Effects of complement factor D deficiency on the renal disease of MRL/lpr mice

BACKGROUND: The alternative complement pathway (AP) is activated in individuals with lupus nephritis and in murine models of systemic lupus erythematosus, including MRL/lpr mice. A previous study from our laboratory evaluated the development of renal disease in MRL/lpr mice genetically deficient in factor B (Bf-/-), a protein necessary for AP activation. MRL/lpr Bf-/- mice developed less renal disease and had improved survival; however, these mice were also a different major histocompatibility complex (MHC) haplotype (H-2b) than their wild-type littermates (H-2k) due to the gene for Bf being located in the MHC gene complex. We undertook the current study to determine if the decreased renal disease in MRL/lpr Bf-/- mice was due to the lack of AP activation or the H-2b haplotype by studying the effects of factor D (Df) deficiency, a critical protein for AP activation, on disease development in MRL/lpr mice. METHODS: Df-deficient mice were backcrossed with MRL/lpr mice for four to nine generations. MRL/lpr H-2k Df-/-, Df+/-, and Df+/+ littermates were evaluated for disease development. Lack of AP activation in MRL/lpr Df-/- mice was determined by the zymosan assay. Serum creatinine levels were measured using a creatinine kit. Proteinuria and autoantibody levels were determined by enzyme-linked immunosorbent assay (ELISA). Sections from one kidney were stained with fluorescein isothiocyanate (FITC) alpha-murine C3 or alpha-murine IgG to detect C3 and IgG deposition. The remaining kidney was cut in half with one half fixed, sectioned, and stained with hematoxylin and eosin and periodic acid-Schiff (PAS) to evaluate pathology and another half fixed in glutaraldehyde and examined via electron microscopy. RESULTS: MRL/lpr Df-/- mice had similar glomerular IgG deposition, proteinuria and autoantibody levels, as Df+/+ and Df+/- littermates. However, glomerular C3 deposition, serum creatinine levels, and pathologic renal disease were significantly reduced in Df-/- mice. Despite the lack of renal disease in Df-/- mice, life span was not impacted by factor D deficiency. CONCLUSION: The absence of Df and AP activation is protective against the development of proliferative renal disease in MRL/lpr mice suggesting the similar effect of Bf deficiency in MRL/lpr mice was also due to the lack of AP activation.     

Retinoic acid treatment protects MRL/lpr lupus mice from the development of glomerular disease

BACKGROUND: Retinoic acid (tRA) is an active metabolite of vitamin A with potent anti-inflammatory properties. We analyzed the effects of tRA on the development of lupus nephritis in MRL/lpr mice. METHODS: MRL/lpr mice received chow supplemented with vehicle or tRA (daily 10 mg/kg) from 8 to 14 weeks until their sacrifice. MRL/wt mice served as an additional control. RESULTS: tRA-treated MRL/lpr mice showed reduced lymphoadenopathy and splenomegaly as compared to vehicle-treated controls. Treatment reduced proteinuria to almost basal levels. Plasma IgG and anti-DNA antibodies increased comparably in both vehicle and tRA-treated mice. Vehicle-treated mice showed characteristic renal lesions. In contrast tRA-treated mice showed almost normal glomerular histology with a pronounced reduction in endocapillary cell proliferation. T-cell and macrophage infiltrates were reduced after tRA treatment within glomeruli and interstitium as compared to vehicle-treated animals. In spite of this, immune complex and complement deposition were comparable in both groups. Adoptively transferred T cells from vehicle-treated to tRA-treated MRL/lpr mice did not induce renal lesions or proteinuria. These beneficial effects of tRA treatment were associated with reduced renal expression of chemokines and inflammatory cytokines. Surprisingly, renal transforming growth factor-beta (TGF-beta) mRNA levels of tRA-treated mice were elevated, possibly indicating that TGF-beta acts as an anti-inflammatory signal in this lupus model. CONCLUSION: tRA treatment reduces lymphoproliferation and glomerulonephritis in MRL/lpr mice. This occurs in spite of unaltered anti-DNA titers and glomerular immune complex deposition, and cannot be overcome by T-cell transfer from nephritic MRL/lpr mice.     

Administration of a soluble recombinant complement C3 inhibitor protects against renal disease in MRL/lpr mice

Complement receptor 1-related gene/protein y (Crry) in rodents is a potent membrane complement regulator that inhibits complement C3 activation by both classical and alternative pathways. To clarify the role of complement in lupus nephritis, MRL/lpr mice were given Crry as a recombinant protein (Crry-Ig) from 12 to 24 wk of age. Control groups were given saline or normal mouse IgG. Sera and urine were collected biweekly. Only 1 of 20 (5%) Crry-Ig-treated mice developed renal failure (BUN > 50 mg/dl) compared with 18 of 38 (47.4%) mice in control groups (P = 0.001). BUN levels at 24 wk were reduced from 68.8 +/- 9.7 mg/dl in control groups to 38.5 +/- 3.9 mg/dl in the Crry-Ig-treated group (P < 0.01). Urinary albumin excretion at 24 wk was also significantly reduced from 5.3 +/- 1.4 mg/mg creatinine in the control groups to 0.5 +/- 0.2 mg/mg creatinine in the Crry-Ig-treated group (P < 0.05). Of the histologic data at 24 wk, there was a significant reduction in scores for glomerulosclerosis and C3d, IgG, IgG3, and IgA staining intensity in glomeruli in complement-inhibited animals. Crry-Ig-treated animals were also protected from vasculitic lesions. Although there was no effect on relevant autoimmune manifestations such as anti-double stranded DNA titers or cryoglobulin IgG3 levels, circulating immune complex levels were markedly higher in complement-inhibited animals. Thus, inhibition of complement activation with Crry-Ig significantly reduces renal disease in MRL/lpr lupus mice. The data support the strategy of using recombinant complement C3 inhibitors to treat human lupus nephritis.     

Steroid-responsive cochlear dysfunction in the MRL/lpr autoimmune mouse.

Corticosteroids historically have been used to treat autoimmune sensorineural hearing loss, although little is known of how steroids restore normal inner ear function. Therefore, to identify a potential model for this field of research, this study examined the effects of prednisolone on auditory brain stem response thresholds in the MRL/lpr mouse model of autoimmune sensorineural hearing loss. Mice treated with prednisolone after auditory threshold elevations demonstrated significant improvement and stabilization of thresholds compared with untreated controls. MRL/lpr mice treated with steroids before the onset of autoimmune disease and cochlear dysfunction demonstrated decreased serum immune complexes, higher survival rates, and lower auditory thresholds compared with untreated controls. These positive results suggest the autoimmune mouse may be useful for studies of steroid-responsive mechanisms of the cochlea in autoimmune sensorineural hearing loss, as well as any hearing disorder in which steroid therapy is currently used.     

Modulation of renal disease in MRL/lpr mice by pharmacologic inhibition of inducible nitric oxide synthase

BACKGROUND: MRL-MPJFaslpr (MRL/lpr) mice spontaneously develop lupus-like disease characterized by immune complex glomerulonephritis and overproduction of nitric oxide (NO). Blocking NO production pharmacologically by a non-specific nitric oxide synthase (NOS) inhibitor ameliorated renal disease in MRL/lpr mice while genetically deficient inducible NOS (iNOS) mice developed proliferative glomerulonephritis similar to wild-type controls.METHODS: To clarify the role of iNOS in the pathogenesis of nephritis in MRL/lpr mice, we treated mice with two different NOS inhibitors. Either NG-monomethyl-l-arginine (L-NMMA), a nonspecific NOS inhibitor, or l-N6-(1-iminoethyl)lysine (L-NIL), an iNOS specific inhibitor, was administered in the drinking water from 10 through 22 weeks of age with disease progression monitored over time. Control mice received water alone.RESULTS: Both L-NMMA and L-NIL blocked NO production effectively in MRL/lpr mice. As expected, neither L-NNMA nor L-NIL had an effect on antibody production, immune complex deposition or complement activation. Although both NOS inhibitors decreased protein excretion, L-NMMA was more effective than L-NIL. Pathologic renal disease was significantly decreased at 19 weeks in both treatment groups. At 22 weeks the L-NIL treated mice, but not the L-NMMA mice, had significantly reduced renal disease scores compared to controls.CONCLUSION: These results indicate that specific inhibition of iNOS blocks the development of pathologic renal disease in MRL/lpr mice.     

Chemokine receptor Ccr2 deficiency reduces renal disease and prolongs survival in MRL/lpr lupus-prone mice

MRL/MpJ-Fas(lpr)/J (MRL/lpr) mice represent a well-established mouse model of human systemic lupus erythematosus. MRL/lpr mice homozygous for the spontaneous lymphoproliferation mutation (lpr) are characterized by systemic autoimmunity, massive lymphadenopathy associated with proliferation of aberrant T cells, splenomegaly, hypergammaglobulinemia, arthritis, and fatal immune complex-mediated glomerulonephritis. It was reported previously that steady-state mRNA levels for the chemokine (C-C motif) receptor 2 (Ccr2) continuously increase in kidneys of MRL/lpr mice. For examining the role of Ccr2 for development and progression of immune complex-mediated glomerulonephritis, Ccr2-deficient mice were generated and backcrossed onto the MRL/lpr genetic background. Ccr2-deficient MRL/lpr mice developed less lymphadenopathy, had less proteinuria, had reduced lesion scores, and had less infiltration by T cells and macrophages in the glomerular and tubulointerstitial compartment. Ccr2-deficient MRL/lpr mice survived significantly longer than MRL/lpr wild-type mice despite similar levels of circulating immunoglobulins and comparable immune complex depositions in the glomeruli of both groups. Anti-dsDNA antibody levels, however, were reduced in the absence of Ccr2. The frequency of CD8+ T cells in peripheral blood was significantly lower in Ccr2-deficient MRL/lpr mice. Thus Ccr2 deficiency influenced not only monocyte/macrophage and T cell infiltration in the kidney but also the systemic T cell response in MRL/lpr mice. These data suggest an important role for Ccr2 both in the general development of autoimmunity and in the renal involvement of the lupus-like disease. These results identify Ccr2 as an additional possible target for the treatment of lupus nephritis.     

Compartmental distribution of complement activation products in artificial kidneys

The compartmental distribution of the human anaphylatoxins C3a and C5a has been defined during simulated hemodialysis performed with various types of hemodialyzers. New cuprophan hollow fiber dialyzers were found to activate human complement very readily in vitro, while re-used cuprophan dialyzers displayed only modest complement activating potential. The C3a and C5a antigens, formed as a result of complement activation in these dialyzers, accumulated predominantly in the blood path and were not adsorbed extensively on the membrane surface or transported into the dialysate compartment. Cellulose acetate membranes also produced complement activation in vitro, but to a lesser degree than new cuprophan hollow fibers. However, these membranes exhibited a significant capacity to bind the anaphylatoxins to their surface. Polyacrylonitrile membranes appeared to be unique in that they not only failed to activate complement significantly, but they rapidly adsorbed large quantities of C3a and C5a. These findings demonstrate that hemodialysis membranes may differ with regard to their complement activating potential as well as their ability to remove circulating anaphylatoxins from the blood path. Clinical measurements of anaphylatoxin production during hemodialysis reflect these dynamic events.     

Nephritogenic cytokines and disease in MRL-Fas(lpr) kidneys are dependent on multiple T-cell subsets

BACKGROUND: Renal parenchymal cells produce cytokines, colony-stimulating factor-1 (CSF-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha), which recruit autoreactive T cells and, in turn, elicit renal injury in MRL-Fas(lpr) mice. METHODS: To determine whether select T-cell populations regulate intrarenal nephritogenic cytokines (CSF-1, GM-CSF, and TNF-alpha) and renal disease, we compared MRL-Fas(lpr) mice that are genetically deficient in T-cell receptor (TCR) alpha beta T cells, CD4 T cells, and major histocompatibility complex class I (MHC class I), lacking CD8 and double negative (DN) T cells, with wild-type mice. To identify the T cells instrumental in downstream (effector) events, we delivered CSF-1 or GM-CSF into the kidney via gene transfer in these select T-cell-deficient and wild-type strains. RESULTS: Intrarenal CSF-1, GM-CSF, and TNF-alpha were absent or dramatically reduced in TCR alpha beta, CD4, and class I-deficient MRL-Fas(lpr) strains as compared with wild-type mice. In addition, the decrease in CSF-1, GM-CSF, and TNF-alpha was associated with a reduced kidney leukocytic infiltrates and spontaneous autoimmune nephritis. Intrarenal ex vivo retroviral gene transfer of CSF-1 and GM-CSF failed to elicit nephritis in these T-cell-deficient MRL strains (TCR alpha beta, CD4, CD8/DN) as compared with wild-type mice. CONCLUSIONS: Multiple T-cell populations initiate renal disease by increasing intrarenal nephritogenic cytokines, CSF-1, GM-CSF, and TNF-alpha. CSF-1 and GM-CSF recruit additional CD4 and CD8 and DN T cells, which augment downstream events, resulting in progressive autoimmune renal disease. We suggest that MRL-Fas(lpr) kidney disease is driven by a T-cell amplification feedback loop dependent on multiple T-cell populations.     

Increased survival and reduced renal injury in MRL/lpr mice treated with a novel sphingosine-1-phosphate receptor agonist

Agonists of the type 1 sphingosine-1-phosphate (S1P) receptor inhibit lymphocyte migration, causing their sequestration in lymphoid tissue. The S1P agonist FTY720 prolongs the survival of organ allografts and blocks T-cell mediated autoimmune diseases in experimental models; however, it is a non-selective agonist of four of the five S1P receptors. In this study female MRL/lpr mice, which develop an aggressive form of spontaneous autoimmune kidney disease, were treated with a more selective agonist of the type 1 receptor (KRP-203) or vehicle at 12 or 16 weeks of age. Eighty percent of the mice treated at 12 weeks, before the onset of visible disease, survived to the 24 weeks end point with decreased tubulointerstitial disease and significantly fewer infiltrating CD4(+) and CD8(+) T-cells. Only half of the control vehicle-treated mice survived. All of the mice treated at 16 weeks survived with reduced proteinuria. Mice in both groups had significant reductions in circulating lymphocytes. Mice receiving KRP-203 for 8-12 weeks had significant reductions in T-cells and consequently less adenopathy. Ex vivo treatment of lymphocytes from MRL/lpr mice with KRP-203 enhanced their apoptosis. Our study indicates that KRP-203 attenuates kidney injury in MRL/lpr mice, in part, by reducing T-cell infiltrates.     

Spiegelmer inhibition of CCL2/MCP-1 ameliorates lupus nephritis in MRL-(Fas)lpr mice

The monocyte chemoattractant protein CCL2 is crucial for monocyte and T cell recruitment from the vascular to the extravascular compartment at sites of inflammation. CCL2 is expressed in human lupus nephritis and was shown to mediate experimental lupus; therefore, CCL2 antagonists may be beneficial for therapy. This study describes the l-enantiomeric RNA oligonucleotide mNOX-E36, a so-called Spiegelmer that binds murine CCL2 with high affinity and neutralizes its action in vitro and in vivo. The mirror image configuration of the Spiegelmer confers nuclease resistance and thus excellent biostability. mNOX-E36 does not induce type I IFN via Toll-like receptor-7 or cytosolic RNA receptors, as recently shown for certain synthetic D-RNA. Autoimmune-prone MRL(lpr/lpr) mice that were treated with a polyethylene glycol form of mNOX-E36 from weeks 14 to 24 of age showed prolonged survival associated with a robust improvement of lupus nephritis, peribronchial inflammation, and lupus-like inflammatory skin lesions. Thus, mNOX-E36-based inhibition of CCL2 represents a novel strategy for the treatment of autoimmune tissue injury, such as lupus nephritis.     

IgM‐dependent activation of the lectin pathway of complement in xenotransplantation

Background: The classical pathway is the dominant initiator of complement activation in xenotransplantation. By amplification of C3b generation, the alternative pathway is also critical. However, little attention has been paid up to date to the involvement of the lectin pathway in xenograft rejection. Natural IgM, containing anti‐Gal, is a major initiator of classical pathway complement activation, but recently it has been shown that during ischemia/reperfusion injury, IgM also induces lectin pathway activation. Thus, the present study was focused on lectin pathway as well as interaction of IgM and MBL in a pig‐to‐human in vitro xenotransplantation model. Methods: Cell ELISA using porcine aortic endothelial cells (PAEC) and normal human serum (NHS) was used to assess activation of the different complement pathways. To confirm activation of the lectin pathway and to analyze the role of natural IgM in it's activation, co‐localized deposition of MBL/MASP2 with C3b/c, C4b/c & C6 and IgM with MBL & MASP2 was investigated by immunofluorescence (IF)/confocal microscopy on PAEC. Influence of IgM presence on MBL binding to PAEC was tested using IgM depleted/repleted and anti‐Gal immunoabsorbed NHS. Finally, tissue samples from ex vivo xenoperfusion of pig limbs with whole human blood were tested for IgM mediated lectin pathway activation by IF staining. Results: Activation of all the three pathways of complement system was observed in vitro as indicated by IgM, C1q, MBL and Factor Bb binding on PAEC. MBL deposition was co‐localized with MASP2, C3b/c, C4b/c and C6, suggesting a predominant role of the lectin pathway in xenograft rejection. IgM co‐localization with MBL and MASP2 as well as dose‐dependently increased deposition of MBL on PAEC in the presence of human polyclonal IgM, further supports the idea that upon deposition of IgM a binding site for MBL is exposed. In addition, co‐localized deposition of MBL with IgM, C4b/c and C6 was also observed on ex vivo xenoperfusion samples. Conclusion: The lectin pathway of complement activation was shown to be involved in xenotransplantation. Co‐localization of MBL / MASP2 with IgM and complement proteins indicate that lectin pathway activation in xenotransplantation is dependent on antigen recognition by naturalIgM. These findings suggest that, similar to ischemia/reperfusion injury, the lectin pathway has a functional role in endothelial damage in xenotransplantation.     

三氧化二砷及5-氮胞苷对狼疮鼠的免疫状态和基因甲基化的影响

目的：探讨三氧化二砷（arsenic trioxide，ATO）及5-氮胞苷（5-Aza）对MRL／lpr狼疮小鼠免疫状况和基因甲基化的影响。方法：12周龄MRL／lpr小鼠36只，随机分为3组分别给ATO、5-Aza与生理盐水（NS），疗程为60d。分别检测治疗前后抗dsDNA抗体，治疗后用高效液相方法检测脾脏、淋巴结、胸腺和血液DNA甲基化水平。结果：（1）治疗后ATO组血清抗dsDNA抗体明显低于NS组与5-Aza组（P〈0．01）；（2）治疗后ATO组脾脏及淋巴结重量较NS组与5-Aza组有明显减轻（P〈0．05）；（3）治疗后ATO组脾脏及淋巴结的甲基化水平较Ns组有明显升高（P〈0．05），而血液及胸腺甲基化水平两组间差异无统计学意义，治疗后5-Aza组脾脏、淋巴结及血液的甲基化水平较NS组明显下降（P〈0．05）。结论：ATO能够降低小鼠外周血抗dsDNA抗体水平并抑制淋巴组织的增生，明显提高脾脏和淋巴结的基因甲基化水平。     

Acute renal failure in endotoxemia is dependent on caspase activation

In previous work, it was demonstrated that apoptosis occurs in the kidney during LPS-induced acute renal failure (ARF). However, the relative importance of apoptosis in LPS-induced ARF remained unproven. Because the caspase enzyme cascade is responsible for carrying out apoptosis, it was hypothesized that treatment with a caspase inhibitor would protect mice from LPS-induced ARF. C57BL/6 mice received an injection of LPS and were treated with either the broad-spectrum caspase inhibitor z-VAD-fmk or vehicle and compared with unmanipulated mice. LPS induced a significant increase in caspase-3 activity in vehicle-treated mice, which was significantly inhibited by z-VAD. Mice that were treated with z-VAD were protected from ARF and demonstrated significantly less apoptosis as measured by both terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining and DNA laddering. Although apoptosis is classically described as a noninflammatory process, z-VAD treatment significantly attenuated multiple markers of inflammation, such as renal neutrophil infiltration and renal expression of the neutrophil chemotactic factor macrophage inflammatory protein-2. Thus, caspase inhibition may protect against LPS-induced ARF not only by preventing apoptotic cell death but also by inhibiting inflammation. These data raise the possibility that apoptotic kidney cells may actually be a source of this local inflammation, contributing to subsequent nonapoptotic renal injury.     

Targeted inhibition of complement activation prevents features of preeclampsia in mice

Preeclampsia is a major cause of maternal and neonatal morbidity and mortality. In mouse models, complement activation in the placenta is associated with abnormal placental development and miscarriage, and inhibiting complement prevents fetal injury. We mated two mouse strains, DBA/2 and CBA/J, expecting that the pregnancies might show features of preeclampsia and of immunologically mediated pregnancy loss. Along with placental dysfunction, these matings resulted in proteinuria, elevated BUN, fibrin deposition, and glomerular endotheliosis. We blocked placental complement activation throughout pregnancy by administering a single dose of the C3 inhibitor CR2-Crry given on day 5 of the pregnancy. This procedure specifically targets the sites of complement activation without inducing any systemic effects. Placental complement inhibition prevented oxidative stress and placental dysfunction, as well as proteinuria and renal pathologic features of preeclampsia. Thus, local blockade of complement activation at the maternal-fetal interface rescues preeclampsia in mice, and identifies new treatments. Hence, complement triggers a feed-forward cycle of placental damage, antiangiogenic factor production, and maternal vascular damage in patients.     

Aggregation of human platelets induced by porcine endothelial cells is dependent upon both activation of complement and thrombin generation

Abstract: The binding of human xenoreactive antibody (XNA) to porcine endothelium with complement (C) activation via the classical pathways is considered the major event triggering hyperacute rejection (HAR) with microvascular thrombosis in vivo. As C components are linked to key events in blood coagulation, we have examined pathways whereby activation of complement by endothelial cells results in xenogeneic platelet activation in vitro. Methods: Cultured porcine aortic endothelial cells (pEC), human aortic endothelial cells (HAEC) or human umbilical vein endothelial cells (HUVEC) were prepared in suspension (5 × 10⁶/ml) using EDTA-collagenase. Human platelet rich plasma (PRP) and washed platelets with platelet poor plasma (PPP) were prepared from control, drug free, volunteer donors. All aggregation tests used a two-sample, four-channel, model 560 Ca Lumi-Aggregometer (Chronolog Corporation, Havertown, PA). Selected assays for complement (C3a; C5b-C9; CH50; and AP50) were then performed on supernatant fluids. To test the effects of complement inhibition and thrombin antagonists, the following agents were pre-incubated with PRP (or PPP) in various titrations for 10 min at 37°C prior to combination with pEC in the aggregometer: soluble complement receptor typel (sCR1); Cobra Venom Factor (CVF), heparin, hirudin, and anti-CD31 (anti-PECAM). In addition, pEC, HAEC, and HUVEC were incubated with 10–20% human PPP; supernatant fluids were harvested at various time points and used for platelet activation assays and for functional tests of thrombin or levels of thrombin-antithrombin complexes (TAT). Results: pEC but not HAEC/HUVEC resulted in activation of PRP or washed platelets only in the presence of supplemental PPP. Platelet activation could be inhibited by pre-incubation of samples with CVF (2–5 IU/ml to deplete complement components) and to a variable extent with sCRl (1–2 mg/ml). Complement assays confirmed activation of C3 by the classical pathway with reduction of the CH50 while C6 deficient samples also supported platelet activation. Aggregation of platelets was inhibited by preincubation of PRP with concentrations of hirudin (1 unit/ml) and heparin (5–10 units/ml) sufficient to neutralize thrombin generated. Supernatant fluids containing PPP removed from pEC were found to have increased levels of thrombin and thrombin-antithrombin complexes and could also activate platelets in a hirudin sensitive manner. Discussion: Platelet and pEC combinations underwent aggregation only in the presence of complement components. This process appeared independent, at least in part, of the assembly of terminal complement components. Consequent pEC generation of thrombin appeared adequate to trigger platelet aggregation which could in turn be inhibited by hirudin or heparin in vitro.