In vitro role of IL-1 in heightened IgG, anti-DNA, and nephritogenic idiotype production by B cells derived from the murine MRL/lpr lupus model

The MRL/lpr murine model resembles human lupus both in its serologic and immunopathologic features, and is characterized by high-level IgG and autoantibody production. The precise mechanisms for this B cell hyperactivity are poorly understood. This study explored the role of IL-1 in determining high-level IgG and autoantibody production in the MRL/lpr murine lupus model by blocking IL-1 activity with a recombinant IL-1 receptor antagonist (IL-1Ra). IgG and autoantibody production (anti-DNA ab and Id-H130 activity) by B cells derived from MRL/lpr mice was significantly suppressed by treating B cell cultures with IL-1Ra. In contrast, IgG and autoantibody production by B cells derived from young MRL/lpr, MLR/++, or normal C3H/HeJ mice showed virtually no suppression with IL-1Ra. Collectively, these findings indicate that IL-1 may be an important factor in determining the heightened production of IgG, anti-DNA, and id-H130 antibody production in lupus-prone MRL/lpr mice. Furthermore, heightened IL-1 activity appears to be influenced by both age and the presence of the lpr mutation.     

Effect of cyclic AMP level reduction on human neutrophil responses to formylated peptides

In an attempt to elucidate the mechanism of development of organ-specific autoimmune lesions resembling human Sj?gren's syndrome of MRL/lpr mice, we have analyzed local cytokine gene expressions and organ-specific autoantibody production in vivo. We have demonstrated that a major proportion of T cells bearing CD4 and V(beta)8 molecules are essentially responsible for triggering the autoimmunity in the salivary glands of MRL/lpr mice. The local cytokine gene expressions including interferon(IFN)-gamma, IL-12(p40) mRNAs were observed during the course of murine Sjogren's syndrome in MRL/lpr autoimmune strain. In particular, a high level of local expressions of IL-12 mRNA was detected earlier in the proinflammatory stage of autoimmune lesions. A significant level of local expression of MHC class-II(I-Ak) mRNA was detected before the onset of inflammatory lesions in the salivary glands, and I-Ak-positive epithelial duct cells were frequently observed in the salivary glands of MRL/lpr mice. In addition, we found the salivary gland-specific autoantibody in sera from MRL/lpr mice with early phase of autoimmune lesions by immunoblot analysis. These results suggest that cytokine gene stimulation and autoantibody production are essentially involved in the development of organ-specific autoimmune lesions in Sj?gren's syndrome of MRL/lpr mice.     

Dysregulated cytokine expression in vivo in prediseased and diseased autoimmune-prone MRL mice

Macrophages (m?) from prediseased autoimmune-prone MRL/ + and MRL/lpr mice produce markedly decreased levels of IL-1 in vitro in response to LPS. In contrast, tissues from diseased MRL/lpr mice overexpress IL-1 in vivo. To determine whether IL-1 underproduction in the MRL strains is solely an in vitro phenomenon, we compared in vivo cytokine mRNA expression from prediseased age-matched MRL/ + and MRL/lpr mice to that from normal BALB/c and C3HeB/FeJ mice. Like m? in vitro, whole organ RNA from the spleen, liver, and kidney of MRL/ + and MRL/lpr mice showed down-regulation of IL-1 RNA following intraperitoneal injection of LPS. This abnormality in inducible IL-1 expression was present in all MRL mice, irrespective of disease stage or the presence of the lpr gene. On the other hand, only diseased MRL/lpr mice displayed elevated and constitutive expression of IL-1 in their livers and kidneys. We suggest that inducible expression is most indicative of the intrinsic, or genetic, capacity of cells to produce cytokine, whereas constitutive expression reflects extracellular disease-related inflammatory stimuli present only in the diseased MRL/lpr strains. By restricting our studies to prediseased MRL mice, we have tried to eliminate the effects of disease and to focus on the predisposing genetic background. The existence both in vitro and in vivo of a defect in inducible IL-1 expression by prediseased MRL mice suggests that the molecular abnormality underlying this defect may be a part of this predisposing background to autoimmunity.     

Fas-mediated cytotoxicity is not required for rejection of murine nonvascularized heterotopic cardiac allografts

BACKGROUND: Using mice with loss-of-function mutations in the Fas and Fas ligand (FasL) genes (lpr and gld, respectively) in transplantation experiments has resulted in contradictory findings concerning the role of Fas/FasL-mediated cytotoxicity in allograft rejection. The observation that these mutant mice develop an abnormal lymphocyte phenotype with increasing age that is hyporesponsive in vitro led us to examine the possibility that this characteristic might explain seemingly discordant observations in the literature. Therefore, to distinguish between the effects of Fas/FasL pathway disruption and the effects of immune senescence on in vivo cytotoxicity and allograft rejection, we evaluated the survival of cardiac allografts in gld, lpr, and wild-type mice of varying ages. METHODS: Six- to 21-week-old C3H, C3H/HeJ-Fasl(gld), C57B1/6, and B6.MRL-Fas(lpr) recipients were transplanted with heterotopic, nonvascularized cardiac allografts from neonatal Balb/c, C3H, C57Bl/6, and B6.MRL-Fas(lpr) donors. Mixed lymphocyte reactions were performed in naive gld, lpr, and wild-type animals, 6 and 12 weeks of age. Rejected allografts in gld, lpr, and wild-type recipients and functioning syngeneic transplants were evaluated for intragraft apoptosis by a DNA fragmentation detection assay. RESULTS: Graft survival was not significantly different between 6-week-old gld and lpr recipients and their respective wild-type controls. However, allograft rejection was delayed significantly in older (13-week) gld mice compared with age-matched wild-type mice (P=0.02) or young (6-week) gld animals (P=0.04). Similarly, 21-week-old lpr mice exhibited prolonged graft survival compared with 6-week-old lpr animals (P=0.01). Reduced alloreactive proliferative responses in 12-week-old gld and lpr mice were observed when compared with age-matched wild-type strains. Rejecting allografts displayed a similar level of intragraft apoptotic cells regardless of mutant or wild-type phenotype or age of recipient. CONCLUSIONS: The findings of this study confirm that Fas/FasL-mediated cytotoxicity is not required for murine cardiac allograft rejection. Our findings also demonstrate that the observed delayed graft rejection in lpr and gld mice is a consequence of an age-related alteration of the immune system, specific to gld and lpr mice and associated with an in vivo and in vitro hyporeactivity to alloantigens.     

Efficient peripheral clonal elimination of B lymphocytes in MRL/lpr mice bearing autoantibody transgenes

Peripheral B cell tolerance was studied in mice of the autoimmune-prone, Fas-deficient MRL/ lpr.H-2(d) genetic background by introducing a transgene that directs expression of membrane-bound H-2Kb antigen to liver and kidney (MT-Kb) and a second transgene encoding antibody reactive with this antigen (3-83mu delta, anti-Kk,b). Control immunoglobulin transgenic (Ig-Tg) MRL/lpr.H-2(d) mice lacking the Kb antigen had large numbers of splenic and lymph node B cells bearing the transgene-encoded specificity, whereas B cells of the double transgenic (Dbl-Tg) MRL/lpr.H-2(d) mice were deleted as efficiently as in Dbl-Tg mice of a nonautoimmune B10.D2 genetic background. In spite of the severely restricted peripheral B cell repertoire of the Ig-Tg MRL/lpr.H-2(d) mice, and notwithstanding deletion of the autospecific B cell population in the Dbl-Tg MRL/lpr.H-2(d) mice, both types of mice developed lymphoproliferation and exhibited elevated levels of IgG anti-chromatin autoantibodies. Interestingly, Dbl-Tg MRL/lpr.H-2(d) mice had a shorter lifespan than Ig-Tg MRL/lpr.H-2(d) mice, apparently as an indirect result of their relative B cell lymphopenia. These data suggest that in MRL/lpr mice peripheral B cell tolerance is not globally defective, but that certain B cells with receptors specific for nuclear antigens are regulated differently than are cells reactive to membrane autoantigens.     

Up-regulation by ammonium trichloro(dioxoethylene-0,0') tellurate (AS101) of Fas/Apo-1 expression on B16 melanoma cells: implications for the antitumor effects of AS101

It was recently reported that human and mouse melanoma cells express Fas ligand (FasL) but almost no Fas, which may contribute to their immune privilege. AS101 (ammonium trichloro(dioxoethylene-0,0')tellurate), a synthetic immunomodulator with minimal toxicity, was found to have antitumor effects in various tumor models. Our present study shows that AS101 has direct and indirect effects on tumor cells; AS101 inhibits the clonogenicity of B16 melanoma cells in vitro. Moreover, wild-type P53 expression, which is required for induction of Apo-1 expression, increased significantly in AS101-treated cells. We therefore investigated Fas expression in AS101-treated B16 cells and found that Fas, but not FasL, expression was significantly increased; moreover, Fas receptors were functional. Longer incubation with AS101 resulted in spontaneous apoptosis triggered by the Fas-FasL system. To explore the relationship of these results to the antitumor effects of AS101, we injected B16-F10 mouse melanoma cells into syngeneic C57BL/6 mice carrying the lpr mutation in the Fas gene and to gld mutant mice that lack functional FasL. Tumor development in control groups was lowest in the lpr mice, while no difference was observed between gld and wild-type mice. Among the AS101-treated groups, the most pronounced effect appeared in the lpr mice, while the lowest was seen in the gld mutant mice. Our study suggests that AS101 may render melanoma tumor cells more sensitive to Fas/FasL-induced apoptosis and may therefore have clinical potential.     

gp130, the cytokine common signal-transducer of interleukin-6 cytokine family, is downregulated in T cells in vivo by interleukin-6

gp130 is a common signal-transducing receptor component for the interleukin-6 (IL-6) family of cytokines. To investigate the expression of gp130 in T-cell subsets and its regulation, anti-murine gp130 monoclonal antibody (MoAb) was used for flow cytometric analysis. In normal mice, gp130 was differentially expressed in thymocyte and splenic T-cell subpopulations defined by CD4/CD8 expression. In aged MRL/lpr mice, although gp130 expression was detectable in splenic CD4(+) or CD8(+) T cells, gp130 expression was significantly downregulated. Because serum levels of IL-6 and soluble IL-6 receptor (sIL-6R) are elevated in these mice, we examined the possibility that the downregulation of gp130 expression on splenic T cells might be produced in response to continuous activation of gp130 by high levels of serum IL-6. In transgenic mice overexpressing IL-6, gp130 expression in the splenic T cells was significantly decreased. After stimulation with IL-6 in vitro, the level of gp130 on CD4(+) or CD8(+) splenic T cells from normal mice was significantly decreased. These results suggest that the expression of gp130 in splenic T cells could be downregulated by the IL-6 stimulation under physiological or pathological circumstances.     

Genotype effects on the antioxidant enzymes activity and mRNA expression in liver and kidney tissues of autoimmune-prone MRL/MpJ-lpr/lpr mice

Congeneic pairs of MRL/lpr and MRL/++ (+/+) mice differ in incidence of autoantibodies, lymphoproliferative disease and survival, characteristics that are linked to immunological abnormalities. MRL/lpr mice have a significantly shorter life span compared to +/+ mice. Because a weak antioxidant defense and an increased generation of free radicals are generally implicated in the severity of many autoimmune disease, the present study was undertaken to compare the influence of genotype on lipid composition, lipid peroxidation and expression of mRNA, and activity of antioxidant enzymes such as catalase (CAT), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in the livers and kidneys of these mice. The expression of SOD, GSH-Px and CAT mRNAs was significantly higher (P < 0.05) in the livers of +/+ mice, while in the kidneys only SOD expression was found significantly higher in +/+ mice when compared to MRL/lpr mice. Further, the activity of cytosolic SOD and GSH-Px was also found significantly higher (P < 0.001) in the livers of +/+ mice. Both livers and kidneys of MRL/lpr mice exhibited significantly higher levels of arachidonic acid (20:4(n-6)), significantly higher generation of thiobarbituric acid reactive substances (TBARS) and higher estimated peroxidation index than the +/+ mice. In addition, the MRL/lpr mice had higher levels of serum anti-cardiolipin antibodies. In summary, the results from the present study indicate that besides several immune-related abnormalities, the MRL/lpr mice may exhibit their inability to cope with oxidative stress due to a poor antioxidant defense system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fas- and FasL-deficient mice are resistant to induction of autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an organ-specific autoimmune disease inducible in susceptible animals by myelin Ag-specific CD4+ Th1 cells. The mechanisms by which these cells induce inflammation and demyelination in the central nervous system (CNS) are incompletely understood. To determine the roles of Fas and FasL in the involvement of CNS autoimmune injury, we determined susceptibility to EAE of Fas-or FasL-deficient mice. Compared with wild-type mice, mice expressing lpr (Fas) and gld (FasL) mutations were relatively resistant to the development of clinical EAE, and this correlated with fewer inflammatory infiltrates and cells undergoing apoptosis in the CNS of the mutant mice. The gld and lpr mice, however, developed significant T cell responses with production of Th1 cytokines in response to the encephalitogenic myelin peptide. These results suggest that the Fas/FasL pathway plays a critical role in the development of EAE probably by mediating apoptosis within the target tissue.     

A new strategy for treatment of autoimmune diseases in chimeric resistant MRL/lpr mice

A new strategy for the treatment of autoimmune diseases in chimeric resistant MRL/lpr mice is established. The strategy includes injection of cyclophosphamide (CY), fractionated irradiation (5 Gy x 2), bone grafts (to recruit stromal cells), and two transplantations of whole bone marrow cells (WBMCs) from allogeneic normal C57BL/6 mice (CY/2X/Bone/2BMT). MRL/lpr mice, thus treated, survived more than 40 weeks (1 mouse survived for >40 weeks, 7 for >50 weeks, and 4 for >60 weeks after these treatments). Immunohistological studies showed that the mice were completely free from both lymphadenopathy and autoimmune diseases such as systemic lupus erythematosis and rheumatoid arthritis. The levels of autoantibodies (IgM/IgG rheumatoid factors and IgM/IgG anti-ssDNA antibodies [Abs]) in the treated mice decreased to those in the normal mice. In addition, successful cooperation among T cells, B cells, and antigen-presenting cells (APCs) was observed. Abnormal T cells with immunophenotypes of B220+/Thy-1+/CD3+/CD4-/CD8- present in untreated MRL/lpr mice disappeared, and the hematolymphoid cells of the treated mice were of donor origin. However, the mice that had been irradiated with 8.5 Gy and then reconstituted with T-cell-depleted BMCs plus bone grafts died within 2 weeks due to the side effect of irradiation. The depletion of CD8+ cells (not CD4+ cells) from WBMCs resulted in graft failure; 60% of the recipient mice, thus treated, died within 2 weeks, and all recipients died by 15 weeks. Furthermore, limiting dilution assays showed that approximately more than 0.5% of T cells contained in the BMCs are necessary not only for engraftment of BMCs but also for long-term disease-free survival of the recipients. In contrast, recipients that had received CD4-depleted BMCs with CY plus fractionated irradiation (5Gy x 2) survived for more than 40 weeks without showing graft-versus-host reaction (GVHR). This indicates that CD8(+)cells in the BMCs are essential for the successful engraftment of the donor-type hematolymphoid cells.     

MRL/lpr and MRL+/+ macrophage DNA synthesis in the absence and the presence of colony-stimulating factor-1 and granulocyte-macrophage colony-stimulating factor

Macrophage accumulation and proliferation as well as altered macrophage properties have been observed in autoimmune MRL mice. To determine whether there might be innate differences in the proliferative responses, we examined the DNA synthesis responses of peritoneal macrophages and macrophages derived in vitro from bone marrow precursors (bone marrow-derived macrophages (BMM)). Murine peritoneal exudate macrophages normally require the addition of macrophage CSF (CSF-1) to enter cell cycle in vitro. In contrast, we have found that many thioglycollate-induced adherent peritoneal macrophages, but not resident peritoneal macrophages, from both MRL/lpr and MRL+/+ mice atypically underwent DNA synthesis even in the absence of added CSF-1. They also responded very well to granulocyte-macrophage CSF. These findings may help to explain the appearance of increased macrophage numbers in MRL lesions. In contrast to a previous report, it was found that MRL/lpr and MRL+/+ BMM did not have an enhanced response to CSF-1 and that modulation of CSF-1 receptor expression was not more rapid in MRL BMM. We also found no evidence for abnormal CSF-1 internalization and degradation or for the lpr mutation to have any enhanced effect on BMM survival in the absence of CSF-1. TNF-alpha lowered the DNA synthesis response to CSF-1 of MRL/lpr BMM rather than enhanced it, as has been reported. Our data suggest that the enhanced accumulation of macrophages in the MRL/lpr kidney cannot be explained by a proposed model of enhanced responsiveness of MRL/lpr BMM to CSF-1, including a contribution by TNF-alpha.     

Bruton's tyrosine kinase regulates apoptosis and JNK/SAPK kinase activity

Mast cells derived from Bruton's tyrosine kinase (Btk)-defective xid or btk null mice showed greater expansion in culture containing interleukin-3 (IL-3) than those from wild-type (wt) mice. Although the proliferative response to IL-3 was not significantly different between the wt and xid mast cells, xid and btk null mast cells died by apoptosis more slowly than their wt counterparts upon IL-3 deprivation. Consistent with these findings, the apoptosis-linked c-Jun N-terminal kinase/stress-activated protein kinase (JNK) activity was compromised in these btk-mutated cells upon Fc(epsilon)RI crosslinking or upon stimulation with IL-3 or with stem cell factor. p38 activity was less severely, but significantly, affected by btk mutation, whereas extracellular signal-regulated kinases were not affected by the same mutation. Btk-mediated regulation of apoptosis and JNK activity was confirmed by reconstitution of btk null mutant mast cells with the wt btk cDNA. Furthermore, growth factor withdrawal induced the activation and sustained activity of JNK in wt mast cells, while JNK activity was consistently lower in btk-mutated mast cells. These results support the notion that Btk regulates apoptosis through the JNK activation.     

Enhanced Th2-like responses in IL-1 type 1 receptor-deficient mice

IL-1 has a number of effects on T cell growth but a specific role for IL-1 in T cell responses in vivo has not been elucidated. In this study the role of IL-1 in Th1/Th2 responses was examined in mice deficient for the IL-1 type 1 receptor (IL-1RI-/-) during cutaneous Leishmania major infection or following immunization with keyhole limpet hemocyanin (KLH). After inoculation of L. major stationary phase promastigotes into the hind footpad, both IL-1RI-/- and wild-type (WT) mice developed small lesions which resolved spontaneously. Lymph node cells from infected IL-1RI-/- mice produced significantly more IL-4 and IL-10 than those from WT mice following antigenic stimulation in vitro. Splenocytes from IL-1RI-/- and WT mice showed similar levels of antigen-induced proliferation. In contrast, splenocyte cultures from the IL-1RI-/- mice contained significantly more IL-4 than those from WT mice. Similar results were also obtained after immunization with KLH. While lymph node cells from both IL-1RI-/- and WT mice displayed similar levels of KLH-specific proliferation, those from IL-1RI-/- mice produced significantly more IL-4 than those from WT mice. Conversely, antigen-stimulated lymph node cells from WT mice secreted significantly greater amounts of IFN-gamma as compared with those from IL-1RI-/- mice. These data indicate that while IL-1 is not required for mounting an immune response or antigen-dependent proliferation, it appears to be required for normal regulation of Th1/Th2 responses and may function to negatively regulate IL-4 expression.     

T-lymphocyte downregulation after acute viral infection is not dependent on CD95 (Fas) receptor-ligand interactions

Infection of mice with lymphocytic choriomeningitis virus (LCMV) causes a major expansion of CD8+ T cells followed by a period of immune downregulation that coincides with the induction of lymphocyte apoptosis in the mouse spleen. CD95 (Fas) and its ligand are important for regulating peripheral T-lymphocyte numbers and can mediate apoptosis of mature T lymphocytes. We infected CD95- and CD95L-deficient mice (lpr and gld, respectively) with LCMV to determine if the immune downregulation that occurred following resolution of the LCMV infection was due to a CD95-dependent apoptotic mechanism. Lymphocytes from LCMV-infected lpr and gld mice were capable of normal T-cell expansion and cytolytic function but were, in contrast to activated cells from normal virus-infected mice, relatively more resistant to T-cell receptor-induced apoptosis in vitro. However, in vivo there were significant numbers of apoptotic cells in the spleens of lpr and gld mice recovering from the infection, and the T-cell number and cytolytic activity decreased to normal postinfection levels. Thus, CD95 is not required for the immune downregulation of the CD8+-T-lymphocyte response following acute LCMV infection.     

Anti-IL-12 antibody prevents the development and progression of collagen-induced arthritis in IFN-gamma receptor-deficient mice

In several models of inflammation, including collagen-induced arthritis (CIA), the disease-promoting effect of IL-12 has been attributed to its well-known ability to produce IFN-gamma. However, IFN-gamma receptor knockout (IFN-gammaR KO) mice of the DBA/1 strain have been reported to be more susceptible to CIA than corresponding wild-type mice, indicating the existence of an IFN-gamma-mediated protective pathway in this model. In the present study the development of CIA was found to be completely prevented by pretreatment with a neutralizing anti-IL-12 antibody, not only in wild-type, but significantly also in IFN-gammaR KO mice. In both strains of mice, the protective effect of anti-IL-12 was associated with lower production of anti-collagen type II antibodies. In vivo stimulation with anti-CD3 antibody in arthritic IFN-gammaR KO mice resulted in production of higher levels of circulating IFN-gamma, TNF and IL-2 than in corresponding control mice that had not received the arthritis-inducing immunization. This was not the case in arthritis-developing wild-type mice. Furthermore, the protective effect of anti-IL-12 antibody in mutant, but not in wild-type mice, was associated with lower circulating IFN-gamma, TNF and IL-2 and higher IL-4 and IL-5 cytokine levels following an anti-CD3 challenge. The data indicate that IL-12 promotes the development of arthritis independently of its ability to induce or favor production of IFN-gamma. In fact, any IFN-gamma produced in the course of the disease process rather exerts a protective effect. Furthermore, our study suggests that, in the absence of a functional IFN-gamma system, endogenous IL-12 exerts its disease-promoting effect by favoring production of other Th1-associated cytokines (IL-2 and TNF), by inhibiting development of IL-4- and IL-5-producing T cells and by stimulating production of anti-collagen autoantibodies.     

Host modifier genes affect mouse autoimmunity induced by the lpr gene

A defect in apoptotic signal transmission through CD95 is an essential genetic mechanism for lymphoproliferation and autoimmunities in lpr or gld mice. However, disease manifestations are largely affected by the host genetic background. To identify and map such host genes modifying lpr gene effect, ie, the lpr modifier (Lprm) genes, 82 MRL/lpr x (MRL/lpr x C3H/lpr) F1 mice were subjected to immunopathological and genetical analyses. High-grade vasculitis and glomerulonephritis among backcross mice were observed in separate groups of mice. Microsatellite analysis revealed that there were two host genes affecting the occurrence of vasculitis, Lprm1 (chromosome 4) and Lprm2 (chromosome 3). A recessive MRL allele at Lprm1 enhanced vasculitis to occur in both sexes, whereas that of Lprm2 inhibited its development selectively in females. Genotype combinations of these two genes explained the severity of vasculitis in crosses of MRL/lpr and C3H/lpr mice and also the vasculitis-prone recombinant inbred strain McH5/lpr. A recessive MRL allele at Lprm3 (chromosome 14) suppressed glomerulonephritis. The weight of the spleen was increased by a recessive MRL allele at Lprm4 (chromosome 5) yielding a logarithm of odds score of 2.02 in a quantitative trait locus analysis. In contrast, the weight of axillary lymph nodes was increased by a recessive MRL allele at a locus on chromosome 2, but its presence was not supported by the quantitative trait locus analysis. The titer of anti-dsDNA autoantibody was controlled by the locus Lprm5 on chromosome 16, which had an logarithm of odds score of 3.41. Possible candidate genes for Lprm genes deduced from their map locations are discussed and compared with the autoimmunity genes reported thus far. In conclusion, autoimmune disease manifestations by the lpr mutation are affected by multiple host genes separately.     

Differential contribution of M(r) 120 kDa rasGTPase-activating protein and neurofibromatosis type 1 gene product during the transition from growth phase to arrested state in human fibroblasts accompanied by a unique rasGTPase-activating activity

Production of IgG3 in MRL/Mp-lpr/lpr (MRL/lpr) lupus mice is one of the major factors to develop glomerulonephritis (GN) in these mice. To examine molecular characteristics of IgG3 responsible for GN in these mice, hybridoma clones producing IgG3 antibodies were prepared from one unmanipulated MRL/lpr mouse. Two clones, 2B11.3 and 7B6.8, were nephritogenic; that is, they caused severe glomerular lesions when injected to normal mice, moreover with a different histopathological manifestation. The 2B11.3 clone generated diffuse cell-proliferative lesions, while those induced by the 7B6.8 clone resembled wire loop lesions in human lupus nephritis. The cDNA sequence analysis of 7B6.8 antibody and the other IgG3 antibody, 1G3, non-nephritogenic, revealed that the C regions of the heavy and light kappa chains were completely the same between them. Furthermore, they were identical in deduced amino acid sequences to those from non-autoimmune BALB/c mice, indicating no allelic difference of Igh-8 between these two strains. The V regions of 2B11.3 and 7B6.8 antibodies were composed of different sets of VH, D, JH, Vk and Jk. Although both of the VH belonged to the J558 family, they seemed to use a different VH germline gene. These findings suggest that GN in MRL/lpr mice is generated by the expansion of clonally different B cells producing particular antibodies possibly with a different pathogenetic potency.     

Small bowel enteropathy: role of intraepithelial lymphocytes and of cytokines (IL-12, IFN-gamma, TNF) in the induction of epithelial cell death and renewal

The small bowel mucosa contains within its villus epithelium a large number of intraepithelial lymphocytes (IEL) which upon activation are cytotoxic and release large quantities of IFN-gamma and TNF; these activities are increased by in vitro exposure to IL-12. Mice injected with IL-12 develop severe damage of the villus epithelial cells, in form of apoptosis, necrosis and a third distinct form of cell death, characterized ultrastructurally by progressive cell shrinkage. These lesions are accompanied by a compensatory acceleration of the epithelial renewal, a hallmark of epithelial injury. Use of a variety of mutant mice showed that these lesions require the presence of IEL (all populations being involved, thymus-dependent as well as natural killer-T cell IEL) and the release of IFN-gamma. The critical role of IFN-gamma may result in part from its capacity to induce on epithelial cells the expression of target molecules involved in the different cytotoxic pathways used by IEL. However, injection of IFN-gamma into mutant mice lacking IEL showed that IFN-gamma can directly induce villus epithelial damage as well. On the other hand, injection of TNF induces fulminant apoptosis of villus epithelial cells, starting at the top of the villi; however TNF is not required for IL-12-induced enteropathy, which is unmodified in mutant mice lacking TNF. We propose that, when activated by their cognate ligands and/or IL-12 produced by cells in the lamina propria, IEL eliminate infected and senescent epithelial cells through a combination of cytotoxicity and of IFN-gamma and TNF release. This insures the rapid epithelial renewal of the villi, which in turn helps maintain the functional integrity of the barrier.     

Solitary pancreas allografts. The role of percutaneous biopsy and standardized histologic grading of rejection

Murine models such as NZB/W F1, NZB.H-2bm12 and MRL.lpr/lpr mice have provided greater insight into the pathogenic mechanisms of lupus. To understand further the roles of T cells and cytokines in the pathogenesis of murine lupus, 11 cloned anti-DNA antibodies augmenting autoreactive T cell lines were derived from NZB/W F1 mice. All these autoreactive cells responded to syngeneic splenic cells and helped syngeneic B cells to produce anti-DNA antibodies, especially the IgG antibody. Ten out of 11 autoreactive T cell lines expressed neither CD4 nor CD8 cell surface markers on their surface. In addition, the cytokine production pattern of these autoreactive T cell lines was predominantly of type 0 (Th0) or type 2 T helper cells (Th2). To further investigate the role of accessory molecules in the activation of these autoreactive T cell lines, expression of IL-2R and heat-stable antigen (HSA) on these autoreactive T cells was analysed. Results suggest that the HSA played a critical role in the activation and function of these double-negative cloned autoreactive T cells.