c-fms blockade reverses glomerular macrophage infiltration and halts development of crescentic anti-GBM glomerulonephritis in the rat

Depletion and adoptive transfer studies have demonstrated that macrophages induce glomerular lesions in experimental anti-glomerular basement membrane (anti-GBM) glomerulonephritis. However, there is no current therapeutic strategy that can rapidly and selectively remove these cells from the glomerulus in order to halt disease development. This study examined whether inhibition of the receptor for macrophage colony-stimulating factor (known as c-fms), which is selectively expressed by monocyte/macrophages, can eliminate the macrophage infiltrate in a rat model of crescentic anti-GBM glomerulonephritis. Wistar-Kyoto rats were treated with 10 or 30 mg/kg bid of fms-I (a selective c-fms kinase inhibitor) from the time of anti-GBM serum injection until being killed 1, 5 or 14 days later. fms-I treatment had only a minor effect upon the glomerular macrophage infiltrate on day 1 and did not prevent the subsequent induction of proteinuria. However, fms-I treatment reduced the glomerular macrophage infiltrate by 60% at day 5 and completely reversed the macrophage infiltrate by day 14. In addition, fms-I treatment downregulated the glomerular expression of pro-inflammatory molecules (TNF-α, NOS2, MMP-12, CCL2 and IL-12) on days 1 and 5, suggesting a suppression of the macrophage M1-type response. Despite a significant early loss of glomerular podocytes, ongoing proteinuria and glomerular tuft adhesions to Bowman's capsule, the reversal of the macrophage infiltrate prevented the development of glomerulosclerosis, crescent formation, tubulointerstitial damage and renal dysfunction. In conclusion, this study has identified c-fms kinase inhibition as a selective approach to target infiltrating macrophages in acute glomerular injury, which may have therapeutic potential in rapidly progressive crescentic glomerulonephritis.     

Macrophage-derived MT1-MMP and increased MMP-2 activity are associated with glomerular damage in crescentic glomerulonephritis

Membrane-type matrix metalloproteinases (MT-MMPs) have been shown to activate pro-MMP-2 on the cell surface and are suggested to be key enzymes in tissue remodelling under various physiological and pathological conditions. To investigate the role of MT-MMP in progressive renal injury, the gene expression and enzymatic activity of MT-MMP were examined in crescentic glomerulonephritis induced by anti-glomerular basement membrane (GBM) antibody in WKY rats. Isolated glomeruli were subjected to RNA and protein extraction 0, 1, 3, 7, 14, and 28 days after intravenous injection of rabbit anti-GBM antibody. Semiquantitative RT-PCR analysis revealed that among the three members of the MT-MMP family, mRNA expression of MT2-MMP remained unchanged and that of MT3-MMP was not observed in glomeruli during the development of nephritis. However, MT1-MMP gene expression increased from day 3 and reached maximum levels at day 7 (5.5+/-0.7-fold increase over day 0), closely associated with macrophage accumulation, crescent formation, and increased proteinuria. Gelatin zymography showed that the active from of MMP-2 emerged from day 7 and remained during the experimental period accompanied by increased proMMP-2, while no active form of MMP-2 was found in control rats. Using an antisense cRNA probe, intense signals of MT1-MMP mRNA were observed mostly in cells within the crescent and in some cells in the mesangial areas. Most of these cells were ED-1-positive macrophages, based on immunostaining of sequential sections. These results suggested that in the MT-MMP family, MT1-MMP was induced in infiltrating macrophages during the development of crescentic glomerulonephritis and possibly contributed to pathological degradation of glomerular extracellular matrices through the activation of proMMP-2.     

Leukocyte-derived interleukin-1beta interacts with renal interleukin-1 receptor I to promote renal tumor necrosis factor and glomerular injury in murine crescentic glomerulonephritis

The involvement of proinflammatory cytokines interleukin (IL)-1 and tumor necrosis factor (TNF) in crescentic glomerulonephritis (GN) is well established. Recently the requirement of intrinsic renal cell participation via their production of TNF in crescentic GN was demonstrated. The current studies address the relative contributions of leukocyte and intrinsic renal cell-derived IL-1beta in the induction of TNF production and glomerular injury by studying bone marrow chimeric mice. Leukocyte-derived IL-1beta was critical in the development of crescentic renal injury because IL-1beta(-/-)-->WT (absent leukocyte IL-1beta) chimeric mice had significantly attenuated TNF expression and were protected from the development of crescentic GN. In contrast, WT-->IL-1beta(-/-) chimeric mice (intact leukocyte but absent renal IL-1beta) developed similar TNF expression and crescentic GN to wild-type mice. To determine the cellular target for IL-1 in this model, IL-RI chimeric mice were studied. IL-1RI(-/-)-->WT chimeric (absent leukocyte IL-1RI expression) mice showed no attenuation of crescentic GN, whereas in the absence of renal IL-1RI (WT-->IL-1RI(-/-) chimeras), glomerular TNF expression and the development of crescentic GN were significantly decreased. These studies demonstrate that leukocytes are the major cellular source of IL-1beta, and that IL-1beta acts principally via the IL-1RI on intrinsic renal cells to induce TNF expression and crescentic glomerular injury.     

Role of glomerular epithelial cell injury in the pathogenesis of glomerular scarring in the rat remnant kidney model.

We investigated the roles of glomerular epithelial cell (GEC) pathology and dysfunction in the pathogenesis of glomerular scarring and attempted to separate them from direct hypertensive injury in the 5/6 nephrectomy (RK) model of glomerular injury. Male WKY rats weighing 200 g were studied 6 weeks after RK, when approximately one-half had developed systemic hypertension (systolic blood pressure > or = 150 mm Hg) (HT), and one-half were normotensive (NT). The incidence of glomerular necrosis and scarring was greatest in the HT rats (P = 0.0259), and vascular necrosis was only seen in 4 of 11 HT rats. The RK group had increased glomerular diameters (HT, 174 mu mean; NT, 171 mu; sham, 142 mu; P = 0.0014 by analysis of variance). There was foot process effacement in the HT and NT groups (HT, 104 filtration slits/100 mu glomerular basement membrane; NT, 112 mu; sham, 143 mu; P < 0.005 by analysis of variance), but GEC separation from the glomerular basement membrane was not significant in either HT or NT rats. GEC function was determined from protamine-heparin aggregate disappearance curves, and the curves, representing GEC endocytosis, were not different in either HT or NT groups compared with the sham-operated groups. These findings suggest that GEC function is preserved in RK, and the changes in glomerular size and GEC morphology are nonlethal and adaptive. The morphological appearance of the acute glomerular and vascular lesions and their presence only in HT animals is consistent with a hypertensive pathogenesis. The glomerular sclerosis seen in both HT and NT may result from either resolution of acute lesions with scarring and/or adaptive changes in glomerular structure and cellular functions other than the GEC clearance function we studied.     

Generation and characterization of the murine monoclonal antibody M-KID 2 to VLA-3 integrin.

Through the analysis of the antigenic phenotype of a recently established human renal carcinoma cell line (KJ29), we have demonstrated that alpha 3 subunit of the integrin family is selectively expressed by the plastic adherent cell subpopulation. Because of the scanty availability of monoclonal antibodies to this adhesion molecule, we have used KJ29 cell line as immunogen to raise novel murine monoclonal antibodies. We isolated an hybridoma secreting the mAb M-KID 2 of the IGg1k isotype that immunoprecipitates from intrinsically [35S]-Methionine labeled KJ29 cells, an heterodimer of 130/130 and 110/150 Kd, in reducing and nonreducing conditions respectively. This reactivity was completely abolished by immunodepletion of the cell extract with a polyclonal anti alpha 3 chain antiserum. Treatment of M-KID 2 immunoprecipitates with various solutions of pH ranging from 2 to 10.5, to dissociate alpha 3 from beta 1 chains, showed a retention of both alpha 3 beta 1 chains thus indicating that the epitope identified by mAb M-Kid 2 is likely to be constituted by the alpha 3 beta 1 heterodimer. Furthermore immunohistochemical studies on selected frozen and paraffin embedded tissues with mAb M-Kid 2 have provided staining pattern indicating the recognition of Vla-3. These findings demonstrate that mAb M-KID 2 can represent a valuable reagent for the study of Vla-3 integrin in normal and pathologic conditions.     

抗中性粒细胞胞浆抗体检测在类风湿性关节炎并发新月体性肾小球肾炎中的临床意义

目的:探讨类风湿性关节炎(rheumatoid arthritis,RA)并发新月体性肾小球肾炎(crescenticglomerulonephritis,CrGN)抗中性粒细胞胞浆抗体(antineutrophil cytoplasmic antibody,ANCA)检测的临床意义。方法:回顾性分析2000年1月至2010年12月萍乡市人民医院诊治的5例RA并发CrGN患者(RA并发CrGN组)、86例RA(RA组)和16例ANCA相关性小血管炎患者(ANCA相关性小血管炎组)的ANCA检测结果及临床病理特征。结果:RA并发CrGN组中,5例都为中年妇女,且具有7~14年的RA病龄,最初的临床症状都为血尿,从血尿发展到具有明显组织病变平均时间为17个月,ANCA检测均为核周型ANCA(pANCA)阳性,靶抗原为髓过氧化物酶(myeloperoxidase,MPO),被检肾小球平均有31%形成新月体,同时也存在肾小球硬化和间质性纤维化等症状;RA组ANCA检测均为阴性,ANCA相关性小血管炎组ANCA检测阳性,阳性率为87.5%(14/16),且均为pANCA;与ANCA相关性小血管炎组相比,RA并发CrGN组确诊年龄明显小于前者(P<0.05)。结论:RA并发CrGN发病年龄趋于年轻化,疾病的发展过程趋于漫长并且伴随轻微肾功能损伤,ANCA检测对RA并发CrGN患者早期发现、治疗具有重要的意义。     

Ultrastructural localization of vascular cell adhesion molecule-1 in proliferative and crescentic glomerulonephritis

Recent studies have demonstrated an important role of vascular cell adhesion molecule-1 (VCAM-1) in the pathogenesis of nephritis. In the present study, renal biopsy specimens from patients with proliferative and crescentic glomerulonephritis were subjected to immunoelectron microscopy using an anti-VCAM-1 monoclonal antibody. In control normal kidney tissue, VCAM-1 expression was restricted to the free surface of parietal epithelial cells. In diseased glomeruli, VCAM-1 was expressed on the free surface of parietal and visceral epithelial cells, on the luminal surface of capillary endothelial cells, on infiltrating monocyte/macrophage-like cells, on mesangial cells, and in the matrix of the expanded mesangium. There was also VCAM-1 expression on almost all cell types in the crescents, including macrophage-like cells, fibroblast-like cells, and epithelial cells. Some cells also showed VCAM-1 positivity in the rough endoplasmic reticulum and the perinuclear space. Both the glomerular capillary lumen and urinary spaces of Bowman's capsule contained positive reaction products, which were often associated with exocytosis by the surrounding cells. VCAM-1 was predominantly expressed on the basal and lateral surfaces of a few proximal tubules, but it could not be localized ultrastructurally. These findings suggest that production and secretion of VCAM-1 by both infiltrating monocyte/macrophages and resident glomerular cells may be related to the pathogenesis of proliferative and crescentic glomerulonephritis.     

Parenteral administration of an activating monoclonal antibody to the alpha1beta1 integrin in dogs

In mice, monoclonal antibody (mAb) to the alpha1 integrin abrogate gastro-intestinal damage during graft-versus-host-disease (GVHD), suggesting anti alpha1 mAb as candidates for treatment in humans as well. Our current data show that one such reagent, mAb 1B3.1, when immobilized to plastic wells via rabbit- anti murine (ram) immunoglobulin (Ig) induces a protein kinase-dependent spreading of activated human T cells. Furthermore, it significantly increases the proliferative response, and expression of interleukin-2 (IL-2) receptors (R) and CD69, of resting T cells, expressing minimal integrin on the cell surface, to sub-optimal stimulation by anti-CD3 mAb. We found, in addition, that mAb 1B3.1 a) immuno-precipitates alpha1beta1 integrins from cell-surface iodinated canine epithelial cells b) is highly reactive with canine T cells after their activation and c) inhibits adhesion of canine T cells to collagen IV. Despite the potential ability of the mAb to co-activate T cells in vitro, two dogs that received 4 injections of 0.5-0.3 mg/Kg of mAb 1B3.1 remained healthy, developing only marginal transient lymphopenia. Injection of 0.75mg/Kg in a third dog induced a more marked lymphopenia, and an additional dose of 1.0 mg/Kg 2 weeks later was followed by gastrointestinal hemorrhage. importantly, the lymphopenia was associated with a greater and more persistent decrease of CD8+ than of CD4+ T cells, leading to an increase in the CD4/CD8 ratio 24 hours after the injection. Thus, despite it's co-activating effects in vitro, administration of this mAb in vivo is feasible when appropriately dosed, and may have immuno-modulatory effects.     

厄贝沙坦对肾硬化大鼠肾小管间质中MMP-9/TIMP-1表达的影响

为探讨基质金属蛋白酶9（MMP－9）及其抑制剂（TIMP－1）在单侧肾切除后，重复注射阿霉素复制肾小球硬化大鼠肾小管－间质的表达，以及厄贝沙坦对其的影响和可能的保护作用机制。将肾小球硬化大鼠分为厄贝沙坦治疗组和对照组，设假手术组为正常对照。检测厄贝沙坦治疗4周后，肾功能和病理改变，并用免疫组化或原位杂交的方法分别检测肾小球和肾小管－间质中MMP－9／TIMP－1，转化生长因子β1（TGF－β1）的表达。结果表明肾小球硬化组肾小管中MMP－9，TIMP－1，TGF－β1显著增加。厄贝沙坦组中肾小管中上述指标表达下降。提示厄贝沙坦在延缓肾小球硬化同时，在减轻肾小管－间质纤维化方面有一定保护作用。     

The evolution of crescentic nephritis and alveolar haemorrhage following induction of autoimmunity to glomerular basement membrane in an experimental model of Goodpasture's disease

Goodpasture's, or anti-glomerular basement membrane (GBM), disease presents with rapidly progressive glomerulonephritis and lung haemorrhage, and is caused by autoimmunity to the NC1 domain of the alpha3 chain of type IV collagen (alpha3(IV)NC1). This study examines the development of crescentic nephritis and alveolar haemorrhage in a model of Goodpasture's disease, experimental autoimmune glomerulonephritis (EAG), induced in WKY rats by immunization with rat GBM in adjuvant. An increase in circulating anti-GBM antibodies and albuminuria was observed by week 2, which increased further by weeks 3 and 4, while a decrease in creatinine clearance was observed by week 2, which decreased further by weeks 3 and 4. The kidneys of animals with EAG showed linear deposits of IgG on the GBM and a transient glomerular infiltration by CD4+ T cells at week 2. By week 3 there were large deposits of fibrin in Bowman's space, and glomerular infiltration by CD8+ T cells and macrophages, accompanied by focal necrotizing glomerulonephritis with crescent formation. Ultrastructural studies showed glomerular endothelial cell swelling and epithelial cell foot process effacement at week 2. As the lesion progressed, capillary loops became occluded and the mesangium became expanded by mononuclear cells. By week 3 there was detachment of the endothelium from the GBM, and accumulation of fibrin beneath the disrupted endothelial cells and in Bowman's space. Occasional breaks were observed in the continuity of the basement membrane, and cytoplasmic projections from infiltrating mononuclear cells could be seen crossing the capillary wall between the lumen and the crescent. The lungs of animals with EAG showed patchy binding of IgG to the alveolar basement membrane (ABM) at week 2, and infiltration of the interstitium by CD8+ T cells and macrophages by weeks 3 and 4, accompanied by both interstitial and alveolar haemorrhage. Ultrastructural studies showed focal mononuclear cell infiltrates in alveolar walls at week 2. Occasional breaks were observed in the basement membrane and adjacent endothelium by weeks 3 and 4, together with accumulation of surfactant and erythrocytes within the alveolar spaces. This study defines for the first time the relationship between the immunological and pathological events during the evolution of EAG, and provides the basis for further work on the pathogenesis of Goodpasture's disease.     

Expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in human crescentic glomerulonephritis

AIMS: In glomerulonephritis, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) may play important roles in the formation of crescents. These studies are designed to evaluate the expression patterns of ICAM-1 and VCAM-1 in human crescentic glomerulonephritis and to determine the cellular origin of adhesion molecules in the crescentic lesions. METHODS AND RESULTS: We examined the expression of ICAM-1 and VCAM-1 proteins in renal biopsies with cellular (n=7), fibrocellular (n=9) or fibrous (n=4) crescentic glomerulonephritis, and six controls by immunohistochemistry. mRNA expression of ICAM-1 and VCAM-1 was further evaluated by RNA in-situ hybridization. Cytokeratin or CD68 immunohistochemistry was performed on the same sections, where in-situ hybridization had been carried out. In cellular crescents, ICAM-1 and VCAM-1 proteins were over-expressed to a similar extent. Of the three types of crescents, the extent of ICAM-1 immunopositivity was the greatest in the cellular crescents and decreased towards the fibrous crescents (P < 0.05). Yet the extent of VCAM-1 immunoreactivity was not different between the types. Fibrous crescents still contained some epithelial cells and showed only VCAM-1 expression. In the glomeruli with cellular or fibrocellular crescents, the extent of ICAM-1 immunopositivity in the glomerular tufts was significantly larger than that of VCAM-1 (P < 0.05). In an in-situ hybridization study, the mRNA expression patterns of ICAM-1 and VCAM-1 paralleled their protein expressions. A double-labelling study showed that the signal for ICAM-1 and VCAM-1 mRNAs was mainly present in cytokeratin-positive and CD68-negative cells in the crescentic lesions. CONCLUSIONS: These results suggest that glomerular parietal epithelial cells in cellular crescents up-regulate both ICAM-1 and VCAM-1, and that some epithelial cells retained in fibrous crescents persistently over-express VCAM-1, but not ICAM-1. They also suggest that ICAM-1 is involved in early leucocyte recruitment into glomeruli in crescentic glomerulonephritis.     

Blockade of integrin VLA-4 prevents inflammation and matrix metalloproteinase expression in a murine model of accelerated collagen-induced arthritis

DBA/1LacJ mice were immunized with type II collagen and boosted with bacterial lipopolysaccharide (LPS) 17 days later to induce accelerated arthritis. Clinical signs of inflammation were observed as early as Day 20. Matrix metalloproteinases MMP-2, -3, -9, and -13, but not MMP-12, mRNA levels were increased on Day 24. Administration of anti-VLA-4 antibody (mAb; 8 mg/kg/day for 3 days) at the time of LPS treatment strikingly inhibited arthritis-induced paw inflammation and histological scores, but not the increase in MMP expression. A higher dose of mAb (20 mg/kg/day for 4 days) inhibited pathology and normalized the levels of MMP mRNAs. In conclusion, the pathophysiology of this accelerated model of arthritis is VLA-4-dependent, and VLA-4-mediated events have a role in inflammation-induced MMP expression. Inhibition of arthritis-induced increases in MMP expression is not necessary to reduce pathology. This model is well suited for identifying agents that block integrin VLA-4 in vivo.     

STAT4 deficiency reduces autoantibody production and glomerulonephritis in a mouse model of lupus

To determine the respective role of the IL-12 and IL-4 pathways in the pathogenesis of systemic lupus erythematosus, we bred the Stat4 and Stat6 null alleles onto the lupus-prone mouse B6.TC, which is a congenic derivative of NZM2410. This model is characterized by abnormal splenocyte expansion, distribution and architecture, T cell activation, peripheral B cell development, production of anti-nuclear antibodies, and proliferative glomerulonephritis. STAT4 deficiency normalized the expression of each of these disease markers toward or to C57BL/6 levels. In contrast, STAT6 deficiency impacted splenocyte expansion and architecture, T cell activation, and anti-nuclear autoantibody production, but without any significant effect on B cell development or renal pathology. These results show that the IL-12/STAT4 pathway is involved in multiple disease-associated phenotypes in the B6.TC mouse. In contrast, the IL-4/STAT6 pathway regulates only a subset of disease markers that did not affect renal pathology.     

Treatment of murine immune complex glomerulonephritis with prostaglandin E2: dose--response of immune complex deposition, antibody synthesis, and glomerular damage

A model of immune complex glomerulonephritis (ICGN) produced in mice by the daily injection of apoferritin was employed to study the effect of treatment with various doses of prostaglandin E2 (PGE2) on glomerular damage, immune complex deposition, proteinuria, and serum anti-apoferritin antibody. Administration of PGE2, 200 micrograms twice daily, resulted in a significant decrease in glomerular damage and immune complex deposition, prevented the development of proteinuria, and significantly reduced serum levels of anti-apoferritin antibody. PGE2, 100 micrograms twice daily, resulted in a decrease in immune complex deposition as assessed by immunofluorescence microscopy, but this dosage did not significantly alter glomerular damage, proteinuria, or antibody levels. PGE2 dosages of 50 and 25 micrograms twice daily had no effect on any of these parameters. The protective effect of PGE2 on the development of ICGN occurred only at dosages that were associated with decreased anti-apoferritin antibody.