Invariant chain degradation is a critical step in MHC class II maturation for antigen presentation. It has been shown that different APCs use different cysteine proteases for this process in the thymus and in the periphery. Cat L is necessary for Ii degradation in mouse cortical epithelial cells (13
), whereas Cat S is essential for antigen presentation in peripheral APCs (10
). The differential expression of cathepsins in functionally distinct APCs opens the possibility for a selective therapeutic targeting of thymic versus peripheral antigen presentation. This highlights the necessity of an unambiguous identification of the proteases involved. We have recently described human Cat V as a novel member of the papain family (14
). Although this protease shares a protein sequence identity as high as 80 % with human Cat L, its tissue distribution is strikingly different from that of Cat L. Although human Cat L was found in most tissues, the expression of Cat V was restricted to thymus and testis. Using quantitative real-time PCR, we have analyzed the mRNA expression profiles of human Cats V, L, and S in normal thymus and PBMCs. In agreement with previous reports, we found that Cat S was expressed at much higher levels in peripheral blood monocytes, whereas Cat L revealed similar expression levels in thymus and PBMCs and Cat V was found only in thymus. Moreover, absolute quantification of thymic mRNA expression showed that Cat V is expressed at 75-fold higher levels than that of Cat L (data not shown). Furthermore, the expression of Cat V was restricted to TECs, indicating that in this compartment of the human thymus, Cat V, but not Cat L, is the predominant cysteine protease. Using monoclonal antibodies that allowed discriminating between Cats V and L expression, we confirmed the selective expression of Cat V in cortical thymic stroma. Cat L expression was restricted to vesiculated medullary macrophage-like cells and a few cortical mononuclear cells. It should be noted that the antibodies used do not permit the exclusion of Cat V in these macrophage-like cells.
To demonstrate that Cat V is enzymatically active in thymus lysates and at the same time to judge the presence of other papain-like cysteine proteases in these samples, the active site-directed labeling with the peptidyl epoxid inhibitor, DCG-04, was used. Because Cats V and S cannot be separated by SDS-PAGE, we selectively blocked the labeling of Cat S by preincubating the thymic extract with the potent Cat S inhibitor, LHVS (at 1 nM for 20 minutes) (10
). Under these conditions, Cat V remains labeled. That the labeled bands represent cathepsins was demonstrated by the elimination of all DCG-04 labeling at higher LHVS concentrations. At 1 μM inhibitor concentration, LHVS inhibits Cats V, L, S, K, and B. Thus, we suggest, that the DCG-04–labeled band with an apparent molecular mass of 30 kDa in the presence of 1 nM LHVS represents active human Cat V in thymus extracts. Ideally, one would want to analyze lysosomal fractions from pure epithelial cells, which do not contain Cat S, but to date, it is technically close to impossible to isolate these cells with enough purity in the amounts necessary for organelle separation. TECs that have been in culture for 1 week are already phenotypically very different from the freshly isolated cells: Cat V expression is reduced, and Cat L starts to appear (our unpublished observations).
Cortical TECs are mainly responsible for positive selection of T cells, whereas B cells, dendritic cells, and medullary epithelial cells seem to be involved in the negative selection process (27
). Mechanistic differences of antigen processing and presentation by cortical and medullary TECs have been documented in murine cell lines (28
). Moreover, distinct profiles of self-peptides were characterized in murine cortical and medullary epithelial cell lines (30
), suggesting that a different array of proteases operates in the two compartments. The lack of Cat L expression in the human cortical stroma is in sharp contrast to the reported expression of Cat L in mouse thymus (13
Given the fact that Cat V is expressed mostly in the thymic cortex, the high homology between murine Cat L and human Cat V, and the results from the Cat L KO mice, we propose that, in humans, Cat V has overtaken the role of Cat L in the positive selection of thymocytes. This assumption is supported by our finding that Cat V selectively degrades Ii complexed to MHC and generates a 3-kDa peptide identical to CLIP in size, with similar efficiency to Cat S. The immunologically relevant degradation of Ii by Cat V is reflected by the efficient loading of antigenic peptide to DR3-αβ complexes in the presence of HLA-DM (31
). In contrast, human Cat L activity revealed no such substrate specificity. Although no significant degradation of the αβ-complex was observed at enzyme concentrations as high as 250 nM for the Cats V and S, Cat L degraded the complex already at 50 nM. Although the concentrations of cathepsins in MHC compartments are unknown, concentrations of Cats L and B have been calculated as high as 1 mM in lysosomes (32
). At least for Cat L, this would very likely destroy class II complexes rather than leading to productive antigen presentation. Thus, similar to Cats S, L, and F, we have found that Cat V promotes the degradation of the intermediate product Ii p10 into CLIP.
MG is a prototypic autoimmune disease targeting acetylcholine receptors of skeletal muscle (reviewed in ref. 33
). MG is almost invariably associated with thymic pathologies such as thymitis and thymic tumors, suggesting an intrinsic thymic defect in T cell selection as an underlying cause for MG (34
). It has been found that among the various types of thymomas, those associated with MG are characterized by prominent thymopoesis (26
) and an increased proportion of recent thymic emigrant T cells are found in the periphery of MG patients (36
). It is thus reasonable to assume that an altered molecular microenvironment in the thymus promotes the maturation of thymocytes (38
) including those with an autoimmune potential (39
). Increased numbers of thymocytes may bypass the physiologic check points of selection that usually involves interaction with self-peptide–loaded MHC molecules on specialized thymic APC. We have demonstrated a selective overexpression of Cat V in thymi of MG patients both with thymitis and thymoma when compared with normal controls and non-MG–associated thymomas, both by real-time PCR and immunohistochemical analysis. It must be noted that the non-neoplastic part of a thymoma often shows thymitis. In contrast, expression of other cathepsins was not significantly different between patients and controls. Of note, even samples from non-MG–associated thymomas that were selected for their low thymocyte contents showed no difference in the expression of Cat V compared with control thymus. Although the immediate effects of higher levels of Cat V in the cortical epithelial cells are unknown, a possible outcome would be an imbalance of the positive/negative selection mechanisms, leading to a higher number of autoreactive cells in the periphery. In analogy with these data, the Cat S KO mice were found less susceptible to collagen-induced arthritis than their WT littermates (11
), and although the mechanism for this resistance is not known, it is plausible to think of different kinetics of antigen presentation in the peripheral APC caused by the lack of Cat S.
In summary, we have demonstrated that Cat V adds to the list of proteases that degrade Ii, and that it is crucial for Ii degradation in human cortical TECs. The overexpression of Cat V in MG-associated thymic abnormalities may allow the chain of events that lead to autoimmune disease.
Note added in proof.
While this paper was being reviewed, an article implicating Cat S and not Cat V in the degradation of Ii in the human thymus was published by Bania et al. (40
). In their article, however, the data on thymus is restricted to RT-PCR performed on a cell line derived from cortical epithelium (P1.4D6) and a thymic tumor, which could explain the differences with the data generated from normal donors in our laboratory.