Bacterial antigens in reactive arthritis and spondarthritis. Rational use of laboratory testing in diagnosis and follow-up

An aetiological diagnosis of reactive arthritis is based on the demonstration of recent or ongoing infection with the causative bacterium. This may be done by serological demonstration of antibacterial antibodies, demonstration of the causative microorganism at an extra-articular site or by identification of bacterial nucleic acids or antigens in joint material from patients with aseptic arthritis. The finding of elevated titres of bacteria-specific IgG- and IgA-class antibodies may indicate recent or persistent infection, but has some limitations due to the prevalence of such antibodies among apparently healthy individuals and the persistence of such antibodies after the infection. While Chlamydia can be demonstrated in urogenital specimens in at least one-third of patients with Chlamydia-induced arthritis, the triggering microorganisms are usually no longer detectable in post-dysenteric reactive arthritides. Assays involving molecular amplifications have been successfully used to demonstrate bacterial nucleic acids in joint specimens from patients with reactive arthritis. In addition, bacterial antigens have been detected by immunofluorescence tests. Even though examination of synovial fluid and synovial membrane specimens for bacterial DNA by the polymerase chain reaction is increasingly used to diagnose reactive arthritis, such assays have not been standardized and are not generally available. While some problems remain, these techniques will facilitate the exact diagnosis of reactive arthritides in the near future.     

Longitudinal investigation of bacterium-specific synovial lymphocyte proliferation in reactive arthritis and lyme arthritis

BACKGROUND: Antigen-specific lymphocyte proliferation of synovial fluid mononuclear cells (SF MNC) has been reported repeatedly in reactive arthritis and Lyme arthritis; however, less information is available on serial investigations of SF MNC in the same patients. METHODS: In this study, the synovial lymphocyte proliferation to Yersinia, Chlamydia, Shigella and Borrelia burgdorferi was investigated sequentially at different time points in 28 patients with reactive arthritis, undifferentiated oligoarthritis or Lyme arthritis responding to one of these bacteria. RESULTS: The same bacterium was always recognized in arthritis triggered by Chlamydia, Shigella or Borrelia, with much variation in the proliferative response. Only the Yersinia-specific responses changed specificity, suggesting that the proliferative response to Yersinia is non-specific in some patients. CONCLUSIONS: Our data support the concept of a local antigen-specific T-cell response in reactive arthritis or Lyme arthritis but not the concept suggested by others that a switch to an autoimmune response takes place in long-standing disease.     

Do minocycline and other tetracyclines have a place in rheumatology?

Tetracyclines are a family of antimicrobials with activity against a broad range of organisms including those that develop intracellularly. Links have been reported between some infections and some inflammatory joint diseases, with the most notable example involving mycoplasmas and rheumatoid arthritis. Reactive arthritides are known to be triggered by organisms found in the gastrointestinal or genitourinary tract, and antigenic material from these organisms has recently been demonstrated in synovial tissue from patients with reactive arthritis. These facts led to the hypothesis that tetracyclines may be useful in rheumatoid arthritis and reactive arthritis. Two controlled studies found that minocycline benefited rheumatoid arthritis patients when it was given either as an adjunct to another second-line treatment or as the only slow-acting drug. Lymecycline has been found to expedite recovery from reactive arthritis due to Chlamydia trachomatis, and tetracycline to decrease the incidence of reactive arthritis due to sexually-transmitted diseases. The safety profiles of these treatments were acceptable in all available studies but require further investigation during long-term administration. The benefits may be related to the immunomodulating effects of tetracyclines and/or to their ability to inhibit metalloproteases such as collagenases. Whether tetracycline therapy influences the course of radiologic lesions in rheumatoid arthritis remains unknown. However, minocycline therapy has given sufficient proof of its efficacy to make it an attractive alternative in rheumatoid arthritis.     

2-deoxy-D-glucose-induced metabolic stress enhances resistance to Listeria monocytogenes infection in mice

OBJECTIVE: Bacteria are considered to be important in the pathogenesis of several forms of arthritis. The goal of this study was to apply the 16S ribosomal RNA (rRNA)-polymerase chain reaction method for the detection of bacterial DNA in synovial fluid (SF) and synovial tissue (ST) from inflamed joints. METHODS: Samples from 5 patients with septic arthritis and from 7 with osteoarthritis or arthritis secondary to joint trauma were used as controls. Samples from 6 patients with spondylarthropathy (SpA) and from 20 with undifferentiated arthritis (UA) were analyzed for the presence of bacterial DNA using universal 16S rRNA primers. Automated sequencing and comparative data analysis were performed to identify the species. RESULTS: In the positive control group, the bacterial species cultured from the synovium could be identified in all cases. No bacterial DNA was detected in the SF and ST from patients in the negative control group. In 4 of 6 patients with SpA and 7 of 20 with UA, the analysis of joint samples revealed the presence of bacterial DNA. Sequence analysis indicated the presence of multiple species, which was confirmed by sequencing of cloned products. CONCLUSION: When the the above techniques were used with a stringent regimen, we were able to demonstrate that it is possible to collect and analyze joint samples without contaminating bacterial DNA. The accumulation of phagocytic cells that contain bacterial DNA of various species could play a role in the pathogenesis of both SpA and UA.     

Synovial fluid cell analysis

Arthrocentesis has to be considered as a part of the clinical examination. A reasonable amount of aspirated synovial fluid is the best argument in favour of an objective articular disorder. Moreover some very simple evaluations are very helpful to make a diagnosis and to distinguish some particularities of rheumatic diseases. Such evaluations have to include both bacterial and synovial fluid analysis. Moreover, when performing synovial fluid analysis, a search for microcrystals is also performed. Haemarthrosis can easily be distinguished from a traumatic tap if the investigator is observing carefully the synovial fluid entering in the syringe. The diseases responsible for haemarthrosis differ with the age of patients: chondrocalcinosis, together with osteoarthrosis, is the most frequent aetiology in the elderly; disorders of haemostasis and synovial tumours are mostly observed in children and young adults. Paucicellular (< 1000 cells/mm3) synovial fluid is observed in different 'mechanical' disorders. In the case of purulent synovial fluid the primary diagnosis is septic arthritis. However, the most common aetiology is probably crystal-induced acute arthritis. Differential cell count analysis performed in case of 'inflammatory' (> 1000 or 2000 cells/mm3) synovial fluid usually shows a predominance of polymorphonuclear cells. However, high cellularity may sometimes be associated with a predominance of other cells, i.e. lymphocytes, monocytes, eosinophils. In this situation, such a simple evaluation (differential cell count analysis) is very helpful in making a diagnosis, e.g. eosinophilic arthritis, or to distinguish some particularities of rheumatic diseases, e.g. absence of cartilage breakdown in case of lymphocytic arthritis.     

T helper 1 response is dominant and localized to the synovial fluid in patients with Lyme arthritis

Cytokines produced by subsets of CD4+ T helper cells responding to an infection influences the efficiency with which the host is able to mount a protective immune response. In an attempt to elucidate the population of active cells involved in the propagation of Lyme arthritis we have utilized intracellular cytokine staining to analyze the polyclonal immune response at the single cell level. We have determined the Th phenotype in the synovial fluid of patients with a variety of chronic inflammatory arthritides, including patients representative of the spectrum of Lyme arthritis. Th1 cells dominate the immune response in the synovial fluid of patients with Lyme as well as those with rheumatoid or other types of chronic inflammatory arthritis. In addition, the severity of Lyme arthritis directly correlates with the ratio of Th1 to Th2 cells in the synovial fluid, such that the larger the effusion, the higher the ratio (r = 0.67, p < 0.05). These results suggest that Th1 cells play a direct role in the pathogenesis of the inflammatory process seen in Lyme arthritis, and that Th2 cells modulate the pro-inflammatory response generated by Th1 cells in the joint. Finally, we identify Th1 cells specific for outer surface protein A of Borrelia burgdorferi, the agent of Lyme disease. These cells are restricted to patients with Lyme arthritis and are localized to the joint. Furthermore, they persist in patients with prolonged antibiotic treatment-resistant Lyme arthritis, suggesting the possibility of an autoimmune process.     

Survival and degradation of Salmonella enterica serotype Enteritidis in intestinal epithelial cells in vitro

The survival and fate of Salmonella enterica serotype Enteritidis in Henle-407 human intestinal epithelial cells was investigated during prolonged incubation to evaluate the persistence of causative microbes and the relationship to patients developing reactive arthritis. Most of the bacteria were killed and degraded quite soon after infection of the cells, but there were still live bacteria inside the cells for up to 14 days. These results suggest that in patients developing reactive arthritis the salmonellae could persist in the epithelial cells and spread within the host to the joint and be present there at the time of the inflammatory response. Production of marked amounts of nitric oxide was observed as a novel response to salmonella infection in human intestinal epithelial cells. The present experimental procedure appears to be a suitable model to further investigate host-bacteria interaction in HLA-B27 positive cells from patients developing reactive arthritis.     

The specific antibacterial proliferation of reactive arthritis synovial T cells is not due to their higher proportion of CD45RO+ cells compared to peripheral blood

OBJECTIVE: To determine the role of synovial fluid (SF) compared to peripheral blood (PB) CD45RO+ T cells in patients with reactive arthritis (ReA) and undifferentiated oligoarthritis. METHODS: We examined SF and PB of 8 patients with a specific lymphocyte proliferation to Yersinia enterocolitica (n = 5) and Chlamydia trachomatis (n = 3). After depletion of the CD45RA+ T cell subset by dynabeads, the remaining T cells (> 95% CD45RO+) from PB and SF of these patients were again stimulated with these bacterial antigens. RESULTS: The mean stimulation index (SI) of these 8 patients with ReA (n = 5) and undifferentiated oligoarthritis (n = 3) was 30.3 +/- 21.86 in SF compared to 1.36 +/- 0.75 in PB. The enrichment of CD45RO+ cells influenced the antigen specific proliferative response of T cells neither in PB (SI = 1.75 +/- 1.35) nor in SF (26.1 +/- 24.05); the initial difference remained unchanged. CONCLUSION: Our findings suggest that the antigen specific lymphocyte proliferation obtained with SF cells is not due to abundance of nonspecific CD45RO+ T cells but can rather be taken as an indication of specific recognition of local bacterial antigens in ReA.     

Monocytes that have ingested Yersinia enterocolitica serotype O:3 acquire enhanced capacity to bind to nonstimulated vascular endothelial cells via P-selectin

Reactive arthritis is usually a self-limiting polyarthritis which develops after certain gastrointestinal or urogenital infections. Microbial antigens found in the inflamed joints are thought to play a key role in the development of this disease. It is not known how antigens of the pathogenic organisms migrate from the mucosal tissues into the joints. The data presented here show that mononuclear phagocytes which mediate the dissemination of several intracellular pathogens acquire an enhanced capacity to bind to nonstimulated vascular endothelial cells after phagocytosis of Yersinia enterocolitica O:3, one of the causative organisms of reactive arthritis. The increased binding to previously nonstimulated endothelial cells was mediated by P-selectin, whose translocation to the endothelial cell surface was induced by monocytes with intracellular Yersinia bacteria. These results suggest that mononuclear phagocytes may be responsible for the dissemination of bacterial antigens and the initiation of the joint inflammation in reactive arthritis.     

Synovial fluid lactic acid in septic arthritis

Lactic acid concentrations in the synovial fluid of 71 patients with inflammatory arthritis were determined by an enzyme method. In 63 samples from 54 patients with a variety of non-septic arthritides, including rheumatoid arthritis, reactive arthritis and gout, the concentration of lactic acid was never greater than 10.2 mmol/l, whereas all twelve patients with septic arthritis had concentrations of 11 mmol/l or greater. Two patients with gonococcal arthritis did not have raised lactic acid concentrations. The enzyme method of lactic acid estimation is an accurate reproducible means of differentiating septic from nonseptic arthritis prior to the isolation of the infecting organism. However, caution is necessary when interpreting the results in those patients who have recently received antibiotic therapy, or in whom gonococcal arthritis is suspected.     

Inflammatory infiltrate and interleukin-8 expression in the synovium of psoriatic arthritis--an immunohistochemical and mRNA analysis

Psoriatic arthritis is an inflammatory arthropathy that ultimately can lead to joint destruction. In this study, we investigated the immunophenotypes of the inflammatory cells and the expression of interleukin-8 (IL-8), which is the hallmark chemoattractant cytokine of psoriasis in synovial membranes from patients exhibiting active psoriatic synovitis (n = 9). The tissue samples were examined by immunohistochemistry, Western blot analysis and in situ hybridisation. The inflammatory infiltrate consisted predominantly of CD3+ T lymphocytes, with a higher proportion of CD4+ than CD8+ T lymphocytes in six cases. CD3+ T lymphocytes were focally distributed near small blood vessels and the enlarged synovial intima. CD1+ interdigitating reticulum cells were not detected. CD22+ B lymphocytes and plasma cells were found in small aggregates without KiM4+ follicular dendritic cells. KiM8+ macrophages were located in the synovial intima and were distributed in a diffuse pattern near the synovial lining cells. CD15+ neutrophil granulocytes were detected in four cases. They were preferentially located in the vicinity of blood vessels and the synovial intima. IL-8 was found at a high level in the synovial lining cells and to a lesser extent in cells located in the perivascular areas. Immunofluorescence double staining showed IL-8 to be expressed in KiM8+ multinucleated giant cells, KiM8+ macrophages and CD3+ T lymphocytes. IL-8 receptor A was demonstrated in the synovial lining and in macrophages and lymphocytes. IL-8 was detected by immunoblot analysis of the synovial tissue at 8.4 kD. Employing in situ hybridisation, IL-8 mRNA was strongly and preferentially expressed in the synovial intima, as well as in macrophages and lymphocytes. The immunophenotype of the psoriatic arthritis inflammatory cells shows great similarity to the inflammatory infiltrate found in the synovial tissue of patients with rheumatoid arthritis. The preferential expression of IL-8 and IL-8 mRNA in the enlarged synovial intima and in lymphocytes and macrophages suggests that IL-8 exerts its action through activated mononuclear cells and T lymphocytes. It seems to play a role in regulating leucocyte traffic into the enlarged synovial intima and may contribute to the aggressive synovitis of patients with psoriatic arthritis.     

A single nonamer from the Yersinia 60-kDa heat shock protein is the target of HLA-B27-restricted CTL response in Yersinia-induced reactive arthritis

The reason for the high association of HLA-B27 with diseases such as ankylosing spondylitis and reactive arthritis is not clear. In reactive arthritis, the triggering bacteria are known, thus allowing investigation of their interaction with HLA-B27. CTL lines derived from five patients with Yersinia-induced reactive arthritis were raised by repeated stimulation in vitro with either Yersinia-infected autologous macrophages (four patients) or pooled peptides (three patients) having the HLA-B27-binding motif. The peptides were derived from five Yersinia proteins and from the chlamydial 57-kDa heat shock protein (hsp). Cytotoxicity of T cell lines was then tested against these peptides. Lytic activity was obtained with T cells stimulated with viable Yersinia or pooled peptides. Targets successfully used for lysis were cells pulsed with peptides from the Yersinia 60-kDa hsp, but not cells pulsed with peptides from other Yersinia proteins or the chlamydial hsp. T cell lines raised with 60-kDa peptides also lysed targets infected with Yersinia. Most interestingly, all three CTL lines tested (one raised with Yersinia; two with pool of peptides) recognized only one single peptide (321-329) of seven tested from the Yersinia hsp60. Cytotoxicity occurred only when target cells were matched for HLA-B27. This identification of an immunogenic peptide derived from an arthritogenic bacterium and presented by HLA-B27 opens the way for future investigation of the role of T cells specific for this peptide or cross-reacting peptides, in the immunopathology of HLA-B27-associated diseases.     

Multiple T cell expansions are found in the blood and synovial fluid of patients with reactive arthritis

OBJECTIVE: To look for evidence of T lymphocyte expansions in the blood and synovial fluid (SF) of patients with reactive arthritis (ReA). METHODS: Paired peripheral blood and synovial samples from 10 patients with ReA were studied by dual color flow cytometry using T cell receptor (TCR) V beta specific and CD4 or CD8 specific antibodies. Two synovial CD8 expansions were studied by 3 color flow cytometry. Peripheral blood samples from 13 healthy, age matched individuals were studied as controls. RESULTS: Statistically significant expansions were observed in all patients, occurring in blood and SF CD4 and CD8 compartments, but were most common in the synovial CD8 compartment. Expansions studied in further detail displayed an activated "memory" phenotype. A synovial BV22S1/CD8 expansion was seen in 5/6 patients with sexually acquired ReA. CONCLUSION: Multiple T lymphocyte expansions are found in both the blood and SF of patients with ReA. Expansions were most commonly found in the synovial CD8 compartment, where they appeared to express both activation and memory markers. This indicates that T lymphocytes (and in particular cytotoxic T cells) may play a pathogenic role in ReA. These findings are consistent with either an antigen or a superantigen driven response.     

Role of macrophages in elevated IgA and IL-6 production by Peyer''s patch cultures following acute oral vomitoxin exposure

Concepts about reactive arthritis are changing and must embrace consideration of the fact that bacteria or their products are present in the joint, not just at the portal of entry in the gastrointestinal (GI) or genitourinary (GU) tracts. With chlamydia-associated disease, atypical elementary bodies can be seen in synovium by electron microscopy, and nucleic acids, including RNA, can be found. It is not yet clear if bacterial nucleic acids are present in postenteric reactive arthritis and whether disease courses are predictably different after GI or GU infection. How bacteria are disseminated to joints and local factors, including cytokines that influence their persistence, are under study. Treatment with antibiotics may help some chlamydia-associated reactive arthritis but is not invariably effective.     

Septic arthritis caused by Streptococcus pneumoniae

BACKGROUND: Streptococcus pneumoniae is an uncommon agent of infective arthritis. In this report three cases of pneumococcal arthritis are described. METHODS: Retrospective review of synovial fluids processed in our laboratory yielding bacteria. The study period was from January 1991 to December 1995. The clinical records of patients with the clinical and microbiological diagnosis of septic arthritis were reviewed. RESULTS: Twenty-eight out of a total of 43 clinical records had the clinical and microbiological diagnosis of septic arthritis and three (11%) were caused by Streptococcus pneumoniae. The infective source in two of these three cases was probably the respiratory tract, and the most common location was the knee. CONCLUSIONS: In our cases immunosuppression seemed to be the major risk factor involved in the development of pneumococcal arthritis.     

Patients with rheumatoid arthritis: study of the correlation between density of glucocorticoid receptors in synovial cells and clinical response to steroidal treatment

BACKGROUND: The target cellular response to glucocorticoids is proportional to the concentrations or affinity of specific receptors to these substances. AIM: To look for a correlation between glucocorticoid receptor concentrations in synovial wall cells and the clinical response to steroidal treatment in patients with rheumatoid arthritis. PATIENTS AND METHODS: Twenty eight patients with rheumatoid arthritis were studied. Each subject was subjected to a synovial biopsy, in which a dry radioautographic technique for diffusible compounds was used. Patients were treated afterwards with three 500 mg intravenous pulses of methilprednisolone. RESULTS: A mean of 44.8% of synovial cells (range 30.1-62.8%) had binding sites for 3H dexamethasone. All patients had a significant clinical improvement after methylprednisolone. Multiple regression analysis did not show a correlation between clinical response and glucocorticoid receptor concentration. CONCLUSIONS: The lack of association between glucocorticoid receptor concentrations and clinical response could be due to the large steroid dose used, that saturated all available receptors.     

Polyarticular blastomycotic arthritis

Blastomycotic arthritis usually presents as acute monoarticular septic arthritis resembling a bacterial process. Only 2 cases in the literature have subsequently developed arthritis in a second joint. We describe the case of an elderly woman with polyarticular blastomycotic arthritis associated with lung, skin, and bone involvement. The organism was identified in the synovial fluid and skin biopsies. Our patient underwent open drainage and aspiration of the involved joints and received antimycotic therapy.     

Synthesis and degradation of hyaluronate by synovia from patients with rheumatoid arthritis

OBJECTIVE: Hyaluronate degradation was analyzed in cultures of healthy tissue and tissue obtained from patients with rheumatoid arthritis. METHODS: Arthritic and healthy synovial tissues were incubated in culture with [3H]glucosamine. Labelled hyaluronate was extracted and its size determined by gel filtration. The production of low molecular weight hyaluronate was analyzed by pulse-chase experiments. Radical production was measured by a cytochrome C reduction assay. RESULTS: Healthy tissues and some arthritic tissues that did not contain significant amounts of granulocytes produced high molecular weight hyaluronate. In contrast, arthritic tissue infiltrated with granulocytes released low molecular weight hyaluronate. Pulse-chase experiments suggested that hyaluronate was degraded in these arthritic tissues. Exogenous hyaluronate was degraded only by intact tissue, but not by cells in culture obtained from synovial membranes of synovial fluids. Hyaluronate degradation was accompanied by massive oxygen radical production. Radical scavengers protected hyaluronate from degradation in synovial tissue. Some protection was achieved by superoxide and catalase or by methionine and complete protection by the iron chelators diethyltriaminepentacetic acid or deferoxamine mesylate. CONCLUSION: Degradation of hyaluronate in arthritic synovial tissue may be inhibited in tissue culture by radical scavengers.     

Potent CD14-mediated signalling of human leukocytes by Escherichia coli can be mediated by interaction of whole bacteria and host cells without extensive prior release of endotoxin

How invading microorganisms are detected by the host has not been well defined. We have compared the abilities of Escherichia coli and lipopolysaccharides (LPS) purified from these bacteria to prime isolated neutrophils for phorbol myristate acetate-stimulated arachidonate release, to trigger respiratory burst in 1% blood, and to increase steady-state levels of tumor necrosis factor alpha mRNA in whole blood. In all three assays, bacteria were > or = 10-fold more potent than equivalent amounts of LPS and could trigger maximal cellular responses at ratios as low as one bacterium per 20 to 200 leukocytes. Both E. coli and LPS-triggered responses were enhanced by LPS-binding protein and inhibited by an anti-CD14 monoclonal antibody and the bactericidal/permeability-increasing protein (BPI). However, whereas O polysaccharide did not affect the potency of isolated LPS, intact E. coli carrying long-chain LPS (O111:B4) was less potent than rough E. coli (J5). Furthermore, material collected by filtration or centrifugation of bacteria incubated under conditions used to trigger arachidonate release or chemiluminescence was 5- or 30-fold less active, respectively, than whole bacterial suspensions. Extracellular BPI (not bound to bacteria) inhibited bacterial signalling, but BPI bound to bacteria was much more potent. Taken together, these findings indicate that E. coli cells can strongly signal their presence to human leukocytes not only by shedding LPS into surrounding fluids but also by exposing endotoxin at or near their surface during direct interaction with host cells.