts. Results indicated that mice bearing mutations in the Fas Ag (lpr) displayed the most severe disease, whereas the FasL-defective gld mouse displayed an intermediate phenotype. It was further demonstrated that increased disease was due to lack of Fas expression on bone marrow-derived cells. Of interest, although virus persisted slightly longer in the corneas of mice bearing lpr and gld mutations, the persistence of infectious virus in the trigeminal ganglia was the same for all strains infected. Further, B6 mice bearing lpr and gld mutations were also more resistant to virus-induced mortality than were wild-type B6 mice. Thus, neither disease nor mortality correlated with viral replication in these mice. Collectively, the findings indicate that the presence of FasL on the cornea restricts the entry of Fas(+) bone marrow-derived inflammatory cells and thus reduces the severity of HSK.     

Constitutive activation of the Fas ligand gene in mouse lymphoproliferative disorders.

Mice homozygous for lpr (lymphoproliferation) or gld (generalized lymphoproliferative disease) develop lymphadenopathy and splenomegaly and suffer from autoimmune disease. The lpr mice have a defect in a cell-surface receptor, Fas, that mediates apoptosis, while gld mice have a mutation in the Fas ligand (FasL). Northern hybridization with the FasL cDNA as probe indicated that the cells accumulating in lpr and gld mice abundantly express the FasL mRNA without stimulation. By means of in situ hybridization and immunohistochemistry, we identified the cells expressing the FasL mRNA as CD4-CD8- double negative T cells. The T cells from lpr mice were specifically cytotoxic against Fas-expressing cells. Since FasL is normally expressed in activated mature T cells these results indicate that the double negative T cells accumulating in lpr and gld mice are activated once, and support the notion that the Fas/FasL system is involved in activation-induced suicide of T cells. Furthermore, the graft-versus host disease caused by transfer of lpr bone marrow to wild-type mice can be explained by the constitutive expression of the FasL in lpr-derived T cells.     

Thymocyte apoptosis by T-2 toxin in vivo in mice is independent of Fas/Fas ligand system

To find whether Fas/Fas ligand (FasL) pathway is involved in T-2 toxin (T-2)-mediated thymocyte apoptosis, we used lpr/lpr (lpr) and gld/gld (gld) mice, whose Fas and FasL proteins, respectively, are functionally deficient. Based on the DNA fragmentation profile in gel electrophoresis and measurement of apoptotic cell percent by flow cytometry, the levels of thymocyte apoptosis in lpr and gld mice that had received T-2 showed that both lpr and gld mice had undergone apoptosis essentially to the same magnitude as those of corresponding wild type mice (+/+). These results strongly suggest that T-2-induced thymocyte apoptosis in vivo in mice is independent of the Fas/FasL pathway.     

Influence of gender and the endocrine environment on the distribution of androgen receptors in the lacrimal gland

Androgens are known to regulate both the structure and function of lacrimal tissue in a variety of species. To explore the endocrine basis for this hormone action, the following study was designed to: (1) determine the cellular distribution of androgen receptors in the lacrimal gland; and (2) examine the influence of gender and the endocrine environment on the glandular content of these binding sites. Lacrimal glands were obtained from intact, castrated, hypophysectomized, diabetic or sham-operated male or female adult rats, mice or hamsters, as well as from orchiectomized rats exposed to placebo compounds or physiological levels of testosterone. The cellular of androgen receptors was evaluated by utilizing an immunoperoxidase protocol, in which a purified rabbit polyclonal antibody to the rat androgen receptor was used as the first antibody. Our findings with lacrimal glands showed that: (1) androgen receptors are located almost exclusively in nuclei of epithelial cells; (2) the cellular distribution or intranuclear density of these binding sites is far more extensive in glands of males, as compared to females; (3) orchiectomy or hypophysectomy, but not sham-surgery or diabetes, lead to a dramatic reduction in the immunocytochemical expression of androgen receptors; and (4) testosterone administration to orchiectomized rats induces a marked increase in androgen receptor content, relative to that in placebo-exposed glands. Our results also reveal that a 10 kb androgen receptor mRNA exists in the rat lacrimal gland. Overall, these findings demonstrate that gender and the endocrine system may significantly influence the distribution of androgen binding sites in rat lacrimal tissue. Moreover, our results show that androgens up-regulate their own lacrimal gland receptors.     

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.     

Increase in potential activities of protein phosphatases PP1 and PP2A in lymphoid tissues of autoimmune MRL/MpJ-lpr/lpr mice.

The differential assay conditions for protein phosphatases PP1, PP2A, and PP2C were extensively studied by using crude extracts from mouse lymphoid tissues as enzyme sources. Under these conditions, the protein phosphatase activities were measured in MRL/MpJ-lpr/lpr mice (MRL/lpr mice), autoimmune-prone mice, and MRL/MpJ(-)+/+ mice (MRL/+/+ mice) and C3H/HeJ mice as the controls. In MRL/lpr mice, significant alterations in protein phosphatase activities from those in the control mice were demonstrated. In spleen and liver from MRL/lpr mice, potential activities of PP1 and PP2A were distinctly elevated over those of the control mice. These elevations appeared to be due to accumulation of the abnormal lymphocytes that emerged in MRL/lpr mice. Although the PP1 activity in MRL/lpr lymph nodes was lower than those of normal spleen and thymus, it was greatly increased by Co(2+)-trypsin treatment so that the PP1 activity of MRL/lpr lymph nodes was the highest among those of all the tissues examined. In contrast, PP2C activity showed no remarkable alteration in the autoimmune disease model mice as compared with that in the control mice. These results demonstrated a specific elevation in potency of protein dephosphorylation in the tissues of MRL/lpr mice, suggesting a new explanation for the defect in signal transduction in this disease.