Tumor necrosis factor α (TNFα) is an important regulator of physiologic and inflammatory immune responses. Although TNFα antagonists have remarkable therapeutic benefits in several autoimmune diseases including rheumatoid arthritis, psoriasis, and Crohn's disease, they also have been associated with the development of antinuclear antibodies and even clinical systemic lupus erythematosus (SLE), multiple sclerosis, and other demyelinating diseases. TNFα is protective in the early stages of lupus in some mouse models but is overexpressed in the inflamed target organs of both mice and humans with lupus (1, 2). Consistent with both the protective and proinflammatory roles of TNFα, blockade of TNFα improved proteinuria in patients with treatment-refractory lupus nephritis but increased anti–double-stranded DNA (anti-dsDNA) and anticardiolipin autoantibody titers (3).
In this study, we determined the clinical effects of TNF receptor type II (TNFRII) Ig in a mouse model of lupus nephritis in which an increase in serum levels of TNFα and renal production of TNFα occurred concomitantly with the onset of renal disease. TNFα inhibition introduced after the development of autoantibodies and at the onset of clinical nephritis stabilized the nephritis and resulted in a long period of relapsing and remitting proteinuria that was associated with marked improvement in survival. Mechanistic studies revealed no effect of TNFα inhibition on autoantibody production or lymphocyte activation in the spleen or on renal autoantibody deposition; however, a marked decrease in renal periglomerular and interstitial accumulation of F4/80high renal macrophages was observed. Real-time polymerase chain reaction (PCR) analysis revealed a significant decrease in renal expression of chemokines CCL2, CCL5, and CCL9, the endothelial activation marker vascular cell adhesion molecule 1 (VCAM-1), genes involved in tissue remodeling, and the markers of proximal tubule damage lipocalin 2 and hepatitis A virus cellular receptor 1 (HAVCR-1) (kidney injury molecule 1; KIM-1). We conclude that TNFα inhibition decreases the renal inflammatory response to immune complex deposition. This decrease is associated with decreased accumulation of periglomerular and interstitial renal macrophages, which play a role in tissue remodeling, and decreased damage of renal tubular cells. In contrast, perivascular aggregates containing CD11chigh dendritic cells as well as B cells and T cells accumulate in the kidneys in a TNFα-independent manner and are not sufficient to induce terminal renal damage.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
TNFα is a pleiotropic cytokine that has both protective and pathogenic functions in SLE. Lupus-prone NZB/NZW mice have a defect in TNFα production. In young mice, further depletion of TNFα exacerbates disease, whereas TNFα replacement is protective (1, 12). The relevance of this mouse model to human biology has been borne out by the finding that antinuclear antibodies develop in up to 50% of patients treated with TNFα inhibitors, antibodies to DNA develop in 15%, and clinical SLE (including SLE nephritis) develops in 1 of 500 patients treated with TNFα inhibitors (13, 14). The mechanism for the pathogenic role of TNFα blockers in the induction of autoimmunity has not been elucidated, but it is clearly a class effect of all TNFα blockers. The prevailing hypotheses include increased production of type I IFN (15), blockade of the antiinflammatory effects of TNFα (16), failure to adequately regulate activated T cells including Th17 T cells (17, 18), and failure to induce cytotoxic T lymphocytes that can kill autoreactive B cells (19).
In NZB/NZW mice older than age 28 weeks, TNFα replacement is no longer effective, and low-dose TNFα has even been shown to exacerbate disease in older NZB/NZW mice that have high serum levels of TNFα (1, 12). TNFα levels are similarly elevated in the serum of patients with active SLE (20), and TNFα is expressed in the kidneys of both mice (6) and humans with lupus nephritis (3). The renal source of this cytokine is not entirely clear, with infiltrating mononuclear phagocytes, T cells, and glomerular mesangial cells all having been suggested to produce TNFα. The expression of TNF receptors, particularly TNFRI, is markedly up-regulated in the glomeruli of patients with proliferative lupus nephritis (21), and soluble TNFRI is shed in the urine (22). Regardless of the precise source of TNFα in human lupus glomerulonephritis, a short course of infliximab has had remarkable and long-lasting therapeutic benefit in open-label studies in a small number of SLE patients with treatment-refractory nephritis. Nevertheless, general enthusiasm for this approach has been tempered by the observation of transient increases in autoantibodies, severe infusion reactions following multiple doses of infliximab, and concerns about concomitant treatment with other immunosuppressive agents. An increased rate of bacterial infection and one case of lymphoma were observed in patients receiving continuous anti-TNFα therapy (3).
In the current study, we show that treatment with TNFRII, which is analogous to etanercept, in a lupus model in which renal TNFα expression occurs concomitantly with nephritis onset protects the kidneys against damage and prolongs survival. Systemic lymphocyte activation, induction of class-switched anti-dsDNA autoantibodies, and renal immunoglobulin deposition, all of which occur in the first 4 weeks after the initiation of IFNα-induced lupus in this model, were not altered by TNFα inhibition, but the renal inflammatory response to immune complex deposition was dramatically reduced.
The onset of proteinuria in lupus-prone mice is associated with the expansion and activation of a dominant population of resident renal mononuclear phagocytes that have variably been referred to as intrinsic renal macrophages or resident renal dendritic cells, due to their mixed function as both antigen-presenting cells and phagocytic cells (23). At nephritis onset, these cells markedly up-regulate their expression of CD11b, and they accumulate in both the interstitium and the periglomerular space (10, 24). Proteinuria onset and disease progression in NZB/NZW mice are also characterized by the renal influx of CD11chigh myeloid dendritic cells that accumulate exclusively in large lymphoid aggregates that also contain B cells and T cells and are located in the hilum and perivascular regions (8). In this study, we demonstrated that the recruitment of renal F4/80high macrophages to the periglomerular region and the renal interstitium is TNFα dependent, whereas the accumulation of myeloid dendritic cells, B cells, and T cells in perivascular or perihilar lymphoid aggregates and the accumulation of F4/80high cells in a cuff around these aggregates occur independently of TNFα.
Recruitment of inflammatory cells to the lupus kidney in response to immune complex deposition depends on several factors. In NZB/NZW mice, engagement of Fc receptors on circulating myeloid cells by glomerular immune complexes is required for renal damage to occur (25). Fc receptor crosslinking on monocytes induces TNFα production (26), providing a direct link between renal immune complex deposition and TNFα production by renal mononuclear phagocytes. Complement activation and consequent activation of inflammatory cascades also contribute to renal damage. Endothelial cell activation by immune complexes and cytokines results in expression of integrins that help to capture circulating cells. The onset of proteinuria in NZB/NZW mice is associated with up-regulation of the endothelial cell–derived adhesion molecules E-selectin, P-selectin, and VCAM-1 (8). Elaboration of chemokines that attract cells expressing the corresponding receptors facilitates transmigration of inflammatory cells into the renal parenchyma and precedes or coincides with the onset of clinical nephritis. In NZB/NZW mice, we have observed renal expression of chemokines that can attract macrophages and dendritic cells (8). Similarly, in MRL/lpr mice, expression of a similar set of chemokines in glomeruli is an early feature of lupus nephritis that precedes inflammatory cell infiltration (27). Of these chemokines, CCL2 has definitively been shown to be pathogenic (28) and, as shown here, is one of several chemokines whose up-regulation is dependent on TNFα.
We further show that renal IL-1β production is not decreased by TNFα blockade. IL-1β is produced by both intrinsic renal cells and macrophages/dendritic cells and has a proinflammatory function in acute immune complex–mediated renal inflammatory disease, being involved in crescent formation and inflammatory cell recruitment. In the model of acute nephrotoxic nephritis, leukocyte-derived IL-1β expression is required for the induction of glomerular TNFα expression and for the subsequent recruitment of macrophages to the kidneys (29). Our results are consistent with these findings. Similarly, CXCL13, which is highly expressed by CD11chigh dendritic cells in lymphoid aggregates (8), is not decreased by TNFα inhibition.
As shown here, TNFα inhibition markedly decreased the recruitment of periglomerular and interstitial F4/80high macrophages that amplify tissue injury (10, 24). Periglomerular macrophages may be attracted by locally produced chemokines or may be activated by inflammation mediators to proliferate in situ. In addition, because effluent blood flow from the glomerulus provides the sole blood supply for peritubular capillaries, glomerular hypertension and hypertrophy compromise peritubular blood flow, resulting in hypoxia, tubular activation (including chemokine secretion), and tubular epithelial cell death. This induces resident macrophage activation and peritubular inflammation and is associated with tissue remodeling, fibrosis (30), and finally, irreversible renal damage. Damaged tissue, cytokines, and other inflammation mediators amplify the inflammatory process.
We have previously shown in NZB/NZW mice that several markers of inflammation are up-regulated during renal disease, normalize upon remission induction, and remain at low levels even late after remission induction, when glomerular damage is recurring but interstitial damage has not yet recurred (8). Using protein analysis, we showed that the expression of CCL9, VCAM-1, and lipocalin 2 was decreased in TNFRII Ig–treated mice compared with controls. Lipocalin 2 is a highly sensitive marker for renal tubule damage whose expression increases before any increase in the serum creatinine level, and it has proinflammatory functions (31, 32). Furthermore, oxidative damage is also decreased in TNFRII Ig–treated kidneys, confirming attenuation of end-stage renal disease. In contrast, TNF inhibition does not alter the systemic inflammatory response, as evidenced by failure to regulate lymphocyte activation or to prevent the accumulation of renal perivascular and hilar lymphoid infiltrates. These infiltrates may be attracted by soluble circulating mediators and vascular activation/injury, independently of glomerular immune complex deposition and/or the resulting peritubular hypoxia, and do not appear to be sufficient to mediate end-stage renal failure.
The results of this study show that TNFα is a critical cytokine in the renal effector response to glomerular immune complex deposition, and that regulation of this response is highly therapeutic even in the IFNα-induced lupus model, in which systemic inflammation is difficult to control (6). Judicious inhibition of TNFα may be a potential therapeutic option for rapid control of renal damage in patients who have active lupus nephritis with increased renal expression of TNF and its receptors.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Davidson had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Bethunaickan, Sahu, Tang, Huang, Ramanujam, Davidson.
Acquisition of data. Bethunaickan, Sahu, Liu, Tang, Huang, Edegbe, Tao, Ramanujam, Madaio, Davidson.
Analysis and interpretation of data. Bethunaickan, Sahu, Liu, Tang, Huang, Tao, Madaio, Davidson.