Glomerular macrophages produce reactive oxygen species in experimental glomerulonephritis

The production of reactive oxygen species by intraglomerular macrophages was assessed in a macrophage dependent model of diffuse proliferative glomerulonephritis in rabbits. Glomerular macrophages were obtained from isolated nephritic glomeruli by short term (60 min) culture. Control macrophage populations were simultaneously obtained from peripheral blood (blood monocytes) and lung lavage fluid (alveolar macrophages). Superoxide anion (O2-), hydrogen peroxide (H2O2) and hydroxyl radical (OH.) production was assessed. Glomerular macrophage production of O2- (48.9 +/- 5.5 nmol/hr/10(6) cells), H2O2 (4.4 +/- 2.5 nmol/hr/10(6) cells) and OH. (57.8 +/- 4.7 U/hr/10(6) cells) was significantly greater than the production of reactive oxygen species seen with control monocyte populations: alveolar macrophages, O2- 9.8 +/- 2.0 nmol/hr/10(6) cells; H2O2 0.6 +/- 0.3 nmol/hr/10(6) cells; OH. 11.0 +/- 1.8 U/hr/10(6) cells; blood monocytes, O2- 8.6 +/- 1.4 nmol/hr/10(6) cells; OH. 9.9 +/- 1.2 U/hr/10(6) cells, (all P less than 0.05 cf. glom macs). Hydrogen peroxide production by blood monocytes (1.6 +/- 0.9 nmol/hr/10(6) cells) was less than glomerular macrophages, however this difference was not statistically significant. The enhanced production of reactive oxygen species by glomerular macrophages in this macrophage dependent model of glomerulonephritis suggests that these mononuclear cells are locally activated within the glomerulus following recruitment from the circulation. Reactive oxygen species production by glomerular macrophages may contribute to their ability to induce glomerular basement membrane injury in this disease.     

Interleukin-10 inhibits macrophage-induced glomerular injury

The ability of interleukin-10 (IL-10) to inhibit macrophage recruitment, activation, and proliferation in vivo was studied in a macrophage-mediated, but T cell-independent, passive anti-glomerular basement membrane antibody-induced model of glomerulonephritis (GN) in rats. Treatment with recombinant murine IL-10 resulted in dose-dependent reductions in proteinuria (high dose: 16 +/- 1 mg/24 h; low dose: 30 +/- 2 mg/24 h; control treatment: 69 +/- 6 mg/24 h; normal: 7 +/- 1 mg/24 h) and glomerular macrophage recruitment (high dose: 1.8 +/- 0.1 macrophages per glomerular cross section [c/gcs]; low dose: 5.5 +/- 0.2 c/gcs; control treatment: 12.1 +/- 0.6 c/gcs). Macrophage and intrinsic glomerular cell proliferation were reduced at both doses of IL-10, as was glomerular expression of P-selectin and monocyte chemoattractant protein-1. IL-10 treatment also resulted in a dose-dependent reduction of macrophage activation as indicated by MHC class II and IL-1beta expression. Glomerular nitrite production by isolated cultured glomeruli was reduced after IL-10 treatment in vivo (high dose: 2.3 +/- 2.3 nmol/10(4) glomeruli per 72 h; low dose: 28 +/- 5 nmol/10(4) glomeruli per 72 h; control treatment: 82 +/- 11 nmol/10(4) glomeruli per 72 h). Tumor necrosis factor-alpha production was abolished by high-dose treatment and reduced by the lower dose (3.8 +/- 3.8 pg/10(4) glomeruli per 72 h; control treatment: 249 +/- 23 pg/10(4) glomeruli per 72 h). These studies demonstrate that IL-10 directly attenuates glomerular macrophage recruitment, activation, and proliferation in vivo and can significantly attenuate macrophage-mediated GN independent of any effects on T cells.     

T lymphocyte participation in antibody-induced experimental glomerulonephritis

Macrophage accumulation is a feature of some aggressive forms of human and experimental glomerulonephritis (GN). Both antibody Fc components and T cells may cause macrophage accumulation; however, there has been no previous demonstration of T cells at the site of injury in GN, although some indirect evidence of their possible participation has been reported. Specific monoclonal anti-rat T lymphocyte antibodies W3/13, W3/25, and Ox8 were used to demonstrate T cells within the glomeruli of rats with an augmented autologous anti-GBM GN, by indirect immunofluorescence. The injury in this model has been shown to be mediated by macrophages. The T cell infiltrate consisted mainly of T helper cells, was maximal 24 hr after induction of the disease and clearly preceded the peak influx of macrophages and glomerular damage. Suppression of T cell function using cyclosporin prevented T cell accumulation and the subsequent macrophage-induced injury. Glomerular T cells were not seen in passively induced GN. These studies support a role for cell-mediated immunity in attracting macrophages and initiating injury in experimental anti-GBM antibody-induced GN.     

Differential effects of steroids on leukocyte-mediated glomerulonephritis in the rabbit

The effects of steroids on the development of injury in two models of experimental glomerulonephritis (GN), (one mediated by neutrophils, the other by macrophages) were compared. The neutrophil-associated lesion [initiated by heterologous antiglomerular basement membrane (GBM) antibody] was characterized by the development of an exudative endocapillary GN with heavy neutrophil accumulation [mean, 6.9 neutrophils/glomerular cross section (N/GCS) +/- 2.9 SD], minor macrophage infiltration [7.9 macrophages/glomerulus (M/G) +/- 2.2 SD] and heavy proteinuria (1905 mg/24 hr +/- 520 SD). Steroid-treated (methylprednisolone, 2 mg/kg/12 hr i.v.) rabbits developed a marked monocytopenia, mild neutrophilia, and significant reduction in glomerular macrophage accumulation (0.3 M/G 0.02 SD). However, neutrophil accumulation (6.1 N/CGS +/- 2.5 SD), histological appearances, and proteinuria (1820 mg/hr +/- 490 SD) were unaffected. The macrophage-associated model of GN was induced by passive autologous rabbit anti-sheep IgG 15 hr after the injection of a subnephritogenic dose of the same anti-GBM antibody. The glomerular lesion was characterized by a diffuse endocapillary proliferative GN with heavy macrophage infiltration (54 M/G +/- 8 SD), insignificant neutrophil accumulation (0.8 N/GCS 0.02 SD), and the regular development of proteinuria (420 mg/24 hr +/- 80 SD). Steroid-treated rabbits developed a mild neutrophilia and a significant monocytopenia associated with abrogation of glomerular macrophage accumulation (2.3 M/G +/- 0.8 SD). This was associated with the prevention of the development of GN and proteinuria (22 +/- 9.5 SD). Thus, steroids produce monocytopenia and prevent glomerular macrophage accumulation and associated injury whereas neutrophil accumulation and injury is unaffected. These data suggest steroids may have widely varying effects on the outcome of leukocyte-associated experimental GN depending on the nature of the infiltrating cells.     

Lymphokine (MIF) production by glomerular T-lymphocytes in experimental glomerulonephritis

Glomerular T-lymphocyte infiltration has recently been demonstrated to precede glomerular macrophage influx in a pre-immunized model of anti-glomerular basement-membrane antibody-induced glomerulonephritis (antiGBM-GN). In the current study, the functional role of these glomerular T-lymphocytes in directing macrophage localization was sought by measuring their production of macrophage migration inhibition factor (MIF). MIF activity in supernatants from cultured isolated glomeruli was measured in a conventional capillary tube bioassay. Glomerular T-lymphocytes (OX19 positive cells) were maximal (1.95 +/- 0.19 cells/glomerular cross section, c/gcs) 24 hours after injection of antiGBM antibody into sensitized animals. Seventy-two hours after antibody injection, T-lymphocyte numbers were reduced (1.02 +/- 0.14 c/gcs) while macrophage accumulation was maximal (at 24 hrs 4.2 +/- 1.3 macrophages/glomerulus (m/g), at 72 hrs 19.8 +/- 3.7 m/g). MIF activity was only detected in supernatants from T-lymphocyte infiltrated glomeruli (12 hrs 40.81 +/- 4.32% migration inhibition, 24 hrs 45.11 +/- 4.11% migration inhibition, 48 hrs 38.24 +/- 3.53% migration inhibition, 72 hrs 20.86 +/- 3.85% migration inhibition, all P less than 0.05). Control glomeruli from normal animals, pre-immunized animals given normal sheep globulin, pre-immunized animals given anti-GBM antibody and Cyclosporin A, and non-pre-immunized animals given antiGBM antibody did not contain glomerular T-lymphocytes, and their supernatants contained no MIF activity. This data indicates that the glomerular T-lymphocytes in pre-immunized antiGBM-GN are sensitized cells which release MIF and thus may direct glomerular macrophage localization in this model of antibody-induced glomerulonephritis.     

Endogenous interleukin-10 regulates Th1 responses that induce crescentic glomerulonephritis

BACKGROUND: Interleukin (IL)-10 plays a pivotal role in regulating the Th1/Th2 predominance of immune responses. Exogenously administered IL-10 suppresses nephritogenic Th1 responses, inhibits macrophage function, and attenuates crescentic glomerulonephritis (GN). To determine the role of endogenous IL-10, the development of the nephritogenic immune response and crescentic GN was compared in IL-10-deficient (IL-10-/-) and normal (IL-10+/+) C57BL/6 mice. METHODS: GN was initiated in sensitized mice by the intravenous administration of sheep antimouse glomerular basement membrane globulin. Renal injury was evaluated 21 days later. RESULTS: Following the administration of anti-glomerular basement membrane globulin, normal (IL-10+/+) C57BL/6 mice developed proliferative GN with occasional crescents, glomerular CD4+ T-cell and macrophage accumulation, and fibrin deposition. Using an identical induction protocol, IL-10-/-mice developed more severe GN. Crescent formation (IL-10-/-, 23 +/- 2% of glomeruli; IL-10+/+, 5 +/- 2%), glomerular CD4+ T cells [IL-10-/-, 1. 0 +/- 0.2 cells per glomerular cross-section (c/gcs); IL-10 +/+, 0.3 +/- 0.05 c/gcs], glomerular macrophages (IL-10-/-, 4.8 +/- 0.3 c/gcs; IL-10 +/+, 1.7 +/- 0.2 c/gcs), fibrin deposition [fibrin score (range 0 to 3+); IL-10-/-, 1.10 +/- 0.04; IL-10+/+, 0.6 +/- 0. 07], and serum creatinine (IL-10-/-, 30 +/- 2 micromol/L; IL-10 +/+, 23 +/- 1 micromol/L) were all significantly increased in IL-10-/- mice (P < 0.05). Circulating antibody (IL-10-/-, 1.05 +/- 0.16 OD units; IL-10+/+, 0.63 +/- 0.08 OD units) and cutaneous delayed-type hypersensitivity (skin swelling; IL-10-/-, 0.21 +/- 0.03 mm; IL-10+/+, 0.12 +/- 0.02 mm) to the nephritogenic antigen (sheep globulin) were also increased (both P < 0.05). Interferon-gamma production by cultured splenocytes was increased (IL-10-/- 7.9 +/- 2. 5 ng/4 x 106 cells, IL-10+/+ 0.28 +/- 0.09 ng/4 x 106 cells, P < 0. 05), but IL-4 production was unchanged. CONCLUSIONS: Endogenous IL-10 counter-regulates nephritogenic Th1 responses and attenuates crescentic GN.     

Inducible co-stimulatory molecule ligand is protective during the induction and effector phases of crescentic glomerulonephritis

The inducible co-stimulatory molecule (ICOS)/ICOS ligand (ICOSL) co-stimulatory pathway is critical in T cell activation, differentiation, and effector function. Its role was investigated in a model of Th1-driven crescentic glomerulonephritis (GN). GN was induced by sensitizing mice to sheep globulin (day 0) and challenging them with sheep anti-mouse glomerular basement membrane antibody (Ab; day 10). Disease and immune responses were assessed on day 20. For testing the role of ICOSL in the induction of GN, control or anti-ICOSL mAb were administered from days 0 to 8. For examining the role of ICOSL in the effector phase of GN, treatment lasted from days 10 to 18. Blockade of ICOSL during the induction of GN increased glomerular accumulation of CD4+ T cells and macrophages and augmented renal injury. These results correlated with attenuated splenocyte production of protective Th2 cytokines IL-4 and IL-10 and decreased apoptosis of splenic CD4+ T cells. ICOSL was upregulated within glomeruli of mice with GN. Inhibition of ICOSL during the effector phase of GN enhanced glomerular T cell and macrophage accumulation and augmented disease, without affecting the systemic immune response (cytokine production, T cell apoptosis/proliferation, Ab levels). Increased presence of leukocytes in glomeruli of mice that received anti-ICOSL mAb was associated with enhanced cellular proliferation and upregulation of P-selectin and intercellular adhesion molecule-1 within glomeruli. These studies demonstrate that ICOSL is protective during the induction of GN by augmenting Th2 responses and CD4+ T cell apoptosis. They also show that ICOSL is upregulated in nephritic glomeruli, where it locally reduces accumulation of T cells and macrophages and attenuates renal injury.     

Increased oxygen radical and eicosanoid formation in immune-mediated mesangial cell injury.

To evaluate whether monocytes/macrophages derived from glomeruli could be a source of increased eicosanoid and free oxygen radical formation in glomerular disease, monocytes/macrophage (M/M) were isolated from nephritic glomeruli and their in vitro generation of eicosanoids and superoxides were measured. Glomerular immune injury was induced by i.v. injection of a rabbit-anti-rat thymocyte antiserum (ATS). Kidneys were removed two, five, and 24 hours, and three and eight days after ATS. Adhesive glomerular macrophages were obtained by isolation of glomeruli, enzymatic digestion and incubation of the single cell suspensions in culture dishes. O2-production was evaluated by superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome C; PGE2 and TxB2 release was assessed by direct RIA. Glomerular macrophage infiltration was maximal 24 hours after intravenous antibody (35.9 +/- 5.1 M/M per glomerulus). In vitro production of superoxide was significantly enhanced (P less than 0.001) five hours after ATS administration (51.6 +/- 4.4 nmol O2/10(6) MM/hr), when compared with M/M from controls (30.4 +/- 2.0 nmol O2/10(6) MM/hr). TxB2 formation of glomerular M/M was increased (P less than 0.001) two hours and five hours after ATS administration (1056 +/- 75 and 1182 +/- 112 pg TxB2/10(6) MM/hr) compared with controls (390 +/- 34 pg TxB2/10(6) MM/hr). PGE2 synthesis, however, was decreased (P less than 0.01) at five hours after ATS (629 +/- 43 pg PGE2/10(6) MM/hr) compared with controls (950 +/- 125 pg PGE2/10(6) MM/hr). Furthermore, there was release of leukotriene B4 (LTB4) in monocytes of nephritic glomeruli five hours after ATS administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Important role for macrophages in induction of crescentic anti-GBM glomerulonephritis in WKY rats.

BACKGROUND: A crucial role for CD8(+) cells in induction of crescentic anti-glomerular basement membrane (GBM) glomerulonephritis (GN) in WKY rats was demonstrated in studies showing that depletion of CD8(+) cells completely suppressed glomerular accumulation of monocytes/macrophages (Mo/Mphi), crescent formation and proteinuria. Because these studies did not definitively identify CD8(+) cells as the cause of tissue injury, we examined the roles of Mo/Mphi in the development of anti-GBM GN. METHODS: We examined correlations between the amount of urinary protein and the numbers of glomerular CD8(+) cells or Mo/Mphi in rats after administrating different doses of anti-GBM antibody (5.0, 7.5, 10.0 and 25.0 microl/100 g body weight). The roles of Mo/Mphi in induction of GN were examined in animals by depleting Mo/Mphi in the glomerulus. To do this, rats were injected intravenously with liposome-encapsulated dichloromethylene diphosphonate (liposome-MDP) from day 3 to day 7 after anti-GBM antibody injection and they were then sacrificed at day 8. RESULTS: Liposome-MDP treatment significantly reduced the number of ED-1(+) Mo/Mphi accumulated in glomeruli from 32.1 +/- 1.2 to 1.4 +/- 0.3/glomerular cross-section (mean +/- SD, P < 0.01), and the amount of urinary protein from 103.8 +/- 19.8 to 31.8 +/- 15.9 mg/day (P < 0.01), as well as the incidence of crescentic glomeruli from 91.3 +/- 2.7 to 23.3 +/- 7.6% (P < 0.01) at day 8. This treatment also reduced the number of CD8(+) cells accumulating in the glomeruli from 5.4 +/- 0.7 to 0.5 +/- 0.1/glomerular cross-section (P < 0.01). Upregulation of glomerular intercellular adhesion molecule 1 (ICAM-1) and monocyte chemoattractant protein 1 (MCP-1) mRNA expression was suppressed by Mo/Mphi depletion. CONCLUSION: These results indicate that Mo/Mphi play an important role in the induction of crescentic anti-GBM GN and glomerular injury.     

Stimulation of PAI-1 in rabbit anti-GBM glomerulonephritis

SUMMARY: It has previously been shown in human disease and animal models of glomerulonephritis (GN) that fibrin deposition is associated with a net reduction of glomerular fibrinolytic activity as a result of reduced expression of plasminogen activators and increased expression of plasminogen activator inhibitor type 1 (PAI-1). Conditioned media (CM) prepared from cultured glomeruli of normal rabbits and rabbits 24 (Day 1) and 96 (Day 4) h after induction of anti-GBM GN were compared for their effects on the synthesis of fibrinolytic molecules in human endothelial cells (EC). Only CM from Day 4 GN rabbits showed PAI-1 protein stimulatory activity of up to 148% ( P <0.05; n = 3) above that of untreated EC. This was also seen at the mRNA level. Glomerulonephritis Day 4 CM showed significantly higher amounts of tumour necrosis factor (TNF) and thrombin and transforming growth factor-β (TGF-β) bioactivity in comparison to glomerular CM from normal rabbits. After high performance liquid chromatography (HPLC) of Day 4 GN CM, PAI-1 stimulatory activity was found to correlate with the presence of interleukin 1 (IL-1), TNF and TGF-β. These results suggest a correlation between severity of anti-GBM GN in a rabbit model, increased PAI-1 synthesis and increased expression of TNF and TGF-β. This may potentiate glomerular fibrin and extracellular matrix deposition in anti-GBM GN, leading to glomerular crescent formation and eventual renal failure.     

Stimulation of PAI-1 in rabbit anti-GBM glomerulonephritis

It has previously been shown in human disease and animal models of glomerulonephritis (GN) that fibrin deposition is associated with a net reduction of glomerular fibrinolytic activity as a result of reduced expression of plasminogen activators and increased expression of plasminogen activator inhibitor type 1 (PAI-1). Conditioned media (CM) prepared from cultured glomeruli of normal rabbits and rabbits 24 (Day 1) and 96 (Day 4) h after induction of anti-GBM GN were compared for their effects on the synthesis of fibrinolytic molecules in human endothelial cells (EC). Only CM from Day 4 GN rabbits showed PAI-1 protein stimulatory activity of up to 148% ( P <0.05; n =3) above that of untreated EC. This was also seen at the mRNA level. Glomerulonephritis Day 4 CM showed significantly higher amounts of tumour necrosis factor (TNF) and thrombin and transforming growth factor-β (TGF-β) bioactivity in comparison to glomerular CM from normal rabbits. After high performance liquid chromatography (HPLC) of Day 4 GN CM, PAI-1 stimulatory activity was found to correlate with the presence of interleukin 1 (IL-1), TNF and TGF-β. These results suggest a correlation between severity of anti-GBM GN in a rabbit model, increased PAI-1 synthesis and increased expression of TNF and TGF-β. This may potentiate glomerular fibrin and extracellular matrix deposition in anti-GBM GN, leading to glomerular crescent formation and eventual renal failure.     

Adoptive transfer studies demonstrate that macrophages can induce proteinuria and mesangial cell proliferation

BACKGROUND: Glomerular macrophage accumulation is a feature of proliferative human and experimental glomerulonephritis. However, our understanding of the role of macrophages in the induction of renal injury is based upon indirect evidence from depletion studies, most of which lack specificity for this cell type. Therefore, an adoptive transfer approach was used to directly assess the potential of macrophages to induce renal injury. METHODS: Accelerated anti-glomerular basement membrane (anti-GBM) disease was induced in rats by immunization with sheep IgG (day -5), followed by administration of sheep anti-rat GBM serum (day 0), with animals killed on day 2. To facilitate the adoptive transfer studies, immunized animals were made leukopenic by cyclophosphamide (CyPh) given on day -2. Bone marrow-derived (BM) or NR8383 macrophages were transferred by tail vein injection 24 hours after injection of anti-GBM serum, with animals killed 3 or 24 hours after transfer. RESULTS: Pretreatment with CyPh prevented glomerular leukocyte accumulation and completely inhibited proteinuria, glomerular cell proliferation and hypercellularity in accelerated anti-GBM disease. Adoptive transfer led to significant glomerular accumulation of BM or NR8383 macrophages within 3 hours of injection, and this was still evident 24 hours later. Adoptive transfer of BM or NR8383 macrophages induced proteinuria (63 +/- 16 BM vs. 5 +/- 2 mg/24 h CyPh control; P < 0.001), glomerular cell proliferation (5.1 +/- 1.2 BM vs. 0.5 +/- 0.1 PCNA+ cells/gcs CyPh; P < 0.001) and glomerular hypercellularity (51.2 +/- 2.0 BM vs. 41.9 +/- 0.9 nuclei/gcs CyPh; P < 0.001). The degree of renal injury correlated with the number of transferred glomerular macrophages. Two-color immunostaining demonstrated that most glomerular proliferative cell nuclear antigen+ (PCNA+) proliferating cells were OX-7+ mesangial cells. CyPh treatment did not prevent up-regulation of glomerular intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1) expression or an increase in urinary monocyte chemoattractant protein-1 (MCP-1) excretion. CONCLUSION: This study provides the first direct evidence that macrophages can induce renal injury in terms of proteinuria and mesangial cell proliferation.     

IL-18 translational inhibition restricts IFN-gamma expression in crescentic glomerulonephritis

BACKGROUND: Interleukin-18 (IL-18), a potent inducer of interferon gamma (IFN-gamma) production, is a cytokine involved in the cell-mediated immune response that is expressed during inflammatory and pathologic conditions. IFN-gamma plays a role in the development of some models of glomerulonephritis (GN); however, the role of IL-18 in the production of IFN-gamma during these pathologies has not been studied. METHODS: Rat IL-18 cDNA was isolated and the regulation of IL-18 gene expression was studied. IFN-gamma and IL-18 expression were determined in anti-glomerular basement membrane (GBM) antibody (Ab)-induced GN. Recombinant active IL-18 (rIL-18) was used to further identify its effect on IFN-gamma production during this GN. Glomerular injury and levels of IFN-gamma were assayed in Wistar Kyoto (WKY) rats with anti-GBM GN in the presence or absence of rIL-18. RESULTS: Rat IL-18, similar to the mouse clone, requires processing by the IL-1beta converting enzyme to become activated. A rat IL-18 5'-untranslated region (UTR) translational inhibitor was identified that strongly inhibited the synthesis of IL-18. This translational inhibitor with different lengths (180 and 130 bp) was highly expressed during GN and correlated with minimal IFN-gamma mRNA expression. Injection of recombinant active IL-18 in WKY rats with anti-GBM GN was associated with an increase of glomerular IFN-gamma levels, proliferating cell nuclear antigen (PCNA)-ED1+ cells, and PCNA-CD8+ cells, with worsening of glomerular injury. CONCLUSION: These data suggest that the translational control of IL-18 expression by its 5'-UTR limits the production of IL-18, resulting in restricted expression of mRNA and protein IFN-gamma in this model of GN. Furthermore, it was suggested that possible IL-18/IFN-gamma induction of local proliferation of macrophages and CD8+ cells might be an important mechanism for amplifying CD8+-mediated macrophage-dependent GN.     

A pathogenetic role for mast cells in experimental crescentic glomerulonephritis

Mast cells infiltrate kidneys of humans with crescentic glomerulonephritis (GN), and the degree of infiltrate correlates with outcome. However, a functional role for mast cells in the pathogenesis of GN remains speculative. GN was induced by intravenous administration of sheep anti-mouse glomerular basement membrane globulin. After 21 d, systemic immune responses and disease severity were analyzed in wild-type, mast cell-deficient (W/Wv), and bone marrow-derived mast cell-reconstituted W/Wv mice (BMMC-->W/Wv). There were no significant differences in the humoral response toward the nephritogenic antigen or in memory T cell number among the three groups; however, antigen-stimulated T cell IFN-gamma production was significantly elevated in BMMC-->W/Wv mice. Dermal delayed-type hypersensitivity in W/Wv mice was reduced compared with wild-type and BMMC-->W/Wv mice. No mast cells were detected in kidneys of W/Wv mice with GN, whereas in BMMC-->W/Wv mice, the numbers of renal mast cells were similar to wild-type mice with GN. W/Wv mice were protected from the development of crescentic GN, exhibiting reduced crescent formation (10 +/- 1% c.f. 36 +/- 2% in wild type), glomerular influx of T cells/macrophages, and interstitial infiltrate compared with wild-type mice. In contrast, BMMC-->W/Wv demonstrated a similar severity of GN as wild-type mice (35 +/- 2% crescentic glomeruli), accompanied by a prominent inflammatory cell infiltrate into glomeruli and interstitial areas. Glomerular expression of intercellular adhesion molecule-1 and P-selectin were reduced in W/Wv mice but restored to wild-type levels in BMMC-->W/Wv mice. These findings suggest that renal mast cells mediate crescentic GN by facilitating effector cell recruitment into glomeruli via augmentation of adhesion molecule expression.     

Glomerular expression of CD80 and CD86 is required for leukocyte accumulation and injury in crescentic glomerulonephritis

The participation of renal expression of CD80 and CD86 in the immunopathogenesis of crescentic Th1-mediated anti-glomerular basement membrane (anti-GBM) glomerulonephritis (GN) has not been assessed. Immunohistochemical staining demonstrated prominent upregulation of both molecules in glomeruli of mice with anti-GBM GN, suggesting a potential role for the local expression of CD80 and CD86 in nephritogenic effector T cell responses. For testing this hypothesis, control or inhibitory anti-CD80 and/or anti-CD86 mAb were administered to mice during the effector phase of the disease but after the establishment of a systemic immune response. Anti-CD80 or anti-CD86 mAb treatment had no effect on the development of GN or infiltration of leukocytes into glomeruli; however, administration of anti-CD80/86 mAb attenuated glomerular accumulation of CD4+ T cells and macrophages, crescent formation, and proteinuria, correlating with reduced antigen-specific skin delayed-type hypersensitivity. Attenuated glomerular infiltration of leukocytes in mice that were treated with anti-CD80/86 mAb was associated with decreased intraglomerular expression of adhesion molecules P-selectin and intercellular adhesion molecule-1, as well as attenuated renal mRNA levels of proinflammatory cytokines IFN-gamma and migration inhibitory factor, without reducing chemokine and chemokine receptor expression in the kidney or intraglomerular apoptosis and proliferation. The systemic Th1/Th2 balance (assessed by splenocyte production of IFN-gamma and IL-4 and circulating levels of IgG1 and IgG2a) was not affected by the inhibition of CD80 and CD86. These studies show that CD80 and CD86 are expressed in glomeruli of mice with crescentic anti-GBM GN, in which they play a critical role in facilitating accumulation of Th1 effectors and macrophages, thus exacerbating renal injury.     

Glomeruli synthesize nitrite in active Heymann nephritis; the source is infiltrating macrophages.

Glomeruli synthesize nitrite (NO2-) in experimental nephrotoxic nephritis, a model of glomerulonephritis where infiltrating macrophages are pathogenic. NO2- synthesis was studied in active Heymann nephritis (AHN), a model of membranous glomerulonephritis in which macrophages have not been implicated. Active Heymann nephritis (AHN) was induced with purified renal tubular epithelial antigen and adjuvants. Glomeruli isolated at seven to eight weeks after induction (proteinuria 183 +/- 28 mg/24 hr, N = 6; adjuvant controls, 1.2 +/- 0.8 mg/24 hr, N = 6) produced NO2- in culture spontaneously (7.1 +/- 1.4, adjuvant controls 2.1 +/- 0.9 nmol/2000 g/48 hours; P = 0.021) and in increased amount following LPS stimulation (12.1 +/- 2.8, controls 4.2 +/- 1.6 nmol/2000 g/48 hours; P = 0.047). Synthesis was inhibited by L-NMMA, a competitive inhibitor of NO synthase. Enzymic digestion of glomeruli plus staining with mouse anti-rat macrophage monoclonal antibody ED1 showed macrophage infiltration (32 +/- 6, adjuvant controls 14 +/- 2 macrophages/glomerulus; P = 0.002). Whole body irradiation (XR) suppressed NO2- production (LPS stimulated: 1.0 +/- 0.4, N = 5; non-XR controls 7.2 +/- 4.6 nmol/2000 g/48 hours; N = 5, P = 0.016) and macrophage infiltration (1.1 +/- 0.5; non-XR controls 30 +/- 12 macrophages/glomerulus; P = 0.008) but had no effect on proteinuria. Irradiation with renal shielding confirmed the close correlation between glomerular NO2- synthesis and glomerular macrophage numbers (rs = 0.837, P less than 0.001). These results show that macrophages infiltrate glomeruli in AHN; they are the source of NO2- in this model. Neither macrophages nor NO2- are the cause of proteinuria.     

Glomeruli synthesize nitrite in experimental nephrotoxic nephritis

Activated macrophages synthesize nitric oxide (NO) from L-arginine. In culture, the major stable end product is nitrite (NO2). Activated macrophages accumulate in glomeruli and are responsible for injury in experimental immune complex glomerulonephritis. We examined NO2- production by isolated glomeruli and urinary NO2- in accelerated nephrotoxic nephritis in the rat. Normal glomeruli did not produce NO2- spontaneously or when stimulated with lipopolysaccharide (LPS) (1 microgram/ml) or A23187 (2 microgram/ml). Cultured mesangial cells at first or seventh passage did not produce NO2- spontaneously or when stimulated. Nephritic glomeruli spontaneously produced NO2 at all times studied; this production was maximal at 24 hours after induction of glomerulonephritis (158.4 +/- 8.4 nmol/48 hr/ml, N = 3). The production of NO2- was inhibited 75 to 100% by NG-monomethyl-L-arginine (L-NMMA), and this inhibition was reversed by L-arginine, indicating NO2- production from L-arginine via NO. The production of NO2- was increased by LPS (1 microgram/ml) at 2, 7 and 21 days. NO2- was undetectable in normal rat urine; however, it was present in urine of rats with glomerulonephritis (Day 0 to 1:8161 +/- 2605 nmol/24 hr. N = 12). The production of NO in nephritic glomeruli may have implications for both the mechanism of glomerular injury and glomerular hemodynamics.     

Interleukin-4 ameliorates crescentic glomerulonephritis in Wistar Kyoto rats

BACKGROUND: Activated macrophages play a central role in crescentic glomerulonephritis. Interleukin-4 (IL-4) down-regulates many macrophage proinflammatory activities. We therefore studied the effect of IL-4 on glomerular injury in a model of crescentic glomerulonephritis in the Wistar Kyoto rat. METHODS: Glomerulonephritis was induced by i.v. administration of rabbit antirat glomerular basement membrane antiserum (nephrotoxic serum, NTS). In experiment 1, IL-4 was given from two hours before NTS until day 6. In experiment 2, rats were treated from day 0 to 7 and were then monitored until killed on day 28. In experiment 3, IL-4 was given from day 4 to 7. RESULTS: Continuous IL-4 treatment (experiment 1) significantly (P = 0.001) reduced proteinuria (3 +/- 1 mg per 24 hr vs. 56 +/- 7), fibrinoid necrosis (0.06 +/- 0.04 quadrants/glomulus vs. 1.2 +/- 0.1), macrophage infiltration (6.7 +/- 2.6 cells/glom vs. 33 +/- 2.5), CD8+ cells (1.5 +/- 0.6 cells/glom vs. 6.2 +/- 1.1), inducible nitric oxide synthase positive cells (0.04 +/- 0.04 cells/glom vs. 3.7 +/- 0.6), proliferating cell nuclear antigen positive cells (3.2 +/- 1 cells/glom vs. 15 +/- 2.3), and glomerular intercellular adhesion molecule-1 expression. Follow-up after seven days of treatment (experiment 2) showed that at four weeks, creatinine clearance was higher in treated rats (1.1 +/- 0.1 ml/min vs. 0.4 +/- 01, P = 0.011), and both glomerular scarring (P = 0.006) and tubular atrophy (P = 0.006) were less. Delayed treatment (experiment 3) reduced proteinuria (41 +/- 5 mg per 24 hr vs. 97 +/- 9, P = 0.004) and fibrinoid necrosis (0.39 +/- 0.05 quadrants/glom vs. 1.6 +/- 0.1, P = 0.004). There was no difference in macrophage infiltration, but inducible nitric oxide synthase positive cells were reduced (0.6 +/- 0.1 cells/glom vs. 1.8 +/- 0.4, P = 0.01) as were ED3+ cells (0.18 +/- 0.06 cells/glom vs. 1.86 +/- 0.21, P = 0.004). CONCLUSION: In this model of crescentic glomerulonephritis, early IL-4 treatment abolished proteinuria and markedly reduced glomerular inflammation. If treatment was stopped after seven days, there was continuing benefit on glomerular and tubulointerstitial scarring and creatinine clearance at four weeks. If treatment was delayed until inflammation was established, there was still a reduction of injury, but without an alteration of macrophage numbers, suggesting that IL-4 may be acting, in part, to reduce macrophage activation.     

Granulocyte macrophage colony-stimulating factor expression by both renal parenchymal and immune cells mediates murine crescentic glomerulonephritis

GM-CSF has previously been demonstrated to be important in crescentic glomerulonephritis (GN). As both renal parenchymal cells and infiltrating inflammatory cells produce GM-CSF, their separate contributions to inflammatory renal injury were investigated by creation of two different types of GM-CSF chimeric mice: (1) GM-CSF-deficient (GM-CSF-/-)-->wild-type (WT) chimeras with leukocytes that are unable to produce GM-CSF and (2) WT-->GM-CSF-/- chimeras with deficient renal cell GM-CSF expression. Crescentic anti-glomerular basement membrane GN was induced in WT, GM-CSF(-/-)-->WT chimeras, WT-->GM-CSF-/- chimeras, and GM-CSF-/- mice by planting an antigen (sheep globulin) in their glomeruli. WT mice developed severe crescentic GN, whereas GM-CSF-/- were protected from development of disease. Glomerular T cell recruitment, CD40+ glomerular cells, and renal IFN-gamma and TNF expression were similar in both chimeras and WT mice but significantly reduced in GM-CSF-/- mice, indicating that either leukocyte or renal sources of GM-CSF are sufficient to drive these aspects of the inflammatory response. Restricted expression of GM-CSF revealed a major role for renal cell-derived GM-CSF but a minor role for leukocyte-derived GM-CSF in the formation of cellular crescents; glomerular MHC II expression; serum creatinine; and monocyte chemoattractant protein-1, vascular cellular adhesion molecule, and IL-1beta expression. Glomerular macrophage accumulation, proteinuria, and interstitial infiltrate were equivalent in both chimeric groups but intermediate between WT and GM-CSF-/-, indicating that both sources are required for the full development of glomerular injury in crescentic GN.     

Angiopoietin correlates with glomerular capillary loss in anti-glomerular basement membrane glomerulonephritis

BACKGROUND: Emerging evidence suggests that endothelial turnover occurs in several glomerular diseases and correlates with resolution or progression of glomerular lesions. We hypothesized that the growth factors modulating embryonic kidney endothelial cell survival and capillary morphogenesis may be implicated in capillary loss that occurs in immune-mediated glomerulonephritis (GN). METHODS: GN was induced in C57BL/6 mice by intravenous administration of sheep anti-mouse glomerular basement membrane (GBM) globulin and assessed with markers of vascularity in glomerular lesions, correlating these with expression of specific vascular growth factors. RESULTS: As assessed by periodic acid Schiff staining, 14 +/- 4% (mean +/- SD) glomeruli were affected by sclerosis at 14 days after globulin administration, and 33 +/- 5% were affected at 21 days. By 21 days, a significant increase of plasma creatinine and urinary protein occurred. P-selectin expression was increased in glomerular capillaries 14 days after disease induction, and capillary loss, as assessed by immunohistochemistry for platelet-endothelial cell adhesion molecule, vascular endothelial growth factor (VEGF) receptor 2 and the angiopoietin (Ang) receptor Tie-2, was recorded at 14 and 21 days in glomeruli affected by proliferative crescents and/or sclerosis. VEGF-A immunostaining, evident in control glomeruli, was qualitatively diminished in glomeruli with lesions. Ang-1 immunostaining was detected in control glomeruli and was diminished at 14 days after administration of anti-mouse GBM globulin; instead, Ang-1 was immunolocalized to distal tubules. In contrast, Ang-2 immunostaining was barely detectable in control glomeruli but was prominent in disease glomeruli. In GN mice, rare apoptotic glomerular endothelia were detected by electron microscopy and in situ end-labeling, but such cells were not seen in controls. CONCLUSIONS: Loss of glomerular capillaries during the course of anti-GBM GN in mice was temporally associated with decreases in endothelial survival molecules VEGF-A and Ang-1, and with up-regulation of Ang-2, an antagonist of Ang-1. A changing balance of these growth factors may contribute to decreased glomerular vascularity in crescentic GN.