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Cleavage of denatured natural collagen type II by neutrophil gelatinase B reveals enzyme specificity, post-translational modifications in the substrate, and the formation of remnant epitopes in rheumatoid arthritis

PHILIPPE E. VAN DEN STEEN^*, PAUL PROOST^*, BERNARD GRILLET^*, DAVID D. BRAND, ANDREW H. KANG, JO VAN DAMME^* and GHISLAIN OPDENAKKER^*

^* Rega Institute for Medical Research, Laboratory of Molecular Immunology, University of Leuven, 3000 Leuven, Belgium, and
Veterans Affairs Medical Center, Research Service, University of Tennessee, Memphis, Tennessee 38104, USA

¹Correspondence: G. Opdenakker, Rega Institute for Medical Research, Laboratory of Molecular Immunology, University of Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium. E-mail: Ghislain.Opdenakker{at}rega.kuleuven.ac.be

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
During acute inflammation, leukocytes release proteolytic enzymes including matrix metalloproteinases (MMPs), but the physiopathological mechanisms and consequences of this process are not yet fully understood. Neutrophils, the predominant leukocyte type, produce neutrophil collagenase (MMP-8) and gelatinase B (MMP-9) but not the tissue inhibitors of MMPs. After stimulation, these cells also activate MMPs chemically. In arthritic diseases, neutrophils undergo great chemoattraction to the synovium, are activated by interleukin-8, and are stimulated to release gelatinase B in vivo. Production levels and net activities of gelatinase B were found to be absent in degenerative osteoarthritis but significantly increased in rheumatoid arthritis. The cleavage sites in cartilage type II collagen by gelatinase B were determined by a combination of reverse phase high-performance liquid chromatography, Edman degradation, and mass spectrometry analysis. The analysis revealed the site specificity of proline and lysine hydroxylations and O-linked glycosylation, the cleavage specificities by gelatinase B, and the preferential absence and presence of post-translational modifications at P2^' and P5^', respectively. Furthermore, gelatinase B leaves the immunodominant peptides intact, which are known from studies with (autoreactive) T cells. Lysine hydroxylation was detected at a critical position for T-cell activation. These data lend support to the thesis that extracellular proteolysis and other post-translational modifications of antigenic peptides may be critical in the establishment and perpetuation of autoimmune processes. Van den Steen, P.E., Proost, P., Grillet, B., Brand, D.D., Kang, A.H., Van Damme, J., Opdenakker, G. Cleavage of denatured natural collagen type II by neutrophil gelatinase B reveals enzyme specificity, post-translational modifications in the substrate, and the formation of remnant epitopes in rheumatoid arthritis.

Key Words: autoimmunity • gelatinase B • neutrophil • rheumatoid arthritis • glycosylation

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
THE ETIOLOGY OF autoimmune diseases remains obscure, although predisposing genetic factors or environmental agents have been shown to play a role in these human diseases and the animal models thereof. Aside from disease associations and biological and biochemical studies of rheumatoid arthritis and multiple sclerosis, animal model studies led to the development of the REGA, or Remnant Epitopes Generate Autoimmunity, model for autoimmune diseases (1) . This model describes the central role of cytokines and proteases in autoimmune diseases. When an etiological agent causes a local inflammation, immune cells produce cytokines, chemokines, and proteases, which enhance the inflammatory response and attract and activate other immune cells. The inflammation is further regulated by anti-inflammatory cytokines and protease inhibitors. The proteolytic activity, such as matrix metalloproteinases (MMPs), generated during the inflammation degrades various local proteins, and the resulting peptides (remnant epitopes) are further processed and presented through major histocompatibility complex class II molecules (MHC II) and activate autoreactive T cells. These T cells are present in a latent state in a large number of healthy individuals (2) . Knowledge about the peptides that are left intact (the remnant epitopes just mentioned) is crucial for understanding autoimmunity. For instance, multiple sclerosis is associated with an elevated level of the protease gelatinase B (MMP-9) in the cerebrospinal fluid of patients, and gelatinase B degrades human myelin basic protein into immunodominant peptides (1) . It has also been shown that inflammation and cytokines enhance the presentation of antigenic peptides by antigen-presenting cells (3) , and extracellular processing and loading of peptides onto MHC II were recently found to contribute 40–65% of the total antigen presentation (4 , 5) . In addition, dendritic cells produce gelatinase B, which participates in the extracellular processing of antigens (6) . Also, deficiency in the MHC II-related HLA-DM may enhance the presentation of auto-immunodominant epitopes and the occurrence of autoreactive T cells (7 , 8) . Although our discovery of an association between gelatinase B levels and arthritic disease (9) foreshadowed our studies of multiple sclerosis and was later corroborated by other groups (10 , 11) , the REGA model (1) was not yet studied at the biochemical level in rheumatoid arthritis. One of the key target substrates of MMPs in the joints is collagen type II. Extensive immunological studies exist on the immunodominant epitopes in bovine collagen II (12 13 14 15) . These studies provide detailed information on T-cell reactivity and helper functions in the generation of autoantibodies against collagen II. However, collagen II is extensively post-translationally modified, which implies that both T-cell activation by an MHC-peptide complex and binding of autoantibodies to collagen may depend on or at least be influenced by such post-translational modifications (16 17 18 19) .

From the biochemical and clinical points of view, it has been documented that in autoimmunity of the joint, i.e., rheumatoid arthritis, the chemokine interleukin-8/CXC chemokine ligand-8 (IL-8/CXCL-8) is increased in the synovial fluid (20 21 22) . IL-8/CXCL-8 was found to be essential for the recruitment of neutrophils in a rabbit model of rheumatoid arthritis (23) . Also, the level of gelatinase B is increased in synovial fluid and serum of patients with rheumatoid arthritis, in contrast to the situation in nonimmune osteoarthritis (9 10 11 , 24) . We show here a positive correlation among the neutrophil chemoattractant IL-8/CXCL-8, neutrophil cell counts, and gelatinase B levels in the synovial fluid of various arthritic patients. This result is consistent with studies demonstrating that IL-8/CXCL-8 induces the release of gelatinase B from neutrophils in vitro (25) . Gelatinase B is secreted as a proenzyme, kept inactive by the interaction of a conserved cysteine in the propeptide with the catalytic Zn²⁺. Removal of the propeptide results in the activation of gelatinase B according to the cysteine-switch mechanism (26 , 27) , and the activated gelatinase B can be inhibited by the tissue inhibitors of metalloproteinases (TIMPs) (28) . Therefore, it was not yet clear whether the net activity of gelatinase B is increased in synovial fluid of arthritic patients. We describe here that net gelatinase B activity is increased in rheumatoid arthritic joints, whereas in osteoarthritic synovial fluid, no gelatinase B activity was detected. This finding provides a mechanistic concept and a biochemical parameter to permit differentiation between autoimmune and nonautoimmune (degenerative) arthritis. Furthermore, pure natural gelatinase B and type II collagen were used to define the position of the post-translational modifications and the cleavage sites by gelatinase B. Our data show that gelatinase B may contribute to the generation of the auto-immunodominant remnant epitopes as proposed in the REGA model. In addition, at least one of these immunodominant epitopes was found to be modified by lysine hydroxylation. Furthermore, the identification of the positions of the cleavage sites helps to define the sequence specificity of gelatinase B. This information may lead in the future to the design of specific peptidomimetic inhibitors for the treatment of, for example, rheumatoid arthritis.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Synovial fluid samples
Samples of synovial fluid were collected in dry tubes from patients with hydrops of the knee. The patients were scored on the basis of the American College of Rheumatologists criteria (29) . After the samples were centrifuged at 1700g for 10 min, they were immediately frozen at -20°C until analysis. At the same time, fresh synovial fluid collected in EDTA tubes was analyzed for neutrophil counts.

Analysis of IL-8/CXCL-8 and gelatinase B in synovial fluid
IL-8/CXCL-8 in synovial fluid was determined by using the radioimmunoassay described by Rampart et al. (21) . The detection limit (20% inhibition of the ¹²⁵I-labeled IL-8/CXCL-8 binding) was 30 pg/100 µl; 50% displacement occurred at 140 pg/100 µl. The intra-assay and interassay coefficients of variation were 4% and 7%, respectively.

Gelatinase B levels in 5 µl synovial fluid samples were measured by gelatin zymography, and quantification was done using scanning densitometry as described earlier (30) . Net gelatinase activity in 1 µl of synovial fluid was determined by using the fluorescent-activated substrate conversion (FASC) assay (31) . One FASC unit was defined as the activity that resulted in 50% cleavage of the fluorescent substrate. For samples containing high activity levels, dilution series were tested to quantify the activity in the most sensitive test range.

Purification of natural gelatinase B from human neutrophils
Progelatinase B from human neutrophils was purified to more than 99% homogeneity as demonstrated by electrophoresis and Edman degradation analysis (32) . It was activated with stromelysin-1 at a molar ratio of 1:100 (stromelysin-1 to gelatinase B), as described recently (32) .

Preparation of bovine type II collagen
Type II collagen was extracted from fetal bovine cartilage by limited proteolysis with pepsin, as described previously in detail (33 , 34) . The articular cartilage was dissected, diced into cubes of 2–5 mm, and homogenized in a Waring blender. All operations were performed at 4°C. The homogenate was treated with 0.05 M Tris/4 M guanidine, pH 7, to remove proteoglycans, and collagen was solubilized by pepsin digestion. The solubilized collagen was then purified by several cycles of differential salt precipitation and DE 52 (Whatman, Maidstone, UK) chromatography. The purity of the final collagen preparation was monitored by amino acid analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and CNBr peptide mapping.

Cleavage of bovine collagen type II by natural gelatinase B
Bovine collagen type II was denatured and incubated at 1 mg/ml with activated gelatinase B (enzyme/substrate molar ratio of 1:1000) in assay buffer (100 mM Tris-HCl pH 7.4, 100 mM NaCl, 10 mM CaCl₂, 0.01% Tween 20) at 37°C for 4 h (unless otherwise indicated). As a control experiment, denatured collagen type II was incubated with stromelysin-1 (molar ratio of 1:100,000) under similar conditions. Inhibition studies were done by addition of 100 mM EDTA, 3.6 mM o-phenantroline, 0.2 mg/ml E64, 67 µg/ml aprotinin (Sigma Chemical, St. Louis, MO), or 6 µg/ml recombinant human TIMP-1 (Calbiochem, La Jolla, CA) to the reaction mixture. Degradation of collagen was visualized by SDS-PAGE and staining analysis of the reaction mixture. Alternatively, a similar digest was done with omission of Tween 20 in the assay buffer, and the resulting fragments were separated by reverse phase high-performance liquid chromatography (RP-HPLC). The amino-terminal sequence of each fragment in each fraction was analyzed by Edman degradation on a pulsed liquid protein sequencer model 477A, equipped with a 120A analyzer (Applied Biosystems, Foster City, CA). Each fraction was also analyzed by electrospray ion trap mass spectrometry (Esquire-LC, Bruker Daltonic, Bremen, Germany), yielding the exact length of each fragment. The number of post-translational modifications (hydroxylation, glycosylation) on each fragment was derived from the mass spectrometry (MS) data, and the presence of hexose residues was confirmed in MS/MS mode. The positions of the modifications were found by Edman degradation, as the modified amino acid derivatives elute from the HPLC column at different positions compared with the unmodified derivatives. The numbering of the protein sequence is according to that of the mature processed protein, i.e., the amino-terminal and carboxy-terminal propeptides are not included.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
IL-8/CXCL-8, neutrophils, and gelatinase B in vivo
Arthritic diseases are characterized by inflammation of the synovium. Because of its accessibility, synovial fluid can be used, much like cerebrospinal fluid in multiple sclerosis, to obtain insight into the arthritic disease process. First, correlative evidence was sought for infiltrating cell numbers, specific cytokines, and proteases. Figure 1 A shows a significant correlation between the levels of the major leukocyte cell type in arthritic synovial fluid, the neutrophil, and IL-8/CXCL8, the major neutrophil chemoattractant and activator in humans. Because IL-8/CXCL-8 induces the release of gelatinase B from neutrophils (25) , it was anticipated and shown that in synovial fluid the gelatinase B amounts correlate significantly with the number of neutrophils, the predominant cell type in rheumatoid arthritis and the major producer cell type of gelatinase B (Fig. 1B ).

View larger version (17K): [in this window] [in a new window]   Figure 1. IL-8, neutrophils, and gelatinase B in synovial fluid. A) IL-8/CXCL-8 levels and neutrophil cell counts were analyzed in the synovial fluid of patients with various arthritic diseases. A significant correlation was obtained (n = 27, R = 0.51, P = 0.0071). B) The levels of gelatinase B in the synovial fluid of patients with arthritis, as measured by zymography and quantified by scanning densitometry, were found to be significantly correlated with the synovial neutrophil counts (n = 27, R = 0.85, P = 0.00027).

Gelatinolytic activity as evidenced by substrate zymography does not reveal an insight into the net enzymatic activity (35) . Because neutrophils produce only a specific collagenase (MMP-8) and gelatinase B (MMP-9) and neither gelatinase A nor tissue inhibitors of metalloproteinases, one would expect net collagenolytic and gelatinolytic activity if the MMPs are activated. Such activation is executed chemically by oxygen radicals of the activated neutrophils (36) . When the recently developed FASC assay with gelatin as substrate is used, net gelatinolytic activity can be measured in crude samples from patients with various arthritic diseases. Whereas no net gelatinolytic activity was found in the synovial fluid of any of the 17 patients with osteoarthritis, 41 of 167 patients with rheumatoid arthritis showed net gelatinolytic activity (varying from 0 to 90,000 FASC U/µl), indicating a significantly higher occurrence of net gelatinolytic activity in rheumatoid arthritis (P = 0.02 using the ² test). Analysis of a small number of available serial samples collected from individual patients at different clinical investigations (time intervals between sampling were from 35 days to 2.3 years) indicated that the net gelatinolytic activity varied greatly (from 0 to 32,000 FASC U/µl) during the evolution of the disease in individuals. This finding may explain why net activity is not present in the synovial fluid of all patients with rheumatoid arthritis (data not shown). These results also demonstrate that net gelatinolytic activity in synovial fluid may be a useful biological marker to allow discrimination of degenerative from autoimmune diseases. Because no gelatinase A (MMP-2) is produced by neutrophils but net gelatinolytic activity is present in neutrophil supernatants and fluids in rheumatoid arthritis, we studied the effect of the gelatinolytic activity on the predominant synovial substrate, denatured collagen II.

Cleavage of collagen type II by neutrophil gelatinase B
Purified gelatinase B, activated with a 1:100 molar ratio of stromelysin-1, was incubated with denatured bovine collagen type II at a ratio of 1:1000. Because stromelysin-1 is also known to degrade gelatin (37) , control experiments were done with a stromelysin-1/gelatin ratio of 1:100,000, to show that under these conditions no cleavage of gelatin occurred by stromelysin-1. The resulting degradation at various time intervals is shown in Fig. 2 . Various inhibition experiments were performed to ensure that the degradation of the collagen was by gelatinase B and not by another contaminating protease (Fig. 3 ). Addition of the metalloprotease inhibitors EDTA or o-phenantroline inhibited the cleavage completely, whereas the thiol and serine protease inhibitors E64 and aprotinin did not influence the reaction. Also, TIMP-1, which is specific for MMPs, inhibited the cleavage.

View larger version (61K): [in this window] [in a new window]   Figure 2. Degradation of bovine collagen type II by human neutrophil gelatinase B. Denatured bovine collagen type II was incubated with stromelysin-1-activated gelatinase B at 37°C. As a control, denatured type II collagen was incubated with stromelysin-1 without gelatinase B. Samples were taken at the indicated time intervals and analyzed by SDS-PAGE and Coomassie blue staining. S indicates the molecular weight standard; -, incubation with stromelysin-1 alone; +, incubation with gelatinase B, activated with stromelysin-1.

View larger version (52K): [in this window] [in a new window]   Figure 3. Inhibition of the clipping of collagen II by neutrophil gelatinase B. Bovine collagen II was incubated with activated gelatinase B in the presence or absence of the protease inhibitors EDTA, o-phenantroline (PHEN), E64, aprotinin, and TIMP-1. S indicates the molecular weight standard; 0, no incubation; -, incubation with stromelysin-1 alone; +, incubation with activated gelatinase B. Respective inhibitors are indicated at the top of each lane.

Determination of the cleavage sites of gelatinase B in collagen type II
Collagen fragments resulting from the cleavage of bovine type II collagen by gelatinase B were separated by RP-HPLC (Fig. 4 ). The different fractions were analyzed by Edman degradation and mass spectrometry (Table 1 ). With the use of Edman degradation, the amino-terminal sequences of the fragments present in the RP-HPLC fractions were determined. This information was combined with the exact mass determinations by mass spectrometry to define the carboxy-terminal end of each fragment. Examples of the latter are illustrated for fractions 31 and 47 in Fig. 5 . At least 24 cleavage sites were identified, as indicated in Fig. 6 . Most important, the previously identified immunodominant remnant epitopes (12 13 14 15) were shown to be left intact by gelatinase B. Such a cleavage pattern indicates that gelatinase B may assist in the generation of the immunodominant peptides. These data thus agree with a functional role of chemokines and proteases (1) in autoimmune arthritis.

View larger version (16K): [in this window] [in a new window]   Figure 4. Separation of the degradation fragments of collagen type II by RP-HPLC. Denatured bovine collagen type II was incubated with activated gelatinase B, and the collagen fragments were separated by RP-HPLC (C8 Aquapore column, Applied Biosystems). Absorbance was measured at 220 nm. The resulting fractions were collected for analysis by Edman degradation to determine the amino-terminal sequence. In addition, all fragments in the different fractions were analyzed by mass spectrometry, and the numbers of post-translational modifications were determined by comparison with the theoretical mass (see Table 1 ). Arrows indicate locations of the immunodominant peptides.

View larger version (24K): [in this window] [in a new window]   Figure 5. Mass spectrometry of collagen II fragments. Each fraction of the RP-HPLC separation of the collagen peptides (see Fig. 4 ) was analyzed by electrospray ion trap mass spectrometry. As examples, two mass spectra are shown. The upper panel gives the mass spectrum of fraction 47. Different charge states of different components were observed. Component A (4280.7 Da; see Table 1 ) corresponds to peptide 801–845 with seven hydroxylations, component B (4264.7 Da) is the same peptide with six hydroxylations, and component C (3341.2 Da) is peptide 483–518 with five hydroxylations. The lower panel gives the mass spectrum of fraction 31. Peptide 357–380 was detected, with four hydroxylations (component A, 2277.9 Da), five hydroxylations (component B, 2262.0 Da), five hydroxylations and one hexose (component C, 2440.5 Da), and five hydroxylations and two hexoses (component D, 2602.5 Da). The peak at 850.6 Da (m/Z) is a contaminant originating from the plastic of the test tubes used.

View larger version (73K): [in this window] [in a new window]   Figure 6. Localization of the cleavage sites of gelatinase B and the post-translational modifications in processed bovine collagen type II. At least 24 different gelatinase B cleavage sites (indicated by ) were determined in collagen type II, as indicated in Table 1 . The cleavage at position 15–16 (indicated by ) remains uncertain, as the amino-terminal fragment (positions 1–15) was not detected by amino-terminal sequencing and by mass spectrometry. The amino-terminal sequence of uncleaved collagen II was blocked for Edman degradation. Mass spectrometry also allowed the determination of the number of hydroxylations and the glycosylations on each fragment, both of which were further corroborated and localized by amino-terminal sequencing. Partial modifications are indicated between brackets, and glycosylation is indicated as hexoses (Hex). Underlined residues indicate sequences determined by Edman degradation; amino acids on a black background indicate positions different in the bovine versus the human sequence. Boxed sequences indicate the immunodominant T-cell epitopes. The protein sequences that were not corroborated by Edman degradation were copied from the data in the gene and protein libraries, when available for bovine collagen II. Otherwise the sequence of human collagen II, which has 98% identity with bovine collagen II, was used, and these residues are indicated in italics. Uncertain (possibly polymorphic) positions in the bovine collagen II sequence are similarly indicated in italics.

Determination of sites of post-translational modification
Type II collagen is modified by hydroxylation of proline and lysine residues. In addition, hydroxylysine can be further modified by O-linked glycosylation [(Glc1–2)Galß1-O-Lys]. Hydroxyproline and hydroxylysine derivatives elute at specific positions during RP-HPLC analysis after Edman degradation, which allowed us to pinpoint the exact localizations of these post-translational modifications in the primary sequence of collagen type II. Glycosylated hydroxylysine derivatives resulted in a blank signal. Furthermore, the mass spectrometry data gave information on the number of hydroxylations and glycosylations on each peptide (Fig. 5) . This type of analysis allowed us to determine the site specificity of the most extensive set of post-translational modifications on bovine collagen so far, as shown in Fig. 6 . As for the function of T-cell reactivity profiles against type II collagen, the immunodominant epitope was found to contain a hydroxylated lysine residue in its center.

Alignment of the cleavage sites of gelatinase B in denatured bovine collagen II
The 24 cleavage sites were aligned in Table 2 . This comparison showed that gelatinase B cleaved collagen II always after a Gly residue (P1 position). At P1^', there is a clear preference for hydrophobic residues, and at P3 for Pro. Only 4% of the amino acids at P2^' were found to be post-translationally modified, whereas 71% of the residues at P5^' showed this modification. These modification results are significantly different (P = 0.00046 and P = 0.0045, respectively, ² test) from the 40% modification on residues before Gly, as was observed in collagen II in general (Fig. 6) .

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
One of the challenges of the postgenome era is the study of the proteome and how it is altered in disease states. A rather clear view of the immunological basis of autoimmune diseases and the molecular events that are implicated in the disease process already exists as the REGA model, in which cytokines, chemokines, and protease activities are key elements (1) . For particular autoimmune diseases, e.g., rheumatoid arthritis, proof of this concept is still missing. In addition, not only the proteome (of the intact proteins) but also the complete knowledge of all the post-translational modifications of the proteins is crucial for understanding T-cell activation and antibody formation and reactivity. Among post-translational modifications, proteolysis and glycosylation are the best known examples. Additional examples include phosphorylation, isoprenylation, glycosylphosphatidylinositol anchoring, and transglutamination.

In this study, we first corroborated the finding of a positive correlation between the major neutrophil chemoattractant IL-8/CXCL-8 and neutrophil cell counts in the synovial fluid of patients with arthritis (20 , 21) . We extended these data with the relationship between neutrophil counts and gelatinase B levels. This analysis indicates that IL-8/CXCL-8, produced, for example, by fibroblasts and chondrocytes in the synovium (38) , attracts neutrophils and induces the release of gelatinase B from their granules in vivo, as was previously shown in vitro (25) . Gelatinase B is important for transendothelial migration of neutrophils into tissues (39) and potentiates the activities of IL-8/CXCL-8 (32) , thus contributing to a stronger inflammatory reaction. In each rheumatoid synovial fluid sample, a high level of gelatinase B was present as measured by zymography. Zymography measures not only activated gelatinase B levels, but also progelatinase B and gelatinase B in a noncovalent complex with its inhibitor TIMP-1, because SDS activates the proenzyme and dissociates the TIMP-gelatinase complexes. Therefore, we showed further here that, in the rheumatoid joint, not only were gelatinase B levels elevated, but also net gelatinase activity was present in 24% of the analyzed patients (n = 167). This incidence was significantly higher (P<0.02) than that for patients with osteoarthritis, in whose synovial fluids no net gelatinase activity was detected. This finding is consistent with the fact that rheumatoid arthritis is a prototypic joint autoimmune disease, whereas osteoarthritis is instead a degenerative (metabolic) disease (40) . The analysis of net gelatinase activity thus provides a useful tool for discriminating between the two types of arthritic diseases.

Neutrophils produce a specific collagenase (MMP-8) and gelatinase B (MMP-9), but no gelatinase A (MMP-2) or TIMPs (35) . When the neutrophils are activated, for example, by IL-8/CXCL-8, they can chemically activate MMPs (36) by the cysteine switch mechanism (26 , 27) . Collagenases cleave collagens at a single site and generate the typical one-third and two-thirds fragments, leading to further unwinding of the triple helix. This result allows additional cleavage of the collagen chains by gelatinase B (41) . Therefore, it was relevant to analyze how neutrophil gelatinase B cleaves denatured collagen II.

For this study, we took advantage of the electrophoretic purity of both natural collagen II (33 , 34) and neutrophil gelatinase B (32) , as well as in-depth knowledge of the immunodominant epitopes of collagen II (12 13 14 15) . From the technical point of view, we used a combination of gelatinase B digests, fragment separation by RP-HPLC, mass spectrometry on an ion trap apparatus, and Edman degradation. These methods allowed detailed study of the cleavage sites of gelatinase B and the elucidation of the post-translational modifications by hydroxylation of prolines and lysines and by attachment of mono- and disaccharides in collagen type II. In total, 24 different cleavage sites were identified, and the remnant epitopes generated by gelatinase B were determined. The two immunodominant epitopes of collagen II were left intact by gelatinase B cleavage, and the positions of the cleavage sites indicate that gelatinase B may be at least in part responsible for the generation of the (previously identified) immunodominant peptides. This indicates that the REGA model is applicable to rheumatoid arthritis and that gelatinase B is a key target in this autoimmune disease. Gelatinase B is obviously not a crucial enzyme in osteoarthritis, in which no net gelatinase activity was detected. The peptides generated by gelatinase B cleavage in rheumatoid arthritis are possibly loaded directly onto MHC II on the surface of antigen-presenting cells, or perhaps further processing can take place outside the cell by secreted proteases or inside the cell after internalization (4 , 42) . The role of neutrophil gelatinase B in the physiopathology of rheumatoid arthritis is shown schematically in Fig. 7 . These data imply that patients with rheumatoid arthritis may benefit from treatment with specific gelatinase B inhibition, but osteoarthritis patients presumably would not. The known beneficial effect of the inhibition of tumor necrosis factor (TNF-) by a neutralizing antibody may be in part mediated by a reduction of the levels of IL-8/CXCL-8 and of the recruitment of neutrophils in the synovium (43) . However, TNF- treatment also has side effects (44) , which may be circumvented by more specific inhibition of the final effector molecules such as gelatinase B.

View larger version (33K): [in this window] [in a new window]   Figure 7. The REGA model in rheumatoid arthritis. A trigger (e.g., a primary cytokine in rheumatoid arthritis or a bacterial product in septic arthritis) induces the production of (other) cytokines and the chemokine IL-8/CXCL-8 by chondrocytes in the joint (38) . IL-8/CXCL-8 induces influx and activation of neutrophils. These cells degranulate MMP-8 and MMP-9 without inhibitor. This results in net proteolytic activity and degradation of collagen II into immunodominant remnant epitopes. Because IL-8/CXCL-8 is also a lymphocyte chemoattractant (48) , T cells are randomly attracted and eventually encounter the remnant epitope or its derivatives in the context of MHC II. These T cells are activated and produce interferon and IL-2. Interferon activates macrophages to release IL-1, TNF-, and IL-8/CXCL-8, and these primary cytokines and chemokines keep the process going as a vicious circle (1) . The circle is further reinforced by the observation that active MMP-9 potentiates IL-8/CXCL-8 10-fold by amino-terminal truncation (32) . SF, synovial fluid; C, cartilage.

A large number of post-translational modifications were identified and their positions determined in collagen II. It is interesting that the peptide starting at position 273 was shown to be hydroxylated on lysine 283, which is part of an immunodominant epitope (12) . This indicates that peptide and alanine scanning experiments for T-cell activation, with the use of synthetic unmodified peptides, should be interpreted with caution. Therefore, we favor the idea of studying antigen presentation in MHC and T-cell activation with natural rather than synthetic peptides. This notion is further reinforced by other studies in which post-translational modifications, such as glycosylation, have been shown to play an important role in the processes underlying autoimmunity (17 , 45) . This result was also found for glycosylation of collagen (16) . In addition, here we aligned and compared the cleavage sites in collagen II (Table 2) . Gelatinase B clippings in denatured collagen II occurred after Gly (the P1 position), in accordance with the earlier documented finding that gelatinase B has a clear preference for small amino acids at P1 (46) . At P1^', hydrophobic residues were preferred, and this confirms previous results (46) . At P3, there was a clear preference for Pro. In collagen II, 40% of the residues before a Gly were post-translationally modified [hydroxyproline or (glycosylated) hydroxylysine]. It was striking to note, however, that at position P2^', which is in front of a Gly, these modifications were not well tolerated (only 4%), in contrast to position P5^', where there was a clear preference for these modifications (71%). Gelatinase A has also a preference for hydroxyproline at P5^' (47) . These results may be instrumental in the design and development of specific peptidomimetics with inhibitory activity against gelatinase B.

Net gelatinase B activity, elevated in the joints of patients with autoimmune rheumatoid arthritis (in contrast with osteoarthritis), helps to release the immunodominant epitopes of type II collagen and may thus contribute to the pathogenesis. Therefore, selective inhibition of gelatinase B should be considered as a potential therapy in rheumatoid arthritis. The largest set of post-translational modifications on bovine type II collagen was identified, and it was shown that such modifications may affect the cleavages by gelatinase B. Together with the fact that these modifications influence the recognition by T lymphocytes, our data also emphasize the role of post-translational modifications in the pathogenesis or evolution of autoimmunity.

	ACKNOWLEDGMENTS

 
The authors thank Ilse Van Aelst and Els Koyen for their contributions with zymography and FASC analysis. P. E. V. D. S. is a research assistant and P. P. is a postdoctoral fellow of the Fund for Scientific Research, Belgium (F.W.O.-Vlaanderen). Financial support of the Charcot Foundation, the Fortis Insurances AB, the Geconcerteerde OnderzoeksActies 2001–2006, and the F.W.O.-Vlaanderen is gratefully acknowledged.

Received for publication September 5, 2001. Revision received November 30, 2001.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Opdenakker, G., Van Damme, J. (1994) Cytokine-regulated proteases in autoimmune diseases. Immunol. Today 15,103-107[CrossRef][Medline]
2. Sun, J. B., Olsson, T., Wang, W. Z., Xiao, B. G., Kostulas, V., Fredrikson, S., Ekre, H. P., Link, H. (1991) Autoreactive T and B cells responding to myelin proteolipid protein in multiple sclerosis and controls. Eur. J. Immunol. 21,1461-1468[Medline]
3. Inaba, K., Turley, S., Iyoda, T., Yamaide, F., Shimoyama, S., Reis e Sousa, C., Germain, R. N., Mellman, I., Steinman, R. M. (2000) The formation of immunogenic major histocompatibility complex class II-peptide ligands in lysosomal compartments of dendritic cells is regulated by inflammatory stimuli. J. Exp. Med. 191,927-936[Abstract/Free Full Text]
4. Santambrogio, L., Sato, A. K., Carven, G. J., Belyanskaya, S. L., Strominger, J. L., Stern, L. J. (1999) Extracellular antigen processing and presentation by immature dendritic cells. Proc. Natl. Acad. Sci. USA 96,15056-15061[Abstract/Free Full Text]
5. Arndt, S. O., Vogt, A. B., Markovic-Plese, S., Martin, R., Moldenhauer, G., Wolpl, A., Sun, Y., Schadendorf, D., Hämmerling, G. J., Kropshofer, H. (2000) Functional HLA-DM on the surface of B cells and immature dendritic cells. EMBO J 19,1241-1251[CrossRef][Medline]
6. Bartholomé, E. J., Van Aelst, I., Koyen, E., Kiss, R., Willems, F., Goldman, M., Opdenakker, G. (2001) Human monocyte-derived dendritic cells produce bioactive gelatinase B: inhibition by interferon-ß. J. Interferon Cytokine Res. 21,495-501[CrossRef][Medline]
7. Fung-Leung, W. P., Surh, C. D., Liljedahl, M., Pang, J., Leturcq, D., Peterson, P. A., Webb, S. R., Karlsson, L. (1996) Antigen presentation and T cell development in H2-M-deficient mice. Science 271,1278-1281[Abstract]
8. Miyazaki, T., Wolf, P., Tourne, S., Waltzinger, C., Dierich, A., Barois, N., Ploegh, H., Benoist, C., Mathis, D. (1996) Mice lacking H2-M complexes, enigmatic elements of the MHC class II peptide-loading pathway. Cell 84,531-541[CrossRef][Medline]
9. Opdenakker, G., Masure, S., Grillet, B., Van Damme, J. (1991) Cytokine-mediated regulation of human leukocyte gelatinases and role in arthritis. Lymphokine Cytokine Res 10,317-324[Medline]
10. Sopata, I., Wize, J., Filipowicz-Sosnowska, A., Stanislawska-Biernat, E., Brzezinska, B., Maslinski, S. (1995) Neutrophil gelatinase levels in plasma and synovial fluid of patients with rheumatic diseases. Rheumatol. Int. 15,9-14[CrossRef][Medline]
11. Ahrens, D., Koch, A. E., Pope, R. M., Stein-Picarella, M., Niedbala, M. J. (1996) Expression of matrix metalloproteinase 9 (96-kd gelatinase B) in human rheumatoid arthritis. Arthritis Rheum 39,1576-1587[Medline]
12. Brand, D. D., Myers, L. K., Terato, K., Whittington, K. B., Stuart, J. M., Kang, A. H., Rosloniec, E. F. (1994) Characterization of the T cell determinants in the induction of autoimmune arthritis by bovine 1(II)-CB11 in H-2q mice. J. Immunol. 152,3088-3097[Abstract]
13. Myers, L. K., Miyahara, H., Terato, K., Seyer, J. M., Stuart, J. M., Kang, A. H. (1995) Collagen-induced arthritis in B10.RIII mice (H-2r): identification of an arthritogenic T-cell determinant. Immunology 84,509-513[Medline]
14. Rosloniec, E. F., Whittington, K. B., Brand, D. D., Myers, L. K., Stuart, J. M. (1996) Identification of MHC class II and TCR binding residues in the type II collagen immunodominant determinant mediating collagen-induced arthritis. Cell. Immunol. 172,21-28[CrossRef][Medline]
15. Andersson, E. C., Hansen, B. E., Jacobsen, H., Madsen, L. S., Andersen, C. B., Engberg, J., Rothbard, J. B., McDevitt, G. S., Malmstrom, V., Holmdahl, R., Svejgaard, A., Fugger, L. (1998) Definition of MHC and T cell receptor contacts in the HLA-DR4 restricted immunodominant epitope in type II collagen and characterization of collagen-induced arthritis in HLA-DR4 and human CD4 transgenic mice. Proc. Natl. Acad. Sci. USA 95,7574-7579[Abstract/Free Full Text]
16. Michaëlsson, E., Malmstrom, V., Reis, S., Engstrom, A., Burkhardt, H., Holmdahl, R. (1994) T cell recognition of carbohydrates on type II collagen. J. Exp. Med. 180,745-749[Abstract/Free Full Text]
17. Haurum, J. S., Arsequell, G., Lellouch, A. C., Wong, S. Y., Dwek, R. A., McMichael, A. J., Elliott, T. (1994) Recognition of carbohydrate by major histocompatibility complex class I-restricted, glycopeptide-specific cytotoxic T lymphocytes. J. Exp. Med. 180,739-744[Abstract/Free Full Text]
18. Ishioka, G. Y., Lamont, A. G., Thomson, D., Bulbow, N., Gaeta, F. C., Sette, A., Grey, H. M. (1992) MHC interaction and T cell recognition of carbohydrates and glycopeptides. J. Immunol. 148,2446-2451[Abstract]
19. Shikhman, A. R., Cunningham, M. W. (1994) Immunological mimicry between N-acetyl-beta-D-glucosamine and cytokeratin peptides. Evidence for a microbially driven anti-keratin antibody response. J. Immunol. 152,4375-4387[Abstract]
20. Peichl, P., Ceska, M., Effenberger, F., Haberhauer, G., Broell, H., Lindley, I. J. (1991) Presence of NAP-1/IL-8 in synovial fluids indicates a possible pathogenic role in rheumatoid arthritis. Scand. J. Immunol. 34,333-339[CrossRef][Medline]
21. Rampart, M., Herman, A. G., Grillet, B., Opdenakker, G., Van Damme, J. (1992) Development and application of a radioimmunoassay for interleukin-8: detection of interleukin-8 in synovial fluids from patients with inflammatory joint disease. Lab. Invest. 66,512-518[Medline]
22. Seitz, M., Dewald, B., Ceska, M., Gerber, N., Baggiolini, M. (1992) Interleukin-8 in inflammatory rheumatic diseases: synovial fluid levels, relation to rheumatoid factors, production by mononuclear cells, and effects of gold sodium thiomalate and methotrexate. Rheumatol. Int. 12,159-164[CrossRef][Medline]
23. Akahoshi, T., Endo, H., Kondo, H., Kashiwazaki, S., Kasahara, T., Mukaida, N., Harada, A., Matsushima, K. (1994) Essential involvement of interleukin-8 in neutrophil recruitment in rabbits with acute experimental arthritis induced by lipopolysaccharide and interleukin-1. Lymphokine Cytokine Res 13,113-116[Medline]
24. Gruber, B. L., Sorbi, D., French, D. L., Marchese, M. J., Nuovo, G. J., Kew, R. R., Arbeit, L. A. (1996) Markedly elevated serum MMP-9 (gelatinase B) levels in rheumatoid arthritis: a potentially useful laboratory marker. Clin. Immunol. Immunopathol. 78,161-171[CrossRef][Medline]
25. Masure, S., Proost, P., Van Damme, J., Opdenakker, G. (1991) Purification and identification of 91-kDa neutrophil gelatinase. Release by the activating peptide interleukin-8. Eur. J. Biochem. 198,391-398[Medline]
26. Van Wart, H. E., Birkedal-Hansen, H. (1990) The cysteine switch: a principle of regulation of metalloproteinase activity with potential applicability to the entire matrix metalloproteinase gene family. Proc. Natl. Acad. Sci. USA 87,5578-5582[Abstract/Free Full Text]
27. Kleifeld, O., Van den Steen, P. E., Frenkel, A., Cheng, F., Jiang, H. L., Opdenakker, G., Sagi, I. (2000) Structural characterization of the catalytic active site in the latent and active natural gelatinase B from human neutrophils. J. Biol. Chem. 275,34335-34343[Abstract/Free Full Text]
28. Murphy, G., Willenbrock, F. (1995) Tissue inhibitors of matrix metalloendopeptidases. Methods Enzymol 248,496-510[Medline]
29. Arnett, F. C., Edworthy, S. M., Bloch, D. A., McShane, D. J., Fries, J. F., Cooper, N. S., Healey, L. A., Kaplan, S. R., Liang, M. H., Luthra, H. S. (1988) The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 31,315-324[Medline]
30. Masure, S., Billiau, A., Van Damme, J., Opdenakker, G. (1990) Human hepatoma cells produce an 85 kDa gelatinase regulated by phorbol 12-myristate 13-acetate. Biochim. Biophys. Acta 1054,317-325[Medline]
31. St-Pierre, Y., Desrosiers, M., Tremblay, P., Estève, P.-O., Opdenakker, G. (1996) Flow cytometric analysis of gelatinase B (MMP-9) activity using immobilized fluorescent substrate on microspheres. Cytometry 25,374-380[CrossRef][Medline]
32. Van den Steen, P. E., Proost, P., Wuyts, A., Van Damme, J., Opdenakker, G. (2000) Neutrophil gelatinase B potentiates interleukin-8 tenfold by aminoterminal processing, whereas it degrades CTAP-III, PF-4, and GRO-alpha and leaves RANTES and MCP-2 intact. Blood 96,2673-2681[Abstract/Free Full Text]
33. Miller, E. J. (1972) Structural studies on cartilage collagen employing limited cleavage and solubilization with pepsin. Biochemistry 11,4903-4909[CrossRef][Medline]
34. Rosloniec, E. F., Kang, A. H., Myers, L. K., Cremer, M. A. (1997) Collagen-induced Arthritis. Coligan, J. J. eds. Current Protocols in Immunology ,15.5.5-24 Wiley & Sons New York.
35. Opdenakker, G., Van den Steen, P. E., Dubois, B., Nelissen, I., Van Coillie, E., Masure, S., Proost, P., Van Damme, J. (2001) Gelatinase B functions as regulator and effector in leukocyte biology. J. Leukoc. Biol. 69,851-859[Abstract/Free Full Text]
36. Peppin, G. J., Weiss, S. J. (1986) Activation of the endogenous metalloproteinase, gelatinase, by triggered human neutrophils. Proc. Natl. Acad. Sci. USA 83,4322-4326[Abstract/Free Full Text]
37. Chin, J. R., Murphy, G., Werb, Z. (1985) Stromelysin, a connective tissue-degrading metalloendopeptidase secreted by stimulated rabbit synovial fibroblasts in parallel with collagenase. Biosynthesis, isolation, characterization, and substrates. J. Biol. Chem. 260,12367-12376[Abstract/Free Full Text]
38. Van Damme, J., Bunning, R. A., Conings, R., Graham, R., Russell, G., Opdenakker, G. (1990) Characterization of granulocyte chemotactic activity from human cytokine-stimulated chondrocytes as interleukin-8. Cytokine 2,106-111[CrossRef][Medline]
39. D’Haese, A., Wuyts, A., Dillen, C., Dubois, B., Billiau, A., Heremans, H., Van Damme, J., Arnold, B., Opdenakker, G. (2000) In vivo neutrophil recruitment by granulocyte chemotactic protein-2 is assisted by gelatinase B/MMP-9 in the mouse. J. Interferon Cytokine Res. 20,667-674[CrossRef][Medline]
40. Clague, R. B., Morgan, K., Collins, I., Pattrick, M., Doherty, M. (1991) Absence of autoimmunity to type II collagen in generalised nodal osteoarthritis. Ann. Rheum. Dis. 50,769-771[Abstract/Free Full Text]
41. Goldberg, G. I., Strongin, A., Collier, I. E., Genrich, L. T., Marmer, B. L. (1992) Interaction of 92-kDa type IV collagenase with the tissue inhibitor of metalloproteinases prevents dimerization, complex formation with interstitial collagenase, and activation of the proenzyme with stromelysin. J. Biol. Chem. 267,4583-4591[Abstract/Free Full Text]
42. Vergelli, M., Pinet, V., Vogt, A. B., Kalbus, M., Malnati, M., Riccio, P., Long, E. O., Martin, R. (1997) HLA-DR-restricted presentation of purified myelin basic protein is independent of intracellular processing. Eur. J. Immunol. 27,941-951[Medline]
43. Taylor, P. C., Peters, A. M., Paleolog, E., Chapman, P. T., Elliott, M. J., McCloskey, R., Feldmann, M., Maini, R. N. (2000) Reduction of chemokine levels and leukocyte traffic to joints by tumor necrosis factor- blockade in patients with rheumatoid arthritis. Arthritis Rheum 43,38-47[CrossRef][Medline]
44. Nunez, M. O., Ripoll, N. C., Carneros, M. J., Gonzalez, L. V., Gregorio, M. H. (2001) Reactivation tuberculosis in a patient with anti-TNF- treatment. Am. J. Gastroenterol. 96,1665-1666[Medline]
45. Rudd, P. M., Elliott, T., Cresswell, P., Wilson, I. A., Dwek, R. A. (2001) Glycosylation and the immune system. Science 291,2370-2376[Abstract/Free Full Text]
46. Netzel-Arnett, S., Sang, Q. X., Moore, W. G., Navre, M., Birkedal-Hansen, H., Van Wart, H. E. (1993) Comparative sequence specificities of human 72- and 92-kDa gelatinases (type IV collagenases) and PUMP (matrilysin). Biochemistry 32,6427-6432[CrossRef][Medline]
47. Seltzer, J. L., Akers, K. T., Weingarten, H., Grant, G. A., McCourt, D. W., Eisen, A. Z. (1990) Cleavage specificity of human skin type IV collagenase (gelatinase). Identification of cleavage sites in type I gelatin, with confirmation using synthetic peptides. J. Biol. Chem. 265,20409-20413[Abstract/Free Full Text]
48. Larsen, C. G., Anderson, A. O., Appella, E., Oppenheim, J. J., Matsushima, K. (1989) The neutrophil-activating protein (NAP-1) is also chemotactic for T lymphocytes. Science 243,1464-1466[Abstract/Free Full Text]

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