The impact of glycosylation on HLADR1restricted T cell recognition of type II collagen in a mouse model.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 54, No. 2, February 2006, pp 482–491 DOI 10.1002/art.21565 © 2006, American College of Rheumatology The Impact of Glycosylation on HLA–DR1–Restricted T Cell Recognition of Type II Collagen in a Mouse Model Alexei von Delwig,1 Daniel M. Altmann,2 John D. Isaacs,1 Clifford V. Harding,3 Rikard Holmdahl,4 Norman McKie,1 and John H. Robinson1 Objective. Type II collagen (CII) is a candidate autoantigen implicated in the pathogenesis of rheumatoid arthritis (RA). Posttranslational glycosylation of CII could alter intracellular antigen processing, leading to the development of autoimmune T cell responses. To address this possibility, we studied the intracellular processing of CII for presentation of the arthritogenic glycosylated epitope CII259–273 to CD4 T cells in macrophages from HLA–DR1–transgenic mice. Methods. HLA–DR1–transgenic mice were generated on a class II major histocompatibility complex– deficient background, and T cell hybridomas specific for the glycosylated and nonglycosylated epitope CII259–273 were developed. Subcellular fractionation of macrophages was used to localize CII degradation to particular compartments and to identify the catalytic subtype of proteinases involved. Results. We showed that the glycosylated CII259–273 epitope required more extensive processing than did the nonglycosylated form of the same epitope. Dense fractions containing lysosomes were primarily engaged in the processing of CII for antigen presentation, since these compartments contained 1) enzyme activity that generated antigenic CII fragments bearing the arthritogenic glycosylated epitope, 2) the antigenic CII fragments themselves, 3) CII peptide–receptive HLA–DR1 molecules, and 4) peptide/HLA–DR1 complexes that could directly activate T cell hybridomas. Degradation of CII by dense fractions occurred optimally at pH 4.5 and was abrogated by inhibitors of serine and cysteine proteinases. Conclusion. Processing of the arthritogenic glycosylated CII259–273 epitope, which is implicated in the induction of autoimmune arthritis, is more stringently regulated than is processing of the nonglycosylated form of the same epitope. Mechanisms of intracellular processing of the glycosylated epitope may constitute novel therapeutic targets for the treatment of RA. Inflammation of peripheral joints with destruction and remodeling of the articular cartilage are characteristic features of rheumatoid arthritis (RA). Type II collagen (CII; ␣1[II]3) forms the backbone of the heteropolymeric fibrils that constitute 80–85% of the total collagen content of articular cartilage (1). Several lines of evidence suggest that CII is a candidate autoantigen in RA. Indeed, immunization of susceptible strains of mice with CII in adjuvant induces CII-specific T cells and B cells and results in the development of autoimmune collagen-induced arthritis (CIA) (2,3). Mice expressing the Aq class II major histocompatibility complex (MHC) molecule (4,5), as well as transgenic mice expressing human HLA–DRB*0101 and DRB*0401 genes (6–8) (which are associated with RA in humans ), have been shown to be susceptible to CIA. The strong genetic link between RA and the MHC supports the role of T cells in the pathogenesis of RA, particularly during disease initiation and perpetuation (10). CIIspecific T cell responses in RA patients also correlate with inflammatory activity and radiographic severity in RA (11), and these responses are directed toward the immunodominant arthritogenic region CII259–273 (12–14). It has been proposed that posttranslational modification of self proteins is involved in autoimmunity via Supported by the Arthritis Research Campaign, UK (grant MP/R0619). 1 Alexei von Delwig, PhD, DSc, John D. Isaacs, PhD, FRCP, Norman McKie, BSc, PhD, John H. Robinson, BSc, PhD: University of Newcastle upon Tyne, Newcastle upon Tyne, UK; 2Daniel M. Altmann, BSc, PhD: Imperial College School of Medicine, Hammersmith Hospital, London, UK; 3Clifford V. Harding, MD, PhD: Case Western Reserve University, Cleveland, Ohio; 4Rikard Holmdahl, MD, PhD: Lund University, Lund, Sweden. Address correspondence and reprint requests to Alexei von Delwig, PhD, DSc, Musculoskeletal Research Group, Clinical Medical Sciences, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, UK. E-mail: firstname.lastname@example.org. Submitted for publication April 8, 2005; accepted in revised form October 20, 2005. 482 ANTIGEN PROCESSING OF HUMAN TYPE II COLLAGEN IN A MOUSE MODEL changes in T cell and antibody specificity or changes in intracellular antigen processing (15). Posttranslational glycosylation of CII, in particular O-linked glycosylation of the K264 and K270 residues located within the arthritogenic CII259–273 region has been shown to correlate with the enhanced arthritogenicity of CII and the creation of new CII epitopes recognized by autoimmune T cells in mice and in humans with RA (12,16,17). The K264 residue has been reported to bind T cell receptors of autoreactive T cells or to contribute to the binding affinity of CII259–273 for Aq, HLA–DR1, and HLA–DR4 molecules (18), such that glycosylation of this amino acid has dramatic effects on class II MHC binding, T cell recognition, arthritis severity, and production of CIIspecific antibodies (19). In order for CII-specific T cells to become activated, proteolytic processing of CII by professional antigen-presenting cells (APCs) is required (20,21). Previous studies have focused on the presentation of undefined CII epitopes (20,22) as well as the CII259–273 epitope (minimal epitope CII260–270) (23). In particular, it has been shown that the glycosyl side chain of K264 could not be removed by APCs (23,24). However, little information is available on the mechanisms of proteolytic CII processing for presentation of the glycosylated epitope CII259–273, which could be the key to understanding the activation of autoimmune T cells during RA. In this study, we evaluated the intracellular processing of CII in macrophages from HLA–DR1– transgenic mice for presentation of the glycosylated CII259–273 epitope. We found that the glycosylated CII epitope required more extensive processing than did the nonglycosylated form of the same epitope for presentation to CD4 T cells. MATERIALS AND METHODS Antigens. CII purified from normal human cartilage was purchased from MD BioSciences (Zurich, Switzerland). Deglycosylation of CII was performed by treatment with trifluoromethanesulfonic acid:anisol (2:1) for 3 hours on ice, followed by 1 hour of incubation at room temperature, as described elsewhere (16). The glycosylated peptide (GIAGFKGEQGPKGET; where K represents GalHyL264) corresponding to epitope CII259–273 containing GalHyL264 was synthesized using ␤-D-galactopyranosyl-5-hydroxy-L-lysine, as described previously (25). The nonglycosylated peptide pCII259–273 was purchased from GenScript (Piscataway, NJ), and identity was confirmed by mass spectrometry. Animals. Previously described HLA–DR1–transgenic mice (designated C57BL/6J0-0 HLA–DR1) carrying full-length genomic constructs for HLA–DRA1*0101 and HLA– DRB1*0101 (26) were crossed for more than 6 generations to 483 C57BL/6 Ab–null mice (27) and genotyped by polymerase chain reaction for the presence of the HLA transgenes. This newly derived strain of DR1–transgenic mice lacked both human CD4 and endogenous mouse class II MHC molecules and was characterized by 1) a normal distribution of DR1⫹ lymphoid cell types in the spleen, 2) the expression of DR1 molecules on professional APCs, but not on CD4 or CD8 T cells, 3) a normal distribution of T cell receptor (TCR) V␤ family expression among spleen cells, and 4) the susceptibility to CIA (Von Delwig A, et al: unpublished observations). The high magnitude of CII-specific T cell responses in DR1transgenic mice suggested that mouse CD4 did not interfere with antigen presentation by human DR1 molecules. Control C57BL/6 mice were purchased from Harlan UK (Blackthorn, UK). Experiments described herein were performed under the terms of Animals (Scientific Procedures) Act of 1986 and were authorized by the Home Secretary, Home Office, UK. Immunization. CII was solubilized in 0.01M acetic acid at 2.0 mg/ml and then emulsified with an equal volume of Freund’s complete adjuvant (CFA) containing 4.0 mg/ml of Mycobacterium tuberculosis (Chondrex, Redmond, WA). One hundred micrograms of CII emulsified in CFA was injected subcutaneously at the base of the tail. Ten days later, the inguinal lymph nodes were removed for testing CII-specific T cell responses and developing CII-specific T cell lines, as described previously (6). Cells. Culture media ingredients, inhibitors, and enzyme substrates were purchased from Sigma (Poole, UK), except as stated otherwise. Cells were grown in culture medium (RPMI 1640 medium containing 3 mM L-glutamine, 50 M 2-mercaptoethanol, 10% fetal bovine serum, and 30 g/ml of gentamicin). Twenty-two CII-specific DR1-restricted T cell hybridomas were developed by polyethylene glycol fusion of BW5147 (TCR␣–/␤–) cells with CD4 T cell lines from CIIimmunized DR1-transgenic mice. The specificity was confirmed in antigen presentation assays using CII and the synthetic peptides described above. Twenty-one of 22 T cell hybridomas recognized the nonglycosylated epitope, and 1 T cell hybridoma was specific for the glycosylated epitope (data not shown). T cell hybridomas HCII-9.1 (CD3⫹, ␣ / ␤TCR⫹,V␤14⫹; specific for the nonglycosylated peptide) and HCII-9.2 (CD3⫹,␣/␤TCR⫹,V␤8.1/8.2⫹; specific for the glycosylated peptide) were selected for the antigen presentation assays. Macrophages from femoral bone marrow cells obtained from DR1-transgenic mice were grown for 6 days in culture medium supplemented with 5% horse serum, 1 mM sodium pyruvate, 10 mM HEPES, and 7.5% of a supernatant from the L929 cell line as a source of macrophage colonystimulating factor. The human macrophage cell line THP-1 (HLA–DR1/DR2) (TIB202; American Type Culture Collection [ATCC], Rockville, MD) was grown in 96-well plates (Greiner, Stonehouse, UK) and was stimulated for 24 hours with 10 ng/ml of phorbol myristate acetate to promote adherence. In all experiments, APCs were stimulated for 24 hours with 10 units/ml of recombinant interferon-␥ (R&D Systems, Abingdon, UK). Antigen presentation assays. Adherent macrophages (105/well) in 48-well flat-bottomed plates were pulsed with 20 g/ml of CII for 3 hours in the absence or presence of the enzyme inhibitors pepstatin (0.5 mM) (28), phenanthroline 484 (0.1 mM) (29), phenylmethylsulfonyl fluoride (PMSF; 3.0 mM), 20 mM NH4Cl (30), (2S,3S)-trans-epoxysuccinyl-Lleucylamido-3-methylbutane ethyl ester (E-64d; 10 M) (31), N␣-p-tosyl-L-lysine chloromethylketone (TLCK; 250 M) (32), and N-p-tosyl-L-phenylalanine chloromethylketone (TPCK; 2.5 M) (29), with either cycloheximide (20 M) to block protein synthesis (33) or brefeldin A (1.0 g/ml) to disrupt Golgi transport (34). The optimal doses of inhibitors were established in separate dose-response experiments, as reported previously (30). To test antigen presentation of CII digests, 40-l aliquots of each digest were added to fixed THP-1 cells in the presence of the same doses of the enzyme inhibitors PMSF, E-64d, pepstatin, and phenanthroline to prevent further degradation (35) and then incubated for 18 hours at 37°C. To localize peptide/DR1 complexes in subcellular fractions, vesicular compartments present in the Percoll gradient fractions were added to 96-well plates and tested with T cell hybridomas. Synthetic peptides (at 1.0 M) were added to macrophages or aliquots of fractions as a positive control for T cell hybridoma responsiveness. Cells were fixed in 1.0% paraformaldehyde for 5 minutes, washed thoroughly to remove the fixative, and T cell hybridomas HCII-9.1 (specific for the nonglycosylated epitope) and HCII-9.2 (specific for the glycosylated epitope) were added (5 ⫻ 104/well) and incubated for 24 hours at 37°C. To measure antigen-nonspecific responses of T cell hybridomas, a dilution series of rat anti-CD3 antibodies (clone KT3; a generous gift of Dr. C. G. Brooks, University of Newcastle upon Tyne, Newcastle upon Tyne, UK) were coated on 96-well plates, and T cell hybridomas were added as above. The interleukin-2 content of hybridoma supernatants was measured by bioassay as the proliferative response of the cytotoxic T cell line CTLL-2 (3 ⫻ 104/well) (TIB214; ATCC) during 24 hours of incubation in the presence of 14.8 kBq of 3Hthymidine (specific activity 307 MBq/mg; Amersham, Little Chalfont, UK). Cells were harvested onto glass-fiber membranes, and radioactivity was quantitated using a direct Beta Counter (Matrix 9600; Packard, Meriden, CT). Subcellular fractionation. Macrophages (15–25 ⫻ 106) were washed with ice-cold RPMI 1640 followed by phosphate buffered saline, and homogenized in buffer containing 0.25M sucrose, 10 mM HEPES, pH 7.4, using a Dounce tissue grinder (Wheaton, Millville, NJ), to obtain 80–85% cell lysis. In some experiments, macrophages were first incubated with 200 g/ml of CII for 0, 1, 3, and 5 hours at 37°C in culture medium. Intact cells and nuclei were removed by 3 cycles of differential centrifugation (270g for 2 minutes at 4°C), and supernatants containing postnuclear membranes were layered on 27% Percoll (Amersham), followed by centrifugation (36,000g for 60 minutes at 4°C) in a Sorvall type A-1256 fixed-angle rotor (Sorvall, Wilmington, DE). Six fractions (numbered 1–6 from the top of the gradient) of 1.5 ml were collected manually. Percoll gradient fractions were each tested for ␤-hexosaminidase activity as a marker for the presence of lysosomal enzymes (34). The reaction mixture containing 50 l of sample, 150 l of assay buffer (0.1M MES, 0.2% Triton X-100, pH 6.5), and 50 l of chromogenic substrate (pnitrophenyl-N-acetyl-␤-D-glucosaminide formulated fresh at 1.37 mg/ml in water) was incubated for 90 minutes at 37°C, and the absorbance was measured at 405 nm. Percoll gradient VON DELWIG ET AL fractions were also tested for alkaline phosphodiesterase I activity as a marker for the presence of plasma membranes (36). The fractions (50 l) were mixed with 200 l of chromogenic substrate (2 mM thymidine-5⬘-monophospho-pnitrophenyl ester formulated fresh in 40 mM Na2CO3/ NaHCO3 buffer, pH 10.5, containing 0.1% Triton X-100), incubated for 20 minutes at 37°C, and absorbance was measured at 405 nm. Protease activity of Percoll gradient fractions was determined by measuring the release of amino acids and small peptides from azocasein. Percoll gradient fractions (100 l) were mixed with 200 l of 2% azocasein (Sigma) dissolved in 0.1M sodium citrate buffer, pH 5.5, containing 0.1% Triton X-100. After incubation at 37°C for 24 hours, proteolysis was stopped by adding 200 l of 20% cold trichloroacetic acid. After centrifugation at 1,000g for 10 minutes, 200 l of the supernatant was collected, and azocaseinase activity was measured as the absorbance at 405 nm. Percoll gradient fractions were mixed at a 1:1 ratio with 2.0 mg/ml of CII in 50 mM MES buffers, pH 4.5, 5.5, and 6.5, and then incubated for 2.5 or 5 hours at 37°C. In some experiments, the enzyme inhibitors described above were added. Digestion was stopped by heat-inactivation at 100°C for 3 minutes. Digests were mixed with sodium dodecyl sulfate (SDS) nonreducing sampling buffer, analyzed by SDS– polyacrylamide gel electrophoresis (PAGE) on 10% polyacrylamide gels under nondenaturing conditions, and silver-stained using a Silver Stain Plus kit (Bio-Rad, Hertfordshire, UK) according to the manufacturer’s instructions. RESULTS Mechanisms of CII presentation to T cells. To study the mechanisms of processing and presentation of CII in macrophages from HLA–DR1–transgenic mice, T cell hybridomas specific for the glycosylated (HCII-9.2) and nonglycosylated (HCII-9.1) CII259–273 epitope were developed that had no cross-reactivity between the two forms of the epitope. Deglycosylation of CII resulted in the complete loss of presentation to T cell hybridoma HCII-9.2, with little effect on presentation to the HCII9.1 hybridoma (Figures 1A and B). These findings were consistent with previous observations (16). Antigen dose-response titrations showed that T cell hybridoma HCII-9.2 was of low sensitivity to antigen (Figure 1A), as compared with the higher sensitivity of HCII-9.1 (Figure 1B). Both CII-specific T cell hybridomas responded similarly in wells coated with anti-CD3 antibodies (data not shown), suggesting the high and low sensitivity to antigen was related to TCR affinity. When time of exposure to CII was evaluated, responses to the glycosylated epitope were detected after 3 hours (Figure 1C), whereas the nonglycosylated epitope was presented optimally to T cell hybridoma HCII-9.1 after a shorter ANTIGEN PROCESSING OF HUMAN TYPE II COLLAGEN IN A MOUSE MODEL 485 Figure 1. Mechanisms of antigen presentation of type II collagen (CII). A and B, The specificity of T cell hybridomas was tested by pulsing macrophages from HLA–DR1– transgenic mice with different doses of glycosylated and deglycosylated CII, as well as glycosylated or nonglycosylated peptides. C and D, The kinetics of antigen presentation was measured by pulsing macrophages from HLA–DR1–transgenic mice with CII for different periods of time. Ag ⫽ antigen. E and F, The source of HLA–DR1 molecules used for presentation of CII epitopes was studied by pulsing macrophages for 3 hours with different doses of CII in the absence or presence of 20 M cycloheximide or 1 g/ml brefeldin A. After fixation, plates were assayed with T cell hybridoma HCII-9.2, which is specific for the glycosylated epitope (A, C, and E), or T cell hybridoma HCII-9.1, which is specific for the nonglycosylated epitope (B, D, and F). Interleukin-2 production by T cell hybridomas was assayed by 3H-thymidine incorporation into CTLL-2 cells. Values are the mean ⫾ SD cpm of triplicates. Results are representative of 3 experiments. period of 1.5 hours (Figure 1D). In experiments with synthetic peptides, presentation of the glycosylated peptide was also slower (Figure 1C) compared with the nonglycosylated peptide (Figure 1D). The source of class II MHC molecules that presented the glycosylated and nonglycosylated epitope was determined by pretreating macrophages with the protein synthesis inhibitor cycloheximide to deplete newly synthesized class II MHC (33) or with brefeldin A, an inhibitor of class II MHC transport between the endoplasmic reticulum and the Golgi complex (34). Both inhibitors blocked presentation of the glycosylated and nonglycosylated CII epitopes (Figures 1E and F), but not the relevant synthetic peptides (data not shown), suggesting that both forms of the epitope are presented by newly synthesized class II MHC molecules. Presentation of the glycosylated epitope was blocked by raising the endosomal pH with NH4Cl (Fig- ure 2A), whereas presentation of the nonglycosylated epitope was only partly inhibited (Figure 2B), suggesting a different pH dependency for presentation of the 2 forms of the same epitope. When macrophages were pulsed with CII in the presence of inhibitors of the major proteinase families, presentation of the glycosylated CII epitope was completely blocked by inhibitors of cysteine proteinases (E-64d), aspartic proteinases (pepstatin), and metalloproteinases (phenanthroline) (Figure 2A), whereas only partial inhibition was observed in the responses to the nonglycosylated epitope (Figure 2B). Presentation of the epitope in both glycosylated and nonglycosylated forms was also blocked by inhibitors of serine proteinases (PMSF, TPCK, and TLCK) (Figures 2A and B). Synthetic peptide presentation was not affected or was only marginally influenced by the inhibitors used (Figures 2C and D). The data showed different sensitivity to inhibitors of antigen presentation of the 486 Figure 2. Effects of enzyme inhibitors on the intracellular processing of type II collagen (CII) by macrophages. Macrophages from HLA– DR1–transgenic mice were pulsed with different doses of A and B, CII, C, glycosylated synthetic peptide, and D, nonglycosylated synthetic peptide in the presence or absence of the following enzyme inhibitors: 10 M (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester (E-64d), 0.1 mM phenanthroline, 3.0 mM phenylmethylsulfonyl fluoride (PMSF), 2.5 M N-p-tosyl-L-phenylalanine chloromethylketone (TPCK), 0.5 mM pepstatin, 250 M N␣-p-tosyl-L-lysine chloromethylketone (TLCK), or 20 mM NH4Cl. Cells were fixed and then incubated with T cell hybridoma HCII-9.2 (A and C), which is specific for the glycosylated epitope, or T cell hybridoma HCII-9.1 (B and D), which is specific for the nonglycosylated epitope. Interleukin-2 production by T cell hybridomas was assayed by 3H-thymidine incorporation into CTLL-2 cells. Values are the mean ⫾ SD cpm of triplicates. Results are representative of 3 experiments. glycosylated epitope as compared with the nonglycosylated epitope. Subcellular localization of CII antigen processing activity. Macrophages were subjected to subcellular fractionation by Percoll density-gradient centrifugation, and subcellular fractions were analyzed for enzyme activity characteristic of cellular compartments. Plasma membranes were localized to fraction 2 based on the activity of the plasma membrane enzyme alkaline phos- VON DELWIG ET AL phodiesterase I (36), and activity of the lysosomal enzyme ␤-hexosaminidase (37) was confined to dense membrane fractions 5–6 (Figure 3A). We also used an azocasein assay to localize total proteinase activity predominantly to membrane fractions 5–6, which correspond to lysosomes (Figure 3A). In native SDS-PAGE analysis, the band corresponding to the uncleaved CII ␣1(II) chains had an apparent molecular weight of 150 kd, and deglycosylation of CII resulted in the reduction of the apparent molecular mass from ⬃150 kd to ⬃130 kd (Figure 3B). We detected no visible band corresponding to 100 kd, which could be expected for the ␣1(II) chains. Hence, the higher molecular mass of the CII preparation used in this study could be explained in part by its high glycosylation and in part by the unusual pattern of high SDS binding, which increases its apparent molecular mass in SDS-PAGE analysis (38). CII was digested with Percoll gradient fractions as a source of enzyme activity at pH 4.5, 5.5, and 6.5, which are characteristic of lysosomes, late endosomes, and early endosomes, respectively (39). Analysis of the digests on native SDS-PAGE showed that the band corresponding to the uncleaved CII ␣1(II) chains (⬃150 kd) was reduced in density, coincident with the appearance of a number of smaller fragments with a molecular mass between 50 kd and 150 kd, after incubation of CII with dense membrane fractions 5–6, but not with lighter membrane fractions (Figures 3C–E). No visible band corresponding to the nonglycosylated CII (⬃130 kd) was detected, suggesting that the CII preparation from normal human joints used in this study was almost entirely glycosylated, which is consistent with previously published results (16,23). SDS-PAGE of fractions alone did not reveal any bands equivalent to CII or its visible products (results not shown). Collagenolytic activity of the lysosome-rich fractions was pH-dependent, being particularly pronounced at pH 4.5 (Figure 3C). Digests were added to prefixed THP-1 macrophages and assayed with T cell hybridomas to measure presentation of the glycosylated and nonglycosylated CII epitope. Intact CII was not presented by prefixed APCs for specific T cell hybridomas, indicating a requirement for uptake and/or intracellular processing for presentation to T cells (Figures 4A and B). Fractions 5–6 processed CII for presentation of both forms of the CII epitope, while the fractions alone did not stimulate T cell hybridomas (Figures 4A and B). Glycosylated and nonglycosylated peptides were added to Percoll gradient fractions from untreated macrophages and assayed with T cell hybridomas, which also showed presentation in fractions 5–6 (Figures 4C and D). Taken together, these ANTIGEN PROCESSING OF HUMAN TYPE II COLLAGEN IN A MOUSE MODEL 487 CII was digested with membrane fraction 6 at pH 4.5 in the presence of enzyme inhibitors. SDS-PAGE analysis of the digests showed that CII degradation was reduced by inhibitors of cysteine proteinases, as well as by inhibitors of chymotrypsin-like (TPCK) and trypsinlike (TLCK) serine proteinases (Figure 5A). Digestion was marginally affected by inhibitors of aspartic proteinases and metalloproteinases, suggesting that CII was preferentially degraded in macrophages by lysosomal serine and cysteine proteinases. To study productive processing, digests were added to prefixed THP-1 cells to show that presentation of both epitopes was blocked by inhibitors of serine proteinases (Figures 5B and C). A residual level of presentation of the nonglycosylated CII epitope was detectable in the presence of inhibitors of aspartic proteinases, cysteine proteinases, and metalloproteinases, whereas the same inhibitors blocked presentation of the glycosylated epitope (Figures 5B and C). These results are consistent with the data on intracell- Figure 3. Characterization of enzyme activity in subcellular fractions of macrophages. A, Fractions 1–6 from Percoll density gradients were incubated with the chromogenic substrates thymidine-5⬘monophospho-p-nitrophenyl ester (to measure activity of the plasma membrane–associated enzyme alkaline phosphodiesterase I), p-nitrophenyl-N-acetyl-␤-D-glucosaminide (to localize the activity of the lysosomal marker ␤-hexosaminidase), or azocasein (to measure total proteolytic activity). Enzyme activity was measured as the absorbance at 405 nm. Fraction 0 represents 27% Percoll alone. Values are the mean ⫾ SD. B, Analysis of CII before (lane 1) and after (lane 2) deglycosylation. Molecular weight standards are shown in the left lane. Analysis of CII digestion by sodium dodecyl sulfate– polyacrylamide gel electrophoresis of subcellular fractions 1–6 from macrophages were incubated for 5 hours with CII at C, pH 4.5, D, pH 5.5, or E, pH 6.5, and the digests were resolved on 10% polyacrylamide gels. CII was incubated with 27% Percoll as a control; the position of the uncleaved CII is indicated by the arrowhead. Molecular weight standards are shown in the left lane. data show that enzyme activity in fractions enriched for lysosomes productively processed CII for presentation of the CII259–273 epitope in both glycosylated and nonglycosylated forms to approximately the same degree. Figure 4. Antigen presentation of type II collagen (CII) epitopes by fixed THP-1 cells. Subcellular fractions 1–6 of macrophages were incubated with CII at pH 4.5 (A and B) or were pulsed with synthetic peptides (C and D). After incubation, digests were added to prefixed THP-1 cells and assayed with T cell hybridoma HCII-9.2 (A and C), which is specific for the glycosylated epitope, or T cell hybridoma HCII-9.1 (B and D), which is specific for the nonglycosylated epitope. As controls, Percoll gradient fractions were incubated in the absence of CII or CII was incubated in the absence of fractions before adding to the THP-1 cells. Interleukin-2 production by T cell hybridomas was assayed by 3H-thymidine incorporation into CTLL-2 cells. Values are the mean ⫾ SD cpm of triplicates. Results are representative of 3 experiments. 488 VON DELWIG ET AL ular processing shown in Figure 2. The differences in sensitivity of SDS-PAGE and T cell assays to aspartic proteinase and metalloproteinase inhibitors may reflect the greater sensitivity of the T cell readout compared with SDS-PAGE in detecting low doses of CII fragments. Subcellular localization of peptide/HLA–DR1 complexes. Macrophages were pulsed with soluble CII, subjected to subcellular fractionation, and fractions were assayed with T cell hybridomas HCII-9.2 and HCII-9.1 to localize peptide/HLA–DR1 complexes. Complexes of HLA–DR1 with the glycosylated and nonglycosylated Figure 6. Subcellular localization of peptide/HLA–DR1 complexes in macrophages. Macrophages were pulsed with type II collagen for the indicated times and then subjected to subcellular fractionation. Fractions were assayed with A, T cell hybridoma HCII-9.2, which is specific for the glycosylated epitope, or B, T cell hybridoma HCII-9.1, which is specific for the nonglycosylated epitope. Interleukin-2 production by T cell hybridomas was assayed by 3H-thymidine incorporation into CTLL-2 cells. Values are the mean ⫾ SD cpm of triplicates. Results are representative of 3 experiments. Figure 5. Effects of enzyme inhibitors on type II collagen (CII) digestion. A, CII was digested by lysosomal fraction 6 at pH 4.5 in the presence or absence of the following enzyme inhibitors: 0.5 mM pepstatin, 0.1 mM phenanthroline, 10 M (2S,3S)-trans-epoxysuccinylL-leucylamido-3-methylbutane ethyl ester (E-64d), 2.5 M N-p-tosylL-phenylalanine chloromethylketone (TPCK), 250 M N␣-p-tosyl-Llysine chloromethylketone (TLCK), or 3.0 mM phenylmethylsulfonyl fluoride (PMSF). The digests were resolved on 10% polyacrylamide gels. Fraction 6 alone served as a control; the position of the uncleaved CII is indicated by the arrowhead. Molecular weight standards are shown in the left lane. Digests obtained in the presence of inhibitors were also added to fixed THP-1 cells and assayed with B, T cell hybridoma HCII-9.2, which is specific for the glycosylated epitope, or C, T cell hybridoma HCII-9.1, which is specific for the nonglycosylated epitope. Interleukin-2 production by T cell hybridomas was assayed by 3 H-thymidine incorporation into CTLL-2 cells. Values are the mean ⫾ SD cpm of triplicates. Results are representative of 3 experiments. CII epitope were detected after 3 hours in dense membrane fractions 5–6, which correspond to lysosomes, with a low level of presentation in fractions 2–3, which contain plasma membranes (Figures 6A and B). Peptide/ HLA–DR1 complexes were more abundant in fractions 5–6 after 5 hours, suggesting an accumulation of complexes in lysosomes. DISCUSSION In this study, we addressed whether posttranslational glycosylation affected intracellular processing of human CII for presentation of the glycosylated epitope CII259–273, as compared with the nonglycosylated epitope, to specific CD4 T cell hybridomas in HLA– DR1–transgenic mice. Posttranslational glycosylation of CII259–273 epitope created a novel T cell epitope that was ANTIGEN PROCESSING OF HUMAN TYPE II COLLAGEN IN A MOUSE MODEL recognized by T cells from HLA–DR1–transgenic mice. Immunizations of DR1-transgenic mice with CII yielded a high recovery of T cell hybridomas specific for the nonglycosylated CII epitope and a low recovery of T cell hybridomas specific for the glycosylated CII epitope. This result could be due to the lower affinity of DR1 for the glycosylated epitope. Indeed, glycosylation of the CII epitope has been reported to decrease CII peptide affinity for Aq molecules (40). The result could also be due to either a higher frequency of T cells specific for the nonglycosylated epitope or a greater availability of the nonglycosylated epitope after antigen processing. Our experimental system used bone marrow– derived macrophages, since these APCs were previously shown to preferentially present CII to T cells (22,24). We demonstrated that CII required intracellular processing for subsequent presentation of both the glycosylated and nonglycosylated CII epitope to T cells, with notable differences in processing requirements between the 2 forms of the same epitope. First, the glycosylated epitope was presented more slowly than the nonglycosylated epitope, suggesting that glycosylation retarded the rate of formation of peptide/HLA–DR1 complexes, which is consistent with the demonstration that glycosylation of the CII epitope decreases CII peptide affinity for Aq molecules (40). Second, the intracellular processing of CII for presentation of the glycosylated epitope was more sensitive than the nonglycosylated epitope to treatment of macrophages with inhibitors of cysteine proteinases, aspartic proteinases, and metalloproteinases, which is consistent with the interpretation that CII was differentially processed for presentation of the glycosylated and nonglycosylated epitopes. In addition, both forms of the epitope were profoundly sensitive to inhibitors of serine proteinases, which together with the results of our previous study (30), suggests a major role of this class of enzyme in antigen processing. Overall, the pattern of sensitivity to enzyme inhibitors suggested that presentation of the glycosylated epitope required more extensive processing than did presentation of the nonglycosylated epitope. Our data therefore support the previous proposal that posttranslational glycosylation of autoantigens confers resistance to proteolysis, with implications for the development of autoimmunity (15). The T cell response to both forms of the epitope was dependent upon intact protein synthesis and Golgi transport, which are required for delivery of newly synthesized class II MHC to peptide-loading compartments of APCs, suggesting engagement of the so-called classic pathway of class II MHC antigen presentation (39). 489 To localize stages of CII degradation to particular intracellular compartments, macrophages were subjected to subcellular fractionation, and the fractions were used as a source of enzyme activity to digest CII. Dense membrane fractions corresponding to lysosomes digested CII at low pH to generate fragments containing the glycosylated CII epitope that could be presented by prefixed APCs. Degradation of CII was blocked by inhibitors of serine and cysteine proteinases, which is consistent with previous studies implicating cysteine proteinases (cathepsins B, K, L, N, and S [41–43]) as well as serine proteinases (44) in intracellular CII degradation and antigen presentation (20). Inhibitors of serine proteinases, cysteine proteinases, aspartic proteinases, and metalloproteinases reduced the productive processing of the glycosylated epitope, and to a lesser extent, the nonglycosylated epitope, which again, is consistent with differential processing of CII for presentation of the glycosylated and nonglycosylated CII epitope. We studied the subcellular localization of HLA– DR1 complexed with the glycosylated and nonglycosylated CII epitope after intracellular antigen processing of CII in macrophages and showed that complexes were present in lysosomes even after a 5-hour pulse with CII, which indicates a slow egress of these complexes from antigen-processing compartments to the plasma membrane. This may be a general mechanism of antigen presentation that is necessary for the sustained activation of autoimmune T cells, which has been shown to require prolonged antigen presentation on the surface of APCs (45). When Percoll gradient fractions from untreated macrophages were pulsed with the glycosylated and nonglycosylated peptides, the formation of peptide/ HLA–DR1 complexes was also detected in dense membrane fractions corresponding to lysosomes, suggesting the presence of peptide-receptive HLA–DR1 molecules in lysosomal compartments. Differential processing of glycosylated and nonglycosylated CII may reflect crucial differences between RA patients and normal individuals. It has been reported that CII exists largely in the glycosylated form in healthy cartilage, whereas RA cartilage is characterized by the presence of both glycosylated and nonglycosylated forms of CII (23). Those investigators also showed that APCs have a limited capacity to deglycosylate CII, suggesting low glycosidase activity, which would favor the differences we observed resulting from antigen processing of distinct pools of glycosylated and nonglycosylated CII. However, it is also possible that deglycosidases, such as lysosomal ␤-galactosidase, influence the 490 VON DELWIG ET AL induction of arthritis by limiting the availability of the glycosylated epitope for presentation to arthritogenic T cells. In this context, it is interesting that a proportion of mice transgenic for ␤-galactosidase show increased susceptibility to CIA (46). Posttranslational glycosylation of CII has been implicated in the balance between tolerance and autoimmunity in mice and in humans (15), although the particular role of T cells against the glycosylated and nonglycosylated CII epitope remains poorly understood. Immune tolerance has been shown to be more pronounced, although not complete, for the nonglycosylated CII epitope, resulting in the appearance of T cells against both the glycosylated and the nonglycosylated CII259–273 epitope in the peripheral blood of RA patients (12). We hypothesize that the requirements for extensive processing of the glycosylated arthritogenic epitope shown herein, the low glycosylation status of collagen at an early age (47), and the inability of some APCs to process CII (21) may result in a paucity of glycosylated peptide/HLA–DR1 complexes in the thymus, the inefficiency of negative selection, and the escape of glycosylated CII–specific T cells into periphery. 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