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Rheumatology 2001; 40: 801-805
© 2001 British Society for Rheumatology

Original Papers

Indirect evidence of intra-articular immunoglobulin G synthesis in patients with Chlamydia trachomatis reactive arthritis

S. Bas, P. Muzzin1, T. Fulpius, N. Buchs and T. L. Vischer

Division of Rheumatology, Department of Internal Medicine, University Hospital, Geneva,
1 Medical Biochemistry Department, Geneva Medical School, Geneva, Switzerland


   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objectives. To investigate whether B-cell stimulation occurs in joints of Chlamydia trachomatis reactive arthritis patients by comparing the immunoglobulin G (IgG) anti-C. trachomatis antibody responses in serum and synovial fluid (SF).

Methods. The number and spectrum of C. trachomatis antigens recognized by paired serum and SF samples from 16 patients with C. trachomatis reactive arthritis and 20 patients with other inflammatory arthropathies independent of this bacteria, were studied by immunoblotting. The responses to five different Chlamydia antigens were also determined in enzyme-linked immunosorbent assays.

Results. In C. trachomatis reactive arthritis patients, a higher number of C. trachomatis antigens was recognized by SF (17.6±5.1) than by serum (11.1±6.3) IgG and a higher intensity of SF IgG binding to the outer membrane protein 2 (OMP2) was observed.

Conclusions. These results suggest an intra-articular IgG production and a possible role of some Chlamydia antigens like OMP2 in the pathogenesis of C. trachomatis reactive arthritis.

KEY WORDS: Chlamydia trachomatis, Reactive arthritis, IgG, Joint, Synovial fluid, Immunoblotting, Outer membrane protein 2.


   Introduction
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 Abstract
 Introduction
Patients and methods
 Results
 Discussion
 References
 
Chlamydia trachomatis is a common human pathogen which causes reactive arthritis in a low percentage of patients [1]. Persistence of chlamydial antigens [2] or repeated infections [3] might play a role in the pathogenesis of the disease. The possibility that this bacterium also plays a role in other arthropathies could also be considered, as the presence of C. trachomatis antigens, DNA and RNA in joints can also occur in patients with other diagnoses [4–9]. However, the articular presence of Chlamydia-derived antigens or nucleic acids does not prove its role in the pathogenesis, as bacteria could be mere bystanders [10, 11]. We therefore investigated whether intra-articular B-cell stimulation occurs in C. trachomatis sexually acquired reactive arthritis (SARA) but not in inflammatory arthropathies attributed to other causes, infectious or not.

In a previous study, with an enzyme-linked immunosorbent assay (ELISA) using purified elementary bodies of Chlamydia as the antigen, we have shown that intra-articular immunoglobulin A (IgA) production, in response to local chlamydial antigen, was likely to occur in patients with C. trachomatis SARA [12]. In the present study, intra-articular IgG production was investigated by comparing, in paired serum and synovial fluid (SF), the number and specificity of the responses to 27 different C. trachomatis antigens by immunoblotting and the binding intensity to five different Chlamydia antigens by ELISA. These antigens were three recombinant proteins [cysteine-rich protein outer membrane protein 2 (OMP2), heat shock protein (hsp) 60, polypeptide encoded by open reading frame 3 of plasmid (pgp3)], synthetic peptides [derived from species-specific epitopes in the variable domain IV of the major outer membrane protein (MOMP) (Labsystems, Finland)] and fragment of the total lipopolysaccharide (LPS) (Medac, Germany) [13].


   Patients and methods
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 Abstract
 Introduction
 Patients and methods
Results
 Discussion
 References
 
Patients
Diagnosis was taken from the chart at the time of sample collection. The patients were divided into the following groups:

Chlamydia trachomatis SARA (n=16): asymmetrical mono/oligoarthritis with urethritis and evidence of urogenital C. trachomatis infection [two had a positive urethral/endocervical C. trachomatis antigen detection by direct immunofluorescence, 12 had a positive urethral/endocervical C. trachomatis culture, two had a positive urethral C. trachomatis DNA amplification with the Amplicor test of Roche Diagnostic Systems (Branchburg, NJ, USA)]; HLA-B27: eight positive, seven negative patients, one not determined; median age: 26 yr.

Inflammatory arthropathies independent of C. trachomatis (n=20): rheumatoid factor positive (n=5) or negative (n=1) rheumatoid arthritis, gout (n=4), Lyme arthritis (diagnosis confirmed by Western blot) (n=5), non-specific post-dysenteric reactive arthritis (n=2), Salmonella enteritidis reactive arthritis (n=2) and septic arthritis (n=1); median age: 43 yr.

Immunoblot analysis of sera and SF
For antigen detection, samples were diluted 1:100 (serum) or 1:50 (SF). Otherwise, the immunoblot analysis was performed as previously described [13, 14].

Recombinant protein preparations and measurement of optical densities (OD) obtained for IgG antibodies to OMP2, hsp60, pgp3, MOMP and LPS by ELISA
Serum and SF samples were diluted 1:100. Other conditions were as previously described [13].

Statistical analysis
Where appropriate, the results were analysed by Student's t-test and {chi}2 test.


   Results
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 Abstract
 Introduction
 Patients and methods
 Results
Discussion
 References
 
Number of C. trachomatis antigens recognized in immunoblotting, by paired serum and SF IgG antibodies from reactive arthritis and control patients
Because the serum IgG concentration was about twice the SF concentration, immunoblotting was performed with SF diluted 1/50, whereas the sera were diluted 1/100. An example of immunoblot evaluations of IgG responses to C. trachomatis antigens, obtained with paired serum and SF from a C. trachomatis SARA patient and from a Salmonella enteritidis reactive arthritis patient is presented in Fig. 1Go.



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  		FIG. 1. Immunoblot evaluation of IgG responses to C. trachomatis antigens, obtained with serum (A) and the paired SF (B) from a C. trachomatis SARA patient and with serum (C) and the paired SF (D) from a Salmonella enteritidis reactive arthritis patient.

 
In the C. trachomatis reactive arthritis group, three patients had serum and SF IgG recognizing an equal number of fractions, whereas the other 13 SF IgG recognized more fractions upon immunoblotting (Fig. 2AGo). The mean number of antigens bound by SF IgG was significantly (P=0.00034) higher (17.6±5.1) than by serum IgG (11.1±6.3), whereas no significant difference was observed for the control group (SF: 7.8±6.4; serum: 7.8±6.7). No relationship was observed between the increase in number of SF responses, the presence of HLA-B27, the disease duration or the number of active joints.



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  		FIG. 2. Plots of the number of serum and SF IgG responses to C. trachomatis antigens in immunoblots and of OD responses to OMP2 C. trachomatis antigen in enzyme immunoassay, for patients with C. trachomatis reactive arthritis (A and C) or inflammatory arthropathies independent of C. trachomatis (B and D).

 

Chlamydia trachomatis antigens recognized more frequently in immunoblotting by SF than by serum IgG from reactive arthritis patients
The binding pattern varied from one sample to the other, in both groups of patients. However, in C. trachomatis SARA, three antigens of 64, 32 and 6 kDa were recognized significantly more often by SF than by serum IgG when high-intensity responses were considered. The 64-kDa antigen was the most frequently recognized (31% of serum and 75% of SF samples, P=0.013).

OD responses obtained in ELISA for serum and SF IgG binding to OMP2, hsp60, pgp3, MOMP and LPS, for reactive arthritis and control patients
These responses were measured in the paired samples still available, i.e. 11 for the C. trachomatis reactive arthritis group and 13 for control patients, except IgG anti-OMP2 which was determined for five and 11 patients, respectively.

A significantly higher mean OD in SF (mean±standard deviation: 1.14±0.9) than in serum (0.65±0.54) (P=0.05) was only observed for IgG anti-OMP2 from C. trachomatis reactive patients and was mainly observed for three of these patients (Fig. 1CGo). No significant difference was observed for IgG anti-OMP2 from control patients (serum: 0.48±0.52 and SF: 0.24±0.32) or for IgG binding to other antigens in both groups.


   Discussion
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
References
 
The highest number of IgG reactions observed in SF from several patients with C. trachomatis SARA could reflect differences between systemic and intra-articular immune system activation. Because germinal centre-like structures have recently been found in the synovial membrane of a patient with post-Chlamydia reactive arthritis [15], a local B-cell activation and differentiation might be considered. As the persisting synovitis in reactive arthritis could be attributable to sequestered antigens, the 64-, 32- and 6-kDa antigens significantly more frequently recognized by SF than by serum IgG may play a role in immunopathology but these antigens were not identified. However, in ELISA, a significantly higher response to OMP2 was only observed in SF from patients with C. trachomatis SARA and not in controls. These results can be related to the antigens identified as targets for the synovial CD4+ T-cell response: hsp60, the 18-kDa histone-like protein (Hc1), an unidentified 30-kDa antigen, MOMP and OMP2 [11, 16–19].

In control samples with no apparent link to C. trachomatis, chlamydial antigens were also bound by IgG, both in serum and SF. This could correspond to cross-binding reactions with antigenic proteins from other micro-organisms. However, five patients could have previously encountered C. trachomatis as they had a high number (>=10) of serum IgG responses [13]. As they did not have more SF than serum IgG responses, these SF antibodies could have been either plasma derived or locally produced if some B cells had been recruited non-specifically to the synovium. Therefore, no argument for a role of Chlamydia antigens in aggravation of the disease can be provided in these patients. Other factors such as the genetic background or the amount of microbial constituents present in the joint could also be crucial.

In conclusion, this study provides indirect evidence that specific IgG antibody production occurs in the joints of C. trachomatis SARA patients, as we and others have previously reported for IgA [12, 20, 21]. Using two different methods, immunoblotting and ELISA, some antigens were more often or more intensively recognized by IgG from SF than from serum. Thus, Chlamydia antigens of 64, 32 and 6 kDa and OMP2 might be immunodominant and play a role in immunopathology via T cell-mediated effector mechanisms or via antibodies forming immune complexes or by autoantigen recognition.


   Acknowledgments
 
The technical assistance of Yvette Froment, Ursula Spenato and Monique Stucker is gratefully acknowledged. This study was supported by grant no. 32–47299.96 from the Fonds National Suisse de la Recherche Scientifique.


   Notes
 
Correspondence to: S. Bas. Back


   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

  1. Keat A, Thomas BJ, Taylor-Robinson D. Chlamydial infection in the aetiology of arthritis. Br Med Bull 1983;39:168–74.[Free Full Text]
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Submitted 20 July 2000; Accepted 16 February 2001


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