TNF is an important mediator of chronic inflammatory immune-mediated disorders and plays an essential role in the pathogenesis of experimental GN (4
). In this study, we have defined differential roles for the 2 TNFRs, TNFR1 and TNFR2, in the development of accelerated nephrotoxic nephritis, a model of progressive immune complex–mediated GN dependent on complement and αβ and γδ T cells (33
). TNFR1 has an immunosuppressive role likely through regulation of the humoral response and T cell apoptosis/accumulation, the latter being dependent on the expression of TNFR1 on cells other than the T cell itself. In sharp contrast, TNFR2 is induced on the glomerular endothelium of the nephritic kidney and its expression is required on intrinsic cells for complement deposition and the initiation and progression of GN.
The nearly complete protection of TNFR2-deficient mice in GN suggests that TNFR2 plays a nonredundant role in mediating renal injury, and this finding is the first demonstration to our knowledge of an essential proinflammatory function of TNFR2 in glomerular disease. TNFR2-deficient mice have normal spleen and lymph node architecture, and the germinal center reaction and formation of follicular dendritic cell networks are intact (26
). Delayed-type hypersensitivity responses in TNFR2-deficient mice are comparable to those in wild-type mice (38
). Furthermore, TNFR2-deficient mice mounted a systemic immune response toward the nephrotoxic antibody, and the reconstitution studies in αβ T cell–deficient mice suggest that TNFR2-deficient T cells are competent to induce GN. Together, these data demonstrate that humoral and cell-mediated immunity are intact in mice lacking TNFR2. Thus, the observed protection in TNFR2-deficient mice subjected to GN suggests that TNFR2 is required for the effector phase of nephrotoxic nephritis.
TNFR2 is expressed on monocytes/macrophages and on peripheral T cells in blood and lymphoid organs. It is also present on infiltrating renal T cells during GN but is absent on renal macrophages. The latter observation suggests that macrophage-expressed TNFR2 in the kidney is unlikely to mediate local renal injury. Although it was hypothesized that recruitment of activated macrophages into the kidney may require TNFR2 expression on their surface, this was also not essential, since macrophage accumulation was not significantly reduced in BM chimeras deficient in TNFR2 in circulating cells. TNFR2 expression on T cells was also not essential for disease progression.
TNFR2 expression was induced in intrinsic renal cells of nephritic kidneys, and confocal microscopy revealed renal TNFR2 protein expression localized to glomerular endothelial cells. In addition, endothelial cell–expressed TNFR2 was present in postcapillary venules of the cortical interstitium. Thus, renal TNFR2 expression localized both with the site of the primary glomerular damage and the site of secondary interstitial nephritis that accompanies the glomerular lesion. Indeed, our findings are consistent with the previously reported glomerular expression of TNFR2 in human glomerulopathies (24
). The observed pattern of TNFR2 expression was highly specific to the disease process. TNFR2 protein was not detected in normal mouse kidney. Moreover, TNFR2 expression was restricted to tubular epithelial cells and was undetectable in glomeruli or interstitial venules in mice with tubular damage resulting from obstructive nephropathy. Importantly, analyses of GN in mice chimeric for TNFR2 expression indicate that TNFR2 expression on intrinsic cells, likely glomerular endothelial cells, is critical for the development of all parameters of GN. However, our data cannot rule out a possible role for intrinsic cells other than renal cells (e.g., activation of complement by extrarenal endothelial cells) in mediating GN. In contrast, leukocyte-expressed TNFR2 only partly contributes to renal injury.
How does renal cell–expressed TNFR2 mediate proteinuria and glomerular damage? One mechanism is the promotion of complement deposition, as suggested by our studies, in which a marked attenuation of glomerular complement deposition was observed in both TNFR2-deficient mice and mice lacking TNFR2 expression on intrinsic cells. We propose that endothelial TNFR2 could contribute to complement activation/deposition. Interestingly, endothelial cells activated by TNF promote complement deposition in areas of exposed subendothelial matrix in vitro, which is initiated by cytokine-induced retraction of these cells (39
). TNFR2 could be involved in this process. Complement fixation on exposed subendothelial basement membrane in vivo promotes glomerular injury and inflammation (35
). Moreover, complement is important in the development of proteinuria in our model of accelerated nephrotoxic nephritis, as C5-deficient mice developed minimal albuminuria at days 14 and 21 (33
). Complement-mediated injury may occur through the generation of deleterious terminal pathway activation products and/or split products, such as C3a, which modulate proinflammatory cytokine expression in monocytes and leukocyte activation and recruitment (39
). It is also possible that TNF-dependent upregulation of vascular adhesion molecules (18
), which would facilitate the infiltration of effector leukocytes into the kidney, is TNFR2 mediated. Nevertheless, it is noteworthy that we detected no differences in renal ICAM-1 expression in nephritic wild-type and TNFR2-deficient mice by immunohistochemistry, and positive signals for E-selectin were not detected in either group of mice at the time points tested (data not shown).
Most classic proinflammatory effects of TNF, including the release of other inflammatory cytokines and the upregulation of vascular adhesion molecules, are mediated through TNFR1 signaling (21
). In our studies, TNFR1-deficient mice exhibited only a delay in the development of GN but were not protected from disease. This was associated with a significant decrease in circulating autologous antibody against rabbit IgG, although its glomerular deposition was equivalent to that observed in wild-type mice. The reduction in autologous antibodies indicates a defect in the systemic immune response toward the injected rabbit anti-GBM antibody. The reduction in circulating autologous antibodies in TNFR1-deficient mice is not responsible for the protection observed in these mice at early time points, since mice lacking B cells and autologous antibody production (μ chain–deficient mice) have proteinuria similar to that of wild-type mice at days 7 and 14 (ref. 34
and data not shown for the serum used in the current experiments). Thus, we propose that the early protection from glomerular injury as well as the decreased humoral response may result from an impaired generation of sensitized T cells in TNFR1-deficient mice. Sensitized T cells not only provide B cell help for the production of autologous antibodies in accelerated nephrotoxic nephritis but are required for the development of GN independently of antibody production (34
). Indeed, TNFR1 is required for T cell priming after subcutaneous injection of antigen (20
), and delayed-type hypersensitivity responses are impaired in TNFR1-deficient mice (38
). Finally, TNFR1-deficient mice, like TNF-deficient animals, are defective in the formation of germinal centers and follicular dendritic cell networks in secondary lymphoid organs (21
). Taken together, these data suggest that an impaired adaptive immune response in TNFR1-deficient mice is responsible for the delay in onset of nephritis observed in these animals.
Despite the initial delay in the development of nephritis, at later time points, TNFR1-deficient mice exhibited disease that was largely comparable to that in wild-type mice. This may be attributed to excessive accumulation of T cells in these mice. Fewer apoptotic T cells were observed in nephritic TNFR1-deficient mice in all organs investigated, and this reduction in apoptosis may account for the increased size of draining paraaortic lymph nodes (data not shown) and the excessive accumulation of renal T cells in nephritic mice. Thus, TNFR1-mediated apoptosis of activated T cells may be a mechanism to control renal T cell responses in GN as reported in a study of lupus-prone C57BL/6-lpr/lpr
mice lacking TNFR1 (41
). Despite the increase in glomerular and interstitial T cell infiltrates in TNFR1-deficient mice, parameters of glomerular injury were not significantly worse compared with those in wild-type mice. As TNFR1 mediates both proinflammatory (T cell priming) and immunosuppressive (apoptosis of activated T cells) responses during nephrotoxic nephritis, the phenotype in TNFR1-deficient mice may reflect the net effect of both T cell–dependent functions. Moreover, it is noteworthy that the extent of renal macrophage but not T cell accumulation correlated with the degree of glomerular injury in wild-type and TNFR1-deficient mice at all time points studied.
Although TNFR1-mediated apoptosis of activated T cells has been described (22
), the adoptive T cell transfer studies suggest that neither the delayed onset of disease nor the excessive renal T cell accumulation and reduced apoptosis of αβ T cells at later time points is dependent on TNFR1 on αβ T cells. We propose that TNFR1-mediated differentiation and activation of APCs (40
) might facilitate the rapid generation of sensitized T cells that are required for disease induction in our model. Apoptosis of T cells during presentation of antigen can be induced by APCs expressing Fas ligand (42
), and we speculate that TNFR1 expressed on APCs such as glomerular mesangial and tubular epithelial cells may have a functional role in this process (15
). Mesangial cells upregulate Fas ligand upon TNF stimulation in vitro (45
). Fas ligand is also expressed in tubular epithelium and in injured glomeruli during immune complex nephritis (46
) and induces apoptosis in Fas-sensitive lymphoid cell lines in vitro (46
In summary, TNFR2 expressed on intrinsic cells, likely glomerular endothelial cells, specifically mediated organ-specific tissue damage in GN, independent of TNFR1. In contrast, TNFR1 deficiency was not protective throughout disease development and actually resulted in increased disease indices such as the accumulation of renal T cells in our model. The successful development and increasing clinical use of anti-TNF therapies for the treatment of chronic autoimmune disease demonstrate that neutralization of endogenous TNF can control progression of disease in humans. However, anti-TNF therapies in inflammatory immune-mediated disorders such as GN may lead to unpredictable outcomes, due to the contrasting proinflammatory and immunosuppressive functions of TNF in these conditions (20
). Indeed, anti-TNF therapies, although beneficial for the majority of patients with rheumatoid arthritis or inflammatory bowel disease, has led to the formation of anti-DNA antibodies and the development of lupus-like syndromes in some patients. Moreover, TNF blockade in multiple sclerosis has frequently caused immune activation and disease exacerbation (21
). Our studies indicate that specific blockade of TNFR2 may be preferable to anti-TNF treatments in human GN, especially in the context of autoimmune disease such as systemic lupus erythematodes. Specific TNFR2 antagonism would preserve immunosuppressive effects of TNF such as apoptosis of autoreactive T cells (21
) but block TNF dependent proinflammatory functions that lead to glomerular dysfunction. Thus, therapeutic blockade of TNFR2 may be a promising strategy in the treatment of human GN.