Lupus nephritis (LN) is a major complication of systemic lupus erythematosus (SLE) and is mediated by glomerular deposition of immune complexes that trigger a number of inflammatory events leading to tissue damage. Although autoantibody production, cytokines, and chemokines are required to promote glomerular inflammation (1–3), the separate pathogenic contributions of these factors to the development of LN have not been fully elucidated.
Impaired T cell function resulting either from an intrinsic defect or as a consequence of an underlying alteration of dendritic cells (DCs) has been demonstrated in experimental and human SLE (4). DCs are considered powerful antigen-presenting cells that activate naive T cells, regulate cytokine production, and interact with T cells, thus promoting autoimmunity.
A defect in the number of circulating DCs in conjunction with high production of serum interferon-α (IFNα) have been described in SLE (5). Two DC subsets, plasmacytoid DCs and myeloid DCs, have been identified on the basis of different antigen expression and levels of maturation, as well as the tendency to migrate toward peripheral tissue (6). Both subsets of DCs express a similar repertoire of adhesion molecules and chemokine receptors; however, most chemokine receptors of plasmacytoid DCs are not functional in circulating cells (7). In addition, myeloid DCs respond to several homeostatic and inflammatory chemokines such as CCR2, CCR5, and CXCR4, whereas responsiveness to CXCL12 by plasmacytoid DCs induces their activation and affects their migration. Although plasmacytoid DCs express high levels of both CCR7 and CXCR3, they fail to migrate in response to any of the specific receptors (8). Thus, additional chemoattractants are possibly involved in plasmacytoid DC trafficking within inflamed tissue.
Tumor necrosis factor α (TNFα) and interleukin-1 (IL-1) have been shown to be capable of stimulating DC activation and trafficking within the damaged skin of patients with SLE (9), whereas serum and glomerular accumulation of IL-18 occurs only in active LN (10). Also, plasmacytoid DCs exert a migratory function owing to the high expression of chemerin or the local accumulation of adenosine, which drives their trafficking toward inflamed tissue (11, 12). In addition, IL-18 receptor (IL-18R) is selectively expressed by peripheral plasmacytoid DCs, which are prone to responding to IL-18 (13). Because IL-18 is detected in the glomeruli of patients with LN, we reasoned that glomerular IL-18 may locally amplify the immune response by attracting the majority of IL-18R–positive cells, including both DCs and T cells. This implies that a defined role should be assigned to plasmacytoid DCs both in promoting a Th1 immune response and in increasing renal damage.
The aim of this study was to investigate circulating peripheral DC subsets in patients with active LN, as well as the levels of Th1 cytokines such as IL-18, IFNγ, and IL-12, to assess their functional roles.
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A defective number and impaired function of DCs have been described in SLE and correlated to high production of IFNα and an imbalance in Th1:Th2 cytokine homeostasis (18). Results from the present study indicate that the number of peripheral immature myeloid DCs and plasmacytoid DCs is decreased in SLE, and that the plasmacytoid DC defect correlates with LN activity. In addition, plasmacytoid DCs accumulate within the glomeruli of patients with LN, and the ability of these DCs to migrate is apparently related to IL-18R stimulation by high local production of IL-18.
The role of subsets of immature DCs in SLE is presently being debated. Previous studies demonstrated a reduced frequency of circulating CD11c-positive myeloid DCs. However, the skew from the typical Th2 immune response, the high IFNα serum levels (18), and the evidence of a role of Th1 cytokines in experimental and human SLE (10, 19, 20) emphasize the involvement of deregulated plasmacytoid DCs in the pathogenesis of SLE. Our results demonstrated a decreased number of circulating DCs in SLE, with a prevalent defect of plasmacytoid DCs in patients with LN. In addition, an inverse correlation between the number of peripheral plasmacytoid DCs and the severity of renal damage was detected. There are different possible explanations for the reduced proportion of peripheral DC subsets in SLE. The number of immature DC progenitors might be reduced, although a deficit of CD34-positive cells has not been demonstrated. DC clearance might be increased due to accelerated apoptosis, or DCs might be actively recruited toward inflamed tissue under the effect of both chemokine and cytokine stimulation, as shown in other autoimmune and inflammatory disorders (21).
Peripheral myeloid DCs and plasmacytoid DCs show differing chemokine repertoires and peculiar migratory behavior (7). Current evidence suggests that peripheral plasmacytoid DCs express receptors that permit their migration into lymphoid tissues, including L-selectins, CXCR4, CCR7, and α4 integrins (8). However, peripheral plasmacytoid DCs may also locate within inflamed peripheral tissue other than lymphoid sites, where they present antigens and stimulate T cell cytotoxicity (9). We observed that peripheral plasmacytoid DCs overexpress different chemokines as compared with myeloid DCs, including CXCR4, CCR5, and CCR7. However, the mechanisms by which peripheral plasmacytoid DCs migrate toward inflamed tissue are still undefined, because they are unresponsive to the above-mentioned molecules (22). In addition, CCL21 was not expressed within glomeruli of patients with LN, thus confirming its lack of involvement in the recruitment of CCR7-positive plasmacytoid DCs. Therefore, additional chemoattractants might be involved in the migratory machinery of plasmacytoid DCs.
We have studied IL-18R as a potential molecule involved in plasmacytoid DC migration. As reported, myeloid DCs produce IL-18, but only plasmacytoid DCs express the functional receptor and migrate in response to IL-18 stimulation (13). Our results are concordant with those of previous studies and confirm that CD123+,BDCA-2+ plasmacytoid DCs from both patients with SLE and control subjects constitutively express IL-18R, whereas myeloid DCs are negative in all instances. Therefore, circulating plasmacytoid DCs might be prone to stimulation induced by IL-18 production.
Human and experimental models have suggested that LN is a largely Th1-mediated disorder, and that high production of serum IL-18 and IFNγ is related to the severity of renal damage (10). IL-18 is mainly produced by macrophages, although myeloid DCs may contribute to elevated levels of IL-18. It is known that peripheral plasmacytoid DCs induce a prevalent Th2 immune response, but under the stimulation of IL-18 they switch T cells toward a Th1 phenotype through IFNγ and IL-12 overproduction (13). Our results also showed deficient production of serum IL-4 and high levels of IL-18, IFNγ, and IL-12 in patients with LN, although the latter abnormality is apparently unrelated to the severity of renal damage. Thus, it is conceivable that inflammatory cells including macrophages may release increased amounts of IL-18 and stimulate plasmacytoid DCs to promote a skewed Th1:Th2 ratio.
Up-regulation of IL-18 was associated with nephritic kidneys in lupus-prone MRL/lpr mice, with levels increasing in parallel with the severity of the disease (23). In addition, IL-18 exerts chemotactic functions, being also influenced by environmental factors that may amplify its inflammatory properties. Finally, IL-18 attracts IL-18R–positive cells such as plasmacytoid DCs and T cells. Thus, we hypothesize that high levels of glomerular IL-18 produced mainly by resident macrophages may recruit plasmacytoid DCs from peripheral blood, leading to subsequent glomerular accumulation.
Our findings emphasize that IL-18 is produced within nephritic glomeruli, and that its striking accumulation in the cytoplasm of resident mononuclear cells reflects its local production. As in other inflamed tissue, macrophages are the prevalent producers of the cytokine, and their number indeed paralleled the severity of renal damage. Therefore, persistent production of IL-18 by activated macrophages in LN may represent a persistent chemotactic trigger for the recruitment of inflammatory cells expressing the functional receptor (24).
T cells are the primary effectors of renal damage, while an accumulation of DCs at different stages of maturation has not been demonstrated. Previous studies (9) revealed the migration of plasmacytoid DCs toward peripheral inflamed tissues, including skin, in patients with SLE (25). This event has been correlated with their ability to transmigrate through high endothelial venules (26), independently of both chemokine and integrin expression. We have hypothesized that in LN, peripheral blood plasmacytoid DCs could be recruited within the kidney as an effect of the persistent inflammatory stimulation induced by IL-18, and that their prevalent glomerular accumulation correlates with the number of IL-18–positive cells. In addition, their ability to locate within glomeruli was also supported by the presence of IL-18R that was overexpressed by resident mononuclear cells, whereas functional chemokine expression was unrelated to the presence of the relative ligands within the kidney, as demonstrated for CCR7/CCL21. The expression of IL-18R by resident cells represents, at least in part, indirect support for the occurrence of plasmacytoid DCs in LN.
The primary role of IL-18 in plasmacytoid DC migration is also indicated by the absence within glomeruli of myeloid DCs that fail to express IL-18R. In vivo evidence suggests that IL-18 shapes the development of Th1 responses, and neutralization of IL-18 did not alter the Th1/Th2 phenotype of allogeneic T cells cocultured with myeloid DCs (27, 28). On the contrary, IL-18 induces chemotaxis of plasmacytoid DCs and enhances their allostimulatory capacity, promoting a prevalent Th1 response. Thus, we speculate that IL-18 may play a major role in the recruitment of IL-18R–positive plasmacytoid DCs within the kidney, and that glomerular plasmacytoid DCs may locally amplify the renal damage through long-term activation of T cells and Th1 cytokine production.
It has been reported that IL-18 induces maturative effects on the human KG-1 myelomonocytic cell line, although cultured monocytes are insensitive to IL-18 in vitro. The expression of CD83 by KG-1 cells confirmed its role as a marker for the identification of mature DCs (29), although its prevalent expression by plasmacytoid DCs and myeloid DCs or both is still controversial (30). CD83 defines myeloid DCs in different experimental and human models, although it was recently shown to be overexpressed by plasmacytoid DCs in other autoimmune diseases (21, 31). Thus, the presence of glomerular CD83-positive cells in severe LN in parallel with BDCA-2–positive plasmacytoid DCs in the absence of myeloid DCs suggested that CD83-positive DCs may be prevalently expressed by mature plasmacytoid DCs that undergo definitive maturation within glomeruli or by already mature DCs that migrate to the kidney in response to triggers of inflammation. IL-18R activation might not be the unique mechanism through which plasmacytoid DCs traffic inside the kidney. Indeed, environmental factors and the activation of adenosine in conjunction with chemerin overexpression may be involved in their activation and migration (12). Thus, the presence of renal plasmacytoid DCs may reflect their defect in the periphery in patients with SLE and supports the high glomerular levels of IL-18 as a primary chemotactic stimulus leading to glomerular accumulation and renal damage in active LN.