HERVE C. GERARD, ZHAO WANG, JUDITH A. WHITTUM-HUDSON, HANI El-GABALAWY, RAFAELA GOLDBACH-MANSKY, THOMAS BARDIN, H. RALPH SCHUMACHER, and ALAN P. HUDSON

ABSTRACT.

Objective. We used a highly quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) assay system to define the steady-state levels of mRNA encoding a large panel of soluble mediators of inflammation in synovial tissues from patients with chronic arthritis infected with Chlamydia trachomatis versus C. pneumoniae.

Methods. RNA/cDNA was prepared from synovial biopsies of 4 patients with chronic arthritis and joint infection with C. trachomatis, 6 with C. pneumoniae at that site, 3 uninfected healthy controls, and 3 patients with undifferentiated oligoarthritis (UO) who were PCR negative for all organisms assayed. Real-time RT-PCR was used to assess relative mRNA levels from 12 cytokine and 2 chemokine genes (IL-1a, IL-1ß, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p35, IL-12p40, IL-15, IFN-g, TNF-a, MCP-1, RANTES). Input loading was normalized to 18S rRNA. Data were obtained for each mRNA from each sample in triplicate in comparison to the same mRNA level in the controls.

Results. In most C. trachomatis infected synovial tissue samples, high levels of IL-10 mRNA were present, with less mRNA for IL-8, IL-15, IFN-g, and TNF-a. Synovial tissues from chronic arthritis patients with synovial C. pneumoniae showed significant levels of mRNA solely for IL-8 and IL-1ß. All other cytokine messengers assessed in each sample from each patient group were at or near control level. One patient with C. pneumoniae showed a high transcript level for RANTES, and one patient with C. trachomatis showed a high transcript level for MCP-1. No patient with UO showed elevated messenger level for any cytokines assayed, but RANTES mRNA was elevated in each.

Conclusion. Our data suggest that while both C. trachomatis and C. pneumoniae have been associated with inflammatory joint disease, each elicits a somewhat different steady-state profile of mRNA encoding relevant cytokines and chemokines during chronic infection of synovial tissue. Precisely how these differing profiles relate to clinical aspects of synovial inflammation will require further study, but the observations confirm and extend data indicating potentially important differences in the pathobiology of these 2 bacterial species. (J Rheumatol 2002;29:1827-35)

Key Indexing Terms:

CYTOKINES
SYNOVIAL INFECTION
CHLAMYDIAE
TRANSCRIPTION

From the Departments of Immunology and Microbiology, Internal Medicine, and Ophthalmology, Wayne State University School of Medicine, and the Department of Veterans Affairs (DVA) Medical Center, Detroit, Michigan; the Arthritis and Rheumatism Branch, NIAMS, National Institutes of Health, Bethesda, Maryland, USA; the Fédération de Rhumatologie, Hôpital Lariboisière, Paris, France; and the Division of Rheumatology, Department of Medicine, University of Pennsylvania School of Medicine, and the DVA Medical Center, Philadelphia, Pennsylvania, USA.

Supported by NIH grants AR-42541 and AI-44055 (APH), AI-44493 (JAW-H), and AR-47186 (HCG), and grants from the DVA Medical Research Service (APH, HRS).

H.C. Gérard, PhD, Research Scientist; Z. Wang, MS, Research Associate; A.P. Hudson, PhD, Professor of Microbiology, Department of Immunology and Microbiology; J.A. Whittum-Hudson, PhD, Professor of Internal Medicine, Immunology, and Ophthalmology, Departments of Internal Medicine, Ophthalmology, and Immunology and Microbiology, Wayne State University School of Medicine; H. El-Gabalawy, MD; R. Goldbach-Mansky, MD, Clinical Investigators, Arthritis and Rheumatism Branch, NIAMS, NIH; T. Bardin, MD, Professeur de Rhumatologie, Hôpital Lariboisière; H.R. Schumacher, MD, Professor of Medicine, University of Pennsylvania School of Medicine, and DVA Medical Center, Philadelphia.

Address reprint requests to Dr. A.P. Hudson, Department of Immunology and Microbiology, Wayne State University School of Medicine, Gordon H. Scott Hall, 540 East Canfield Avenue, Detroit, MI 48201. E-mail: ahudson@med.wayne.edu

Submitted May 30, 2001; revision accepted March 7, 2002.

Synovial presence of any of several bacterial species can generate, or be associated with, inflammatory arthritis. By American College of Rheumatology diagnostic criteria¹, these organisms include the genital pathogen Chlamydia trachomatis, and increasing evidence from several laboratories suggests that Chlamydia pneumoniae may be involved in synovial pathogenesis in some individuals; however, this latter organism's contribution to joint disease has yet to be definitively established^2-4. Inflammation of the joint elicited by antigens from Chlamydia is a function of the proinflammatory molecules produced in response to those antigens, and many groups have described one or a few relevant cytokines, or the mRNA encoding them, in synovial material from patients with Chlamydia associated arthritis and/or in various tissue culture models^5-9. Most such reports, though, have focused on patients relatively early in the disease process⁶. Importantly, no study has examined simultaneously and in quantitative terms the production of a large panel of cytokines, chemokines, or their messengers in joint materials from patients chronically infected with C. trachomatis or C. pneumoniae, and to our knowledge none has provided a large scale, comparative, steady-state analysis of synovial inflammatory mediators elicited by these 2 related organisms during chronic infection. This information would be of significant value, since it would provide insight into an important aspect of host-parasite interaction for these pathogens during persistent synovial infection.

C. trachomatis and C. pneumoniae are both obligate intracellular bacterial pathogens, and as with all Chlamydiae they share many biological characteristics¹⁰. For example, both species undergo a similar biphasic developmental cycle ending with release of new elementary bodies via host cell lysis or exocytosis. Both species seem to prefer epithelial or epithelia-like cells as host, although both are capable of infecting other cell types both in vivo and in vitro. Under some circumstances, these organisms undergo persistent infection, i.e., longterm infection of host cells, and in this state both C. trachomatis and C. pneumoniae display aberrant morphology, unusual transcriptional characteristics, and other attributes not observed during normal active infection^11-15. When these bacteria enter the persistent state in vivo, chronic diseases can result. For C. trachomatis these can include often severe reproductive dysfunction in women, as well as reactive arthritis (ReA) in both sexes^12,16,17. For C. pneumoniae, asthmatic bronchitis, and possibly atherosclerosis and arthritis can result^2-4,18,19.

As suggested by this latter observation, the biology of C. trachomatis and C. pneumoniae does differ in several aspects, despite the common developmental cycle and other characteristics exhibited by both species. C. trachomatis is primarily a pathogen of the urogenital system in developed countries, while its sister-species is a respiratory pathogen; thus, transmission of the 2 to mucosal surfaces involves profoundly different routes²⁰. Moreover, biological characteristics associated with the major outer membrane protein of each species appear to differ, as do various aspects of elementary body morphology, inclusion structure, etc²¹. We reported that some as yet unknown aspect of pathogenesis for C. pneumoniae, but not C. trachomatis, is enhanced in human host cells bearing the e4 allele type at the APOE locus on chromosome 19²². For these and other reasons, the controversial suggestion has been made to reclassify C. pneumoniae into the new Genus Chlamydophila, rather than retaining it within Chlamydia²³.

As indicated, persistent synovial infection with C. trachomatis, and possibly C. pneumoniae as well, can elicit arthritis, although important aspects of the joint disease engendered by the 2 organisms differ^4,12. For example, unpublished data from this group indicate that as many as 45% of patients with chronic ReA are polymerase chain reaction (PCR) positive for C. trachomatis in the joint. In contrast, a lower proportion of synovial samples was shown to be PCR positive for C. pneumoniae; indeed, our results indicated about 13% of synovial tissue samples, and even fewer synovial fluid samples, are so positive⁴. Importantly, while the extraarticular and other attributes of C. trachomatis associated arthritis are well established, we could identify no set of clinical characteristics uniquely associated with the synovial presence of C. pneumoniae. Because of these differences in clinical attributes and biology, because both C. trachomatis and C. pneumoniae are known to elicit a strong inflammatory response at sites of their residence, and because we have only limited information concerning host-parasite interaction for persistent Chlamydiae, we undertook a quantitative, comparative study of a large panel of cytokine and chemokine mRNA in synovial tissues from arthritis patients chronically infected with each of these organisms (but not both at once). For comparison, we included in the analyses synovial tissue samples from patients with undifferentiated oligoarthritis who had no identifiable bacteria in their synovia. The results reveal a major difference in the steady-state pattern of cytokine and chemokine messengers present in synovial tissue chronically infected by the 2 Chlamydia species, and both patterns differ significantly from those of PCR negative patients with undifferentiated oligoarthritis.

MATERIALS AND METHODS

Patient samples. DNA/RNA preparations from patient synovial tissues were selected for analysis from our extensive freezer library of such nucleic acid preparations on the basis of: (1) diagnosis of inflammatory arthritis plus PCR positivity for either C. trachomatis or C. pneumoniae but not both at once (see below); or (2) diagnosis of undifferentiated oligoarthritis plus PCR negativity in each of the Chlamydia directed PCR assay systems and in a pan-bacterial system²⁴; all samples were also selected for analysis on the basis of their having adequate amounts of high quality RNA for assay and relatively long duration of disease. Biopsy samples were originally obtained from patients presenting at the Arthritis Clinics of the DVA Medical Center and the University of Pennsylvania Hospital, Philadelphia, PA, and the Hôpital Lariboisière, Paris, using the Parker-Pearson technique²⁵. RNA preparations derived from synovial biopsies similarly obtained from healthy volunteers were used as controls; the original tissue samples were obtained with informed consent, according to an approved protocol at the NIAMS, National Institutes of Health, Bethesda, MD. At procurement, all samples were immediately snap-frozen at -70°C, transported to the laboratory, and subjected to nucleic acid preparation (see below). A summary of patient characteristics is presented in Table 1. Diagnoses were made according to American College of Rheumatology criteria where possible¹; diagnoses of undifferentiated oligoarthritis followed published criteria²⁶.

Nucleic acid preparations. Total nucleic acids originally were prepared from synovial biopsies as described^4,27. Each preparation was screened first for chromosomal DNA of C. trachomatis and C. pneumoniae by PCR, as described^4,27. Preparations positive in these assays were screened using assays targeting Yersinia, Salmonella, Shigella, and other relevant enteric organisms, and all were negative for these other organisms; samples from patients diagnosed with undifferentiated oligoarthritis were PCR negative in all assays (Table 1), including a pan-bacteria assay system²⁴. Pure RNA was prepared from total nucleic acid preparations by treatment with DNaseI (Promega, Madison, WI, USA)²⁷. Such preparations were confirmed DNA negative by PCR targeting the host ß-actin gene, in the absence of reverse transcription (RT). RT was done using 3 µg RNA from each patient sample incubated with MuLV reverse transcriptase (Life Technologies, Gaithersburg, MD, USA) and random hexamers as primers²⁸. For most RNA preparations in which Chlamydiae were targeted, primary transcripts from the C. trachomatis and C. pneumoniae rRNA operons were assessed^19,27 to confirm viability and metabolic activity of the infecting organisms. Prior to analyses, confirmation of the quality of each cDNA preparation was assessed by standard PCR targeting the host actin gene.

Real-time transcript analyses. We used real-time RT-PCR^29-31 to simultaneously and quantitatively assess relative mRNA levels from 12 cytokine genes (IL-1a, IL-1ß, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p35, IL-12p40, IL-15, IFN-g, TNF-a) in RNA/cDNA from synovial tissue. The control for comparison in the assays was RNA/cDNA from normal uninfected synovia (controls 1-3, Table 1). Real-time PCR on cDNA preparations was performed using the TaqMan Cytokine Gene Expression Plate™ (PE Biosystems, San Jose, CA, USA) as specified by the manufacturer. As supplied, this is a 96 well reaction plate arranged in 12 columns, one for each cytokine. Each column comprises 8 identical wells containing TaqMan primers and probes for assay of one human cytokine mRNA and an 18S rRNA endogenous control; probes used 6-carboxy-fluorescein as the reporter and 6-carboxy-tetramethyl-rhodamine as the quencher at the 5' and 3' ends, respectively. The primer/probe systems for the 12 cytokine cDNA are designed to amplify their target sequences with equal efficiency, allowing comparison among the various messenger levels in each sample. As assayed, each well contained 5 µl of cDNA, 1´ Taqman buffer A, 5.5 mM MgCl₂, 200 µM dATP, dCTP, dGTP, 400 µM dUTP, 0.01 U/µl AmpErase, and 0.025 U/µl AmpliTaq Gold™ DNA polymerase in a total volume of 50 µl. Each well was closed with MicroAmp Optical caps after loading of reagents. For MCP-1 and RANTES mRNA, the SYBR green method for real-time RT-PCR analysis was used³¹, and each assay was done in triplicate as for the cytokines given above; these assays were as described¹¹. Primers for the assays were designed using GeneRunner™ software (Hastings Software, Hastings, NY, USA), and care was taken in the design to insure that each primer system amplified its target sequence with equivalent efficiency. The primers for MCP-1 were: 5'-gtcacctgctgctataacttc-3' and 5'-tgctgctggtgattcttcta-3' to generate an amplification product of 79 bp; primers for RANTES were: 5'-cctcgctgtcatcctcat-3' and 5'-acttgccactggtgtagaaa-3' to generate a 149 bp product. Amplification conditions for all assays were: 2 min at 50°C, 10 min at 95°C, and then 40 cycles of 95°C/15 s, 60°C/1 min. Amplification reactions were performed in a PE Biosystems model 7700 sequence detector. Assays for each cytokine and chemokine mRNA were run in triplicate for each sample, with triplicate controls; data were analyzed using the v. 1.7 software provided by PE Biosystems.

RESULTS

Table 1. Clinical information from patients.

Steady-state cytokine and chemokine mRNA levels in C. trachomatis infected synovial tissue. We used real-time RT-PCR to quantitatively examine mRNA levels encoding 12 relevant cytokines in synovial tissue from each of 4 patients with chronic arthritis infected with C. trachomatis at that site. RNA from each patient sample was positive in RT-PCR assays targeting primary chlamydial rRNA transcripts, indicating viability of the organism in the synovial tissues from which the RNA was prepared (Table 1); in situ hybridization also was done on synovial tissues from each patient, confirming the PCR positivity of each sample for the organism (data not shown). Representative results of the comparative and highly quantitative cytokine transcript assays are shown in Figure 1A, as indexed to mRNA levels from the same cytokine genes in a synovial tissue sample from an uninfected control (designated C1). These data indicate that transcripts encoding IL-10 predominate in most of these infected patient samples. Indeed, in 3/4 samples showing the strongest level of transcript induction, IL-10 mRNA is increased 75 to nearly 250-fold (Patients Ct1, Ct3, Ct4). Two samples showed significant mRNA levels for IL-8 and IL-15 (Patients Ct1, Ct4), and other samples showed varying levels of messenger encoding IFN-g and TNF-a. One patient sample showed an approximately 20-fold higher level of IL-1ß compared to control (Patient Ct2). Interestingly, none of these patients had significant mRNA specifying IL-12 subunits.

Enough cDNA remained from these C. trachomatis infected samples to perform real-time analyses for mRNA from the genes specifying MCP-1 and RANTES, both of which are important in modulation of an inflammatory reaction. Results of these assays are shown in Figure 1B. Only one sample (Patient Ct4) showed high transcript levels from the gene encoding MCP-1, but none of these samples had significant levels of RANTES mRNA.

Steady-state cytokine and chemokine transcript levels in C. pneumoniae infected synovial tissue. RNA/cDNA from 6 synovial tissue samples from chronic arthritis patients infected with C. pneumoniae also were analyzed using the comparative, highly quantitative real-time assay system targeting the 12 cytokine messengers; again, RNA preparations from 4 of these patient samples (Cpn1-Cpn4) were analyzed for primary rRNA transcripts from the organism, and each showed the presence of such transcripts, indicating viability and metabolic activity of the organism in synovial tissue at the time of biopsy (Table 1). Figure 2A shows representative results from the simultaneous cytokine mRNA assays, as indexed to congruent transcript levels in synovial tissue from an uninfected control (C2). In contrast to results for the chronically C. trachomatis infected synovial samples, the predominant cytokine transcripts in the C. pneumoniae infected joint tissues were IL-8 and IL-1ß. Because the assays in Figure 2A were not run simultaneously with those in Figure 1A, the 2 sets of results cannot be compared directly; however, the highest level of transcript induction for IL-8 and IL-1ß in the C. pneumoniae infected patients does appear to be lower than those for IL-10 in the C. trachomatis infected samples. That is, the highest level of induction for IL-8 mRNA was about 50-fold over that of control (Patient Cpn4); for IL-1ß, the highest level of induction was slightly less than 40-fold compared to control (Patient Cpn2). mRNA encoding IL-10 was essentially absent in all C. pneumoniae positive patient samples, as were transcripts encoding all other cytokines; one patient sample (Cpn3) showed a 3-fold increase in TNF-a mRNA.

cDNA remaining from 5 C. pneumoniae infected samples were subjected to real-time analyses for mRNA levels from the 2 chemokine genes. Results of these assays are shown in Figure 2B. Only one patient (Cpn5) showed a high transcript level from either gene, and this was for RANTES rather than MCP-1. Patients Cpn2 and Cpn3 showed low levels of RANTES mRNA, while Patient Cpn1 and Cpn4 showed essentially no transcripts at all from either chemokine gene.

Steady-state cytokine and chemokine transcript levels in PCR negative synovial tissue from patients with undifferentiated oligoarthritis. As an external control, we performed the same extensive real-time RT-PCR analysis on a set of 3 patient samples from individuals diagnosed with undifferentiated oligoarthritis; we chose these particular samples for study because they were PCR negative not only in the Chlamydia directed assays, but also in all PCR assays using a pan-bacterial primer system. As shown in Figure 3A, each of these patient samples showed mRNA levels for all the targeted cytokines that were essentially identical to the control. Similarly, none of these samples showed significant transcript level for MCP-1 (Figure 3B), although RANTES mRNA was elevated nearly 10-fold in one sample (Patient UO3) and 2-3-fold elevated in the other 2 samples.

DISCUSSION

Many methods have been used to define relative mRNA levels from various genes in patient or other samples, including Northern hybridization, RNase protection, and RT-PCR. The first 2 of these methods require relatively large amounts of RNA and thus are not usually suitable for analysis of small specimens. Standard RT-PCR systems have been used to analyze specific cytokine mRNA in synovium^6-8, but this method is only semiquantitative. Recently, real-time RT-PCR techniques have been developed that can overcome many of these limitations. Unlike standard endpoint analytical methods, real-time RT-PCR monitors amplification of target by fluorescence during the earliest portion of the product accumulation curve, the portion in which that accumulation most closely approximates second-order kinetics^29-31. Moreover, this technology allows rapid assessment of large panels of transcripts simultaneously in combination with cDNA from control preparations, permitting quantitative comparison of mRNA levels from many genes in several samples at once. We used real-time RT-PCR to simultaneously and quantitatively evaluate relative steady-state mRNA levels from 12 cytokine and 2 chemokine genes in synovial tissues from patients with chronic arthritis, 4 of which were infected with C. trachomatis, 6 with C. pneumoniae, and 3 whose synovial tissue had no detectable bacteria.

Analyses of the C. trachomatis infected patient samples showed that IL-10 mRNA levels were strongly elevated in the majority of these individuals; messengers encoding IL-8, IFN-g, IL-15, and TNF-a mRNA were also elevated in some patients, although to a lesser extent than IL-10. In these samples, we found virtually no transcripts from the gene encoding RANTES, but one individual showed a high mRNA level from the gene specifying MCP-1. In patients infected with C. pneumoniae IL-8 transcripts were at high level, but no C. pneumoniae infected patient had significant mRNA encoding IL-10. Rather, transcripts for IL-1ß were elevated in these samples. In contrast to C. trachomatis infected patients, no individuals with synovial C. pneumoniae had significant mRNA specifying MCP-1; one patient had a high level of mRNA encoding RANTES. None of the PCR negative patients showed elevated transcript levels for any cytokine assayed or for MCP-1, but each showed an elevated mRNA level for RANTES.

At least in the limited number of patient samples we analyzed, both C. trachomatis and C. pneumoniae elicited to varying extent the production of IL-8 mRNA, encoding a chemoattractant molecule for neutrophils, but the steady-state level of messengers specifying this molecule appeared to be different between groups infected with the 2 organisms. It is of interest that, with the exception of IL-10 in the C. trachomatis group and IL-1ß mRNA in the C. pneumoniae group, transcripts specifying most cytokines other than IL-8 were at relatively low levels in synovia infected with these 2 related bacteria, particularly in those infected with C. pneumoniae. It is especially interesting that C. trachomatis but not C. pneumoniae strongly elicited the messenger specifying IL-10, an attenuator of inflammation and IFN-g production, and that most macrophage derived cytokine mRNA were present in both infected patient groups only at quite low levels.

The data for chemokine production also reflect a differential profile between groups infected with these 2 pathogens. RANTES has been shown recently to be at higher level than MCP-1 in synovial fluid from patients with inflammatory arthritis³², and our data for the 3 PCR negative patients and those PCR positive for synovial C. pneumoniae are consistent with that finding. However, the total lack of messenger specifying RANTES in all patients with synovial C. trachomatis indicates a difference in the details of host-parasite interaction between this organism and its sister-species, at least in the context analyzed. Indeed, we suspect that the differences observed here in steady-state mRNA production for both cytokines and chemokines reflect a difference, and probably an important one, in the details of pathogenesis engendered in the patients harboring persistent synovial C. trachomatis versus C. pneumoniae.

A good deal of variation exists among the patients in the relative level of mRNA from each of the cytokine and chemokine genes assessed; such variation probably results from differences in host genetic background, disease duration, etc. Nonetheless, it may be instructive to examine the cytokine profile observed in these experiments from individual patients in relation to the working diagnoses for those patients. Synovial tissues from patients Ct1, Ct3, and Ct4, individuals with ReA of long duration, each displayed powerful induction of IL-10 mRNA, with some induction of IFN-g and TNF-a mRNA. Patients Ct1 and Ct4 also showed strong induction of IL-8 mRNA, and the latter patient had high levels of MCP-1 mRNA. The mixed induction of pro and antiinflammatory modulators in the chronically C. trachomatis infected patients is consistent with results of somewhat more limited cytokine studies published recently for patient materials^6,7,33, but they are not fully consistent with data from some in vitro studies^5,8. We and others^9,12 suspect that in chronic C. trachomatis associated arthritis, especially in patients with disease of long duration in which the organism is metabolically active, the immunopathogenic response is induced to be mixed Th1 and Th2-type by the organism. Specifically, we suspect that persistently infecting C. trachomatis overtly induce production of the antiinflammatory IL-10, as do Mycobacterium tuberculosis and some viruses³⁴, thus helping to insure its own longterm survival in affected tissues. Along this line, it will be of interest to assess IL-10 levels in relation to those of proinflammatory molecules in patients with chronic C. trachomatis associated ReA during both active and quiescent disease.

In the patients infected with C. pneumoniae, in contrast, the situation for cytokine mRNA appears to be more internally consistent than for the C. trachomatis infected group. That is, each of the 6 patients whose synovial tissues were studied here had inflammatory arthritis, and most showed induction of IL-8 and IL-1ß mRNA, both of which encode proinflammatory molecules, although the relative level of induction of those messengers varied among patients. Patients Cpn3 and Cpn4, for example, each showed elevated transcripts from the IL-8 and IL-1ß genes, and in both patients the former predominated. In patients Cpn4 and Cpn5, however, both of whom showed significant mRNA induction from the same 2 genes, the pattern is different; i.e., in Patient Cpn4 IL-8 transcripts are the most strongly induced, while in Patient Cpn5 IL-1ß mRNA predominates. Of note, Patient Cpn5, with inflammatory arthritis of 96 months' duration, had a significant level of RANTES messenger, in addition to IL-8 and IL-1ß mRNA. Like MCP-1, RANTES is a CC group chemokine with chemoattractant activity for monocytes, basophils, eosinophils, and mast cells³⁵. This molecule is strongly proinflammatory and has been associated with asthma³⁶, sarcoidosis³⁷, arthritis³⁸, and granuloma formation by Mycobacterium bovis³⁹. C. pneumoniae has been associated with several of these clinical entities¹⁸. Moreover, important differences have been noted at the molecular level between the characteristics of chronic infection with C. trachomatis compared to C. pneumoniae²² consistent with elicitation of RANTES mRNA by patients with the latter but not the former species.

At this point neither the clinical nor biological significance of these detailed differences in steady-state host-response characteristics of persistent synovial C. trachomatis and C. pneumoniae infection is clear. More study will be required to confirm whether the observations reported here, especially those relating to elicitation of IL-10 by persistent C. trachomatis and the lack of this mediator in patients persistently infected with C. pneumoniae, represent the situation in all or most cases of chronic Chlamydia infection of the joint. The approach employed in this study will undoubtedly prove to be useful to conduct such studies. Further, it will be important to elucidate the molecular mechanisms that govern host-parasite interaction during persistent synovial Chlamydia infection, since such information is likely to suggest new therapeutic approaches to the treatment of Chlamydia associated arthritis.

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