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Preclinical Testing of Viral Therapeutic Efficacy in Pristane-Induced Lupus Nephritis and Diffuse Alveolar Hemorrhage Mouse Models

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Abstract

Systemic lupus erythematosus (SLE) is a multifactorial and heterogeneous autoimmune disease involving multiple organ systems and tissues. Lupus nephritis occurs in approximately 60% of patients with SLE and is the leading cause of morbidity. Diffuse alveolar hemorrhage (DAH) is a rare but very serious complication of SLE with a greater than 50% associated mortality. The etiology of SLE is unclear but has proposed genetic, hormonal, and environmental aspects. Pristane is a saturated terpenoid alkane and has become the most popular laboratory model for inducing lupus in mice. The pristane model of SLE has the capacity to reproduce many components of the human presentation of the disease. Previous studies have demonstrated that virus-derived immune-modulating proteins have the potential to control inflammatory and autoimmune disorders. Serp-1, a 55 kDa secreted and highly glycosylated immune modulator derived from myxoma virus (MYXV), has potent immunomodulatory activity in models of vasculitis, viral sepsis, collagen-induced arthritis, and transplant rejection. This chapter describes the mouse preclinical pristane lupus model as a method to examine virus-derived protein efficacy for treating autoimmune diseases and specifically lupus nephritis and DAH.

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Objective: In vitro studies suggest that the type I interferon (IFN) signature seen in most lupus patients results from Fcγ receptor-mediated uptake of nucleic acid containing immune complexes by plasmacytoid dendritic cells and engagement of endosomal Toll-like receptors. We re-examined the pathogenesis of the IFN-signature in vivo. Methods: Lupus was induced by injecting mice with pristane. Some mice were treated with normal immunoglobulin or with cobra venom factor (CVF) to deplete complement. The IFN signature was evaluated by PCR. The IFN signature also was determined in C4-deficient patients and controls. Results: Wild type C57BL/6 mice with pristane-induced lupus developed a strong IFN-signature, which was absent in immunoglobulin-deficient (μMT), C3-/-, and CD18-/- mice. Intravenous infusion of normal IgM, but not IgG, restored the IFN signature in μMT mice and the IFN signature in wild type mice was inhibited by depleting complement, suggesting that opsonization by IgM and complement is involved in IFN production. Consistent with that possibility, "natural" IgM antibodies reactive with dead cells were increased in pristane-treated wild type mice vs. untreated controls and in vivo phagocytosis of dead cells was impaired in C3-deficient mice. To examine the clinical relevance, we identified ten C4-deficient patients with lupus-like disease and compared them with 152 C4-intact patients and 21 healthy controls. In comparison with C4-intact patients, C4-deficient patients had a different clinical/serological phenotype and lacked the IFN-signature. Conclusions: These studies define a previously unrecognized role of natural IgM, complement, and complement receptors in generating the IFN signature in lupus. This article is protected by copyright. All rights reserved.
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The complement component C1q is known to play a controversial role in the pathogenesis of systemic lupus erythematosus, but the underlying mechanisms remain poorly understood. Intraperitoneal injection of pristane induces a lupus-like syndrome whose pathogenesis implicates the secretion of type I IFN by CD11b(+) Ly6C(high) inflammatory monocytes in a TLR7-dependent fashion. C1q was also shown to influence the secretion of IFN-α. In this study, we explored whether C1q deficiency could affect pristane-induced lupus. Surprisingly, C1qa(-/-) mice developed lower titers of circulating Abs and milder arthritis compared with the controls. In keeping with the clinical scores, 2 wk after pristane injection the peritoneal recruitment of CD11b(+) Ly6C(high) inflammatory monocytes in C1qa(-/-) mice was impaired. Furthermore, C1q-deficient pristane-primed resident peritoneal macrophages secreted significantly less CCL3, CCL2, CXCL1, and IL-6 when stimulated in vitro with TLR7 ligand. Replenishing C1q in vivo during the pristane-priming phase rectified this defect. Conversely, pristane-primed macrophages from C3-deficient mice did not show impaired cytokine production. These findings demonstrate that C1q deficiency impairs the TLR7-dependent chemokine production by pristane-primed peritoneal macrophages and suggest that C1q, and not C3, is involved in the handling of pristane by phagocytic cells, which is required to trigger disease in this model.
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Background: Lupus nephritis is an autoimmune inflammatory disease and urgently needs effective anti-inflammation therapies. A20, tumor necrosis factor alpha induced protein 3 (TNFAIP3), is a key negative regulator of inflammation, however whether A20 can regulate lupus nephritis has not been clarified. This study aimed at investigating the potential therapeutic effect of A20 on renal inflammation in mouse pristine model oflupus. Methodology/principal findings: Female BALB/c mice were intraperitoneally injected with pristine to establish lupus renal injury. The levels of serum IL-1β, IL-6 and autoantibodies and the degrees of renal injury and CCL2 and F4/80 levels were measured. The levels of the NF-κB and NLRP3 inflammasome activation in peritoneal macrophages were determined. We found that injection with pristine increased the levels of serum IL-1β, IL-6, autoantibodies and CCL20 and F4/80 expression in the kidney and induced renal injury, accompanied by enhancing the NF-κB and NLRP3 inflammasome activation in macrophages of mice. In contrast, treatment with Ad-A20, but not with Ad-control, significantly mitigated pristine-induced inflammatory responses and renal injury,and reduced the NF-κB and NLRP3 inflammasome activation in macrophages in mice. Conclusion/significance: Our data indicated that induction of A20 overexpression inhibited pristane induced lupus inflammation and renal injury in mice and may be a new therapeutic strategy for treatment of lupus nephritis.
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Systemic lupus erythematosus (SLE) is a systemic inflammatory disease, characterized serologically by an autoantibody response to nucleic antigens, and clinically by injury and/or malfunction in any organ system. During their disease course, up to 50% of SLE patients will develop lung disease. Pulmonary manifestations of SLE include pleuritis (with or without effusion), inflammatory and fibrotic forms of interstitial lung disease, alveolar hemorrhage, shrinking lung syndrome, pulmonary hypertension, airways disease, and thromboembolic disease. Two major themes inform our understanding of SLE-associated pulmonary manifestations: first, the presence of specific autoantibodies correlates with the presence of certain pulmonary manifestations and second, vascular injury marks a common pathophysiologic thread among the various SLE-related lung diseases. This review will focus on the clinical presentation, pathogenesis, pathology, management, and prognosis of these SLE-associated lung conditions.
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Innate immunity provides obstacles to successful organ transplantation, which cannot be prevented by cyclosporine (CsA). Here we have determined the potential of a myxoma viral serpin, Serp-1, with proven anti-inflammatory and antiatherogenic actions, to modulate innate immunity and contribute synergistically with CsA in the prevention of acute cardiac allograft rejection. Brown-Norway rat hearts were heterotopically transplanted into Lewis rats and given either a monotherapy treatment of Serp-1, a subtherapeutic dose of CsA, or the two drugs in combination. A brief treatment of Serp-1 alone, or a subtherapeutic dose of CsA, resulted in a marked decrease in intragraft macrophage infiltration and downregulation of toll-like receptor (TLR)-2, TLR4 and MyD88 at 48 hours posttransplantation, which was associated with significantly reduced numbers of mature dendritic cells. A significant reduction in intragraft T-lymphocyte infiltration was observed with both Serp-1 monotherapy and Serp-1 and CsA combination therapy, with the combination treatment achieving indefinite graft survival (>100 days) with normal histology. The CsA monotherapy group displayed partially reduced lymphocyte infiltration compared to the untreated controls, but failed to inhibit early innate immune graft recognition events such as macrophage infiltration and TLR 2, TLR4, and MyD88, and was ultimately unsuccessful in preventing rejection (36.3+/-7.8 days). Observed suppressive effects of Serp-1 on early innate immune response components such as TLR-2 and 4, and on adaptive responses such as T-cell intragraft infiltration suggests that Serp-1 may modulate the transition from innate to adaptive immunity, exhibiting a synergistic effect on allograft survival when used in combination with a subtherapeutic dose of CsA.
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Interferon-γ is required for lupus nephritis in mice treated with the hydrocarbon oil pristane.Background Although the precise mechanisms leading to lupus nephritis remain obscure, both TH1 and TH2 cytokines have been implicated. The present study examined the roles of interleukin (IL)-4 and interferon-γ (IFN-γ) in a novel inducible form of lupus that develops in non-autoimmune mice treated with the hydrocarbon oil pristane.MethodsBALB/c IL-4 or IFN-γ deficient mice (IL-4 −/−, IFNγ −/−) and wild type controls (+/+) received either pristane or phosphate-buffered saline (PBS) IP. Serial sera were analyzed for anti-DNA/chromatin, anti-RNP/Sm, and total immunoglobulin levels. Proteinuria was measured and kidneys were examined by direct immunofluorescence and light microscopy.ResultsRenal disease did not develop in pristane-treated IFN-γ −/− mice, as assessed by the absence of capillary immune deposits, glomerular pathology and proteinuria whereas IL-4 −/− mice developed renal disease similar to +/+ mice. Production of IgG anti-single stranded DNA and anti-chromatin antibodies was abrogated in IFN-γ −/− mice. In contrast, these autoantibodies were produced at similar or higher frequencies and levels by IL-4 −/− versus wild-type mice. The frequency of anti-nRNP/Sm was markedly reduced in IFN-γ −/− mice. IL-4 deficiency had little effect on the production of anti-DNA/chromatin and anti-nRNP/Sm.ConclusionsIFN-γ is essential for the induction of nephritis and anti-DNA/chromatin following pristane exposure in BALB/c mice, suggesting that genetic or environmental factors influencing TH1-TH2 balance could be an important determinant of renal disease in lupus.
Article
Objective To investigate whether the established impaired phagocyte function in systemic lupus erythematosus (SLE) patients also affects apoptotic cell clearance. Accumulation of apoptotic waste as a source for autoantigens that induce and maintain autoimmune responses is discussed.Methods Apoptosis was detected by morphology and propidium iodide staining. In vitro phagocytosis of autologous apoptotic cells in cultured peripheral blood mononuclear cells was evaluated microscopically. Cross-feeding experiments were performed to investigate phagocytosis of heterologous apoptotic cells by in vitro-differentiated macrophages. Furthermore, the effect of annexin V on the phagocytosis of apoptotic cells was investigated.ResultsReduced clearance of apoptotic cells in SLE patients was observed. The defective clearance appeared to reflect phagocyte dysfunction and not an abnormal execution of apoptosis. A similar picture was seen when in vitro-differentiated macrophages from control populations were treated with annexin V.Conclusion Noninflammatory engulfment phagocytosis of apoptotic cells is decreased in SLE patients. Persistently circulating apoptotic waste may encounter inflammatory removal pathways and serve as immunogen for the induction of autoreactive lymphocytes and as antigen for immune complex formation.
Article
Diffuse alveolar hemorrhage is an uncommon yet often fatal complication of systemic lupus erythematosus (SLE). Advances in the treatment of alveolar hemorrhage have been hampered due to the heterogeneity of clinical findings and the lack of suitable animal models. A single intraperitoneal injection of pristane induces a lupus-like syndrome characterized by lupus-related autoantibodies and glomerulonephritis in non-autoimmune prone strains of mice. In addition, C57BL/6 (B6) mice frequently develop alveolar hemorrhage within a few weeks of pristane injection. Immunopathogenesis of pristane-induced alveolar hemorrhage was investigated in the present study. Early (2-4 weeks after injection) mortality due to hemorrhage was unique to C57BL/6 and C57BL/10 strains of mice. Recruitment of the macrophages and neutrophils preceded the hemorrhage by several days and hemorrhage started 3-7 days after pristane injection in some mice, peaked at 2 weeks (84% in B6) and then resolved by 4 weeks in a majority of mice. Alveolar hemorrhage was independent of MyD88-, or TLR7 pathways, in contrast to autoantibody production and glomerulonephritis, and also was independent of FcγR or Fas. Rag1-/- mice had a reduced prevalence of alveolar hemorrhage compared to B6 (P = 0.01) congenics. However, T-cell receptor deficient mice developed alveolar hemorrhage at a rate comparable to wild type controls, while B6 Igμ-/- mice surprisingly had a strikingly reduced prevalence (7% vs 84% in B6, P < 0.0001). Reconstitution of B6 Igμ-/- mice with wild type B cells increased the prevalence to 50% (P = 0.028). Pristane-induced alveolar hemorrhage is a useful model to study the pathogenesis and develop new therapy for this underappreciated and often life-threatening complication of SLE.
Article
Vascular inflammation can lead to plaque instability and acute coronary syndromes (ACS). Viruses produce potent immunomodulating proteins that regulate key inflammatory pathways. A myxoma virus-derived serpin Serp-1 reduces inflammatory cell invasion and plaque growth in vascular injury models. Our objective was to evaluate the safety and efficacy of Serp-1 in patients with ACS undergoing percutaneous coronary intervention. This double-blind pilot trial included 48 ACS patients undergoing percutaneous coronary intervention randomly assigned to Serp-1 at doses of 5 μg/kg (n=19) or 15 μg/kg (n=17) or to placebo (n=12). Serp-1 was given by intravenous bolus immediately before intervention and 24 and 48 hours later. Patients were assessed for safety (primary objective) and efficacy outcomes, including biomarker analysis. In-stent neointimal hyperplasia was evaluated by intravascular ultrasound at 6 months. Key safety outcomes including coagulation parameters and adverse events did not differ between Serp-1 and placebo groups. A dose-dependent reduction in troponin I levels was observed with Serp-1 at 8, 16, 24, and 54 hours (P<0.05) and in creatine kinase-MB levels at 8, 16, and 24 hours after dose (P<0.05). The composite of death, myocardial infarction, or coronary revascularization occurred in 2 of 12 patients with placebo, 5 of 19 in the low-dose group, and none of 17 patients with the high-dose (P=0.058). Intravascular ultrasound did not detect changes in neointimal hyperplasia among groups. This is the first study of a viral serpin demonstrating its safety in ACS patients. The significant reduction in myocardial damage biomarkers supports further assessment of Serp-1 in ACS patients undergoing stent deployment. URL: http://www.clinicaltrials.gov. Unique identifier: NCT00243308.
Article
Tetramethylpentadecane (TMPD, or commonly known as pristane)-induced lupus is a murine model of systemic lupus erythematosus (SLE). Renal disease and autoantibody production strictly depend on signaling through the interferon (IFN)-I receptor. The major source of IFN-I is immature monocytes bearing high levels of the surface marker Ly6C. Interferon production is mediated exclusively by signaling through TLR7 and the adapter protein MyD88. It is likely that endogenous TLR7 ligands such as components of small nuclear ribonucleoprotein complexes are involved in triggering disease. Lupus autoantibodies are produced in ectopic lymphoid tissue developing in response to TMPD. This model is well suited for examining links between dysregulated IFN-I production and the pathogenesis of human SLE, which like TMPD-lupus, is associated with high levels of IFN-I.
Article
The authors describe four patients with systemic lupus erythematosus (SLE) and massive pulmonary hemorrhage in whom open-lung biopsies showed a distinctive small-vessel vasculitis. This lesion is characterized by acute inflammation and necrosis involving capillaries, arterioles, and small muscular arteries and is termed microangiitis to reflect the small size of the affected vessels. The involvement of capillaries is manifested by an infiltrate of necrotic neutrophils within alveolar septa often associated with destruction of the alveolar wall. This capillaritis was present in all cases, while involvement of arterioles and small arteries was seen in three. Immunofluorescence and electron microscopy demonstrated immune complexes in only two. The finding of acute microangiitis in a lung biopsy from a patient with pulmonary hemorrhage should suggest the diagnosis of SLE, and it may be a more reliable diagnostic feature than the demonstration of immune complexes.
Article
The pathogenesis of systemic lupus erythematosus is thought to be primarily under genetic control, with environmental factors playing a secondary role. However, it has been shown recently that intraperitoneal injection of pristane (2,6,10,14-tetramethylpentadecane) induces autoantibodies typical of lupus in BALB/c mice, a strain not usually considered to be genetically susceptible to the disease. In this study, the induction of autoimmune disease by pristane was investigated. BALB/c mice receiving pristane were tested for autoantibody production and histopathological evidence of glomerulonephritis. Six of 11 mice developed IgM anti-single-stranded DNA antibodies shortly after receiving pristane and 4 developed IgM anti-histone antibodies, but anti-double-stranded DNA antibodies were absent. IgG anti-DNA and anti-histone antibodies were absent. In contrast, the lupus-associated anti-nuclear ribonucleoprotein/Sm and anti-Su autoantibodies produced by these mice were predominantly IgG. In addition to autoantibodies, most of the mice developed significant proteinuria. Light microscopy of the kidney showed segmental or diffuse proliferative glomerulonephritis. Electron microscopy showed subepithelial and mesangial immune-complex deposits and epithelial foot process effacement. Immunofluorescence revealed striking glomerular deposition of IgM, IgG, and C3 with a mesangial or mesangiocapillary distribution. Thus, pristane induces immune-complex glomerulonephritis in association with autoantibodies typical of lupus in BALB/c mice. These data support the idea that lupus is produced by an interplay of genetic and environmental factors and that unlike the MRL or (NZB x W)F1 mouse models, in which genetic susceptibility factors are of primary importance, environmental factors are of considerable importance in the autoimmune disease of pristane-treated BALB/c mice.
Article
Intraperitoneal injection of pristane (2,6,10,14 tetramethylpentadecane) is a standard technique for obtaining monoclonal antibody-enriched ascitic fluid. However, pristane also induces plasmacytomas and an erosive arthritis resembling rheumatoid arthritis in BALB/c mice, probably as a consequence of enhanced interleukin 6 production. We report here that the production of autoantibodies characteristic of systemic lupus erythematosus (SLE) is a further consequence of injecting pristane in BALB/c mice. Anti-Su antibodies appeared as early as 1-2 mo after a single injection of 0.5 ml pristane, followed by anti-U1RNP and anti-Sm antibodies after 2-4 mo. Within 6 mo of pristane injection, 9 of 11 BALB/c mice had developed anti-Su, anti-U1RNP, anti-U2RNP, anti-Sm, and possibly anti-U5RNP antibodies. Autoantibodies were not produced by 20 BALB/c mice of the same age and sex that were not injected with pristane. Thus, autoantibodies characteristic of lupus were induced in mice that are not usually considered to be genetically susceptible to the disease. The induction of autoantibodies associated with SLE by pristane may be relevant to understanding the role of abnormal cytokine production in autoantibody production and the pathogenesis of autoimmune disease. Furthermore, the induction of high titer autoantibodies by pristane dictates caution in the use of ascitic fluid as a source of monoclonal antibodies, since the polyclonal antibodies induced by pristane may copurify with the monoclonal antibody secreted by an injected hybridoma.
Article
BALB/c ByJ mice develop a lupus-like syndrome characterized by anti-nRNP/Sm and Su autoantibodies and immune complex glomerulonephritis after a single i.p. pristane injection. In contrast, mercuric chloride induces anti-fibrillarin Abs only in SJL and other H-2s mice, and not in BALB/c (H-2d) mice. In the present study, the specificities of autoantibodies induced by pristane and HgCl2 were compared in SJL and BALB/c mice to examine whether these strains are "programmed" to make different sets of autoantibodies in response to nonspecific immune stimulation. Unexpectedly, the predominant autoantibodies induced by pristane in SJL mice were neither those characteristic of HgCl2-treated SJL mice nor those associated with pristane-induced disease in BALB/c mice but, rather, anti-ribosomal P, another lupus-related specificity. The autoantibodies were strongly reactive with the C-terminal 22 amino acids of the ribosomal P2 protein, indicating that they exhibited similar fine specificities to anti-P Abs in human SLE and MRL/Ipr mice. Like BALB/c mice, pristane-treated SJL mice developed severe glomerulonephritis characterized by proteinuria, mesangial proliferation, and glomerular immune complex deposits. This is the first evidence that the induction of a lupus-like syndrome by pristane is not restricted to BALB/c mice. The predominance of anti-P Abs in SJL mice contrasts sharply with the predominance of anti-nRNP/Sm and Su, in pristane-treated BALB/c mice, even though the renal lesions were similar in both strains. The data suggest that H-2s does not program mice to produce anti-fibrillarin Abs in response to nonspecific immune stimulation, arguing that autoantibody induction by pristane involves Ag-specific mechanisms.
Article
Diffuse alveolar hemorrhage (DAH) complicating systemic lupus erythematosus (SLE) remains a devastating pulmonary complication of this systemic disease. We conducted this study to review the clinicopathologic presentation and the effects of prior treatment, presence of infection, and current treatment on the survival and outcome of patients with DAH and SLE. We reviewed the records of 15 SLE patients who experienced 19 episodes of DAH over a 10-year period in a single tertiary care hospital. These patients were compared with 57 previously reported cases. The 19 episodes of DAH represented 3.7% of the 510 admissions occasioned by various complications of SLE. As previously reported, the majority (66%) were women with a median age of 27 years. The onset was often abrupt: < 3 days in 12 of the episodes. In 3 patients (20%), DAH was the initial manifestation of SLE, compared with 11% in the literature series. In the other patients in the present series, DAH appeared a median of 31 months following the diagnosis of SLE, versus 35 months in the literature series. In only 42% of the episodes in the present series, compared with 66% in the literature series, was hemoptysis present at the time of admission. However, hemoptysis eventually appeared in all 19 episodes. Temperature elevation (> 38 degrees C) was another inconsistent finding, found in only 5 episodes (26%) in the present series. The most constant concurrent systemic finding was lupus nephritis (14/15 patients). This represents a significant increase when compared with the literature series (29/48 patients). In 8 of 10 patients in whom lung tissue was available, pulmonary capillaritis accompanied the DAH. This represents a marked difference in the underlying histologic pattern when compared with the literature series. In those patients, 72% (31/43 patients) had bland pulmonary hemorrhage, and capillaritis was described in only 6 patients. The overall patient mortality rate was 53% in the current series and 50% in the literature series. Factors associated with an increased mortality in the present series include the following: mechanical ventilation (62%) versus no mechanical ventilation (0%); infection (78%) versus no infection (20%); and cyclophosphamide therapy for the acute DAH episode (70%) versus no cyclophosphamide therapy (20%). The incidence of infection in DAH and SLE (9/19 episodes) is far greater than previously reported (7/ 57 episodes). One possible explanation for this difference is the increased use of outpatient immunosuppressive therapy with monthly intravenous cyclophosphamide therapy for lupus nephritis. Eighteen DAH episodes in the present series were treated with intravenous methylprednisolone. When one combines both the current and literature series experience (16 episodes), the use of plasmapheresis does not improve survival. Of the 7 patients in the present series who survived all episodes of DAH, 6 remain alive a median of 50 months post episode and without recurrence of DAH. Diffuse alveolar hemorrhage is an uncommon but lethal complication of SLE. The survival rate remains unchanged from previous reports. The absence of hemoptysis should not exclude this diagnosis, particularly in those patients who experience an acute pulmonary syndrome with new radiographic infiltrates accompanied by falling hematocrit and the presence of a hemorrhagic bronchoalveolar lavage. Evidence for lupus nephritis is present in the great majority of cases. Most cases demonstrate the histologic pattern of pulmonary capillaritis. The mortality is adversely affected by the need for mechanical ventilation, either the presence of infection at the time of admission or the development of infection in the hospital, and the use of cyclophosphamide for treatment of the acute event.
Article
Pristane induces a lupus-like syndrome characterized by autoantibody production and glomerulonephritis in nonautoimmune strains of mice. Although it has been suggested that this syndrome results from nonspecific immune activation, there is little evidence so far that B cells are activated nonspecifically by pristane or that this promotes autoimmunity. In this study, we examined whether polyclonal hypergammaglobulinemia occurs in pristane-induced lupus, and its relationship to the production of anti-DNA, nRNP/Sm, and Su autoantibodies. In conventionally housed mice, there was a marked increase in total IgM and IgG3 2 weeks after i.p. pristane injection, followed by increased IgG1, IgG2a, and IgG2b levels. IgM levels were higher in pristane-treated specific pathogen-free (SPF) mice than in conventionally housed mice, whereas IgG and IgA levels were reduced. Pristane induced anti-nRNP/Sm and Su autoantibodies in SPF mice, but their onset was delayed and levels were lower than those in conventionally housed mice. There was no consistent relationship between total IgG1, 2a, and 2b hypergammaglobulinemia and production of anti-nRNP/Sm and Su autoantibodies. Moreover, the total Ig levels were similar in the anti-nRNP/Sm-positive and -negative groups. In contrast, production of IgM anti-ssDNA antibodies paralleled IgM hypergammaglobulinemia in some, but not all, mice. These studies indicate that pristane-induced lupus is associated with marked hypergammaglobulinemia, the magnitude of which is influenced by the microbial environment. However, anti-nRNP/Sm and Su autoantibody production is at least partly independent of polyclonal B cell activation. The data strongly suggest that pristane-induced lupus is not exclusively the consequence of nonspecific immune stimulation. They also point to the importance of microbial stimulation in the development of hypergammaglobulinemia in this inducible lupus model.
Article
Transplant vasculopathy remains a difficult therapeutic problem, resulting in the majority of late cardiac graft losses. This chronic vascular disease is thought to be triggered by alloantigen-dependent and alloantigen-independent inflammatory factors. Despite improved 1-year survival, the incidence of transplant vasculopathy has not improved with current immunosuppressive protocols. Highly effective strategies have evolved in the large DNA viruses that shield infecting viruses from host inflammatory responses. Serp-1 is a secreted myxoma virus anti-inflammatory serine proteinase inhibitor. Serp-1 inhibits plasminogen activators in a manner similar to plasminogen activator inhibitor (PAI-1), a vascular protein that plays a pivotal regulatory role in vascular wound healing. In this study, we tested the ability of purified Serp-1 protein to ameliorate posttransplant vasculopathy after rat aortic allograft surgery. Serp-1 protein or controls were infused into 98 rats immediately after segmental aortic allograft transplantation. After either late (28 days, 64 rats) or early (12 to 48 hours, 24 rats) follow-up, transplanted aortic segments were harvested for morphological and immunohistochemical analysis. Significant reductions in intimal plaque growth (P<0.002) and mononuclear cell invasion (P<0.033) were detected after Serp-1 infusion at nanogram doses. Serp-1 reduced early macrophage (P<0.0016) and nonspecific lymphocyte (P<0.0179) invasion into medial and adventitial layers and inhibited associated depletion of medial smooth muscle cells (P<0.0006). Infusion of a viral anti-inflammatory serpin, Serp-1, significantly reduces early inflammatory responses and later luminal occlusion in a rat aortic allograft model.
Article
Unlike other agents associated with drug-induced lupus, the isoprenoid alkane pristane induces autoantibodies pathognomonic of lupus, including anti-Sm, anti-dsDNA, and anti-ribosomal P in BALB/c and SJL/J mice. The susceptibility of other strains of mice to pristane-induced lupus is unknown and is the focus of the present study. Anti-nRNP/Sm, anti-Su, and anti-ribosomal P autoantibodies were produced by most strains of mice surveyed within several months of pristane treatment, although there was marked interstrain variability in their frequencies, levels, and times of onset. In sharp contrast, the production of autoantibodies against the double-stranded RNA binding proteins NF45/NF90/p110 was restricted to B6 and B10.S mice. We conclude that pristane selectively induces lupus-specific autoantibodies in virtually any strain of mouse regardless of its genetic background. However, H-2-linked as well as non-H2 genes influenced the expression of individual autoantibody markers. The widespread susceptibility of pristane-treated mice to lupus autoantibody production and the relatively small effect of MHC are unique features of this chemically induced lupus syndrome, with potential implications for understanding the pathogenesis of autoantibodies in idiopathic human systemic lupus erythematosus.
Article
Signalling cross talk provides a molecular basis for modulating a given signalling pathway by another, and it is often critical for regulating cellular responses elicited by cytokines. Previously, we reported on the critical role of the IFN-alpha/beta signalling complex, generated by spontaneously produced IFN-alpha/beta, in efficient IFN-gamma signalling. In the present study, we have demonstrated that the IFN-alpha/beta signalling complex also contributes to efficient IL-6 signalling. In fact, IL-6-induced activation of the Stat1 and Stat3 transcription factors is markedly diminished in the absence of the IFN-alpha/beta signalling complex. The induction of several target genes for these factors is also diminished, both in vitro and in vivo. We provide evidence that the cytoplasmic tyrosine residues of IFNAR-1, which remains phosphorylated by a weak IFN-alpha/beta stimulation, provide docking sites for Stat1 and Stat3 to form homo- or heterodimers following IL-6 stimulation. Furthermore, a chemical cross-linking experiment revealed that IFNAR-1 and gp130, a common signal transducer for the IL-6 family of cytokines, exist in close proximity. The constitutive weak IFN-alpha/beta signal provides a foundation for strong cellular responses to IL-6, IFN-gamma, and possibly other cytokines. Our results also suggest the assembly of cytokine receptor subunits, which may represent a 'receptosome'-like structure, allowing the unique signalling cross talks to occur.
Article
There is strong evidence that Th1 cytokines are essential for disease in murine models of lupus. Interleukin-12 (IL-12) is essential for Th1 cell differentiation and induces interferon-gamma (IFN-gamma) production. Paradoxically, it has been suggested that an IL-12 defect drives the pathogenesis of lupus, although its precise role remains unclear. We investigated the role of IL-12 for lupus-like disease induced by pristane. IL-12p35-deficient (-/-) and control (+/+) BALB/c mice were treated with pristane or phosphate-buffered saline (PBS). Proteinuria was measured and renal pathology evaluated 10 months after treatment. Sera were analyzed for autoantibodies and total immunoglobulin levels. Cytokine expression and production was analyzed. Pristane induced nephritogenic autoantibodies and renal immunoglobulin and complement deposition in both IL-12 -/- and +/+ mice. However, proliferative pathology and proteinuria were absent in IL-12-/- mice, whereas pristane induced severe nephritis in one third of the +/+ mice. As expected, cytokine balance was skewed toward a Th2 response in pristane-treated IL-12 -/- mice. These data indicate that renal immune complex deposition can occur in the absence of IL-12p35, but that structural renal damage requires the presence of IL-12p35 or mediators induced by this molecule, such as IFN-gamma. In contrast to the abrogation of nephritogenic autoantibodies by the lack of IFN-gamma, such antibodies are induced by pristane in IL-12p35-deficient mice. Absence of structural renal disease, despite the presence of nephritogenic autoantibodies in pristane-treated IL-12-/- mice, indicates that antibody deposition alone is not sufficient for the development of lupus nephritis in this model.
Article
In previous studies we have demonstrated that Serp-1, a myxoma virus encoded serine protease inhibitor, dramatically inhibits neointimal hyperplasia in vascular injury and aortic transplant models. Here we examined the effect of peritransplant Serp-1 administration on chronic renal allograft rejection. Rat renal transplants were performed with sequential recipient sacrifice on postoperative days 2, 10 and 140 to examine both the acute and chronic effects of Serp-1 in recipient rats. Serp-1 administration reduced early posttransplant injury (POD 2) with less acute tubular and vascular necrosis. This translated into a reduction of the characteristic late stage changes of chronic rejection (POD 140), with significantly decreased glomerulosclerosis and neointimal hyperplasia. Effects of Serp-1 treatment were already evident as early as POD 2 with markedly decreased levels of TGF-beta mRNA witnessed at both the early and late time points (POD 2, 10 and 140). We have demonstrated that peritransplant Serp-1 viral protein decreased early injury and allowed reduced chronic rejection in a rat renal model. Recipients treated with Serp-1 are associated with a decrease in TGF-beta mRNA levels in the allografts suggesting that the serine protease inhibitor may inhibit TGF-beta transcription and its profibrotic effects.
Article
Both spontaneous and chemically induced rodent models of autoimmune nephritis and autoantibody production have been explored to understand mechanisms involved in human systemic lupus erythematosus (SLE). While it has been known for decades that women are more susceptible than men to SLE, mechanisms underlying this female preponderance remain unclear. One chemically induced model involves injection of hydrocarbon oils such as pristane into otherwise normal mouse strains, which results in the development of autoantibodies and inflammation in organs such as kidney and liver. It is unknown whether lupus-like disease induced by chemicals would exhibit a sex bias in disease susceptibility. Here, we show that SJL/J female mice injected with pristane display greater mortality, kidney disease, serum anti-nuclear and anti-dsDNA antibodies than their male siblings. This is the first evidence that a female sex bias exists in a chemically induced lupus model.
Article
Systemic lupus erythematosus (SLE) is diagnosed according to a spectrum of clinical manifestations and autoantibodies associated with abnormal expression of type I interferon (IFN-I)-stimulated genes (ISGs). The role of IFN-I in the pathogenesis of SLE remains uncertain, partly due to the lack of suitable animal models. The objective of this study was to examine the role of IFN-I signaling in the pathogenesis of murine lupus induced by 2,6,10,14-tetramethylpentadecane (TMPD). IFN-I receptor-deficient (IFNAR(-/-)) 129Sv mice and wild-type (WT) 129Sv control mice were treated intraperitoneally with TMPD. The expression of ISGs was measured by real-time polymerase chain reaction. Autoantibody production was evaluated by immunofluorescence and enzyme-linked immunosorbent assay. Proteinuria and renal glomerular cellularity were measured and renal immune complexes were examined by immunofluorescence. Increased ISG expression was observed in the peripheral blood of TMPD-treated WT mice, but not in the peripheral blood of TMPD-treated IFNAR(-/-) mice. TMPD did not induce lupus-specific autoantibodies (anti-RNP, anti-Sm, anti-double-stranded DNA) in IFNAR(-/-) mice, whereas 129Sv controls developed these specificities. Although glomerular immune complexes were present in IFNAR(-/-) mice, proteinuria and glomerular hypercellularity did not develop, whereas these features of glomerulonephritis were found in the TMPD-treated WT controls. The clinical and serologic manifestations observed in TMPD-treated mice were strongly dependent on IFNAR signaling, which is consistent with the association of increased expression of ISGs with lupus-specific autoantibodies and nephritis in humans. Similar to its proposed role in human SLE, signaling via the IFNAR is central to the pathogenesis of autoantibodies and glomerulonephritis in TMPD-induced lupus. This lupus model is the first animal model shown to recapitulate the "interferon signature" in peripheral blood.
Lentiviral gene transfer to reduce atherosclerosis progression by long-term CC-chemokine inhibition
  • C A Bursill
  • E Mcneill
  • L Wang
  • CA Bursill