INSERM U99 and Service de Néphrologie, Hôpital Henri Mondor, Créteil, France
Keywords: c-maf; minimal-change nephrotic syndrome; NFB; pathogenesis; Proteasome; T cells
Introduction
Minimal-change nephrotic syndrome (MCNS) is a clinical and pathological entity defined by selective proteinuria and hypoalbuminaemia that occurs in the absence of cellular glomerular infiltrates or immunoglobulin deposits. The only detectable abnormalities involve the epithelial visceral cells with effacement of foot processes. These morphological alterations are typical of the nephrotic syndrome but not specific of the MCNS disease [1].
Whereas recent genetic approaches to familial idiopathic nephrotic syndromes have been determining factors in elucidating several molecular aspects of focal glomerular sclerosis [24], our knowledge about the pathogenesis of MCNS is inchoate, despite arguments suggesting a disorder of the immune function. In support of this view, occurrence of the disease in the context of immune challenge initiated by infectious or allergic stimuli, as well as relapse sensitivity to drugs known to inhibit the immune system (glucocorticoids, cyclosporin and cyclophosphamide) are particularly relevant [5]. The possible link between abnormal T-cell response and glomerular disease was postulated 30 years ago. First, Hoyer et al. [6] described early recurrences of nephrotic syndrome following renal transplantation. In two of the three cases reported, early renal biopsies on the native kidney did not detect histological signs of focal glomerular sclerosis although they were prominent during the progression of the disease. Secondly, Lagrue et al. [7] showed that systemic infusion of supernatants of T lymphocytes from patients with MCNS relapse induced proteinuria in rats. These observations have suggested that peripheral immune cells produce a circulating factor, which impairs the glomerular filtration barrier.
Here, we would like to integrate some data deduced from recent molecular approaches on the immunopathogenesis of MCNS in the light of clinical characteristics and immunological and experimental findings.
|
T cells in MCNS
Activation of T cells at the initial step of the immune response is dependent on the interaction with dendritic cells through cell-surface receptors. An expansion of CD4+ and CD8+ T-cell populations, identified by immunophenotyping of peripheral lymphocytes, is easily demonstrated during the nephrotic phase in most cases. Interestingly, it has been reported that an increase of the CD4+ T cells expressing the CD25 marker (IL-2 receptor chain) occurred during the relapse [8]. The CD25 antigen is expressed by at least two subsets of CD4+ T cells: a large population (
50%) that express CD25 upon activation by an immunogen stimulus and a minor population, consisting of natural CD4+ CD25+ T cells (
10%), which plays a regulatory role in the immune response [9]. Experimental studies have shown that natural CD4+ CD25+ T cells, which exit the thymus very early in life, display a suppressor function and are expanded in the context of autoimmune organ specific disease, to circumvent the autoreactive cells. The CD4+ CD 25+ natural T cells do not exhibit a particular cytokine profile and their suppressive function is associated with a down-regulation of IL-2. The role of the CD4+ CD 25+ regulatory T cells in MCNS remains to be clarified.
Activated CD4+ helper T cells (Th) can be differentiated into two functionally and mutually exclusive distinct subsets: type 1 Th (Th1) and type 2 Th (Th2), on the basis of their cytokine profiles [10]. Polarization of uncommitted Th (Th0) into Th1 or Th2 cells involves different mechanisms, of which the cytokine field at the site of antigen encounter plays a driving role. While interferons /ß (IFN-
/ß) and IL-12 are critical mediators of Th1 development, IL-4 was shown to be a potent inducer of Th2 cells from naive T cells. Th1 cells express the IL-12 receptor, and secrete IFN-
, TNF-
and lymphotoxin-
(LT-
, also called TNF-ß). Unlike TNF-
, which is produced primarily by activated macrophages, LT-
is synthesized by activated T and B cells and its biological role has not been clearly established in vivo. Th1 cytokines initiate inflammatory reactions, enhance cell immunity response, and mediate delayed-type hypersensitivity. Th2 cells are prone to synthesize IL-4, IL-5, IL-13, which together contribute to anti-inflammatory responses, help B cells to produce antibodies and protect against extracellular pathogens.
We have recently shown that T lymphocytes from MCNS display a down-regulation of the IL-12 receptor ß2 subunit (IL-12R ß2) during relapse, while the second component of IL-12R, the ß1 chain, was normally expressed [11]. The IL-12R ß2 is selectively expressed by Th1 cells and plays a key role in the transduction of IL-12 signalling through the Jak/Stat pathway. The down-regulation of the IL-12R ß2 is compatible with a lack of IL-12 production during relapse, as reported by Stefanovic et al. [12], and suggests that activated T cells of MCNS were early driven toward Th2 phenotype. Additional evidence came from recent isolation by subtractive cloning of a Th2 specific factor, c-maf, which is strongly induced during relapse [11]; Valanciute A et al., submitted for publication]. These results are in agreement with early reports by Kimata et al. [13] and Yap et al. [14], who found an increased production of IL-13 Th2 cytokine during relapse. IL-13 regulates the switching of immunoglobulin production towards IgE, which is frequently increased in serum of patients with MCNS [15]. Interestingly, Ho et al. [16] have demonstrated that c-maf promotes Th2 and attenuates Th1 differentiation. Patients with MCNS often exhibit a defect in delayed-type hypersensitivity response, suggesting inhibition of Th1-dependent cellular immunity [17]. The inability to mount an effective Th1 response might account for the susceptibility to pneumococcal and other pathogen agents in this disease, known from the outset of the last century [18].
Transcription factors and proteasome activity in MCNS
We recently reported abnormal activities of transcriptionfactors in MCNS, along the same line as Sahali et al. [19] and Cao et al. [20]. We detected high NFB activation in CD4+ T cells during the relapse. NF
B is rapidly activated by a wide variety of pathogenic signals such as viral and bacterial agents, T cell and B cell mitogens, cytokines (TNF-
, LT-
) and oxidative stress [21]. NF
B is involved to various extents in the transcriptional activation of a large set of genes including those encoding IL-1, IL-6, IL-2, IL-8, TNF-
and LT-
, many of them being increased in MCNS relapse. Interestingly, Das et al. [22] identified two NF
B-responsive elements on the IL-13 promoter, thus including this cytokine as a potential downstream target of NF
B. The transcriptional activation of NF
B is tightly regulated by inhibitory proteins called I
B proteins of which I
B
is the most important inhibitor. Following T-cell activation by NF
B inducers such as TNF-
, I
B
is rapidly phosphorylated, then degraded by the proteasome, allowing the nuclear translocation of NF
B, which activates its target's genes, including its proper inhibitor I
B
. In return, I
B
is rapidly re-synthesized and sequestrates NF
B in the cytoplasm, thus switching off the NF
B activity. The fact that NF
B activation is sustained during the relapse, before the initiation of steroid therapy, suggests a break in the NF
B feedback loop in the active phase of the MCNS [19]. The up-regulation of I
B
expression, in T cells and monocytes, following glucocorticoid therapy probably contributes to inhibition of the NF
B activation and the down-regulation of cytokines during the remission. In three patients who developed a steroid-resistant but cyclosporin-sensitive MCNS, we observed a strong NF
B activation during steroid therapy, which diminished upon addition of cyclosporin (Sahali D et al., unpublished data). The break in NF
B autoregulatory loop during relapses, and in some patients with resistance to steroids, is apparently not due to genetic alterations of I
B
, since increased levels of the inhibitor were found in remission. Moreover, we did not detect mutations or deletions in the I
B
promoter in seven patients analysed, including, the three patients described above (Valanciute A, Sobrier ML et al. unpublished data).
Although a close relationship between NFB activation and MCNS relapse seems to prevail, the fact that NF
B activation remains increased in some patients, despite steroid therapy, suggests that additional factors are involved in these transcriptional alterations and might contribute to steroid resistance. These data also suggest that the mechanisms by which glucocorticoids and cyclosporin induce a remission in MCNS might not be similar but rather complementary. This hypothesis is based on experimental findings showing that NF
B activation, in PBMC and T cells from MCNS relapse, is inhibited upon ex vivo addition of the proteasomal inhibitor MG132, which blocks the degradation of phosphorylated form of I
B
[19]. Cyclosporin A exerts an effect similar to MG132 [23,24]. On the other hand, glucocorticoids induce a transactivation of the I
B
gene, which might contribute to increased I
B
expression in remission, the highest levels being observed in patients on steroids. Thus, the glucocorticoids and cyclosporin A act by distinct mechanisms on the stabilization of I
B
and the inhibition of NF
B activation. Conceptually, it is hard to draw a simple model for T-cell activation in MCNS, given our current knowledge, but a working scheme may be proposed (Figure 1
), which will undoubtedly be corrected and completed in the near future.
Alteration of other immune cell subsets in MCNS
Immune dysfunction does not seem to be restricted to CD4+ T cells, but our knowledge concerning the contribution of other T-cell subsets, monocytes, and even B cells, is very limited. Inasmuch that the immune system functions as a complex network, there could well exist other abnormalities that need characterization. As a matter of fact, the bulk of NFB activation detected in relapse is not restricted to CD4+ T cells, but appears more striking in the non-CD4+ mononuclear cell fraction [19]. Frank et al. [25], analysing the genetic polymorphism of the variable region of the TCR ß-chain, found a selective recruitment of some Vß families in peripheral non-CD4+T cells from patients with frequent relapse, suggesting a clonal expansion of CD8+T cells in long-lasting active disease.
Contribution of subtractive and differential cloning to study of MCNS pathogenesis
Initial analysis of subtracted transcripts revealed two important and unexpected findings. First, several transcripts that are selectively up-regulated during the relapse are produced by unusual splicing, given that these forms are undetected in normal subjects. Preliminary data suggest that the protein forms encoded by these new transcripts display functional characteristics distinct from normal spliced proteins (Grimbert P et al., manuscript submitted). Secondly, some genes expressed but not translated in normal subjects exhibit a high protein level during the relapse (Audard V et al., work in progress). These results suggest that both transcriptional and post-transcriptional mechanisms are involved in immunopathogenesis of MCNS, which appears to be a complex disease in which several genes may contribute to immunological disorders.
Perspectives
Evidence can be gathered supporting the hypothesis that T cells, and most probably other immune cells as well, are involved in MCNS disease. Recent data provide one of the first molecular analyses of the signalling pathways preferentially recruited by T cells during the relapse. Although the molecular mechanisms underlying MCNS have yet to be clarified, the strategy developed in our laboratory may allow identification of the genes that play a central role in the immunopathogenesis of this disease. This work opens new perspectives in the molecular characterization of lymphocyte activation pathways in MCNS.
Acknowledgments
We are indebted to Gabriel Richet, Michel Broyer, Patrick Niaudet and Pierre Ronco, who have provided much encouragement throughout the course of this project. We also thank Remy Salomon, Chantal Loirat and Albert Bensman, who have been essential to the research conducted in our laboratory.
Notes
Correspondence and offprint requests to: D. Sahali, Unité INSERM 99, Hôpital Henri Mondor, 51, avenue du Mal de Lattre-de-Tassigny, 94010, Créteil, France. Email: sahali{at}im3.inserm.fr
References