Cellular immune response to human cartilage glycoprotein-39 (HC gp-39)-derived peptides in rheumatoid arthritis and other inflammatory conditions

K. Vos1, A. M. M. Miltenburg4, K. E. van Meijgaarden2, M. van den Heuvel4, D. G. Elferink2, P. J. M. van Galen4, R. A. van Hogezand3, E. van Vliet-Daskalopoulou4, T. H. M. Ottenhoff2, F. C. Breedveld1, A. M. H. Boots4 and R. R. P. de Vries2

1 Departments of Rheumatology,
2 Immunohaematology and Blood Bank and
3 Hepato-Gastroenterology, Leiden University Medical Center, Leiden and
4 Department of Pharmacology, Section Immunology, NV Organon, Oss, The Netherlands


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objective. To study the specificity of the peripheral blood mononuclear cell (PBMC) response to peptides derived from human cartilage glycoprotein-39 (HC gp-39) in patients with rheumatoid arthritis (RA) and the correlation between this response and disease activity.

Methods. RA patients, patients with systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD) or osteoarthritis (OA) and healthy controls were studied. All individuals were typed for HLA-DRB1 and their disease activity score was documented. Proliferation of PBMC was measured following incubation with five different HC gp-39-derived peptides, selected by the use of a DR4 (DRB1*0401) binding motif.

Results. A proliferative response to one of the five peptides (peptide 259–271 at 10 µg/ml) was more often observed in RA patients than in healthy controls (P=0.001). RA patients who expressed DRB1*0401 more often showed a response against this peptide than RA patients who did not express this RA-associated haplotype. This response was not RA-specific since patients with IBD or OA also showed a response significantly more frequently than healthy controls (P=0.02 and P=0.03 respectively). However, the level of the response against peptide 259–271 correlated with disease activity in RA patients but not in patients with IBD or SLE. Increased responses to HC gp-39 263-275 were found in patients with IBD or OA; a trend towards such a response failed to reach significance in RA patients in this study.

Conclusion.  In RA patients as well as in patients with other inflammatory conditions, HC gp-39-derived peptides may be targets of the T-cell-mediated immune response. In the RA patient group the immune response to HC gp-39-derived peptide 259–271 correlated with disease activity.

KEY WORDS: Rheumatoid arthritis, YKL-40, Human cartilage glycoprotein-39, Cellular response, T cells, Autoantigen.


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Rheumatoid arthritis (RA) is an autoimmune disease in which autoreactive T cells are considered to play an important role. Despite extensive research, the autoantigens involved in the initiation or perpetuation of the disease have not been defined. Several antigens of potential interest have been investigated, particularly type II collagen (CII), cartilage proteoglycans, heat shock proteins, and synovial fluid (SF)-derived antigens such as p68 and p205 [110]. Antigen-specific T cells have been isolated from RA patients and immunization with several of these antigens can induce arthritis in rodents. However, except for the SF-derived antigens p68 and p205, T-cell responses to these antigens also occurred in healthy individuals and differences between T-cell responses in healthy individuals and RA patients were quite small.

We have studied immunological responses to the human cartilage-derived antigen HC gp-39, which is identical to YKL-40 [1114]. Interest in HC gp-39 was stimulated by the finding that HC gp-39 messenger RNA is expressed in cartilage obtained from RA patients, whereas healthy adult cartilage does not contain this transcript. Furthermore, HC gp-39-derived peptides that are predicted to bind to DRB1*0401 with the aid of a DRB1*0401 peptide-binding motif may be selectively recognized by peripheral blood mononuclear cells (PBMC) from RA patients [13, 15]. Immunization of BALB/c mice with HC gp-39 protein resulted in inflammatory polyarthritis, suggesting a role for HC gp-39 in joint pathology; this arthritis could be delayed and suppressed by intranasal administration of HC gp-39 prior to immunization [13, 16].

The present study had two objectives: first, to define HC gp-39 responses in a large cohort of RA patients and to compare this reactivity with that found in healthy controls, and secondly, to analyse the disease specificity of this response. To this end, we examined the proliferative response of PBMC against selected HC gp-39 peptides in RA patients, patients with OA, patients with systemic lupus erythematosus (SLE), patients with inflammatory bowel disease (IBD; Crohn's disease and ulcerative colitis) and in healthy controls. The level of the PBMC response was compared with disease activity in patients with RA, SLE and IBD.


    Patients and methods
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients
Patients with RA (n=50), OA (n=51), SLE (n=24) or IBD (n=26) and healthy controls (n=49) participated in the study. The patients were visiting the outpatients clinic of the departments of rheumatology and hepatogastroenterology of Leiden University Medical Center. The healthy controls consisted of blood donors visiting the Leidschenhage Blood Bank. The RA patients (17 men, 33 women) fulfilled the American College of Rheumatology criteria [17]. Disease duration varied from 1 to 56 yr. Patients receiving more than 20 mg prednisone, cyclosporin or any experimental treatment were excluded. Disease activity was measured by assessment of 28 joints for tenderness and swelling, assessment of disease activity on a Visual Analogue Scale (VAS) by the patient and the physician, and by measuring the erythrocyte sedimentation rate (ESR, Westergren method). Extra-articular manifestations were documented and the serum IgM rheumatoid factor concentration was measured by ELISA. A modified disease activity score was calculated as follows: 0.53938 times {surd}(number of tender joints)+0.06465 times number of swollen joints+0.330 times ln ESR+0.224 [18, 19]. The OA group included only patients with primary OA. We noted whether the OA was localized (two or fewer joint areas) or generalized (three or more joint areas) (joint areas were hands and feet, knees, hips and spine). The SLE patients fulfilled four or more of the 11 criteria for the classification of SLE. Disease activity was measured by assessment of the SLE disease activity index (SLEDAI) [20]. The patient group with IBD consisted of patients with active ulcerative colitis or Crohn's disease. All patients with ulcerative colitis had rectal bleeding and diarrhoea, whereas the patients with Crohn's disease had to have a disease activity index (CDAI) above 150 in order to be included in the study [21].

HLA typing
DNA was isolated from blood lymphocytes of all patients and controls. Generic DRB1 typing was performed with a polymerase chain reaction and a biotin-labelled sequence-specific oligonucleotide (SSO) method as described previously [22]. DRB1*04 subtyping was performed by group-specific amplification of DNA from all DRB1*04-positive individuals and hybridization with relevant SSOs [23].

HC gp-39-derived peptides
The peptides derived from HC gp-39 were selected on the basis of a published DRB1*0401 motif and on the basis of binding to DRB1*0401 [13]. The following peptides were tested: HC gp-39 75–87, 103–116, 259–271, 263–275 and 326–338. All peptides were tested at concentrations of 10 and 50 µg/ml.

T-cell proliferation to HC gp-39-derived peptides
Peripheral blood samples were drawn, after the patients had given informed consent, between 9 a.m. and 1 p.m. PBMC were isolated from heparinized venous peripheral blood by standard centrifugation on a Ficoll–Paque gradient.

Five HC gp-39 derived peptides were dissolved in medium (Dulbecco's modified Eagle's minimal essential medium (DMEM)/Ham's-F12; Gibco BRL, Paisley, UK) supplemented with L-glutamine,2-mercaptoethanol and antibiotics. The medium was supplemented with 10% heat-inactivated autologous plasma. Cells were cultured in three to five wells (1.5x105 cells/well) in flat-bottomed microtitre plates (Nunclon, NUNC, Life Technologies, Breda, The Netherlands). Cultures were incubated in a total volume of 200 µl for 7 days (37°C, 5% CO2) and were pulsed during the last 18 h with 3[H]thymidine (3[H]TdR) (Amersham, Den Bosch, The Netherlands; 18.5 kBq/well, specific activity 5 Ci/mmol). Cells were harvested on glass fibre filters using a cell harvester (Skatron Instruments, Lier, Norway). 3[H]TdR incorporation was measured by liquid scintillation counting (beta plate counter). Results were expressed as counts per minute (c.p.m.).

The PBMC proliferation test was considered positive if the geometric mean c.p.m. was above 1000, the stimulation index (geometric mean of antigen-specific counts divided by the geometric mean of background) was >=2 and when the geometric mean c.p.m. of the tested peptide minus the geometric mean c.p.m. of the background was >500. For a positive result, the median c.p.m. of the tested peptide had to be significantly higher than the median c.p.m. of the background (one-sided Wilcoxon test, P=0.05).

Statistical analysis
All tests comparing frequencies of responses between patients or (sub)groups were performed using Fisher's exact test (two-sided, {alpha}=0.05). When comparing stimulation indices between patient groups and controls, the Wilcoxon two-sample test was used. The correlation coefficients were calculated and tested using Spearman's rank order correlation test (two-sided, {alpha}=0.05).


    Results
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The demographic and clinical characteristics of the different patient groups and healthy controls are summarized in Tables 1Go and 2Go. Almost all RA patients had erosive disease and had a long disease duration (median 7.5 yr). The RA patient group was a cross-section of the RA population visiting the outpatient clinic of a tertiary referral centre.


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TABLE 1. Clinical and demographic characteristics of the individuals investigated

 

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TABLE 2. Disease characteristics of RA patients (n=50)

 
The percentages of individuals who showed a positive PBMC response to the various HC gp-39 peptides are shown in Table 3Go. On the basis of dose–response curves using concentrations of peptides from 1 to 100 µg/ml (data not shown), two peptide concentrations (10 and 50 µg/ml) were chosen for this study. The other conditions (time of incubation, the type of microtitre plates, etc) were shown to be optimal for the assay. Only the concentration of peptide in which there was the best discrimination between patients and controls is shown. In all cases this was 10 µg/ml, except for peptide 263–275, to which at a concentration of 10 µg/ml only four individuals responded (one RA patient and three OA patients). For this peptide the results of the proliferation in response to 50 µg/ml are shown.


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TABLE 3. Percentages of patients with proliferation of PBMC upon incubation with HC gp-39 peptides

 
For two peptides there was a significant difference in the frequency of positive responses between the control group and certain patient groups. For peptide 263–275, when tested at 50 µg/ml, there were higher percentages of responders in the RA, OA and IBD patient groups (8.3, 18.4 and 21.7% respectively) compared with the controls (0%). When comparing the percentage of individuals showing a positive response and the stimulation indices of the three patient groups with those of the controls, this difference was statistically significant only for the OA and IBD patient groups.

HC gp-39 peptide 259–271, tested at 10 µg/ml, induced a T-cell response which was significantly higher in the RA patient group than in the control group (positive response in 36.0 and 8.2% respectively; P=0.001). This difference was also significant when comparing the stimulation indices of the response to this peptide (mean for controls 1.38, mean for RA patients 2.74; P=0.02). Noticeably, DRB1*0401-positive RA patients (53.3%) showed a positive T-cell response against this peptide more frequently than the DRB1*0401-negative RA patients (28.6%). This DRB1*0401 effect was highly significant (P=0.001) in the combined data of all patients and controls (Table 4Go). Compared with the control group, the percentage of individuals showing a T-cell response against HC gp-39 (259–271) was also significantly higher in the OA (18.4%) and IBD (21.7%) patient groups (P=0.03 and P=0.02 respectively). The mean stimulation index found for this peptide at 10 µg/ml varied from 1.03 for the SLE patients to 3.57 for the IBD patients. In the RA patients who showed a positive T-cell response to peptide 259–271 (10 µg/ml), there was a significant correlation between the degree of this response and parameters of disease activity (tender joint count, the swollen joint count and the modified disease activity score). No correlation was found with the presence of nodules, the presence of rheumatoid factor or ESR (Table 5Go).


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TABLE 4. Number and percentage of DRB1*0401-positive and DRB1*0401-negative subjects with a positive PBMC response to HC gp-39-derived peptide 259–271

 

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TABLE 5. Correlation between stimulation index to peptide 259–271 and parameters of disease activity in the 18 RA responder patients

 
In the SLE and IBD patients, no correlation was found between the disease activity score and the T-cell responses against the different peptides.


    Discussion
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Although the physiological function of HC gp-39 is still unknown, there is increasing evidence that this protein or its peptide derivatives could play a role in the pathogenesis of RA. HC gp-39 mRNA has been detected in inflamed synovium and in cartilage of RA patients and serum levels of HC gp-39 were found to be elevated in RA patients compared with healthy controls. In a previous study, proliferative responses of PBMC against HC gp-39 peptides were detected in RA patients whereas little reactivity was found in healthy controls [13, 15]. Moreover, immunization with HC gp-39 induced a chronic relapsing arthritis in BALB/c mice which could be suppressed by intranasal administration of the antigen prior to such sensitization [13, 16].

The main goal of the present study was to study T-cell reactivity to these DRB1*0401-binding, HC gp-39-derived peptides in a larger group of RA patients, in other patients with chronic inflammatory disorders with and without joint involvement, and in healthy controls. Furthermore, the relation between T-cell reactivity and disease activity was studied. It was confirmed that in RA patients HC gp-39-derived peptides are a target of the cellular immune response, particularly in patients who are DRB1*0401-positive. The difference in T-cell response to HC gp-39-derived peptides in DRB1*0401-positive and -negative RA patients is in line with the differences in T-cell response to HC gp-39-derived peptides in DR*0401 and DR*0402 transgenic mice [15].

In the RA patient group only peptide 259–271 induced a PBMC response which was significantly higher than that in the healthy controls. Interestingly, in the responders against this peptide, there was a correlation between the degree of the response and disease activity. In the study of Verheijden et al. [13], proliferation of PBMC in response to peptide 259–271 was also higher in RA patients compared with controls, but this difference failed to reach significance. In that study a significantly greater degree of proliferation was found in RA patients compared with the control group for the partially overlapping peptide 263–275. In the present study this peptide was recognized significantly more often by patients with OA and IBD than by controls. The PBMC response to peptide 263–275 in RA patients occurred more frequently than in healthy controls, but the difference failed to reach significance. It seems that these two overlapping peptides can be targets for T-cell populations in chronic inflammatory conditions such as RA. The discrepancies in the results of the two studies may be due to the low number of individuals tested in the former study, to differences in the concentrations of peptides tested, to differences in patient selection criteria and/or variation in the medication used by the patients. The response to HC gp-39-derived peptides is not specific for RA, since increased responses of PBMC against HC gp-39-derived peptides were also found in OA and IBD. This observation does not exclude a pathogenic role of T-cell reactivity to HC gp-39 peptides in RA. It has been shown that HC gp-39 is not chondrocyte-specific and that levels of HC gp-39 are elevated in patient groups without involvement of joints, such as patients with alcoholic cirrhosis, recurrent breast cancer, colorectal cancer and diabetes and patients with IBD, who were selected for not having arthritis [11, 2427]. The fact that in the RA patients a correlation was found between the PBMC response to one of the peptides and disease activity suggests a possible role for the glycoprotein in the pathogenesis of the disease. The immune response to the glycoprotein can play a role in sustaining chronic inflammation. Thus, the protein might be used for tolerance induction in a somewhat broader sense, perhaps by exploiting the phenomenon of bystander suppression [28]. In this respect the data demonstrating suppression of HC gp-39-induced arthritis in BALB/c mice by intranasal administration of HC gp-39 are promising [13, 16].

In RA, few studies have been conducted on the correlation of antigen-specific T-cell reactivity and disease activity. In a study in which T-cell proliferation in response to chondrocyte and fibroblast membranes was tested and various parameters of RA activity were analysed, a correlation was found only between the ESR and the T-cell response to chondrocyte membranes [29]. In the present study there was a correlation between the T-cell response to peptide 259–271 and RA disease activity measured by the tender joint count, swollen joint count and the modified disease activity score.

In the SLE patients, the T-cell response to the various HC gp-39-derived peptides was low. Hyporesponsiveness of T cells in SLE has been described before [30]. The lymphopenia which is often present in SLE patients and/or the use corticosteroids could also be responsible for the observed low T-cell response.

Many studies have been conducted on the proliferative PBMC response to autoantigens in autoimmune disorders. Candidate autoantigens in RA include stress proteins, collagen type II (CII), aggrecan and the SF-derived autoantigens p68 and p205 [110, 31, 32.

In summary, except for the SF-derived autoantigen p68 and the recently described p205, cellular responses to collagen, heat shock protein and proteoglycan, measured with a proliferation assay, appear to occur in a relatively small proportion of RA patients and can also be found in many other arthritic and non-arthritic diseases.

In the present study we found a significant difference between the PBMC response to the well-defined autoantigen HC gp-39 peptide 259–271 in RA patients and that in healthy controls. Furthermore, this peptide, selected on the basis of binding properties to HLA-DRB1*0401, was recognized particularly in DRB1*0401-positive patients and a correlation was observed between disease activity and the level of response to this peptide. In other conditions, such as OA and IBD, HC gp-39-derived peptides may also be targets of the T-cell-mediated immune response.


    Acknowledgments
 
We are grateful to the Regional Blood Bank (Bloedbank Leidschenhage) for providing material from healthy controls.


    Notes
 
Correspondence to: K. Vos, Department of Rheumatology, C4-R, Leiden University Medical Center, P. O. Box, 9600, 2300 RC Leiden, The Netherlands. Back


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 18 October 1999; revised version accepted 12 June 2000.