T-bet deficiency attenuates renal injury in experimental crescentic glomerulonephritis.

T-bet is a transcription factor that is essential for T helper (Th)1 lineage commitment and optimal IFN-gamma production by CD4(+) T cells. We examined the role of T-bet in the development of experimental crescentic glomerulonephritis, which is induced by Th1-predominant, delayed-type hypersensitivity-like responses directed against a nephritogenic antigen. Anti-glomerular basement membrane (GBM) glomerulonephritis was induced in T-bet(-/-) and wild-type C57BL/6 mice. Compared with wild-type controls, renal injury was attenuated in T-bet(-/-) mice with glomerulonephritis, evidenced by less proteinuria, glomerular crescents, and tubulointerstitial inflammation. Accumulation of glomerular CD4(+) T cells and macrophages was decreased, and was associated with reduced intrarenal expression of the potent Th1 chemoattractants CCL5/RANTES and CXCL9/Mig. Supporting the pro-inflammatory nature of T-bet signaling, assessment of systemic immunity confirmed that T-bet(-/-) mice had a reduction in Th1 immunity. The kinetic profile of T-bet mRNA in wild-type mice supported the hypothesis that T-bet deficiency attenuates renal injury in part by shifting the Th1/Th2 balance away from a Th1 phenotype. Expression of renal and splenic IL-17A, characteristically expressed by the Th17 subset of effector T cells, which have been implicated in the pathogenesis of autoimmune disease, was increased in T-bet(-/-) mice. We conclude that T-bet directs Th1 responses that induce renal injury in experimental crescentic glomerulonephritis.

Endogenous myeloperoxidase promotes neutrophil-mediated renal injury, but attenuates T cell immunity inducing crescentic glomerulonephritis.

Myeloperoxidase (MPO) is an enzyme that is found in neutrophils and monocytes/macrophages. Intracellularly, it plays a major role in microbial killing, but extracellularly, it may cause host tissue damage. The role of endogenous MPO was studied during neutrophil-mediated (heterologous) and T helper 1 (Th1)/macrophage-mediated (autologous) phases of crescentic glomerulonephritis. Glomerulonephritis was induced in C57BL/6 wild-type (WT) and MPO-deficient (MPO(-/-)) mice by intravenous injection of sheep anti-mouse glomerular basement membrane globulin. MPO activity was increased in kidneys of WT mice during both the heterologous and autologous phases of glomerulonephritis. During the heterologous phase of glomerulonephritis, proteinuria was decreased, whereas glomerular neutrophil accumulation and P-selectin expression were enhanced in MPO(-/-) mice. In the autologous, crescentic phase of glomerulonephritis, MPO(-/-) mice had increased accumulation of CD4(+) cells and macrophages in glomeruli compared with WT mice. However, no difference in renal injury (crescent formation, proteinuria, and serum creatinine levels) was observed. Neutrophils and macrophages from MPO(-/-) mice exhibited reduced production of reactive oxygen species. Assessment of systemic immunity to sheep globulin showed that MPO(-/-) mice had increased splenic CD4(+) cell proliferation, cytokine production, and dermal delayed-type hypersensitivity, as well as enhanced levels of circulating IgG, IgG1, and IgG3. MPO(-/-) mice also had an augmented Th1:Th2 ratio compared with WT mice (IFN-gamma:IL-4 and IgG3:IgG1 ratios). These results suggest that endogenous MPO locally contributes to glomerular damage during neutrophil-mediated glomerulonephritis, whereas it attenuates initiation of the adaptive immune response inducing crescentic, autologous-phase glomerulonephritis by suppressing T cell proliferation, cytokine production, and Th1:Th2 ratio.

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.

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.

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.

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.

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

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

Intrinsic renal cell expression of CD40 directs Th1 effectors inducing experimental crescentic glomerulonephritis.

Evidence suggests that human and experimental crescentic GN results from Th1-predominant immunity to glomerular antigens. CD40/CD154 signaling plays a key role in initiating Th1 responses and may direct Th1 effector responses. The role of CD40 in the development of GN was assessed in murine experimental anti-glomerular basement membrane GN. In this model, C57BL/6 wild-type (WT) mice sensitized to sheep globulin develop crescentic GN resulting from Th1 effector responses when challenged with sheep globulin planted in glomeruli. CD40-/- mice do not develop immunity in response to sheep globulin and thus fail to develop effector responses or significant GN. CD40 is expressed in nephritic glomeruli, suggesting a potential role for intrarenal CD40-CD154 interactions in injurious effector responses. Immune neutralization of the CD40 ligand (CD154) at the time of challenge significantly reduced accumulation of Th1 effectors and injury. The role of CD40 expression by renal cells was assessed by comparing GN in WT-->CD40-/- chimeras (absent renal but intact bone marrow CD40) and sham chimeric mice (WT-->WT). Both groups developed strong antigen-specific immune responses (antibody and IFN-gamma production). However, WT-->CD40-/- chimeras demonstrated reduced renal monocyte chemotactic protein 1 and IFN-inducible protein 10 mRNA levels and minimal T cell and macrophage influx and were protected from renal injury. Sham chimeric mice developed reduced GFR, with prominent renal expression of monocyte chemotactic protein 1 and IFN-inducible protein 10 mRNA and effector cell accumulation. In conclusion, the expression of CD40 by nonimmune renal cells plays a major role in Th1 effector responses by inducing Th1 chemokine production. Therefore, CD40-CD154 interactions are a potential therapeutic target in GN.