IL-12 drives IFN-gamma-dependent autoimmune kidney disease in MRL-Fas(lpr) mice

IL-12 is secreted by kidney tubular epithelial cells in autoimmune MRL-Fas(lpr) mice before renal injury and increases with advancing disease. Because IL-12 is a potent inducer of IFN-gamma, the purpose of this study was to determine whether local provision of IL-12 elicits IFN-gamma-secreting T cells within the kidney, which, in turn, incites injury in MRL-Fas(lpr) mice. We used an ex vivo retroviral gene transfer strategy to construct IL-12-secreting MRL-Fas(lpr) tubular epithelial cells (IL-12 "carrier cells"), which were implanted under the kidney capsule of MRL-Fas(lpr) mice before renal disease for a sustained period (28 days). IL-12 "carrier cells" generated intrarenal and systemic IL-12. IL-12 fostered a marked, well-demarcated accumulation of CD4, CD8, and double negative (CD4-CD8- B220+) T cells adjacent to the implant site. We detected more IFN-gamma-producing T cells (CD4 > CD8 > CD4-CD8- B220+) at 28 days (73 +/- 14%) as compared with 7 days (20 +/- 8%) after implanting the IL-12 "carrier cells;" the majority of these cells were proliferating (60-70%). By comparison, an increase in systemic IL-12 resulted in a diffuse acceleration of pathology in the contralateral (unimplanted) kidney. IFN-gamma was required for IL-12-incited renal injury, because IL-12 "carrier cells" failed to elicit injury in MRL-Fas(lpr) kidneys genetically deficient in IFN-gamma receptors. Furthermore, IFN-gamma "carrier cells" elicited kidney injury in wild-type MRL-Fas(lpr) mice. Taken together, IL-12 elicits autoimmune injury by fostering the accumulation of IFN-gamma-secreting CD4, CD8, and CD4-CD8- B220+ T cells within the kidney, which, in turn, promote a cascade of events culminating in autoimmune kidney disease in MRL-Fas(lpr) mice.     

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.     

IL-10 regulates murine lupus

MRL/MpJ-Tnfrsf6(lpr) (MRL/MpJ-Fas(lpr); MRL-Fas(lpr)) mice develop a spontaneous lupus syndrome closely resembling human systemic lupus erythematosus. To define the role of IL-10 in the regulation of murine lupus, IL-10 gene-deficient (IL-10(-/-)) MRL-Fas(lpr) (MRL-Fas(lpr) IL-10(-/-)) mice were generated and their disease phenotype was compared with littermates with one or two copies of an intact IL-10 locus (MRL-Fas(lpr) IL-10(+/-) and MRL-Fas(lpr) IL-10(+/+) mice, respectively). MRL-Fas(lpr) IL-10(-/-) mice developed severe lupus, with earlier appearance of skin lesions, increased lymphadenopathy, more severe glomerulonephritis, and higher mortality than their IL-10-intact littermate controls. The increased severity of lupus in MRL-Fas(lpr) IL-10(-/-) mice was closely associated with enhanced IFN-gamma production by both CD4(+) and CD8(+) cells and increased serum concentration of IgG2a anti-dsDNA autoantibodies. The protective effect of IL-10 in this lupus model was further supported by the observation that administration of rIL-10 reduced IgG2a anti-dsDNA autoantibody production in wild-type MRL-Fas(lpr) animals. In summary, our results provide evidence that IL-10 can down-modulate murine lupus through inhibition of pathogenic Th1 cytokine responses. Modulation of the level of IL-10 may be of potential therapeutic benefit for human lupus.     

Programmed death ligand 1 regulates a critical checkpoint for autoimmune myocarditis and pneumonitis in MRL mice

MRL/MpJ-Fas(lpr) (MRL-Fas(lpr)) mice develop a spontaneous T cell and macrophage-dependent autoimmune disease that shares features with human lupus. Interactions via the programmed death 1/programmed death ligand 1 (PD-1/PD-L1) pathway down-regulate immune responses and provide a negative regulatory checkpoint in mediating tolerance and autoimmune disease. Therefore, we tested the hypothesis that the PD-1/PD-L1 pathway suppresses lupus nephritis and the systemic illness in MRL-Fas(lpr) mice. For this purpose, we compared kidney and systemic illness (lymph nodes, spleen, skin, lung, glands) in PD-L1 null (-/-) and PD-L1 intact (wild type, WT) MRL-Fas(lpr) mice. Unexpectedly, PD-L1(-/-);MRL-Fas(lpr) mice died as a result of autoimmune myocarditis and pneumonitis before developing renal disease or the systemic illness. Dense infiltrates, consisting of macrophage and T cells (CD8(+) > CD4(+)), were prominent throughout the heart (atria and ventricles) and localized specifically around vessels in the lung. In addition, once disease was evident, we detected heart specific autoantibodies in PD-L1(-/-);MRL-Fas(lpr) mice. This unique phenotype is dependent on MRL-specific background genes as PD-L1(-/-);MRL(+/+) mice lacking the Fas(lpr) mutation developed autoimmune myocarditis and pneumonitis. Notably, the transfer of PD-L1(-/-);MRL(+/+) bone marrow cells induced myocarditis and pneumonitis in WT;MRL(+/+) mice, despite a dramatic up-regulation of PD-L1 expression on endothelial cells in the heart and lung of WT;MRL(+/+) mice. Taken together, we suggest that PD-L1 expression is central to autoimmune heart and lung disease in lupus-susceptible (MRL) mice.     

Activated protein C attenuates systemic lupus erythematosus and lupus nephritis in MRL-Fas(lpr) mice

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease leading to inflammatory tissue damage in multiple organs (e.g., lupus nephritis). Current treatments including steroids, antimalarials, and immunosuppressive drugs have significant side effects. Activated protein C is a natural protein with anticoagulant and immunomodulatory effects, and its recombinant version has been approved by the U.S. Food and Drug Administration to treat severe sepsis. Given the similarities between overshooting immune activation in sepsis and autoimmunity, we hypothesized that recombinant activated protein C would also suppress SLE and lupus nephritis. To test this concept, autoimmune female MRL-Fas(lpr) mice were injected with either vehicle or recombinant human activated protein C from week 14-18 of age. Activated protein C treatment significantly suppressed lupus nephritis as evidenced by decrease in activity index, glomerular IgG and complement C3 deposits, macrophage counts, as well as intrarenal IL-12 expression. Further, activated protein C attenuated cutaneous lupus and lung disease as compared with vehicle-treated MRL-Fas(lpr) mice. In addition, parameters of systemic autoimmunity, such as plasma cytokine levels of IL-12p40, IL-6, and CCL2/MCP-1, and numbers of B cells and plasma cells in spleen were suppressed by activated protein C. The latter was associated with lower total plasma IgM and IgG levels as well as lower titers of anti-dsDNA IgG and rheumatoid factor. Together, recombinant activated protein C suppresses the abnormal systemic immune activation in SLE of MRL-Fas(lpr) mice, which prevents subsequent kidney, lung, and skin disease. These results implicate that recombinant activated protein C might be useful for the treatment of human SLE.     

Membranous glomerulonephritis development with Th2-type immune deviations in MRL/lpr mice deficient for IL-27 receptor (WSX-1)

MRL/lpr mice develop spontaneous glomerulonephritis that is essentially identical with diffuse proliferative glomerulonephritis (World Health Organization class IV) in human lupus nephritis. Lupus nephritis is one of the most serious complications of systemic lupus erythematosus. Diffuse proliferative glomerulonephritis is associated with autoimmune responses dominated by Th1 cells producing high levels of IFN-gamma. The initial mounting of Th1 responses depends on the function of the WSX-1 gene, which encodes a subunit of the IL-27R with homology to IL-12R. In mice deficient for the WSX-1 gene, proper Th1 differentiation was impaired and abnormal Th2 skewing was observed during infection with some intracellular pathogens. Disruption of the WSX-1 gene dramatically changed the pathophysiology of glomerulonephritis developing in MRL/lpr mice. WSX-1-/- MRL/lpr mice developed disease resembling human membranous glomerulonephritis (World Health Organization class V) with a predominance of IgG1 in glomerular deposits, accompanied by increased IgG1 and IgE in the sera. T cells in WSX-1-/- MRL/lpr mice displayed significantly reduced IFN-gamma production along with elevated IL-4 expression. Loss of WSX-1 thus favors Th2-type autoimmune responses, suggesting that the Th1/Th2 balance may be a pivotal determinant of human lupus nephritis development.     

Aberrant macrophages mediate defective kidney repair that triggers nephritis in lupus-susceptible mice

CSF-1, required for macrophage (M?) survival, proliferation, and activation, is upregulated in the tubular epithelial cells (TECs) during kidney inflammation. CSF-1 mediates M?-dependent destruction in lupus-susceptible mice with nephritis and, paradoxically, M?-dependent renal repair in lupus-resistant mice after transient ischemia/reperfusion injury (I/R). We now report that I/R leads to defective renal repair, nonresolving inflammation, and, in turn, early-onset lupus nephritis in preclinical MRL/MpJ-Faslpr/Fas(lpr) mice (MRL-Fas(lpr) mice). Moreover, defective renal repair is not unique to MRL-Fas(lpr) mice, as flawed healing is a feature of other lupus-susceptible mice (Sle 123) and MRL mice without the Fas(lpr) mutation. Increasing CSF-1 hastens renal healing after I/R in lupus-resistant mice but hinders healing, exacerbates nonresolving inflammation, and triggers more severe early-onset lupus nephritis in MRL-Fas(lpr) mice. Probing further, the time-related balance of M1 "destroyer" M? shifts toward the M2 "healer" phenotype in lupus-resistant mice after I/R, but M1 M? continue to dominate in MRL-Fas(lpr) mice. Moreover, hypoxic TECs release mediators, including CSF-1, that are responsible for stimulating the expansion of M1 M? inherently poised to destroy the kidney in MRL-Fas(lpr) mice. In conclusion, I/R induces CSF-1 in injured TECs that expands aberrant M? (M1 phenotype), mediating defective renal repair and nonresolving inflammation, and thereby hastens the onset of lupus nephritis.     

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.     

Protective role for CCR5 in murine lupus nephritis

Leukocyte infiltration is a characteristic feature of human and experimental lupus nephritis and is closely correlated with loss of renal function. The chemokine receptor CCR5 is expressed on monocyte and T cell subsets and is thought to play an important role in recruiting these cells into inflamed organs. To investigate the functional role of CCR5 in lupus nephritis, CCR5-deficient mice were backcrossed onto the lupus-prone MRL-Fas(lpr) (MRL/lpr) genetic background. Unexpectedly, CCR5(-/-) MRL/lpr mice developed an aggravated course of lupus nephritis in terms of glomerular tissue injury and albuminuria. Deterioration of the nephritis was associated with an overall increase in mononuclear cell infiltration into the kidney, whereas renal leukocyte subtype balance, systemic T cell response, and autoantibody formation were unaffected by CCR5 deficiency. Renal and systemic protein levels of the CCR5 ligand CCL3, which can also attract leukocytes via its alternate receptor CCR1, were significantly increased in nephritic CCR5(-/-) MRL/lpr mice. Further studies revealed that the systemic increase in the CCR5/CCR1 ligand is also observed in nonimmune CCR5(-/-) C57BL/6 mice and that this increase was due to a reduced clearance, rather than an overproduction, of CCL3. Taken together, our data support the hypothesis that CCR5-dependent consumption of its own ligands may act as a negative feedback loop to restrain local chemokine levels within inflamed tissues, thereby limiting inflammatory cell influx.     

Characterization of reciprocal Lmb1-4 interval MRL-Faslpr and C57BL/6-Faslpr congenic mice reveals significant effects from Lmb3

Susceptibility to severe lupus in MRL-Fas(lpr) mice requires not only the lpr mutation but also other predisposing genes. Using (MRL-Fas(lpr) x B6-Fas(lpr))F2 (where B6 represents C57BL/6) intercrosses that utilize the highly susceptible MRL and poorly susceptible B6 backgrounds, we previously mapped CFA-enhanced systemic lupus-like autoimmunity to four loci, named Lmb1-4, on chromosomes 4, 5, 7, and 10. In the current study, we generated and analyzed reciprocal interval congenic mice for susceptibility to CFA-enhanced autoimmunity at all four Lmb loci. Although all loci had at least a slight effect on lymphoproliferation, only Lmb3 demonstrated a major effect on lymphoproliferation and anti-chromatin Ab levels. Further characterization of Lmb3, primarily by comparing MRL-Fas(lpr) with MRL.B6-Lmb3 Fas(lpr) congenic mice, revealed that it also played a significant role in spontaneous lupus, modifying lymphoproliferation, IgG and autoantibody levels, kidney disease, and survival. The less susceptible B6 Lmb3 locus was associated with a marked reduction in numbers of CD4(+) and double-negative (CD4(-)CD8(-)) T cells, particularly in lymph nodes, as well as reduced T cell proliferation and enhanced T cell apoptosis, both in vivo and in vitro. IFN-gamma-producing CD4(+) T cells were also reduced in MRL.B6-Lmb3 Fas(lpr) mice. Further mapping using subinterval congenic mice placed Lmb3 in the telomeric portion of chromosome 7. Thus, Lmb3, primarily through its effects on CD4(+) and double-negative T cells, appears to be a highly penetrant lupus-modifying locus. Identification of the underlying genetic alteration responsible for this quantitative trait locus should provide new insights into lupus-modifying genes.     

Deficiency in beta(2)-microglobulin, but not CD1, accelerates spontaneous lupus skin disease while inhibiting nephritis in MRL-Fas(lpr) nice: an example of disease regulation at the organ level

When mutations that inactivate molecules that function in the immune system have been crossed to murine lupus strains, the result has generally been a uniform up-regulation or down-regulation of autoimmune disease in the end organs. In the current work we report an interesting dissociation of target organ disease in beta(2)-microglobulin (beta(2)m)-deficient MRL-Fas(lpr) (MRL/lpr) mice: lupus skin lesions are accelerated, whereas nephritis is ameliorated. beta(2)m deficiency affects the expression of classical and nonclassical MHC molecules and thus prevents the normal development of CD8- as well as CD1-dependent NK1(+) T cells. To further define the mechanism by which beta(2)m deficiency accelerates skin disease, we studied CD1-deficient MRL/lpr mice. These mice do not have accelerated skin disease, excluding a CD1 or NK1(+) T cell-dependent mechanism of beta(2)m deficiency. The data indicate that the regulation of systemic disease is not solely governed by regulation of initial activation of autoreactive lymphocytes in secondary lymphoid tissue, as this is equally relevant to renal and skin diseases. Rather, regulation of autoimmunity can also occur at the target organ level, explaining the divergence of disease in skin and kidney in beta(2)m-deficient mice.     

Dicer insufficiency and microRNA-155 overexpression in lupus regulatory T cells: an apparent paradox in the setting of an inflammatory milieu

Systemic lupus erythematosus is a chronic autoimmune disease characterized by loss of tolerance to self-Ags and activation of autoreactive T cells. Regulatory T (Treg) cells play a critical role in controlling the activation of autoreactive T cells. In this study, we investigated mechanisms of potential Treg cell defects in systemic lupus erythematosus using MRL-Fas(lpr/lpr) (MRL/lpr) and MRL-Fas(+/+) mouse models. We found a significant increase in CD4(+)CD25(+)Foxp3(+) Treg cells, albeit with an altered phenotype (CD62L(-)CD69(+)) and with a reduced suppressive capacity, in the lymphoid organs of MRL strains compared with non-autoimmune C3H/HeOuj mice. A search for mechanisms underlying the altered Treg cell phenotype in MRL/lpr mice led us to find a profound reduction in Dicer expression and an altered microRNA (miRNA, miR) profile in MRL/lpr Treg cells. Despite having a reduced level of Dicer, MRL/lpr Treg cells exhibited a significant overexpression of several miRNAs, including let-7a, let-7f, miR-16, miR-23a, miR-23b, miR-27a, and miR-155. Using computational approaches, we identified one of the upregulated miRNAs, miR-155, that can target CD62L and may thus confer the altered Treg cell phenotype in MRL/lpr mice. In fact, the induced overexpression of miR-155 in otherwise normal (C3H/HeOuj) Treg cells reduced their CD62L expression, which mimics the altered Treg cell phenotype in MRL/lpr mice. These data suggest a role of Dicer and miR-155 in regulating Treg cell phenotype. Furthermore, simultaneous appearance of Dicer insufficiency and miR-155 overexpression in diseased mice suggests a Dicer-independent alternative mechanism of miRNA regulation under inflammatory conditions.     

Sunlight triggers cutaneous lupus through a CSF-1-dependent mechanism in MRL-Fas(lpr) mice

Sunlight (UVB) triggers cutaneous lupus erythematosus (CLE) and systemic lupus through an unknown mechanism. We tested the hypothesis that UVB triggers CLE through a CSF-1-dependent, macrophage (M?)-mediated mechanism in MRL-Fas(lpr) mice. By constructing mutant MRL-Fas(lpr) strains expressing varying levels of CSF-1 (high, intermediate, none), and use of an ex vivo gene transfer to deliver CSF-1 intradermally, we determined that CSF-1 induces CLE in lupus-susceptible MRL-Fas(lpr) mice, but not in lupus-resistant BALB/c mice. UVB incites an increase in M?s, apoptosis in the skin, and CLE in MRL-Fas(lpr), but not in CSF-1-deficient MRL-Fas(lpr) mice. Furthermore, UVB did not induce CLE in BALB/c mice. Probing further, UVB stimulates CSF-1 expression by keratinocytes leading to recruitment and activation of M?s that, in turn, release mediators, which induce apoptosis in keratinocytes. Thus, sunlight triggers a CSF-1-dependent, M?-mediated destructive inflammation in the skin leading to CLE in lupus-susceptible MRL-Fas(lpr) but not lupus-resistant BALB/c mice. Taken together, CSF-1 is envisioned as the match and lupus susceptibility as the tinder leading to CLE.     

Depletion of autoreactive plasma cells and treatment of lupus nephritis in mice using CEP-33779, a novel, orally active, selective inhibitor of JAK2

Accumulating evidence suggests that autoreactive plasma cells play an important role in systemic lupus erythematosus (SLE). In addition, several proinflammatory cytokines promote autoreactive B cell maturation and autoantibody production. Hence, therapeutic targeting of such cytokine pathways using a selective JAK2 inhibitor, CEP-33779 (JAK2 enzyme IC(50) = 1.3 nM; JAK3 enzyme IC(50)/JAK2 enzyme IC(50) = 65-fold), was tested in two mouse models of SLE. Age-matched, MRL/lpr or BWF1 mice with established SLE or lupus nephritis, respectively, were treated orally with CEP-33779 at 30 mg/kg (MRL/lpr), 55 mg/kg or 100 mg/kg (MRL/lpr and BWF1). Studies included reference standard, dexamethasone (1.5 mg/kg; MRL/lpr), and cyclophosphamide (50 mg/kg; MRL/lpr and BWF1). Treatment with CEP-33779 extended survival and reduced splenomegaly/lymphomegaly. Several serum cytokines were significantly decreased upon treatment including IL-12, IL-17A, IFN-α, IL-1β, and TNF-α. Anti-nuclear Abs and frequencies of autoantigen-specific, Ab-secreting cells declined upon CEP-33779 treatment. Increased serum complement levels were associated with reduced renal JAK2 activity, histopathology, and spleen CD138(+) plasma cells. The selective JAK2 inhibitor CEP-33779 was able to mitigate several immune parameters associated with SLE advancement, including the protection and treatment of mice with lupus nephritis. These data support the possibility of using potent, orally active, small-molecule inhibitors of JAK2 to treat the debilitative disease SLE.     

An IL-12-independent role for CD40-CD154 in mediating effector responses: studies in cell-mediated glomerulonephritis and dermal delayed-type hypersensitivity

Crescentic glomerulonephritis (GN) results from IL-12-driven Th1-directed cell-mediated responses (akin to delayed-type hypersensitivity (DTH)) directed against glomerular Ags. CD40-CD154 interactions are critical for IL-12 production and Th1 polarization of immune responses. Crescentic anti-glomerular basement membrane GN was induced in C57BL/6 (wild-type (WT)) mice (sensitized to sheep globulin) by planting this Ag (as sheep anti-mouse glomerular basement membrane globulin) in their glomeruli. Crescentic GN did not develop in CD40(-/-) mice due to significantly reduced nephritogenic Th1 responses. IL-12 was administered to CD40(-/-) mice with GN to dissect interactions between IL-12 and CD40 in inducing nephritogenic immunity and injury. Administration of IL-12 to CD40(-/-) mice restored Th cell IFN-gamma production, and up-regulated intrarenal chemokines and glomerular T cell and macrophage accumulation compared with WT control mice. Despite this, renal macrophages were not activated and renal injury and dermal DTH were not restored. Thus, CD40-directed IL-12 drives Th1 generation and effector cell recruitment but CD40 is required for activation. To test this hypothesis, activated OT-II OVA-specific CD4(+) cells and OVA(323-339)-loaded nonresponsive APCs were transferred into footpads of WT, CD40(-/-), and macrophage-depleted WT mice. WT mice developed significant DTH compared with CD40(-/-) and macrophage-depleted WT mice. This study demonstrated that CD40-induced IL-12 is required for generation of systemic Th1 immunity to nephritogenic Ags, and that IL-12 enhances Th1 effector cell recruitment to peripheral sites of Ag presentation via generation of local chemokines. Effector cell activation, renal DTH-like injury, and dermal DTH require direct Th1 CD154/macrophage CD40 interactions.     

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.     

Terminal deoxynucleotidyl transferase deficiency decreases autoimmune disease in MRL-Fas(lpr) mice.

The neonatal Ab and TCR repertoires are much less diverse, and also very different from, the adult repertoires due to the delayed onset of terminal deoxynucleotidyl transferase (TdT) expression in ontogeny. TdT adds nontemplated N nucleotides to the junctions of Igs and TCRs, and thus its absence removes one of the major components of junctional diversity in complementarity-determining region 3 (CDR3). We have generated TdT-deficient MRL/lpr, Fas-deficient (MRL-Fas(lpr)) mice, and show that they have an increased lifespan, decreased incidence of skin lesions, and much lower serum levels of anti-dsDNA, anti-chromatin, and IgM rheumatoid factors. The generalized hypergammaglobulinemia characteristic of MRL-Fas(lpr) mice is also greatly reduced, as is the percentage of CD4(-)CD8(-)B220(+) (double-negative) T cells. IgG deposits in the kidney are significantly reduced, although evidence of renal disease is present in many mice at 6 mo. CDR3 regions of both IgH and TCR from peripheral lymphocytes of MRL-Fas(lpr) mice are shorter in the absence of TdT, and there is a paucity of arginines in the IgH CDR3 regions of the MRL-Fas(lpr) TdT(-/-) mice. Because the amelioration of symptoms is so widespread, it is likely that the absence of N regions has more of an affect than merely decreasing the precursor frequency of anti-dsDNA B cells. Hence, either the T or B cell repertoires, or more likely both, require N region diversity to produce the full spectrum of autoimmune lupus disease.     

Inhibition of mesangial cell nitric oxide in MRL/lpr mice by prostaglandin J2 and proliferator activation receptor-gamma agonists

MRL/Mp-lpr/lpr (MRL/lpr) mice develop immune complex glomerulonephritis similar to human lupus. Glomerular mesangial cells are key modulators of the inflammatory response in lupus nephritis. When activated, these cells secrete inflammatory mediators including NO and products of cyclooxygenase perpetuating the local inflammatory response. PGJ2, a product of cyclooxygenase, is a potent in vitro inhibitor of macrophage inflammatory functions and is postulated to function as an in vivo inhibitor of macrophage-mediated inflammatory responses. We hypothesized that in lupus, a defect in PGJ2 production allows the inflammatory response to continue unchecked. To test this hypothesis, mesangial cells were isolated from MRL/lpr and BALB/c mice and stimulated with IL-1beta or LPS plus IFN-gamma. In contrast to the 2- to 3-fold increase in PGJ2 production by stimulated BALB/c mesangial cells, supernatant PGJ2 did not increase in MRL/lpr mesangial cell cultures. NO production in stimulated MRL/lpr and BALB/c mesangial cells, was blocked by PGJ2 and pioglitazone. These studies suggest that abnormalities in PGJ2 production are present in MRL/lpr mice and may be linked to the heightened activation state of mesangial cells in these mice.