title: Interferon-gamma production in response to in vitro stimulation with collagen type II in rheumatoid arthritis is associated with HLA-DRB1(*)0401 and HLA-DQ8.
abstract: BACKGROUND
  Despite much work over past decades, whether antigen-specific immune reactions occur in rheumatoid arthritis (RA) and to what extent such reactions are directed towards joint-specific autoantigens is still questionable. One strong indicator for antigenic involvement in RA is the fact that certain major histocompatibility complex (MHC) class II genotypes [human leucocyte antigen (HLA)-DR4 and HLA-DR1[ predispose for the development of the disease [1]. In the present report, collagen type II (CII) was studied as a putative autoantigen on the basis of both clinical and experimental data that show an increased frequency of antibodies to CII in RA patients [2-4] and that show that CII can induce experimental arthritis [5]. It is evident from the literature that RA peripheral blood mononuclear cells (PBMCs) respond poorly to antigenic stimulation [6-8], and in particular evidence for a partial tolerization to CII has been presented [9]. The strategy of the present work has accordingly been to reinvestigate T-cell reactivity to CII in RA patients, to relate it to the response to commonly used recall antigens and to analyze interferon (IFN)-gamma responses as an alternative to proliferative responses.
  
  
  OBJECTIVE
  To study cellular immune reactivity to CII in patients with RA and in healthy control individuals and to correlate this reactivity to HLA class II genotypes and to the presence of antibodies to CII in serum.
  
  
  METHODS
  Forty-five patients who met the 1987 American college of Rheumatology classification criteria for RA [10] and 25 healthy control individuals of similar age and sex were included. Twenty-six of these patients who had low levels of anti-CII in serum were randomly chosen, whereas 19 patients with high anti-CII levels were identified by enzyme-linked immunosorbent assay (ELISA)-screening of 400 RA sera. Heparinized blood was density gradient separated and PBMCs were cultured at 1 x 10(6)/ml in RPMI-10% fetal calf serum with or without antigenic stimulation: native or denatured CII (100 microgram/ml), killed influenza virus (Vaxigrip, Pasteur Merieux, Lyon, France; diluted 1:1000) or purified protein derivative (PPD; 10 microgram/ml). CII was heat-denatured in 56 degrees C for 30 min. Cell supernatants were collected after 7 days and IFN-gamma contents were analyzed using ELISA. HLA-DR and HLA-DQ genotyping was performed utilizing a polymerase chain reaction-based technique with sequence-specific oligonucleotide probe hybridization. Nonparametric statistical analyses were utilized throughout the study.
  
  
  RESULTS
  PBMCs from both RA patients and healthy control individuals responded with IFN-gamma production to the same degree to stimulation with native and denatured CII (Fig. 1a), giving median stimulation indexes with native CII of 4.6 for RA patients and 5.4 for health control individuals, and with denatured CII of 2.9 for RA patients and 2.6 for healthy control individuals. RA patients with elevated levels of anti-CII had a weaker IFN-gamma response to both native and denatured CII that did healthy control individuals (P-).02 and 0.04, respectively). Stimulation with the standard recall antigens PPD and killed influenza virus yielded a median stimulation index with PPD of 10.0 for RA patients and 51.3 for healthy control individuals and with influenza of 12.3 for RA patients and 25.7 for healthy, control individuals. The RA patients displayed markedly lower responsiveness to both PPD and killed influenza virus than did healthy control individuals (Fig. 1b). IFN-gamma responses to all antigens were abrogated when coincubating with antibodies blocking MHC class II. The low response to PPD and killed influenza virus in RA patients relative to that of healthy control individuals reflects a general downregulation of antigen-induced responsiveness of T cells from RA patients [6-8]. That no difference between the RA group and the control group was recorded CII-induced IFN-gamma production therefore indicates that there may be an underlying increased responsiveness to CII in RA patients, which is obscured by the general downregulation of T-cell responsiveness in these patients. In order to address this possibility, we calculated the fraction between individual values for the CII-induced IFN-gamma production and the PPD-induced and killed influenza virus-induced IFN-gamma production and the PPD-induced and killed influenza virus-induced IFN-gamma production, and compared these fractions. A highly significant difference between the RA and health control groups was apparent after stimulation with both native CII and denatured CII when expressing the response as a fraction of that with PPD (Fig. 2a). Similar data were obtained using killed influenza virus-stimulated IFN-gamma values as the denominator (Fig. 2b).When comparing the compensated IFN-gamma response to denatured CII stimulation between RA patients with different HLA genotypes, highly significant differences were evident, with HLA-DRB1*0401 patients having greater CII responsiveness than patients who lacked this genotype (Fig. 3a). HLA-DQ8 positive patients also displayed a high responsiveness to CII as compared with HLA-DQ8 negative RA patients (Fig. 3b). These associations between the relative T-cell reactivity to denatured CII and HLA class II genotypes were not seen in healthy control individuals. Similar results were achieved using influenza as denominator (P = 0.02 for HLA-DRB1*0401 and P = 0.01 for HLA-DQ8).
  
  
  CONCLUSIONS
  No reports have previously systematically taken the general T-cell hyporesponsiveness in RA into account when investigating specific T-cell responses in this disease. In order to address this issue we used the T-cell responses to PPD and killed influenza virus as reference antigens. This was made on the assumption that exposure to these antigens is similar in age-matched and sex-matched groups of RA patients and healthy control individuals. The concept of a general hyporesponsiveness in RA T cells has been documented in several previous reports, in which both nominal antigens [6,7,8] and mitogens [11,12,13] have been used. The fact that a similar functional downregulation in RA PBMCs was obtained with both PPD and killed influenza virus as reference antigens strengthens the validity of our approach. We identified an association between the IFN-gamma response to CII and HLA-DRB1*0401 and HLA-DQ8 in the RA patient group, which is of obvious interest because both these MHC class II alleles have been associated with high responsiveness to CII in transgenic mice that express these human MHC class II molecules [14,15]. There was no association between high anti-CII levels and shared epitope (HLA-DRB1*0401 or HLA-DRB1*0404).
  
  
  CONCLUSIONS
  CII, a major autoantigen candidate in RA, can elicit an IFN-gamma response in vitro that is associated with HLA-DRB1*0401 and HLA-DQ8 in RA patients. This study, with a partly new methodological approach to a classical problem in RA, has provided some additional support to the notion that CII may be a target autoantigen of importance for a substantial group of RA patients. Continued efforts to identify mechanisms behind the general hyporesponsiveness to antigens in RA, as well as the mechanisms behind the potential partial anergy to CII, may provide us with better opportunities to study the specificity and pathophysiological relevance of anti-CII reactivity in RA.
authors.affiliation: Department of Medicine, Karolinska Hospital, Stockholm, Sweden. louise.berg@cmm.ki.se
authors.forename: L
authors.initials: L
authors.lastname: Berg
authors.forename: J
authors.initials: J
authors.lastname: Rönnelid
authors.forename: C B
authors.initials: CB
authors.lastname: Sanjeevi
authors.forename: J
authors.initials: J
authors.lastname: Lampa
authors.forename: L
authors.initials: L
authors.lastname: Klareskog
mesh_terms: D019138:Antibodies, Blocking / Q000494:pharmacology; D001172:Arthritis, Rheumatoid / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D002469:Cell Separation; D002478:Cells, Cultured; D003094:Collagen / Q000032:analysis / Q000276:immunology / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006683:HLA-DQ Antigens / Q000032:analysis / Q000235:genetics*; D006684:HLA-DR Antigens / Q000032:analysis / Q000235:genetics*; D059811:HLA-DRB1 Chains; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis* / Q000235:genetics; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D008213:Lymphocyte Activation; D008297:Male; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D014373:Tuberculin
chemical_list: D019138:Antibodies, Blocking; D006683:HLA-DQ Antigens; C109756:HLA-DQ8 antigen; D006684:HLA-DR Antigens; D059811:HLA-DRB1 Chains; C553505:HLA-DRB1*04:01 antigen; D014373:Tuberculin; D007371:Interferon-gamma; D003094:Collagen