Cysteine proteases in Langerhans cells limits presentation of cartilage derived type II collagen for autoreactive T cells

Meirav Holmdahl1,2, Anders Grubb1 and Rikard Holmdahl2

1 Department of Clinical Chemistry, Lund University and 2 Department of Medical Inflammation Research, Lund University, 221 84 Lund, Sweden

Correspondence to: M. Holmdahl; E-mail: Meirav.Holmdahl@inflam.lu.se
Transmitting editor: G. J. Hammerlin


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Development of type-II collagen (CII)-induced arthritis (CIA) is dependent on activation of CII-reactive T cells. Dendritic cells (DCs) are believed to play a crucial role in antigen-specific priming of T cells but it is still unclear how the CII-reactive T cells are primed since Langerhans cells (LCs) are poor antigen-presenting cells for CII. In the present study we show that LCs, treated with cysteine protease inhibitors, are able to process and present CII to T-cell hybridomas specific for the immune-dominant glycosylated 259–270 peptide bound to the MHC class II molecule Aq. Interestingly, the self (mouse) CII peptide could also now be efficiently presented. The poor presentation by LCs is a peptide-specific effect, since both bovine CII (bCII) (presenting a different peptide on H-2r) and ovalbumin could be efficiently presented, and blockage of cysteine proteases did not enhance antigen presentation. The enhanced CII-presentation by cysteine protease inhibition is seen mainly in LCs and not in antigen-primed B cells or macrophages. B cell and macrophage presentation of CII occur even without protease inhibition and are only to a minor extent influenced by cysteine protease inhibition. These data suggest that a LC deficiency in processing of the immune-dominant CII epitope in both CIA and RA may limit the exposure of this self-antigen to T cells, but that presentation can be overcome by modulation of the peptide proteolysis during CII processing.

Keywords: antigen degradation, immune dominant CII peptide, MHC class II, mouse, protease inhibition


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Type-II collagen (CII) is the major protein component of cartilage and immune recognition of CII plays a critical role for development of collagen-induced arthritis (CIA), a widely used model for rheumatoid arthritis (RA). Presentation of peptides from the region 260–271 is critical for triggering of CII-specific reactivity in both CIA and RA (1,2). However, we do not fully understand how the priming of CII occurs or the importance of the various antigen-presenting cells (APCs) in the pathogenesis of CIA. CIA is induced after an intradermal immunization with CII in adjuvant and the activation of both CII-specific T cells and the susceptibility to CIA is MHC class II associated (3,4). However, it is still unclear which type of APC has the capacity to process and present CII and subsequently activate T cells. Collagen is handled differently by APCs when compared with other antigens such as ovalbumin (OVA), since macrophages and B cells process and present CII to T cells more efficiently than DCs (57). However, the presence of DCs is believed to play an important role in the pathogenesis of RA (8,9), a disease where CII is recognized as an auto-antigen (2,8,10,11). In order to initiate a T-cell dependent immune response, proteins must undergo proteolysis within the APCs, thus generating immune-dominant peptides that bind to MHC class II and are presented on the surface of the APCs. In addition, co-stimulatory molecules are needed for activating T cells. DCs express high levels of both MHC class II and co-stimulatory molecules and it is widely believed that DCs are the only APCs that can prime naive T cells (12). Lysosomal proteases of the cathepsin family control the processing of antigens and the formation of MHC class II. The cathepsins belong to a large family of aspartyl (e.g. cathepsins D and E) and cysteinal (e.g. legumain, cathepsins B, F, S and L) endosomal proteases that are differentially expressed within antigen-presenting cells (13). CatS is highly expressed in DCs, peritoneal macrophages and in B cells (1416) whereas macrophages express several cathepsins, CatL, CatS and CatF (16,17). CatS has been shown to be essential for invariant chain (Ii) degradation and for normal MHC class II function and it has also been suggested to be involved in antigen processing of certain antigenic epitopes (18). CatS-deficient mice showed an impaired presentation of CII by DCs and macrophages compared to wild type mice and were suggested to be less susceptible to CIA (15). CatF has been shown to be as efficient as CatS in CLIP (Class II-associated invariant chain peptide) generation (17). CatB was shown to interfere in the antigen degradation resulting in impaired antigen presentation (19,20). Other studies have shown that cysteine protease inhibitors, including those inhibiting CatB, enhance rather than inhibit presentation of specific protein antigens, probably because inhibition of certain cathepsins prevents degradation of essential epitopes (7,21). It has also been shown that CatS and CatB can substitute each other (22) and that CatS regulates the CatL activity in B cells (23). The use of specific protease inhibitors such as LHVS (inhibiting CatS), CA074 (inhibiting CatB) and of more general cysteinyl protease inhibitors like E-64 has allowed studies of the role of different cathepsins in antigen presentation.

Langerhans cells, the DCs of the epidermis, acquire exogenous proteins penetrating the skin and migrate to regional lymph nodes where they display antigen-derived peptides for T-cell recognition (2430). On their way to the draining lymph node the LCs have to pass the basement membrane by degrading collagen. We previously showed that DCs do not, or very poorly, present collagen, neither type-I collagen containing the CII immune-dominant peptide (256–270), nor CII (5). We can now provide an explanation for the poor CII-presentation by DCs through investigations of the intracellular processing of CII in Aq-restricted Langerhans cells by using cysteine protease inhibitors. We show that LCs present rat and mouse CII efficiently if treated with cysteine protease inhibitors. This indicates that LCs can process CII intracellularly but cysteine proteases normally destroy the immune-dominant peptide 259–270, preventing its presentation. Thus, cysteine proteases like CatS play a pivotal role not only in degrading invariant chain (Ii) but also in processing of certain antigens such as CII. We further conclude that neither B cell nor macrophage processing of CII is dramatically influenced by cysteine protease inhibition, showing that these proteases play a unique role in LCs for processing of the immunodominant CII peptide.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
B10.Q x DBA/1 F1 mice were housed and bred in the animal facility of Medical Inflammation Research. DBA/1 was originally obtained from Jackson Laboratories (Bar Harbor, ME), and B10.Q originated from professor Jan Klein, Tübingen, Germany.

Antigens
Rat type-II collagen (CII) was prepared from the Swarm chondrosarcoma and bovine CII from calf cartilage by limited pepsin digestion as described (31). Native CII and CII peptides were dissolved and stored in 0.1 mol/l acetic acid. The peptides were synthesized as previously described (1). Ovalbumin (OVA) (Grade V) was purchased from Sigma (Sigma chemical Co., St Louis, MO).

Inhibitors
The aspartic protease inhibitor pepstatin A and the cysteine protease inhibitor E-64 [trans-epoxysuccinyl-L-leucylamido (4-guanidino)-butane] was obtained from Sigma. The CatS inhibitor LHVS (N-morpholinurea-leucine-homophenylalanine-vinylsulfone-phenyl) was a generous gift from H. A. Chapman. CA074 (L-trans-epoxysuccinyl-Ile-Pro-OH propylamid) a CatB inhibitor was obtained from Bachem. LHVS and CA074 were dissolved in dimethylsulfoxide (DMSO) to a final stock concentration of 10 mM. E-64 was dissolved to a final stock concentration of 1 mM in water and stored at –20°C. Table 1 gives a summary of the protease inhibitors used.


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Table 1. Effect on CII presentation by various protease inhibitors
 
Antibodies
Anti mouse B220 antibody RA3-6B2 conjugated to paramagnetic microbeads (Miltenyi Biotech, Bergisch Gladback, Germany) was used to enrich the B cell population, but also in negative selection for collecting macrophages from peritoneum. Anti-CD11c (N418) antibody conjugated to microbeads (Miltenyi Biotech) was used for depleting DCs from the peritoneal cells. Antibodies used for flow cytometry analysis of peritoneal cells but also of epidermal cells were: anti mouse CD11b PE (Mac 1, PharMingen), F4/80 (biotin conjugated F4/80, from our hybridoma collection), MHC class II (FITC-conjugated 7.16.17, PharMingen), B220 (PE and cychrome-conjugated RA3-6B2, PharMingen), CD11c (biotinylated N418, from our hybridoma collection), CD8 a (PE conjugated 53-6.7, PharMingen).

T cell hybridomas and culture medium
MHC Aq-restricted T-cell hybridomas used have been described earlier (1): HCQ.10 responds to CII, galactose-peptide 256–270 and HOB.6 responds to OVA. We made new Ar-restricted T-cell hybridomas by fusion of B10.RIII lymph node cells (LNCs) with the TCR-negative variant of the thymoma BW 5147 (32). This T-cell hybridoma responds to amino acids 607–621 of bCII. All cells were grown in DMEM supplemented with 10% FCS, 10 µM ß-mercaptoethanol, 10 mM HEPES, penicillin, L-glutamine and streptomycin in a humidified incubator at 37°C in 7.5% CO2.

Preparation of antigen presenting cells (APCs)
LNCs were prepared from (B10.Q x DBA/1)F1 mice immunized 11–12 days previously with 50 µg of either OVA or native CII emulsified in CFA H37Ra (Difco, Detroit, MI) in each of the hind paws and at the base of the tail. B cells were separated from LNC using anti-B220 RA3-6B2 microbeads and MACS as described by the manufacturer (Miltenyi Biotec). Peritoneal exudates cells were collected by peritoneal lavage. Since peritoneal exudates contain 40–50% B cells, we used anti-B220 microbeads to deplete B cells and the resulting cell population is referred to as macrophages. Langerhans cells were prepared from the ears as described (33). Briefly, epidermis was separated from dermis after incubation with 0.5% trypsin (Sigma) in PBS for 30 min at 37°C. Single cell suspension was obtained by teasing the epidermis with forceps in the presence of 0.25% deoxyribonuclease (Sigma). The cells were washed once in 20% complete medium and twice in medium containing 10% FCS. The purity of the N418-positive cells was measured by flow cytometry, staining with N418 bio/APC-labeled antibodies, using FACSTM and FACStarTM and CellquestTM Software (BD PharMingen). For the fixation of DCs we used a final concentration of 1% paraformaldehyde (Apoteket, Göteborg, Sweden) in the cell cultures. The cells were fixed during 1 min and were then washed with 10% complete medium before they were stimulated with the CII glycopeptide and co-cultured with CII-specific T-cell hybridomas (HCQ.10).

T-cell proliferation assay
50 x 103 T-cell hybridoma cells were co-cultered with APC, protease inhibitors and antigen in a total volume of 200 µl in flat-bottomed 96-well plates (Nunc, Roskilde, Denmark). In most of the experiments the protease inhibitors or the antigen were titrated and the APC (250 x 103 cells/well) were kept constant. After 24 h in culture, 100 µl aliquots of the supernatants were removed and frozen at –20°C. To measure the content of IL-2 the supernatants were thawed and cultured with 100 x 103 of an indicator murine T cell line (CTLL), dependent on IL-2, in a total volume of 200 µl for 24h, the cells were then pulsed with [3H]Thymidine for an additional 15–18 h. The cells were harvested in a Micromate 196 cell harvester (Canberra Packard, Meriden, CT) and the radioactivity determined in a MatrixTM96 direct ß-counter (Canberra Packard).

All results are mean values of duplicate cultures. All experiments are made using individual mice, three to six animals in each group, and data presented as mean values ±SEM.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The inability by LC to present CII is MHC class II/peptide specific
To investigate a DC population without contamination of other antigen presenting cells such as macrophages and B cells we use freshly prepared epidermal cells containing 3% Langerhans cells. In this epidermal cell preparation all class II-expressing cells also stained for CD11b, CD11c and F4/80 with no detectable B cells or macrophages. We and others have earlier shown that various DC populations from H-2q mice, including Langerhans cells, do not or only very poorly present CII (5,6). In H-2q mice, only the immunodominant CII259–270 peptide, with a central core of 260–267, is presented in an MHC class II (Aq)-restricted way after immunization with CII. T cells mainly recognize various posttranslational modifications of the lysine at position 264, and we have previously shown that the inability to present CII by DC is not dependent on which form of the peptide is recognized (5). In the present study we mainly use the HCQ.10 hybridoma that is specific for the glycopeptide 259–270 with an O-linked galactose bound to hydroxylysine at position 264 i.e. the immune-dominant structure recognized by T cells in CIA (on Aq molecules) and RA (on DR4/DR1 molecules) (2). To determine if the inability of LCs to present CII was collagen- or peptide-specific we needed to isolate T cells to another peptide. Since no other peptide is recognized after immunization with CII in H-2q mice we used H-2r mice, which develop a T-cell response to the bovine-specific CII peptide 607–621. We could show that DCs isolated from H-2r mice efficiently presented bCII to a bCII607-621/Ar-specific T-cell hybridoma (at 0.4 µg/ml). In contrast, extreme concentrations (50 µg/ml) of bovine, chick or rat CII were needed for presentation by LCs isolated from H-2q mice to CII259-270/Aq-restricted T-cells (Fig. 1). This finding indicates that the inability to present CII by LCs is not collagen specific but Aq/CII259-270 specific.



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Fig. 1. Poor CII presentation by Aq-expressing LC. (A) Aq expressing DCs only presents chick (open triangles), bovine (open circles) and rat CII (closed circles) to CII 260–270/Aq restricted T cells at extreme concentrations (50 µg/ml). The LCs were co-cultured with CII259–270/Aq restricted T cells. Proliferation was measured by CTLL. The data represents the mean c.p.m. values ±SEM of individual three mice in each group. To see whether the inability to present CII concerned even other collagens we used in (B) Ar-expressing LCs stimulated with titrated doses of bCII (open circles). The LCs were co-cultured with Ar/607–621 restricted T cells. Proliferation was measured by CTLL. The data represents the mean values ±SEM of six individual mice.

 
LCs present CII if treated with cysteine protease inhibitors
To investigate whether the inability of Aq expressing LC to present CII is due to excessive protease degradation we treated the cells with a series of protease inhibitors. LC from B10.Q x DBA/1 F1 mice were prepared and loaded with 10 µg/ml native CII together with various concentrations of different protease inhibitors co-cultured with the CII reactive T-cell hybridoma (HCQ.10). The results are identical if denatured or native CII was used (data not shown). Only cysteine protease inhibitors, CA074, LHVS and E-64, affected antigen presentation, which resulted in enhanced CII presentation. At higher doses the response is inhibited, an effect most likely due to protection of invariant chain degradation by cathepsin inhibition (34). LHVS works as a selective CatS inhibitor between 3–10 nM. It was found to be the most efficient inhibitor (Fig. 2; Table 1). This argues for CatS playing a role for the CII degradation in LCs, causing the inability of presentation of CII to T cells. However, it is likely that other proteases contribute, as the effect was observed also at higher doses of LHVS. To exclude the possibility that the enhancement of CII presentation was dependent only on rat CII we also tested bovine and chick CII, that share the immunodominant glycopeptides at position 259–270, with essentially the same results (Fig. 2). Thus, a dose-dependent inhibition of cysteine proteases enhanced presentation of CII by Aq-expressing LC.



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Fig. 2. Treatment with cathepsin inhibitors improves the CII presentation by LCs. The concentrations of cathepsin inhibitors were titrated on LCs stimulated with 10 µg/ml of native rat CII and co-cultured with HCQ.10. CA074, cathepsin B inhibitor (open triangles), E-64, inhibits cysteine proteases (closed circles), and LHVS, a CatS inhibitor (open squares). LHVS and CA074 were titrated from a DMSO stock concentration and E64 from a water-based stock. Therefore, as an additional control, titration was performed also from a stock of DMSO without protease inhibitors (open circles). Cathepsin inhibition improves also the presentation of bovine and chick CII in similar to rat CII (data not shown). The data represents the mean c.p.m. values ±SD of four individual mice in each group. *P < 0.05 as determined by both one way analysis of variance (ANOVA) and Mann–Whitney as compared with the control cultures (the corresponding DMSO titrated curve for LHVS and CA074 and the culture without any proteases or DMSO for E64). CTLL was used for measuring proliferation. The data represents the mean values ±SEM of four individual mice in each group.

 
Augmentation of CII presentation by CatS inhibition of LC in vivo
To investigate whether the augmenting effect of cysteine protease inhibition is effective on antigen degradation and presentation by immature LC in vivo, E64 with or without CII was injected under epidermis in the ear of B10.Q x DBA/1 F1 mice. Six hours later epidermal cells were prepared and tested for APC activity to HCQ.10 cells. Notably, injection of CII + E64 only, significantly stimulated the T cells whereas E64 or CII alone had no effect (Fig. 3).



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Fig. 3. Enhanced CII presentation by CatS inhibition of immature LCs in vivo. E-64 alone (open triangles), E-64 together with CII (open squares) or only CII (open circles) was injected intradermally into ears of B10.Q x DBA/1 F1 mice. Six hours after E-64 injection the LCs were prepared and tested as APCs to T-cell hybridoma HCQ.10. CTLL was used for measuring proliferation. The data represent the mean c.p.m. values ± SEM of three individual mice in each group. *P < 0.05 by analysis of variance (ANOVA) or Mann–Whitney compared with CII only.

 
The presentation of the immunodominant CII glycopeptide is not affected by cathepsin inhibition
To ensure that E-64 had any effect on the extracellular processing, we treated both live and formaldehyde-fixed LCs with E-64 (100 µg/ml) and compared them with live LCs not treated with E-64 for presentation of the glycosylated CII peptide. The DCs were stimulated with titrated doses of the glycosylated CII260–270 peptide and co-cultured with the glyco-specific T-cell hybridomas (HCQ.10).

As expected, both live and fixed LCs presented the CII peptide well but we could not see any effect of the E64 treatment (Fig. 4). This observation suggests that cysteine proteases effect on the extracellular processing of CII does not contribute to the observed inhibition of CII processing in LCs.



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Fig. 4. No effect of protease inhibition on LC presentation of the CII 259–273 glycopeptide. Formaldehyde fixated (open triangles) and live LCs treated with E-64 (open squares) and live LCs not treated with E-64 (open circles), were stimulated in vitro with 10 µg/ml of CII peptide and co-cultured with antigen-specific T-cell hybridomas (HCQ.10). CTLL was used for measuring proliferation. The data represent the mean c.p.m. values ± SEM of three individual mice in each group.

 
Influence by cysteine protease inhibition on other antigen presenting cells
To investigate if the enhancement induced by inhibition of cysteine proteases is unique to LCs we tested B cells and macrophages from the same mouse strain, (B10.Q x DBA/1)F1. To obtain antigen-primed B cells, the mice were immunized with either CII or OVA; 12 days later draining lymph node cells (LNC) were collected and enriched by MACS separation using microbeads coupled with anti-B220 antibody. Macrophages were collected by peritoneal lavage and depleted for B cells by B220 negative selection but also for CD11c. The CII-specific HCQ10 and the OVA-specific HOB.6 hybridoma clones were used as responder T cells. Both antigen-primed B cells and macrophages were found to efficiently present both CII (Fig. 5) and OVA (Fig. 6). Cysteine protease inhibition of titrated E64 had no significant influence on the CII- (Fig. 5) or the OVA-presentation (6) by B cells or macrophages. To determine whether E64 could exert an effect on presentation of other concentrations of the antigen we titrated CII and OVA with a fixed concentration of E64. Interestingly a minimal, but significant, enhancing effect was observed at very small concentrations of CII in both B cells and macrophages (Fig. 5) and with OVA on macrophages (Fig. 6). This could possibly be due to a contamination of DCs in the peritoneal cell population or due to a macrophage subset. To exclude DCs we depleted anti-CD11c positive cells and confirmed it by flow cytometric analysis, however, this minimal effect remained (data not shown). Taken together, the augmentation of antigen presentation is specific for LC and possibly also at small antigen concentrations or for a subsets of peritoneal macrophages and B cells.



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Fig. 5. CII can be processed and presented by B cells and macrophages. (A) Titration of the E64 dose on purified B cells (n = 4) and macrophages (n = 4) in similarity with the data on LCs in Fig. 2, showing no effect. To address whether any effect could be detected by using cathepsin inhibitors we also titrated the antigen with a constant optimal dose of E64 (100 µg/ml). (B) CII-primed B cells treated with E-64 (closed triangles) (n = 3) or without (open triangles) (n = 4) and stimulated with titrated doses of CII. (C) Macrophages were treated with E-64 (closed circles) (n = 3) or without (open circles) (n = 3) and stimulated with 10 µg/ml CII. B cells and macrophages were co-cultured with CII-specific T-cell hybridoma HCQ.10. Proliferation was measured by CTLL. The data represent the mean c.p.m. values ± SEM of the indicated numbers of individual mice in each group. *P < 0.05 as tested with Mann–Whitney as compared with the CII-only control group.

 


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Fig. 6. OVA can be processed and presented by B cells and macrophages but cathepsin inhibitors do not enhance the presentation. Mice were immunized with OVA; on day 12 draining lymph node cells were positively selected on B220 expression and used as APCs. The selected B cells were treated with 100 µg/ml E-64 (closed triangles) (n = 3) or not (open triangles) (n = 6), and stimulated with titrated doses of OVA. (B) Macrophages collected from peritoneum and depleted by anti-B220 microbeads were treated with 100 µg/ml E-64 (closed circles) (n = 3) or not (open circles) (n = 4), and stimulated with titrated concentrations of OVA. The B cells and macrophages were co-cultured with OVA-specific T-cell hybridoma HOB.6. Proliferation was measured by CTLL. The data represent the mean c.p.m. values ± SEM of the indicated numbers of individual mice in each group. *P < 0.05 as tested with Mann–Whitney as compared with the OVA only control group.

 
The pronounced augmentation of antigen presentation by cysteine protease inhibition of LCs is Aq/CII260–270 specific
To further investigate the antigen specificity of the observed augmentation of CII-presentation by cysteine protease inhibition of LCs we tested OVA with Aq-restricted T cells and bCII with Ar-restricted T cells. LCs from B10.Q x DBA/1 F1 mice were loaded with titrated doses of OVA, co-cultured with an OVA-specific T-cell hybridoma (HOB.6) and treated with E64, in similarity with the CII experiments. Similarly, the response to bCII was tested using LCs from B10.RIII mice, which were loaded with titrated doses of bCII, co-cultured with T-cell hybridomas specific for Ar/bovCII607–621 and treated with E64. No augmenting effects, similar to what was observed with the Aq-restricted CII response, were seen (Figs 7 and 8). In fact, if anything, E64 treatment led to lower presentation, an effect possibly due to reduced invariant chain degradation. Thus, with both OVA and bCII, no augmentation of antigen presentation was seen using the same doses of antigen and inhibitors as with CII presented by Aq-expressing LCs. Taken together, this argues for a CII peptide-specific effect of LC presentation that is dependent on cysteine proteases. A possible explanation is that the cathepsin inhibition leads to prolonged existence of newly generated peptides in the endosomes, an effect more pronounced for the CII259–270 peptide, and the LCs differ from other APCs in the efficiency of degrading the CII259–270 peptide.



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Fig. 7. Cathepsin inhibition does not enhance bCII presentation by LCs expressing Ar. Firstly, the E64 inhibitor was titrated on a fixed bCII dose in similarity with Fig. 2; (A) (n = 3) Ar-expressing LCs were incubated with 10 µg/ml bCII and co-cultured with 607–621Ar-specific T-cell hybridoma (9D9D8). Secondly, bCII was titrated on a constant E64 dose; (B) bCII was titrated on LCs treated with 100 µg/ml E-64 (closed circles) (n = 3) or not (open circles) (n = 6). Surprisingly, E64 treatment led to lower c.p.m. counts. CTLL was used for measuring proliferation. The data represent the mean c.p.m. values ± SEM of the indicated numbers of individual mice in each group. *P < 0.05 as tested with both ANOVA and Mann–Whitney, as compared with the CII-only control group.

 


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Fig. 8. Cathepsin inhibition does not enhance OVA presentation by LCs. OVA was titrated on DCs while the E-64 concentration was held constant at 100 µg/ml. DCs treated with E-64 (closed circles) (n = 3) or not (open circles) (n= 6) and co-cultured with T-cell hybridoma HOB.6. Proliferation was measured by CTLL. The data represent the mean c.p.m. values ± SEM of the indicated numbers of individual mice in each group.

 
Cysteine protease inhibition leads to presentation of the immunodominant mouse CII peptide
A consequence of a faster degradation rate in LC would be that low affinity peptides are not well presented and cysteine protease inhibition should enhance their presentation. The immunodominant rat CII peptide differs from the mouse at position 266. The mouse has an aspartic acid (D), at this position instead of glutamic acid (E), an exchange that leads to a 13-fold lower affinity to the Aq molecule (Table 2). In similarity with the poor presentation of rat CII, LC could not present mouse CII (Fig. 9). Interestingly, however, inhibition of cysteine proteases led to an efficient presentation of mouse CII to HCQ.10, in fact relatively more efficient than presentation of rat CII.


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Table 2. Binding of the autologous and heterologous immunodominant CII peptide to Aq
 


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Fig. 9. Cathepsin inhibition leads to mouse CII presentation by LC. LC from B10.Q x DBA/1 F1 mice, treated with (closed circles) or without (open circles) 100 µg/ml E-64 stimulated with titrated levels of mouse CII. The LCs were co-cultured with the T-cell hybridoma HCQ.10. CTLL was used for measuring proliferation. The data represent the mean c.p.m. values ± SEM of three individual mice in each group. The *P < 0.05 as tested with both ANOVA and Mann–Whitney as compared with the CII-only control group.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We and others have earlier shown that DCs, which are believed to be the main antigen presenting cells to prime T cells (12), do not efficiently present CII (5,6). In the present study we have further investigated the mechanism behind this inability and we found that LCs both in vitro and in vivo present CII efficiently if cysteine proteases are inhibited. Notably, the most selective CatS inhibitor, LHVS, could be shown to reproduce the effect arguing for a role of CatS. However, the effect of LHVS was even more pronounced in the dose interval in which it inhibits other cathepsins than CatS, arguing for an important role of several unknown proteases. A role for CatS as responsible for degrading the CII peptide is also questioned by the results of Nakagawa et al. (15) who showed that both macrophages and DCs from CatS-deficient mice presented CII less efficiently. This observation is difficult to reconcile with our results but could be due to the fact that in these mice CatS is completely lacking and the inherited deficiency had allowed feedback mechanisms for antigen processing.

Interestingly, LCs can only present mouse CII if cysteine proteases are inhibited. This effect was found to be unique for presentation of the CII260–270 peptide by As. Recognition of the CII260–270 peptide bound to Aq is the critical event for development of collagen-induced arthritis (CIA) in mice and a similar peptide, CII260–273, bound to DR4, is also recognized in RA (2,35). We conclude from these data that LCs do have the capacity to process and present CII but that the CII259–270 peptide is sensitive to cathepsin-mediated degradation. This effect will be more pronounced for presentation of the immunodominant mouse CII259–270 peptide, because it is of slightly lower affinity for binding to Aq, allowing a prolonged degradation time by CatS.

To initiate a T-cell dependent immune response, proteins must undergo proteolysis within the APCs to generate immunodominant peptides that bind to MHC class II and are presented by APCs on the cell surface. It has earlier been shown that cathepsins B, D, E, L and H play important roles in the antigen processing and it has been possible to specifically pinpoint cathepsins F, L and S as responsible for the degradation of Ii. Our data also support the role for cysteine proteases in antigen processing in LCs, but that this effect is more pronounced for a specific peptide (CII259–270) MHC class II (Aq) combination. In addition, this effect is more pronounced in LCs than in macrophages and B cells. Although macrophages and B cells had a small enhanced presentation of low concentrations of antigens when treated with cysteine protease inhibitors this augmentation was not as dramatic as the protease inhibition of LC presentation. This window of activity by cysteine proteases is best explained by a degradative effect of the peptide in the endosomal compartment before loading to the class II molecule. As soon as peptides are loaded they are relatively resistant to degradation. Thus, the protection of an antigenic peptide is expected to result from the balanced effect of the degradation of the Ii chain and the antigenic peptide. In such a scenario a low affinity peptide, like the mouse CII260–270 homologue is more efficiently degraded as compared with the slightly higher affinity rat peptide. The effect of cathepsin inhibitors in LCs could possibly be due to a different specificity of protease activity in the endosomal compartments of LCs as compared with other APCs. Thus, the effect by cathepsins on antigen degradation, as well as on balance with Ii degradation, are likely to vary with antigen, peptide and type of antigen presenting cells (36).

The detected unique window of cathepsin effect on the CII260–270 peptide is likely to be of pathophysiological relevance. In mice expressing Aq, the immunodominant peptide presented and recognized is the CII259–270 peptide and T cells are normally either ignorant to the peptide due to its low affinity to Aq or partially tolerized. Therefore, immunization with homologous CII causes a disease with lower incidence (37,38) as compared with immunization with heterologous CII. In MMC mice expressing mutated CII with aspartic acid replaced by glutamic acid at position 266 leading to higher MHC affinity of the peptide, the T cells are tolerized (39). These mice are nevertheless susceptible to arthritis but the incidence is lower compared to non-transgenic littermates and T-cell reactivity is reduced but far from abolished. In the light of the extensive leakage of CII from cartilage it has been difficult to explain the inability to completely tolerize the T cells. The reduced capacity of LCs to process and present this particular CII epitope, which can be recognized by autoreactive T cells and is crucial for development of CIA, could therefore provide an explanation. A corresponding observation has been made through a series of elegant experiments on presentation of myelin basic protein in H-2s mice in which it could be shown that the cysteine protease asparagine endopeptidase (AEP) destroys the immunodominant myelin basic protein (MBP) peptide in the thymus, prohibiting induction of thymus tolerance (21). Both the destruction of the CII peptide by cysteine proteases and the destruction of the MBP85–99 peptide by AEP highlight the importance of protease processing in regulating T-cell tolerance.

The inability to present CII is a common feature for DCs derived from different sources and maturation stages (5,6). The inability of DCs to present CII is possibly valid also in human RA, which is associated with certain MHC class II genes, namely DRB1*0401 in DR4. In fact it has been found that the peptide CII260–273 binds to DR4 (DRB1*0401) and that transgenic mice expressing human DR4 (DRB*0401) are susceptible to CIA (40,41). It has recently been reported that DCs from human DR4 individuals present human CII weaker than the human CII peptide, compared to macrophages which efficiently presented both the whole human CII and the peptide (42). Different soluble forms of CII were also found in synovial fluid from RA patients. Although these findings do not prove a role for CII as the autoantigen in RA, it shows that such recognition is possible.

The role of DCs is likely to be complex in CIA and RA. The physiological inability of DCs to present CII probably protects against self-reactivity towards CII by increasing the threshold for disease induction since macrophages in general need higher antigen concentrations compared to DCs for eliciting T-cell response. On the other hand, a relative inability of DCs to present CII might also pose a risk since DCs play a crucial role for maintenance of tolerance. Thus, both a subtle increase or decrease of the DC’s capacity to present CII might increase the risk of T-cell activation to CII and thereby development of arthritis.


    Acknowledgements
 
We thank Sandy Liedholm, Isabelle Bohlin and Carlos Palestro for taking excellent care of the animals and Alexandra Treschow for linguistic corrections. The work was supported by grants from the Swedish Rheumatism Association, the Swedish Research Council, King Gustaf V’s 80-year, Crafoord, Påhlsson, Kock and Österlund Foundations.


    Abbreviations
 
AEP—aspargine endopeptidase

APC—antigen presenting cell

Aq and Ar—murine MHC class II molecules of the q and r haplotype, respectively

bCII—bovine type II collagen

CII—type II collagen

Cao74—L-trans-epoxysuccinyl-Ile-Pro-OH propylamide

Cat—cathepsin

CIA—collagen-induced arthritis

CLIP—class-II-associated invariant chain peptide

DC—dendritic cell

E-64—trans-epoxysuccinyl-L-leucylamido (4-guanidino)-butane

Ii—invariant chain

LC—Langerhans cells

LHVS—N-morpholinurealeucine-homophenylalanine-vinylsulfone-phenyl

LNC—lymph node cell

MBP—myelin basic protein

OVA—ovalbumin

RA—rheumatoid arthritis


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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