Cathepsin S Controls MHC Class II-Mediated Antigen Presentation by Epithelial Cells In Vivo
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免疫学杂志 2005年第1期
Abstract
Epithelial cells at environmental interfaces provide protection from potentially harmful agents, including pathogens. In addition to serving as a physical barrier and producing soluble mediators of immunity, such as cytokines or antimicrobial peptides, these cells are thought to function as nonprofessional APCs. In this regard, intestinal epithelial cells are particularly prominent because they express MHC class II molecules at the site of massive antigenic exposure. However, unlike bone marrow-derived professional APC, such as dendritic cells or B cells, little is known about the mechanisms of MHC class II presentation by the nonprofessional APC in vivo. The former use the lysosomal cysteine protease cathepsin S (Cat S), whereas thymic cortical epithelial cells use cathepsin L (Cat L) for invariant chain degradation and MHC class II maturation. Unexpectedly, we found that murine Cat S plays a critical role in invariant chain degradation in intestinal epithelial cells. Furthermore, we report that nonprofessional APC present a class II-bound endogenous peptide to naive CD4 T cells in vivo in a Cat S-dependent fashion. These results suggest that in vivo, both professional and nonprofessional MHC class II-expressing APC use Cat S, but not Cat L, for MHC class II-mediated Ag presentation.
Introduction
Major histocompatibility complex class II molecules are constitutively expressed on so-called professional APCs of bone marrow (BM)5 origin, i.e., B cells, dendritic cells, and macrophages (M), and in specialized thymic epithelium. In addition, MHC class II expression can be induced by bacterial cell products and inflammatory cytokines on different types of epithelial cells at environmental interfaces, including intestinal and skin epithelium (1, 2). The expression of MHC class II molecules on these nonprofessional APC has been implicated in immune-mediated inflammation and progression of or resistance to autoimmunity (3). In particular, it has been suggested that Ag presentation by intestinal epithelial cells (IEC) may play an important role in MHC class II-mediated presentation to immunoregulatory T cells (4, 5). However, little is known about the pathways and molecules involved in the regulation of MHC class II Ag presentation in IEC and their potential roles in Ag presentation to CD4 T cells in vivo.
MHC class II heterodimers are assembled in the endoplasmic reticulum with the assistance of invariant chain (Ii). A portion of Ii termed CLIP binds in the peptide groove of MHC class II molecules, thereby preventing premature loading of peptides. Endosomal localization motifs in the cytoplasmic tail of Ii promote entry of newly synthesized Ii oligomers into the endosomal compartment, where Ii undergoes rapid degradation. Lysosomal proteases degrade Ii until only CLIP remains bound in the MHC class II peptide-binding groove. CLIP removal and loading of peptides derived from foreign and self-protein Ags are mediated by DM, an MHC class II-like accessory molecule, localized to endosomal and lysosomal compartments of MHC class II-positive cells (6).
Until recently, the in vivo roles of specific lysosomal proteases in the MHC class II presentation pathway were poorly understood. Gene targeting studies revealed a critical role for the lysosomal cysteine protease cathepsin S (Cat S) in the late stages of Ii degradation in B cells, dendritic cells, and, to a lesser degree, M (7, 8, 9). As a result, in the absence of Cat S, many MHC class II molecules in the endosomal/lysosomal compartments and on the cell surface are associated with the Ii degradation intermediate p12 (8, 9, 10, 11). In contrast, another essential MHC class II-expressing cell type, thymic cortical epithelium, lacks Cat S expression. Instead, these cells use a different lysosomal cysteine protease, cathepsin L (Cat L), for the late stages of Ii degradation because similar accumulation of Ii fragments bound to MHC class II molecules is observed in Cat L-deficient thymic cortical epithelial cells.
Despite these observations, the role of lysosomal proteases in MHC class II presentation by nonprofessional APC expressed in vivo remains unknown. In this study we have studied the roles of Cat S and Cat L in MHC class II presentation in IEC. Because substantial phenotypic similarity has been reported for thymic epithelium and other types of keratinized epithelium (12), and Cat L is crucial for the late stages of Ii degradation in thymic epithelium and for hair follicle epithelium homeostasis (13, 14), we expected to find a prominent role for Cat L in Ii processing and MHC class II presentation by IEC. Unexpectedly, we observed that Cat S plays a critical role in Ii degradation in and MHC class II presentation mediated by these cells. Furthermore, we demonstrate that Cat S expression is essential for efficient presentation of endogenous peptides to CD4 T cells by nonprofessional APC in vivo. These results indicate that Cat S is a key enzyme regulating MHC class II presentation to CD4 T cells in both professional APC and in one of the most significant nonprofessional APC of epithelial origin, IEC.
Materials and Methods
Mice
C57BL/6 (B6) mice were purchased from Charles River, and Cat S–/–, Cat L–/–, MHC class II–/–, human Ii, H-2M–/–, Ii–/–, H-2M–/–xIi–/–, and Ly5.1+ TEa-B6 mice were maintained under specific pathogen-free conditions at the University of Washington. Cat S–/– and Cat S+/– (H-2bxd, E+) mice were generated by crossing Cat S–/– B6 (H-2b, E–) and B10.D2 (H-2d, E+) mice. All animals were used at 6–10 wk of age. Procedures and care of the animals were in accordance with the University of Washington guidelines provided by the Institutional Animal Care and Use Committee.
Antibodies
The mAbs M5/114 (anti-I-Ab, d, q and anti-Ed, k; ATCC TIB120; American Type Culture Collection) and In-1 (anti-Ii) have been described previously (15, 16). The Y3P mAb recognizes I-Ab (ATCC HB183), MKD6 recognizes I-Ad (ATCC HB3), and the YAe mAb recognizes I-Ab with E peptide (aa 52–68) bound in the peptide groove (17). Anti-CD45-PE, anti-B220-PE, anti-CD11c-FITC, anti-CD11b-PE, anti-CD4-allophycocyanin, and streptavidin-allophycocyanin were purchased from eBioscience, and the polyclonal rabbit antiserum recognizes mouse Cat L (a gift from A. Erickson, University of North Carolina, Chapel Hill, NC). The G8.8 mAb is specific for intestinal and thymic epithelia (18). The 15G4 mAb is specific for MHC class II molecule I-Ab bound to murine, but not human, Ii degradation intermediates p12 and CLIP (data not shown). Streptavidin-HRP conjugate was purchased from Vector Laboratories.
IEC isolation
Intestines from 6- to 10-wk-old mice were dissected, flushed, and cut into pieces as previously described (19). The intestinal tissue was dissociated with a collagenase/dispase/deoxyribonuclease mixture (Roche) and fractionated on a discontinuous Percoll gradient. Any remaining BM-derived cells were removed by negative sorting CD45+ cells on an AutoMACS magnetic cell sorter (Miltenyi Biotec) or positively sorting epithelial cells using the G8.8 mAb on a FACSVantage cell sorter (BD Biosciences).
Immunohistochemistry and immunoelectron microscopy
Small and large intestines from 6- to 10-wk-old mice were snap-frozen, and serial sections (5 μm) were prepared, air-dried, and acetone fixed. Sections were blocked with normal serum and subsequently incubated with optimal dilutions of primary mAb for 1 h at 25°C before washing and incubation with appropriate HRP-conjugated secondary Ab. Control slides were incubated with nonimmune species-matched Ig. Microscopic analysis was performed with a Pro Vis AX70 microscope (Olympus). MHC class II and Ii levels were assessed by staining with the mAbs Y3P and 15G4. These Abs were directly conjugated to digoxigenin-3-o-methyl-carbonyl--aminocarproic acid-N-hydroxysuccinimide ester. Tissue sections were then developed as described by Farr et al. (20).
For immunoelectron microscopy, small intestines from B6 and Cat S–/– mice were fixed in 2% paraformaldehyde and processed for ultrathin cryosectioning and Immunogold labeling as described previously (21). Immunogold labeling was performed with M5/114 and In-1 mAbs and 10-nm protein A-conjugated gold particles.
Cysteine protease active site labeling
IEC (2 x 105) were incubated for 2 h at 37°C with the iodinated cysteine protease inhibitor Cbz-[125I]-Tyr-Ala-CN2 (22), washed, and lysed. Lysates were analyzed by 12% SDS-PAGE.
Immunoblotting
Purified IEC were washed in PBS and lysed in cell lysis buffer (0.5% Nonidet P-40, 0.15 M NaCl, and 50 mM Tris-HCl, pH 7.2) supplemented with a mixture of protease inhibitors (Roche). Debris was removed by centrifugation at 8000 rpm for 10 min. Samples containing the indicated number of cell equivalents were boiled for 5 min in SDS reducing buffer and separated by 12% SDS-PAGE. The proteins were electrophoretically transferred onto nitrocellulose membrane and probed using the indicated primary Ab. Binding was detected using the appropriate HRP-conjugated secondary Ab diluted 1/1000 and was visualized by chemiluminescence (ECL; Amersham Biosciences).
Flow cytometric analysis
To assess MHC class II expression on IEC, B cells, dendritic cells, and M, flow cytometric analysis was performed. B6 and Cat S–/– IEC were stained with digoxigenin-derivatized G8.8 mAb (BD Biosciences) and biotinylated mAbs Y3P and 15G4. Binding of digoxigenin-conjugated Abs was detected with anti-digoxigenin fluorescein-labeled Fab (Roche), and binding of biotinylated class II-specific Abs was detected with streptavidin-allophycocyanin (BD Biosciences); stained cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences). B cells, dendritic cells, and M were analyzed using anti-B220-PE, anti-CD11c-FITC and anti-CD11b-PE, and anti-CD11b-PE (eBioscience), respectively, from splenocytes isolated from B6 and from MHC class II–/–Cat S+/– H-2bxdE+, Cat S+/– H-2bE–, Cat S–/–H-2bxdE+, and Cat S–/–H-2bE– chimeric mice. MHC class II was detected using M5/114 mAb (eBioscience).
T cell hybrid assay
T cell hybrid assays were performed as previously described (10, 23, 24). I-Ab-restricted T hybrids (1.0 x 105/well), specific for IgM (77.1) and 2-microglobulin (2M; 4.1) (23, 24), were cultured in duplicate for 24 h with 2 x 105 B6 or Cat S–/– IEC. In parallel, T cell hybrids were stimulated with 1 x 105 B6 or Cat S–/– splenocytes. IL-2 production was assessed using CTLL-2 proliferation, as determined by the Alamar Blue colorimetric assay. The results are expressed as arbitrary OD units (A570–A600).
In vivo activation of CD4+ T cells
To generate chimeric mice expressing MHC class II molecules on nonprofessional radiation-resistant APC, but not on BM-derived APC, BM from MHC class II–/– mice was transferred into lethally irradiated (1000 rad) Cat S+/– H-2bxdE+, Cat S+/– H-2bE–, Cat S–/–H-2bxdE+, and Cat S–/–H-2bE– mice. Six weeks after BM transplantation, complete reconstitution of host BM-derived APC with MHC class II–/– APC was confirmed by FACS. CD4+ T cells T cells were purified from the lymph nodes and spleens of Ly5.1+ TEa-B6 mice using an AutoMACS magnetic cell sorter (Miltenyi Biotec). These cells were labeled with CFSE (Molecular Probes), and 5 x 106 T cells were i.v. injected into the chimeric mice. Proliferation of donor cells was monitored by flow cytometry. On days 3 and 5 post-transfer, lymph nodes and spleen cells were analyzed by flow cytometry using anti-Ly5.1-PE and anti-CD4 APC.
Restimulation assay
On day 5 after transfer, TEa CD4+ T cells were reisolated from splenocytes. CD4+ T cells (5 x 105) were incubated in the presence of irradiated (2000 rad) 2 x 105 B6 splenocytes and titrating amounts of E52–68 peptide in 96-well, flat-bottom plates. Cell proliferation was measured by labeling cultures with [3H]thymidine (1 μCi/well), and the assay was harvested 24 h later. Data are presented as the mean cpm of [3H]thymidine incorporation in triplicate cultures.
Results
Immunohistochemical analysis of Ii degradation in intestinal epithelium of Cat S- and Cat L-deficient mice
We generated mAb 15G4, specific for MHC class II molecule I-Ab bound to Ii degradation intermediates p12 and CLIP (data not shown), and used this reagent to detect I-Ab-bound Ii degradation fragments in tissues from Cat S–/– and Cat L–/– mice. In initial experiments, frozen sections of thymi from B6, Cat S–/–, Cat L–/–, H-2M–/– (positive control), and H-2M–/–Ii–/– (negative control) mice were stained with digoxigenin-derivatized 15G4 Ab. Previously, we have observed large amounts of p12 bound to I-Ab molecules in Cat L–/– cortical thymic epithelium (14). Indeed, 15G4 stained the cortical region of Cat L–/– thymi in a manner similar to the H-2M–/– positive control. In contrast, only the medullary region of Cat S–/– thymi stained intensely with 15G4, consistent with the function of this enzyme in p12 degradation in BM-derived APC (data not shown). Thus, the 15G4 Ab serves as an excellent tool for analyzing Ii degradation and MHC class II maturation in tissue sections.
Immunohistochemical survey of MHC class II expression in nonlymphoid tissues, including kidney, lung, small and large intestine, and skin, in wild-type B6 mice using 15G4 and pan-I-Ab-specific Y3P Ab revealed intestinal and skin surface epithelia as the main sites of MHC class II expression (data not shown). We limited our studies of the roles of lysosomal cysteine proteinases in nonprofessional APC to IECs.
To study the roles of Cat S and Cat L in IEC, we first stained sections of the small and large intestines from B6, Cat S–/–, and Cat L–/– mice. Unexpectedly, 15G4 also stained the epithelial cell layer in the small and large intestines of Cat S–/–, but not Cat L–/– or B6 mice (Fig. 1, A and B), in addition to intense staining of BM-derived cells in the lamina propria. Although comparable expression of MHC class II was found in the small intestine in all three strains of mice, an increase in MHC class II expression was observed only in Cat S–/– large intestine by staining with the Y3P Ab (Fig. 1B). These results suggest that Cat S, but not Cat L, plays a role in Ii degradation in IEC in both small and large intestines.
To discriminate between the contribution of BM-derived APC and IEC to the increased 15G4 staining in the absence of Cat S, we generated reciprocal BM chimeras between wild-type B6 and Cat S–/– mice. Sections of small and large intestines from B6Cat S–/–, Cat S–/–B6, as well as control Cat S–/–Cat S–/– and B6B6 chimeric mice were stained with 15G4 and Y3P Abs. APC reconstitution in recipient mice by donor BM-derived cells was confirmed by 15G4 staining of spleen sections from chimeric mice (data not shown). Examination of small and large intestinal tissues from the chimeric mice stained with 15G4 Ab corroborated observations made in nonirradiated Cat S–/– mice. In the B6Cat S–/– chimeras, 15G4 intensely stained the epithelium, but not BM-derived APC, in the lamina propria, whereas Cat S–/–B6 chimeras displayed the opposite staining pattern (Fig. 1, C and D). Control sections from B6B6 and Cat S–/–Cat S–/– showed staining similar to that in nonchimeric B6 and Cat S–/– mice, respectively (see Fig. 1, A and B). Notably, elevated levels of MHC class II expression, i.e., increased Y3P staining, were observed in the large intestine of all Cat S–/– BM recipients. Hence, in the absence of Cat S MHC class II-bound Ii degradation intermediates accumulate in IEC in small and large intestinal epithelia.
Analysis of Cat S expression and function in IEC
To examine in more detail the role of Cat S in the MHC class II presentation pathway in non-BM-derived epithelial APCs, we decided to focus on IEC as a prototype nonprofessional APC. The increased 15G4 staining in the intestinal epithelial layer of Cat S–/–, but not Cat L–/–, mice led us to directly investigate the expression of these enzymes, the cellular distribution of total MHC class II molecules, and MHC class II molecules bound to Ii fragments. Therefore, we isolated IEC by collagenase digestion of the small intestine from B6 and Cat S–/– mice. The IEC were subsequently enriched by Percoll density gradient centrifugation. The resulting cell population was subsequently depleted of BM-derived CD45+ cells using magnetic beads or FACS sorting.
To directly evaluate Cat S and Cat L activities, FACS-sorted IEC (purity, 95%) were incubated with the active site inhibitor CN2-125Tyr-Ala-Cbz, and labeled enzymes were visualized after protein separation by PAGE (Fig. 2A). These experiments showed that Cat S activity is readily detectable in IEC, whereas Cat L activity was lacking.
The absence of Cat L activity was unexpected, because Cat L plays a role in Ii degradation in thymic cortical epithelial cells and is involved in hair follicle epithelium homeostasis (18, 19). Our failure to detect Cat L activity could be due to a lack of expression of the protein or inhibition of Cat L activity. Western blot analysis of wild-type B6 and Cat S–/– IEC lysates using a Cat L-specific Ab showed high levels of mature Cat L protein in both B6 and Cat S–/– IEC (Fig. 2B). Recently, we reported that M, which normally express active Cat L, exhibit a marked decrease in Cat L activity upon exposure to the proinflammatory cytokine IFN-, whereas mature protein levels remain high. This down-modulation of Cat L activity is thought to be mediated by a specific inhibitor, probably cystatin F (11). However, cystatin F mRNA was not detectable in IEC (data not shown). Therefore, an as yet unidentified inhibitor might control Cat L activity in IEC.
To characterize the Ii fragments bound to MHC class II molecules in Cat S–/– IEC, we performed Western blot analysis of purified IEC using the In-1 Ab specific for Ii (Fig. 2C). Only Cat S-deficient IEC showed similar accumulation of the p12 Ii fragment as that in B cells, dendritic cells, and M lacking this enzyme (9, 11).
Next, we subjected IEC isolated from B6, Cat S–/–, and MHC class II–/– mice to flow cytometric analysis to examine the cell surface expression of total MHC class II and MHC class II bound to Ii fragments using Y3P or 15G4 Abs. Anti-CD45 and G8.8 mAbs were used to distinguish between BM-derived cells and IEC, respectively (Fig. 2D). Cat S–/– IEC displayed an increase in MHC class II staining compared with B6 and MHC class II–/– IEC. In addition, we observed increased 15G4 staining in Cat S–/– IEC compared with the controls. Thus, flow cytometric analysis revealed increased cell surface expression of Ii fragments bound to MHC class II molecules, in agreement with the immunohistochemical studies.
To investigate intracellular accumulation of Ii fragments bound to MHC class II molecules, we performed immunoelectron microscopic analysis of frozen thin sections of B6 and Cat S–/– IEC. The 15G4 Ab does not reliably stain tissue samples processed for immunoelectron microscopy. Therefore, sections were stained with the In-1 Ab, specific for intact Ii and N-terminal Ii fragments, and with the M5/114 Ab, recognizing I-Ab molecules (Fig. 2E). We observed increased levels of MHC class II and Ii in the lysosomal vesicles of Cat S–/– compared with wild-type B6 IEC. Although Immunogold staining of plasma membrane was not very strong, the majority of cell surface MHC class II molecules were localized to the basolateral membrane (data not shown). As expected, a block of Ii degradation in Cat S-deficient IEC results in accumulation of Ii degradation intermediates in endosomal/lysosomal compartments. This leads to expansion and maturation of endocytic compartments, as shown for several different cell types by Bakke et al. (25, 26, 27). In addition, we have stained IEC sections with anti-lysosome-associated membrane protein Ab and MHC class II and found Ii-positive compartments to be lysosome-associated membrane protein positive (data not shown).
Thus, Cat S deficiency in nonprofessional epithelial APC, i.e., IEC, results in the accumulation of Ii fragments bound to MHC class II molecules on the cell surface and in lysosomal compartments. These data directly implicate Cat S in mediating Ii processing in IEC. The increase in cell surface and intracellular MHC class II molecules found in Cat S–/– IEC may be due to a specific role for this protease in the turnover of MHC class II molecules. Alternatively, Ii p12 fragments may stabilize MHC class II I-Ab molecules if there is a relative paucity of class II binding peptides in IEC, thereby increasing total MHC class levels.
To elucidate the effect of Cat S deficiency on Ag presentation by IEC, we generated chimeric mice using MHC class II–/– donor BM transferred into either B6 or Cat S–/– lethally irradiated mice. We purified IEC from chimeric mice 6–8 wk after BM transfer and cultured them in the presence of self-peptide-specific T cell hybridomas. We used T cell hybridomas specific for the endogenous Ags IgM and 2M and investigated the ability of B6 and Cat S–/– IEC to present these Ags compared with splenocyte controls. B6 and Cat S–/– IEC failed to present 2M peptide, whereas splenocytes from these mice presented the 2M peptide with equal efficiency (Fig. 3, A and C). B6 and Cat S–/– splenocytes also present IgM epitope equally efficiently (Fig. 3B). In contrast, Cat S–/– IEC proficiently presented IgM peptide whereas B6 IEC induced substantially lower levels of stimulation of IgM specific T cell hybridomas (Fig. 3D) despite comparable serum IgM levels in Cat S mutant and wild-type mice (data not shown). Enhanced presentation of the IgM epitope by cathepsin S–/– IEC can also be explained by increased class II expression in these cells. Although we cannot formally exclude this possibility, we believe that this is an unlikely explanation because a similar relative increase in IgM epitope presentation was observed in Cat S–/– M, dendritic cells, and fibroblasts in which total surface class II levels were not affected by Cat S expression (9, 11, 28). This is probably due to a putative Cat S cleavage site within the IgM peptide epitope.
To investigate the role of Cat S in MHC class II presentation of self-peptides by IEC, we analyzed expression of I-Ab bound to a well-characterized self-peptide E52–68 derived from the class II molecule I-E. This complex can be detected by the mAb YAe (17). Therefore, we crossed B10.D2 (H-2d E+) with Cat S–/– (H-2b, E–) animals to generate Cat S–/– H-2bxd E+ mice. IEC as well as splenocytes isolated from Cat S+/– H-2bxd E+, Cat S–/– H-2bxd E+, and MHC class II–/– mice (negative control) were stained with YAe (Fig. 3, E and F). Cat S–/– H-2bxd E+ IEC and splenocytes both show a significant decrease in YAe staining compared with Cat S+/– littermate controls. The negative peak in bimodal YAe staining of unseparated splenocytes corresponds to MHC class II-negative T cells, whereas the majority of YAe-positive cells are B cells. Therefore, Cat S deficiency decreases the expression of E peptide-I-Ab complexes in splenic B cells as well as in IEC. Thus, Cat S is involved in Ii degradation and regulates the expression of self-peptide-MHC class II complexes in IEC.
In vivo presentation of self-peptide-MHC complexes by nonprofessional APC to CD4 T cells is diminished in the absence of Cat S
To elucidate the functional significance of altered presentation of self-peptide-MHC class II complexes by nonprofessional APC lacking Cat S, we examined activation of TEa TCR transgenic T cells recognizing E52–68 peptide bound to I-Ab on non-BM-derived APC. This in vivo E52–68 peptide presentation was assessed using adoptive transfer of CFSE-labeled Ly5.1+ TEa T cells. To eliminate presentation of E52–68 peptide by professional BM-derived APCs, we first transferred MHC class II–/– BM into lethally irradiated (1000 rad) Cat S–/– E+ and Cat S+/– E+ mice and corresponding littermate control H-2b mice lacking E. Flow cytometric analysis of MHC class II expression by B cells, dendritic cells, and macrophages (Fig. 4A) confirmed essentially complete reconstitution of host APC subsets by MHC class II–/– donor BM-derived cells. These chimeric mice were injected i.v. with 5 x 106 purified CSFE-labeled CD4+ Ly5.1+ TEa T cells, and their Ag-driven division was analyzed on days 3 and 5 post-transfer (Fig. 4B). We found that on day 3 the CD4+ T cells in E-positive Cat S+/– recipients underwent three to five divisions. In contrast, in E-positive recipients lacking Cat S, TEa T cells showed delayed proliferation kinetics by one or two rounds of division. This difference became more pronounced on day 5 when almost all TEa CD4 T cells in Cat S+/– recipients have lost CFSE labeling by dividing six or seven times or more, whereas in Cat S–/– hosts the majority of TEa T cells divided only three or four times, making up 6 and 3% of the total CD4 T cells in corresponding recipient mice, respectively (Fig. 4A; data not shown). In agreement with this result, we found that in comparison with class II–/–Cat S–/– E+ recipients, CD4 T cells isolated from class II–/–Cat S+/– E+ recipients of TEa T cells showed 3-fold higher in vitro recall response to E52–68 peptide in the presence of irradiated T cell-depleted B6 splenocytes as APC (Fig. 4C). In contrast, in both heterozygous and homozygous Cat S mutant mice lacking E, used as negative controls, transferred TEa T cells remained undivided, constituting 0.3% of CD4 T cells and generating comparable in vitro recall responses (Fig. 4C). It seems highly unlikely that the observed proliferation is due to remaining host BM-derived APC in chimeric mice because we have not observed MHC class II expression on BM-derived cells, such as B cells and dendritic cells, upon flow cytometric examination (Fig. 4A) or by immunohistochemistry (data not shown).
Therefore, Cat S expression in nonprofessional APC in vivo, e.g., epithelial cells, is essential for optimal Ag presentation and activation of T cells. Together, these data clearly defines Cat S as the leading cysteine protease involved in Ii degradation and Ag presentation in vivo not only in professional, but also in nonprofessional, APC.
Discussion
Previously we have shown the expression of lysosomal cysteine proteases Cat S and Cat L in distinct subsets of professional APC; Cat S is active in B cells and dendritic cells, whereas Cat L activity is detected in thymic cortical epithelial cells. An essential role of these enzymes in MHC class II presentation is illustrated by the profound defect in Ii degradation and MHC class II Ag presentation in their absence (7, 9, 14). M express both enzymes; however, Cat S plays the leading role in MHC class II presentation due to down-regulation of Cat L activity upon induction of MHC class II molecules in response to proinflammatory stimuli (11). In addition, lysosomal cysteine proteinase Cat F has been suggested to contribute to Ii degradation in these cells (29). Thus, there is a striking dichotomy in utilization of the lysosomal cysteine proteinases involved in MHC class II presentation by the cells mediating positive selection of CD4+ T cell in the thymus, i.e., thymic cortical epithelial cells, and by the BM-derived APC involved in thymic negative selection of autoreactive TCR and in presentation of self and foreign Ags in the periphery.
In this study we expand our previous studies of the roles of Cat S and Cat L in MHC class II presentation by professional APC to include IEC, a major peripheral nonprofessional APC of epithelial origin. IEC express MHC class II constitutively due to continuous stimulation by bacterial flora and cytokines (30, 31) and are in contact with a large number of intraepithelial lymphocytes and lamina propria lymphocytes (32). Analysis of the Ag-presenting properties of ex vivo isolated, nontransformed IEC has been a major challenge because these cells are fragile and prone to death upon handling and under tissue culture conditions. In this study for the first time we demonstrate the ability of ex vivo isolated IEC to present endogenous and extracellular self-Ags in context with MHC class II. Furthermore, our studies using Cat S–/– and Cat L–/– mice have brought about the surprising finding that Cat S, but not Cat L, controls Ii degradation and peptide presentation by MHC class II molecules in these cells. In agreement with these functional results, we found only Cat S activity in freshly isolated IEC, whereas Cat L activity was not detected despite the expression of a large amount of mature Cat L protein, as revealed by Western blot analysis (Fig. 3, A and B). It is likely that these findings in mice also apply to humans, because Cat S has been found in the human colonic cancer cell line T84 (33, 34). The lack of Cat L activity in IEC was surprising because Cat L is active in other types of epithelium, e.g., thymic epithelial cells and kidney epithelium (14). Because we were able to detect mature Cat L protein, we suggest that this is due to inhibition of Cat L activity by an as yet unknown inhibitor. Intriguingly, in our recent studies we found that Cat L activity is also inhibited in IFN--induced M and in dendritic cells isolated from mice expressing Cat L as a transgene under the CD11c promoter (11). One likely candidate for such an inhibitor is the recently described cysteine protease inhibitor cystatin F, which has been shown to be expressed in M and dendritic cells (35, 36). Cystatin F mRNA is restricted primarily to hemopoietic cells, and it exhibits high affinity for Cat L. Cystatin F mRNA is up-regulated in maturing dendritic cells (37), and we found cystatin F mRNA levels markedly increased in M upon treatment with IFN-, suggesting that it could be a Cat L inhibitor in these cells (data not shown). However, cystatin F mRNA was undetectable in IEC. Thus, an unknown mechanism responsible for Cat L inhibition in these cells will have to be further investigated.
IEC are probably not the only type of peripheral nonprofessional APC using Cat S for MHC class II presentation. A recent study of human myoblasts stimulated in vitro with IFN- showed induction of Cat S and its role in regulation of MHC class II expression (38). Furthermore, Cat S expression was found in biopsies isolated from patients with myopathies, although M are probably present in the inflamed muscle tissue analyzed in these experiments and could account for this observation (38). Although further in vivo study of the roles of cysteine proteases in MHC class II-positive nonprofessional APC other than IEC is warranted, we predict that the majority of, if not all, nonprofessional APC expressing MHC class II under physiologic conditions in vivo use Cat S.
It is interesting to speculate why Cat S expression in both BM-derived professional APC and peripheral nonprofessional APCs is enforced. One possible explanation for such apparently stringent differential expression of Cat L and Cat S is to diminish the incidence of autoimmunity by avoiding the expression of largely overlapping repertoires of MHC class II-bound peptides in positively selecting thymic cortical epithelial cells and all other major APC types. In this regard, our recent study using a model APC line expressing Cat S or Cat L suggested that repertoires of MHC class II-bound peptides displayed by these cells are only partially overlapping (28). Our analysis of presentation of several known self-peptides by IEC showed that the expression of Cat S has a significant impact on the level of expression of peptide/MHC class II complexes. We found that the expression of the well-characterized I-Ab binding endogenous peptide E52–68 (17) is diminished in both IEC and splenic APC in the absence of Cat S. On the contrary, the expression of another endogenous peptide, IgM377–392, bound to I-Ab (39, 40) was significantly enhanced in Cat S-deficient IEC. Although B cells from Cat S–/– and wild-type mice present IgM equally efficiently, this result is in agreement with our previous findings of increased presentation of this IgM T cell epitope by M and dendritic cells isolated from Cat S-deficient mice compared with those from wild-type mice (9). Cat S-deficient MHC class II-positive fibroblasts expressing or lacking Cat L also exhibited enhanced presentation of this IgM epitope (28). The most likely reason for the negative effect of Cat S on presentation of the IgM epitope is its direct cleavage or degradation of its precursor. Thus, the expression of these two major self-peptide-class II complexes in IEC follows the pattern observed in professional APC. In contrast, a peptide derived from 2-M is not expressed in either wild-type or Cat S mutant IEC, whereas it is expressed at very high levels by BM-derived APC. It is possible that this may be a result of assembly of this peptide-class II complex at an intracellular site other than the late endosomal/lysosomal compartment. This possibility is supported by our observation of significantly increased presentation of this peptide-MHC class II complex by B cells and dendritic cells lacking Ii compared with wild-type APC (40) (S. Kovats and A. Y. Rudensk, unpublished observations). A distinct trafficking pathway(s) responsible for sorting MHC class II molecules to the basolateral membrane in polarized epithelial cell lines (41, 42) may account for the differences between IEC and BM-derived APC in MHC class II sampling of nonlysosomal compartments.
Does the expression of Cat S in nonprofessional APC affect their ability to present Ag to T cells in vivo? Adoptive transfer experiments using CFSE-labeled TEa T cells showed that Cat S deficiency in non-BM-derived APC results in a significantly less efficient activation of T cells in response to the highly abundant endogenous peptide-MHC class II complex. Although these experiments did not evaluate individual contributions of specific types of nonprofessional APC to TEa T cell stimulation, this is the first direct demonstration of naive T cell activation in response to self-peptide-MHC class II complex presented by non-BM-derived APC and an essential role of Cat S in this process. Under physiologic conditions, such activation is likely to lead to tolerance induction. However, in combination with infection and certain genetic factors, for example, associated with impaired central tolerance, recognition of self-Ags on nonprofessional APC may lead to tissue-specific autoimmunity, such as the skin pathology observed in mice expressing MHC class II transgene under keratin 14 promoter (43).
Thus, in addition to its key role in BM-derived professional APCs, Cat S is the principal lysosomal cysteine protease responsible for MHC class II maturation and presentation in vivo in a major nonprofessional APC type.
Acknowledgments
We thank K. Honey for critical review of the manuscript, and C. Plata and N. Li for their excellent care of the mice used in these experiments.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the Howard Hughes Medical Institute and grants from the National Institutes of Health (to A.Y.R.). C.B. is supported by a National Institutes of Health predoctoral training grant. A.B. is supported by the Department of Comparative Medicine.
2 Current address: Department of Comparative Medicine, Oregon Health and Science University, Portland, OR 97239-3098.
3 Current address: Infectious Diseases Service, Department of Medicine, Immunology Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
4 Address correspondence and reprint requests to: Dr. Alexander Y. Rudensky, Department of Immunology, Howard Hughes Medical Institute, University of Washington School of Medicine, Seattle, WA 98195. E-mail address: aruden@u.washington.edu
5 Abbreviations used in this paper: BM, bone marrow; B6, C57BL/6; 2M, 2-microglobulin; Cat L, cathepsin L; Cat S, cathepsin S; IEC, intestinal epithelial cell; Ii, invariant chain; M, macrophage.
Received for publication August 17, 2004. Accepted for publication October 25, 2004.
References
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Epithelial cells at environmental interfaces provide protection from potentially harmful agents, including pathogens. In addition to serving as a physical barrier and producing soluble mediators of immunity, such as cytokines or antimicrobial peptides, these cells are thought to function as nonprofessional APCs. In this regard, intestinal epithelial cells are particularly prominent because they express MHC class II molecules at the site of massive antigenic exposure. However, unlike bone marrow-derived professional APC, such as dendritic cells or B cells, little is known about the mechanisms of MHC class II presentation by the nonprofessional APC in vivo. The former use the lysosomal cysteine protease cathepsin S (Cat S), whereas thymic cortical epithelial cells use cathepsin L (Cat L) for invariant chain degradation and MHC class II maturation. Unexpectedly, we found that murine Cat S plays a critical role in invariant chain degradation in intestinal epithelial cells. Furthermore, we report that nonprofessional APC present a class II-bound endogenous peptide to naive CD4 T cells in vivo in a Cat S-dependent fashion. These results suggest that in vivo, both professional and nonprofessional MHC class II-expressing APC use Cat S, but not Cat L, for MHC class II-mediated Ag presentation.
Introduction
Major histocompatibility complex class II molecules are constitutively expressed on so-called professional APCs of bone marrow (BM)5 origin, i.e., B cells, dendritic cells, and macrophages (M), and in specialized thymic epithelium. In addition, MHC class II expression can be induced by bacterial cell products and inflammatory cytokines on different types of epithelial cells at environmental interfaces, including intestinal and skin epithelium (1, 2). The expression of MHC class II molecules on these nonprofessional APC has been implicated in immune-mediated inflammation and progression of or resistance to autoimmunity (3). In particular, it has been suggested that Ag presentation by intestinal epithelial cells (IEC) may play an important role in MHC class II-mediated presentation to immunoregulatory T cells (4, 5). However, little is known about the pathways and molecules involved in the regulation of MHC class II Ag presentation in IEC and their potential roles in Ag presentation to CD4 T cells in vivo.
MHC class II heterodimers are assembled in the endoplasmic reticulum with the assistance of invariant chain (Ii). A portion of Ii termed CLIP binds in the peptide groove of MHC class II molecules, thereby preventing premature loading of peptides. Endosomal localization motifs in the cytoplasmic tail of Ii promote entry of newly synthesized Ii oligomers into the endosomal compartment, where Ii undergoes rapid degradation. Lysosomal proteases degrade Ii until only CLIP remains bound in the MHC class II peptide-binding groove. CLIP removal and loading of peptides derived from foreign and self-protein Ags are mediated by DM, an MHC class II-like accessory molecule, localized to endosomal and lysosomal compartments of MHC class II-positive cells (6).
Until recently, the in vivo roles of specific lysosomal proteases in the MHC class II presentation pathway were poorly understood. Gene targeting studies revealed a critical role for the lysosomal cysteine protease cathepsin S (Cat S) in the late stages of Ii degradation in B cells, dendritic cells, and, to a lesser degree, M (7, 8, 9). As a result, in the absence of Cat S, many MHC class II molecules in the endosomal/lysosomal compartments and on the cell surface are associated with the Ii degradation intermediate p12 (8, 9, 10, 11). In contrast, another essential MHC class II-expressing cell type, thymic cortical epithelium, lacks Cat S expression. Instead, these cells use a different lysosomal cysteine protease, cathepsin L (Cat L), for the late stages of Ii degradation because similar accumulation of Ii fragments bound to MHC class II molecules is observed in Cat L-deficient thymic cortical epithelial cells.
Despite these observations, the role of lysosomal proteases in MHC class II presentation by nonprofessional APC expressed in vivo remains unknown. In this study we have studied the roles of Cat S and Cat L in MHC class II presentation in IEC. Because substantial phenotypic similarity has been reported for thymic epithelium and other types of keratinized epithelium (12), and Cat L is crucial for the late stages of Ii degradation in thymic epithelium and for hair follicle epithelium homeostasis (13, 14), we expected to find a prominent role for Cat L in Ii processing and MHC class II presentation by IEC. Unexpectedly, we observed that Cat S plays a critical role in Ii degradation in and MHC class II presentation mediated by these cells. Furthermore, we demonstrate that Cat S expression is essential for efficient presentation of endogenous peptides to CD4 T cells by nonprofessional APC in vivo. These results indicate that Cat S is a key enzyme regulating MHC class II presentation to CD4 T cells in both professional APC and in one of the most significant nonprofessional APC of epithelial origin, IEC.
Materials and Methods
Mice
C57BL/6 (B6) mice were purchased from Charles River, and Cat S–/–, Cat L–/–, MHC class II–/–, human Ii, H-2M–/–, Ii–/–, H-2M–/–xIi–/–, and Ly5.1+ TEa-B6 mice were maintained under specific pathogen-free conditions at the University of Washington. Cat S–/– and Cat S+/– (H-2bxd, E+) mice were generated by crossing Cat S–/– B6 (H-2b, E–) and B10.D2 (H-2d, E+) mice. All animals were used at 6–10 wk of age. Procedures and care of the animals were in accordance with the University of Washington guidelines provided by the Institutional Animal Care and Use Committee.
Antibodies
The mAbs M5/114 (anti-I-Ab, d, q and anti-Ed, k; ATCC TIB120; American Type Culture Collection) and In-1 (anti-Ii) have been described previously (15, 16). The Y3P mAb recognizes I-Ab (ATCC HB183), MKD6 recognizes I-Ad (ATCC HB3), and the YAe mAb recognizes I-Ab with E peptide (aa 52–68) bound in the peptide groove (17). Anti-CD45-PE, anti-B220-PE, anti-CD11c-FITC, anti-CD11b-PE, anti-CD4-allophycocyanin, and streptavidin-allophycocyanin were purchased from eBioscience, and the polyclonal rabbit antiserum recognizes mouse Cat L (a gift from A. Erickson, University of North Carolina, Chapel Hill, NC). The G8.8 mAb is specific for intestinal and thymic epithelia (18). The 15G4 mAb is specific for MHC class II molecule I-Ab bound to murine, but not human, Ii degradation intermediates p12 and CLIP (data not shown). Streptavidin-HRP conjugate was purchased from Vector Laboratories.
IEC isolation
Intestines from 6- to 10-wk-old mice were dissected, flushed, and cut into pieces as previously described (19). The intestinal tissue was dissociated with a collagenase/dispase/deoxyribonuclease mixture (Roche) and fractionated on a discontinuous Percoll gradient. Any remaining BM-derived cells were removed by negative sorting CD45+ cells on an AutoMACS magnetic cell sorter (Miltenyi Biotec) or positively sorting epithelial cells using the G8.8 mAb on a FACSVantage cell sorter (BD Biosciences).
Immunohistochemistry and immunoelectron microscopy
Small and large intestines from 6- to 10-wk-old mice were snap-frozen, and serial sections (5 μm) were prepared, air-dried, and acetone fixed. Sections were blocked with normal serum and subsequently incubated with optimal dilutions of primary mAb for 1 h at 25°C before washing and incubation with appropriate HRP-conjugated secondary Ab. Control slides were incubated with nonimmune species-matched Ig. Microscopic analysis was performed with a Pro Vis AX70 microscope (Olympus). MHC class II and Ii levels were assessed by staining with the mAbs Y3P and 15G4. These Abs were directly conjugated to digoxigenin-3-o-methyl-carbonyl--aminocarproic acid-N-hydroxysuccinimide ester. Tissue sections were then developed as described by Farr et al. (20).
For immunoelectron microscopy, small intestines from B6 and Cat S–/– mice were fixed in 2% paraformaldehyde and processed for ultrathin cryosectioning and Immunogold labeling as described previously (21). Immunogold labeling was performed with M5/114 and In-1 mAbs and 10-nm protein A-conjugated gold particles.
Cysteine protease active site labeling
IEC (2 x 105) were incubated for 2 h at 37°C with the iodinated cysteine protease inhibitor Cbz-[125I]-Tyr-Ala-CN2 (22), washed, and lysed. Lysates were analyzed by 12% SDS-PAGE.
Immunoblotting
Purified IEC were washed in PBS and lysed in cell lysis buffer (0.5% Nonidet P-40, 0.15 M NaCl, and 50 mM Tris-HCl, pH 7.2) supplemented with a mixture of protease inhibitors (Roche). Debris was removed by centrifugation at 8000 rpm for 10 min. Samples containing the indicated number of cell equivalents were boiled for 5 min in SDS reducing buffer and separated by 12% SDS-PAGE. The proteins were electrophoretically transferred onto nitrocellulose membrane and probed using the indicated primary Ab. Binding was detected using the appropriate HRP-conjugated secondary Ab diluted 1/1000 and was visualized by chemiluminescence (ECL; Amersham Biosciences).
Flow cytometric analysis
To assess MHC class II expression on IEC, B cells, dendritic cells, and M, flow cytometric analysis was performed. B6 and Cat S–/– IEC were stained with digoxigenin-derivatized G8.8 mAb (BD Biosciences) and biotinylated mAbs Y3P and 15G4. Binding of digoxigenin-conjugated Abs was detected with anti-digoxigenin fluorescein-labeled Fab (Roche), and binding of biotinylated class II-specific Abs was detected with streptavidin-allophycocyanin (BD Biosciences); stained cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences). B cells, dendritic cells, and M were analyzed using anti-B220-PE, anti-CD11c-FITC and anti-CD11b-PE, and anti-CD11b-PE (eBioscience), respectively, from splenocytes isolated from B6 and from MHC class II–/–Cat S+/– H-2bxdE+, Cat S+/– H-2bE–, Cat S–/–H-2bxdE+, and Cat S–/–H-2bE– chimeric mice. MHC class II was detected using M5/114 mAb (eBioscience).
T cell hybrid assay
T cell hybrid assays were performed as previously described (10, 23, 24). I-Ab-restricted T hybrids (1.0 x 105/well), specific for IgM (77.1) and 2-microglobulin (2M; 4.1) (23, 24), were cultured in duplicate for 24 h with 2 x 105 B6 or Cat S–/– IEC. In parallel, T cell hybrids were stimulated with 1 x 105 B6 or Cat S–/– splenocytes. IL-2 production was assessed using CTLL-2 proliferation, as determined by the Alamar Blue colorimetric assay. The results are expressed as arbitrary OD units (A570–A600).
In vivo activation of CD4+ T cells
To generate chimeric mice expressing MHC class II molecules on nonprofessional radiation-resistant APC, but not on BM-derived APC, BM from MHC class II–/– mice was transferred into lethally irradiated (1000 rad) Cat S+/– H-2bxdE+, Cat S+/– H-2bE–, Cat S–/–H-2bxdE+, and Cat S–/–H-2bE– mice. Six weeks after BM transplantation, complete reconstitution of host BM-derived APC with MHC class II–/– APC was confirmed by FACS. CD4+ T cells T cells were purified from the lymph nodes and spleens of Ly5.1+ TEa-B6 mice using an AutoMACS magnetic cell sorter (Miltenyi Biotec). These cells were labeled with CFSE (Molecular Probes), and 5 x 106 T cells were i.v. injected into the chimeric mice. Proliferation of donor cells was monitored by flow cytometry. On days 3 and 5 post-transfer, lymph nodes and spleen cells were analyzed by flow cytometry using anti-Ly5.1-PE and anti-CD4 APC.
Restimulation assay
On day 5 after transfer, TEa CD4+ T cells were reisolated from splenocytes. CD4+ T cells (5 x 105) were incubated in the presence of irradiated (2000 rad) 2 x 105 B6 splenocytes and titrating amounts of E52–68 peptide in 96-well, flat-bottom plates. Cell proliferation was measured by labeling cultures with [3H]thymidine (1 μCi/well), and the assay was harvested 24 h later. Data are presented as the mean cpm of [3H]thymidine incorporation in triplicate cultures.
Results
Immunohistochemical analysis of Ii degradation in intestinal epithelium of Cat S- and Cat L-deficient mice
We generated mAb 15G4, specific for MHC class II molecule I-Ab bound to Ii degradation intermediates p12 and CLIP (data not shown), and used this reagent to detect I-Ab-bound Ii degradation fragments in tissues from Cat S–/– and Cat L–/– mice. In initial experiments, frozen sections of thymi from B6, Cat S–/–, Cat L–/–, H-2M–/– (positive control), and H-2M–/–Ii–/– (negative control) mice were stained with digoxigenin-derivatized 15G4 Ab. Previously, we have observed large amounts of p12 bound to I-Ab molecules in Cat L–/– cortical thymic epithelium (14). Indeed, 15G4 stained the cortical region of Cat L–/– thymi in a manner similar to the H-2M–/– positive control. In contrast, only the medullary region of Cat S–/– thymi stained intensely with 15G4, consistent with the function of this enzyme in p12 degradation in BM-derived APC (data not shown). Thus, the 15G4 Ab serves as an excellent tool for analyzing Ii degradation and MHC class II maturation in tissue sections.
Immunohistochemical survey of MHC class II expression in nonlymphoid tissues, including kidney, lung, small and large intestine, and skin, in wild-type B6 mice using 15G4 and pan-I-Ab-specific Y3P Ab revealed intestinal and skin surface epithelia as the main sites of MHC class II expression (data not shown). We limited our studies of the roles of lysosomal cysteine proteinases in nonprofessional APC to IECs.
To study the roles of Cat S and Cat L in IEC, we first stained sections of the small and large intestines from B6, Cat S–/–, and Cat L–/– mice. Unexpectedly, 15G4 also stained the epithelial cell layer in the small and large intestines of Cat S–/–, but not Cat L–/– or B6 mice (Fig. 1, A and B), in addition to intense staining of BM-derived cells in the lamina propria. Although comparable expression of MHC class II was found in the small intestine in all three strains of mice, an increase in MHC class II expression was observed only in Cat S–/– large intestine by staining with the Y3P Ab (Fig. 1B). These results suggest that Cat S, but not Cat L, plays a role in Ii degradation in IEC in both small and large intestines.
To discriminate between the contribution of BM-derived APC and IEC to the increased 15G4 staining in the absence of Cat S, we generated reciprocal BM chimeras between wild-type B6 and Cat S–/– mice. Sections of small and large intestines from B6Cat S–/–, Cat S–/–B6, as well as control Cat S–/–Cat S–/– and B6B6 chimeric mice were stained with 15G4 and Y3P Abs. APC reconstitution in recipient mice by donor BM-derived cells was confirmed by 15G4 staining of spleen sections from chimeric mice (data not shown). Examination of small and large intestinal tissues from the chimeric mice stained with 15G4 Ab corroborated observations made in nonirradiated Cat S–/– mice. In the B6Cat S–/– chimeras, 15G4 intensely stained the epithelium, but not BM-derived APC, in the lamina propria, whereas Cat S–/–B6 chimeras displayed the opposite staining pattern (Fig. 1, C and D). Control sections from B6B6 and Cat S–/–Cat S–/– showed staining similar to that in nonchimeric B6 and Cat S–/– mice, respectively (see Fig. 1, A and B). Notably, elevated levels of MHC class II expression, i.e., increased Y3P staining, were observed in the large intestine of all Cat S–/– BM recipients. Hence, in the absence of Cat S MHC class II-bound Ii degradation intermediates accumulate in IEC in small and large intestinal epithelia.
Analysis of Cat S expression and function in IEC
To examine in more detail the role of Cat S in the MHC class II presentation pathway in non-BM-derived epithelial APCs, we decided to focus on IEC as a prototype nonprofessional APC. The increased 15G4 staining in the intestinal epithelial layer of Cat S–/–, but not Cat L–/–, mice led us to directly investigate the expression of these enzymes, the cellular distribution of total MHC class II molecules, and MHC class II molecules bound to Ii fragments. Therefore, we isolated IEC by collagenase digestion of the small intestine from B6 and Cat S–/– mice. The IEC were subsequently enriched by Percoll density gradient centrifugation. The resulting cell population was subsequently depleted of BM-derived CD45+ cells using magnetic beads or FACS sorting.
To directly evaluate Cat S and Cat L activities, FACS-sorted IEC (purity, 95%) were incubated with the active site inhibitor CN2-125Tyr-Ala-Cbz, and labeled enzymes were visualized after protein separation by PAGE (Fig. 2A). These experiments showed that Cat S activity is readily detectable in IEC, whereas Cat L activity was lacking.
The absence of Cat L activity was unexpected, because Cat L plays a role in Ii degradation in thymic cortical epithelial cells and is involved in hair follicle epithelium homeostasis (18, 19). Our failure to detect Cat L activity could be due to a lack of expression of the protein or inhibition of Cat L activity. Western blot analysis of wild-type B6 and Cat S–/– IEC lysates using a Cat L-specific Ab showed high levels of mature Cat L protein in both B6 and Cat S–/– IEC (Fig. 2B). Recently, we reported that M, which normally express active Cat L, exhibit a marked decrease in Cat L activity upon exposure to the proinflammatory cytokine IFN-, whereas mature protein levels remain high. This down-modulation of Cat L activity is thought to be mediated by a specific inhibitor, probably cystatin F (11). However, cystatin F mRNA was not detectable in IEC (data not shown). Therefore, an as yet unidentified inhibitor might control Cat L activity in IEC.
To characterize the Ii fragments bound to MHC class II molecules in Cat S–/– IEC, we performed Western blot analysis of purified IEC using the In-1 Ab specific for Ii (Fig. 2C). Only Cat S-deficient IEC showed similar accumulation of the p12 Ii fragment as that in B cells, dendritic cells, and M lacking this enzyme (9, 11).
Next, we subjected IEC isolated from B6, Cat S–/–, and MHC class II–/– mice to flow cytometric analysis to examine the cell surface expression of total MHC class II and MHC class II bound to Ii fragments using Y3P or 15G4 Abs. Anti-CD45 and G8.8 mAbs were used to distinguish between BM-derived cells and IEC, respectively (Fig. 2D). Cat S–/– IEC displayed an increase in MHC class II staining compared with B6 and MHC class II–/– IEC. In addition, we observed increased 15G4 staining in Cat S–/– IEC compared with the controls. Thus, flow cytometric analysis revealed increased cell surface expression of Ii fragments bound to MHC class II molecules, in agreement with the immunohistochemical studies.
To investigate intracellular accumulation of Ii fragments bound to MHC class II molecules, we performed immunoelectron microscopic analysis of frozen thin sections of B6 and Cat S–/– IEC. The 15G4 Ab does not reliably stain tissue samples processed for immunoelectron microscopy. Therefore, sections were stained with the In-1 Ab, specific for intact Ii and N-terminal Ii fragments, and with the M5/114 Ab, recognizing I-Ab molecules (Fig. 2E). We observed increased levels of MHC class II and Ii in the lysosomal vesicles of Cat S–/– compared with wild-type B6 IEC. Although Immunogold staining of plasma membrane was not very strong, the majority of cell surface MHC class II molecules were localized to the basolateral membrane (data not shown). As expected, a block of Ii degradation in Cat S-deficient IEC results in accumulation of Ii degradation intermediates in endosomal/lysosomal compartments. This leads to expansion and maturation of endocytic compartments, as shown for several different cell types by Bakke et al. (25, 26, 27). In addition, we have stained IEC sections with anti-lysosome-associated membrane protein Ab and MHC class II and found Ii-positive compartments to be lysosome-associated membrane protein positive (data not shown).
Thus, Cat S deficiency in nonprofessional epithelial APC, i.e., IEC, results in the accumulation of Ii fragments bound to MHC class II molecules on the cell surface and in lysosomal compartments. These data directly implicate Cat S in mediating Ii processing in IEC. The increase in cell surface and intracellular MHC class II molecules found in Cat S–/– IEC may be due to a specific role for this protease in the turnover of MHC class II molecules. Alternatively, Ii p12 fragments may stabilize MHC class II I-Ab molecules if there is a relative paucity of class II binding peptides in IEC, thereby increasing total MHC class levels.
To elucidate the effect of Cat S deficiency on Ag presentation by IEC, we generated chimeric mice using MHC class II–/– donor BM transferred into either B6 or Cat S–/– lethally irradiated mice. We purified IEC from chimeric mice 6–8 wk after BM transfer and cultured them in the presence of self-peptide-specific T cell hybridomas. We used T cell hybridomas specific for the endogenous Ags IgM and 2M and investigated the ability of B6 and Cat S–/– IEC to present these Ags compared with splenocyte controls. B6 and Cat S–/– IEC failed to present 2M peptide, whereas splenocytes from these mice presented the 2M peptide with equal efficiency (Fig. 3, A and C). B6 and Cat S–/– splenocytes also present IgM epitope equally efficiently (Fig. 3B). In contrast, Cat S–/– IEC proficiently presented IgM peptide whereas B6 IEC induced substantially lower levels of stimulation of IgM specific T cell hybridomas (Fig. 3D) despite comparable serum IgM levels in Cat S mutant and wild-type mice (data not shown). Enhanced presentation of the IgM epitope by cathepsin S–/– IEC can also be explained by increased class II expression in these cells. Although we cannot formally exclude this possibility, we believe that this is an unlikely explanation because a similar relative increase in IgM epitope presentation was observed in Cat S–/– M, dendritic cells, and fibroblasts in which total surface class II levels were not affected by Cat S expression (9, 11, 28). This is probably due to a putative Cat S cleavage site within the IgM peptide epitope.
To investigate the role of Cat S in MHC class II presentation of self-peptides by IEC, we analyzed expression of I-Ab bound to a well-characterized self-peptide E52–68 derived from the class II molecule I-E. This complex can be detected by the mAb YAe (17). Therefore, we crossed B10.D2 (H-2d E+) with Cat S–/– (H-2b, E–) animals to generate Cat S–/– H-2bxd E+ mice. IEC as well as splenocytes isolated from Cat S+/– H-2bxd E+, Cat S–/– H-2bxd E+, and MHC class II–/– mice (negative control) were stained with YAe (Fig. 3, E and F). Cat S–/– H-2bxd E+ IEC and splenocytes both show a significant decrease in YAe staining compared with Cat S+/– littermate controls. The negative peak in bimodal YAe staining of unseparated splenocytes corresponds to MHC class II-negative T cells, whereas the majority of YAe-positive cells are B cells. Therefore, Cat S deficiency decreases the expression of E peptide-I-Ab complexes in splenic B cells as well as in IEC. Thus, Cat S is involved in Ii degradation and regulates the expression of self-peptide-MHC class II complexes in IEC.
In vivo presentation of self-peptide-MHC complexes by nonprofessional APC to CD4 T cells is diminished in the absence of Cat S
To elucidate the functional significance of altered presentation of self-peptide-MHC class II complexes by nonprofessional APC lacking Cat S, we examined activation of TEa TCR transgenic T cells recognizing E52–68 peptide bound to I-Ab on non-BM-derived APC. This in vivo E52–68 peptide presentation was assessed using adoptive transfer of CFSE-labeled Ly5.1+ TEa T cells. To eliminate presentation of E52–68 peptide by professional BM-derived APCs, we first transferred MHC class II–/– BM into lethally irradiated (1000 rad) Cat S–/– E+ and Cat S+/– E+ mice and corresponding littermate control H-2b mice lacking E. Flow cytometric analysis of MHC class II expression by B cells, dendritic cells, and macrophages (Fig. 4A) confirmed essentially complete reconstitution of host APC subsets by MHC class II–/– donor BM-derived cells. These chimeric mice were injected i.v. with 5 x 106 purified CSFE-labeled CD4+ Ly5.1+ TEa T cells, and their Ag-driven division was analyzed on days 3 and 5 post-transfer (Fig. 4B). We found that on day 3 the CD4+ T cells in E-positive Cat S+/– recipients underwent three to five divisions. In contrast, in E-positive recipients lacking Cat S, TEa T cells showed delayed proliferation kinetics by one or two rounds of division. This difference became more pronounced on day 5 when almost all TEa CD4 T cells in Cat S+/– recipients have lost CFSE labeling by dividing six or seven times or more, whereas in Cat S–/– hosts the majority of TEa T cells divided only three or four times, making up 6 and 3% of the total CD4 T cells in corresponding recipient mice, respectively (Fig. 4A; data not shown). In agreement with this result, we found that in comparison with class II–/–Cat S–/– E+ recipients, CD4 T cells isolated from class II–/–Cat S+/– E+ recipients of TEa T cells showed 3-fold higher in vitro recall response to E52–68 peptide in the presence of irradiated T cell-depleted B6 splenocytes as APC (Fig. 4C). In contrast, in both heterozygous and homozygous Cat S mutant mice lacking E, used as negative controls, transferred TEa T cells remained undivided, constituting 0.3% of CD4 T cells and generating comparable in vitro recall responses (Fig. 4C). It seems highly unlikely that the observed proliferation is due to remaining host BM-derived APC in chimeric mice because we have not observed MHC class II expression on BM-derived cells, such as B cells and dendritic cells, upon flow cytometric examination (Fig. 4A) or by immunohistochemistry (data not shown).
Therefore, Cat S expression in nonprofessional APC in vivo, e.g., epithelial cells, is essential for optimal Ag presentation and activation of T cells. Together, these data clearly defines Cat S as the leading cysteine protease involved in Ii degradation and Ag presentation in vivo not only in professional, but also in nonprofessional, APC.
Discussion
Previously we have shown the expression of lysosomal cysteine proteases Cat S and Cat L in distinct subsets of professional APC; Cat S is active in B cells and dendritic cells, whereas Cat L activity is detected in thymic cortical epithelial cells. An essential role of these enzymes in MHC class II presentation is illustrated by the profound defect in Ii degradation and MHC class II Ag presentation in their absence (7, 9, 14). M express both enzymes; however, Cat S plays the leading role in MHC class II presentation due to down-regulation of Cat L activity upon induction of MHC class II molecules in response to proinflammatory stimuli (11). In addition, lysosomal cysteine proteinase Cat F has been suggested to contribute to Ii degradation in these cells (29). Thus, there is a striking dichotomy in utilization of the lysosomal cysteine proteinases involved in MHC class II presentation by the cells mediating positive selection of CD4+ T cell in the thymus, i.e., thymic cortical epithelial cells, and by the BM-derived APC involved in thymic negative selection of autoreactive TCR and in presentation of self and foreign Ags in the periphery.
In this study we expand our previous studies of the roles of Cat S and Cat L in MHC class II presentation by professional APC to include IEC, a major peripheral nonprofessional APC of epithelial origin. IEC express MHC class II constitutively due to continuous stimulation by bacterial flora and cytokines (30, 31) and are in contact with a large number of intraepithelial lymphocytes and lamina propria lymphocytes (32). Analysis of the Ag-presenting properties of ex vivo isolated, nontransformed IEC has been a major challenge because these cells are fragile and prone to death upon handling and under tissue culture conditions. In this study for the first time we demonstrate the ability of ex vivo isolated IEC to present endogenous and extracellular self-Ags in context with MHC class II. Furthermore, our studies using Cat S–/– and Cat L–/– mice have brought about the surprising finding that Cat S, but not Cat L, controls Ii degradation and peptide presentation by MHC class II molecules in these cells. In agreement with these functional results, we found only Cat S activity in freshly isolated IEC, whereas Cat L activity was not detected despite the expression of a large amount of mature Cat L protein, as revealed by Western blot analysis (Fig. 3, A and B). It is likely that these findings in mice also apply to humans, because Cat S has been found in the human colonic cancer cell line T84 (33, 34). The lack of Cat L activity in IEC was surprising because Cat L is active in other types of epithelium, e.g., thymic epithelial cells and kidney epithelium (14). Because we were able to detect mature Cat L protein, we suggest that this is due to inhibition of Cat L activity by an as yet unknown inhibitor. Intriguingly, in our recent studies we found that Cat L activity is also inhibited in IFN--induced M and in dendritic cells isolated from mice expressing Cat L as a transgene under the CD11c promoter (11). One likely candidate for such an inhibitor is the recently described cysteine protease inhibitor cystatin F, which has been shown to be expressed in M and dendritic cells (35, 36). Cystatin F mRNA is restricted primarily to hemopoietic cells, and it exhibits high affinity for Cat L. Cystatin F mRNA is up-regulated in maturing dendritic cells (37), and we found cystatin F mRNA levels markedly increased in M upon treatment with IFN-, suggesting that it could be a Cat L inhibitor in these cells (data not shown). However, cystatin F mRNA was undetectable in IEC. Thus, an unknown mechanism responsible for Cat L inhibition in these cells will have to be further investigated.
IEC are probably not the only type of peripheral nonprofessional APC using Cat S for MHC class II presentation. A recent study of human myoblasts stimulated in vitro with IFN- showed induction of Cat S and its role in regulation of MHC class II expression (38). Furthermore, Cat S expression was found in biopsies isolated from patients with myopathies, although M are probably present in the inflamed muscle tissue analyzed in these experiments and could account for this observation (38). Although further in vivo study of the roles of cysteine proteases in MHC class II-positive nonprofessional APC other than IEC is warranted, we predict that the majority of, if not all, nonprofessional APC expressing MHC class II under physiologic conditions in vivo use Cat S.
It is interesting to speculate why Cat S expression in both BM-derived professional APC and peripheral nonprofessional APCs is enforced. One possible explanation for such apparently stringent differential expression of Cat L and Cat S is to diminish the incidence of autoimmunity by avoiding the expression of largely overlapping repertoires of MHC class II-bound peptides in positively selecting thymic cortical epithelial cells and all other major APC types. In this regard, our recent study using a model APC line expressing Cat S or Cat L suggested that repertoires of MHC class II-bound peptides displayed by these cells are only partially overlapping (28). Our analysis of presentation of several known self-peptides by IEC showed that the expression of Cat S has a significant impact on the level of expression of peptide/MHC class II complexes. We found that the expression of the well-characterized I-Ab binding endogenous peptide E52–68 (17) is diminished in both IEC and splenic APC in the absence of Cat S. On the contrary, the expression of another endogenous peptide, IgM377–392, bound to I-Ab (39, 40) was significantly enhanced in Cat S-deficient IEC. Although B cells from Cat S–/– and wild-type mice present IgM equally efficiently, this result is in agreement with our previous findings of increased presentation of this IgM T cell epitope by M and dendritic cells isolated from Cat S-deficient mice compared with those from wild-type mice (9). Cat S-deficient MHC class II-positive fibroblasts expressing or lacking Cat L also exhibited enhanced presentation of this IgM epitope (28). The most likely reason for the negative effect of Cat S on presentation of the IgM epitope is its direct cleavage or degradation of its precursor. Thus, the expression of these two major self-peptide-class II complexes in IEC follows the pattern observed in professional APC. In contrast, a peptide derived from 2-M is not expressed in either wild-type or Cat S mutant IEC, whereas it is expressed at very high levels by BM-derived APC. It is possible that this may be a result of assembly of this peptide-class II complex at an intracellular site other than the late endosomal/lysosomal compartment. This possibility is supported by our observation of significantly increased presentation of this peptide-MHC class II complex by B cells and dendritic cells lacking Ii compared with wild-type APC (40) (S. Kovats and A. Y. Rudensk, unpublished observations). A distinct trafficking pathway(s) responsible for sorting MHC class II molecules to the basolateral membrane in polarized epithelial cell lines (41, 42) may account for the differences between IEC and BM-derived APC in MHC class II sampling of nonlysosomal compartments.
Does the expression of Cat S in nonprofessional APC affect their ability to present Ag to T cells in vivo? Adoptive transfer experiments using CFSE-labeled TEa T cells showed that Cat S deficiency in non-BM-derived APC results in a significantly less efficient activation of T cells in response to the highly abundant endogenous peptide-MHC class II complex. Although these experiments did not evaluate individual contributions of specific types of nonprofessional APC to TEa T cell stimulation, this is the first direct demonstration of naive T cell activation in response to self-peptide-MHC class II complex presented by non-BM-derived APC and an essential role of Cat S in this process. Under physiologic conditions, such activation is likely to lead to tolerance induction. However, in combination with infection and certain genetic factors, for example, associated with impaired central tolerance, recognition of self-Ags on nonprofessional APC may lead to tissue-specific autoimmunity, such as the skin pathology observed in mice expressing MHC class II transgene under keratin 14 promoter (43).
Thus, in addition to its key role in BM-derived professional APCs, Cat S is the principal lysosomal cysteine protease responsible for MHC class II maturation and presentation in vivo in a major nonprofessional APC type.
Acknowledgments
We thank K. Honey for critical review of the manuscript, and C. Plata and N. Li for their excellent care of the mice used in these experiments.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the Howard Hughes Medical Institute and grants from the National Institutes of Health (to A.Y.R.). C.B. is supported by a National Institutes of Health predoctoral training grant. A.B. is supported by the Department of Comparative Medicine.
2 Current address: Department of Comparative Medicine, Oregon Health and Science University, Portland, OR 97239-3098.
3 Current address: Infectious Diseases Service, Department of Medicine, Immunology Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
4 Address correspondence and reprint requests to: Dr. Alexander Y. Rudensky, Department of Immunology, Howard Hughes Medical Institute, University of Washington School of Medicine, Seattle, WA 98195. E-mail address: aruden@u.washington.edu
5 Abbreviations used in this paper: BM, bone marrow; B6, C57BL/6; 2M, 2-microglobulin; Cat L, cathepsin L; Cat S, cathepsin S; IEC, intestinal epithelial cell; Ii, invariant chain; M, macrophage.
Received for publication August 17, 2004. Accepted for publication October 25, 2004.
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