564 Annals ofthe Rheumatic Diseases 1996; 55: 564-570

Third International Workshop on Reactive Arthritis Ann Rheum Dis: first published as 10.1136/ard.55.8.564 on 1 August 1996. Downloaded from 23-26 September 1995, Berlin, Germany

An overview

Reactive arthritis (ReA) is an asymmetrical inflammatory raised about the antibacterial immune response. It has long oligoarthritis of the lower limbs, associated in some been clear that patients with ReA may not have specific patients with typical spondyloarthropathic features, such antibacterial antibodies in their serum and that, con- as enthesopathy and inflammatory low back pain. When versely, because such antibodies are common in the the arthritis occurs in conjunction with urethritis and general population, positive antibacterial serological tests conjunctivitis the patient is considered to have classical may not be of predictive value in diagnosis. In terms of the Reiter's syndrome; patients may, more rarely, display other cellular immune response, although T cells specific for the systemic features, including iritis, mouth ulcers, kerato- triggering bacterium are found in the ReA joint, their role derma blennorrhagica, neurological and cardiac features. in pathogenesis remains uncertain. With regard to the The disease primarily affects young adults and there is a antigens they recognise, the question has again been raised strong association with the class I major histocompatibility ofwhether the entire pathological process can be attributed complex (MHC) antigen, HLA-B27. Classical ReA is to persistent synovial bacterial antigen or whether non- triggered by two major types of bacterial infection, non- bacterial joint autoantigens, or even HLA-B27 itself, may specific urethritis due to Chlamydia trachomatis, or gastro- have a role, particularly in chronic disease. enteritis due to yersinia, salmonella, shigella, or campylo- In this report, and in the meeting abstracts which follow, bacter. A similar condition associated with B27 has been these and other questions are examined and a state of the described in association with Clostridium difficile infection, art pathogenetic hypothesis is proposed based on dis- though in this case it is unclear whether ReA is triggered cussion at the workshop and other recent research. In the directly by the organism or by secondary changes in bowel final section, diagnostic and treatment guidelines are flora. The concept of ReA can also be expanded to include reviewed together with other points of immediate value for oligoarthritides triggered by a variety of other organisms, clinical practice. including streptococcus, borrelia, brucella, and myco- bacteria. In these cases there is no association with HLA- B27 or with spondyloarthropathic features and, except for Pathogenesis ofreactive arthritis: the role of comparative studies, they will not be further considered in bacteria this paper. The prognosis of ReA is generally considered Unlike most forms of arthritis, in ReA the role of an http://ard.bmj.com/ to be good, at least in comparison with other forms of exogenous trigger is quite certain. However, the mechan- inflammatory arthritis like rheumatoid arthritis; however, ism by which a distant bacterial infection of the gut or most patients are affected for several months and a genitourinary tract can trigger arthritis is unknown; specific minority have chronic or recurrent disease. Treatment is questions which were addressed at the meeting include mainly supportive, though second line antirheumatic whether the mechanism is the same for all the bacterial drugs, especially sulphasalazine and methotrexate, are triggers, whether and in what form the bacteria are present used in severe cases. Antibiotic treatment of the triggering in the joint, and what bacterial antigens are recognised by on September 25, 2021 by guest. Protected copyright. infection has been shown to protect against the develop- the immune response. Zinkemagel (Zurich, Switzerland) ment of arthritis, but the role of long term antibiotic and Kaufnann (Ulm, Germany) discussed pathogenetic treatment of established ReA remains uncertain. mechanisms and made the point that intracellular Unlike most forms of human arthritis, ReA has a known microbes can cause damage either by a direct toxic effect trigger with a clear point of onset, a defined genetic on the cells in which they live (cytopathic effect) or by association, and a well described cellular and humoral initiating an antibacterial immune response which, while immune respone against the triggering pathogen in the ineffective at eradicating the organism, does induce tissue joint. Thus as well as being of interest in itself, analysis of damage (non-cytopathic effect). The most well known pathogenetic mechanisms in ReA should also provide a example of the latter is infection by Mycobacterium useful paradigm for other human inflammatory arthritides tuberculosis, and it is likely that this is the important where these factors are not clearly defined. As is often the mechanism in ReA too. There may be a difference, case, however, further research has raised more questions however, in the time scale over which this takes place than it has answered. Early studies of ReA found that the between the various types of ReA. In ReA triggered by triggering pathogen could not be cultured from the joint, Chlamydia trachomatis (reviewed by Ward, Southampton, hence the concept of ReA in contradistinction to septic United Kingdom), there is clear and increasing evidence arthritis; recent studies, have muddied the water as for persistence of live bacteria within synovial tissue. Not triggering bacterial antigens, and in some cases even only has chlamydial DNA been found by polymerase chain bacterial DNA, have been identified in synovial cells by reaction in the joint (Wilkinson et al and Bas et al presented immunological and molecular techniques. The association at this meeting) but recent studies from Hudson and with HLA-B27 has also become less clear cut, with some Schumacher (presented at the American College of evidence from community based and epidemic studies that Rheumatology Meeting, 1995) show that primary mRNA it acts as a severity rather than a susceptibility marker or transcripts from chlamydia can also be identified. As that it is linked with spondyloarthropathic features rather primary transcripts have an extremely short half life and than synovitis alone. In addition, questions have been the rate of initiation of transcription is dependent on the Third International Workshop on Reactive Arthritis 565 growth rate of the organism, this finding shows that viable, and the second on more general aspects of epidemiology metabolically active chlamydiae must be present, albeit in and prognostic features in ReA, which will be discussed a form or in amounts which preclude their culture. The below. According to these studies, the frequency of HLA- Ann Rheum Dis: first published as 10.1136/ard.55.8.564 on 1 August 1996. Downloaded from story for gastrointestinally triggered ReA may be rather B27 is much less in community based than in hospital different. For some organisms, such as yersinia, the con- based series, suggesting that B27 may determine disease cept of a low level persistent infection is not improbable; severity rather than its initiation. Furthermore, the asso- however, as discussed by Heesemann (Wurzburg, ciation also appeared stronger in those patients who had Germany) and Toivanen (Turku, Finland), it has not so spondyloarthropathic features, such as iritis, enthesitis, far proved possible to identify yersinial DNA either in and sacroiliitis, and in patients who went on to chronic joints from patients with ReA or in animal models of disease. One possible interpretation of these data is that yersinia induced disease despite the presence of yersinia HLA-B27 may have relatively little role in acute ReA antigens by immunofluorescence. One possibility, synovitis. suggested by Toivanen, is that yersiniae do persist, but in Inman (Toronto, Canada) examined further the idea some other organ of the body like the gut, and reach the that HLA-B27 on the surface of a cell reduced the ability joint in showers. Further analysis is required, with ultra- of bacteria, such as salmonella and yersinia, to invade it. sensitive polymerase chain reaction systems, especially as This effect was initially shown in HLA-B27 transfected cell in some cases in animal models Heesemann was able to lines in vitro but does not seem to occur in professional culture yersinia yet polymerase chain reaction for yersinia phagocytes. More recently, in studies with HLA-B27 was negative. In contrast with both chlamydia and yersinia, transgenic mice, Inman's group has shown that bacteria the life cycle of shigella (reviewed by Dehio, Tubingen) is persist extracellularly and can be cultured from the gut and such that persistent low level asymptomatic infection spleen for longer in transgenic mice than in controls; the seems unlikely. One interesting mechanism mentioned by transgenic mice, however, do not develop arthritis nor can Toivanen was that certain organisms, notably Clostridium bacteria be detected in their joints. Inman concluded that difficile, might not induce ReA directly but, because oftheir the modulation of invasion by HLA-B27 is subtle, alteration of the bowel mucosa might enable normal bowel especially since there is no evidence of an overwhelming flora to trigger disease. An alternative approach to con- infection risk in HLA-B27 positive subjects. sidering the role of bacteria, by examining their inter- Probably the most popular current hypothesis for the actions with the cellular immune response, will be association of HLA-B27 with SpA is the arthritogenic discussed further below in the section on T cells. peptide theory in which it is suggested that HLA-B27 is, uniquely among class I MHC molecules, able to present an arthritis-inducing peptide or peptides to cytotoxic Pathogenesis ofreactive arthritis: HLA-B27 and CD8+ T cells; initially it is proposed that the arthritogenic other genes peptide would be derived from the initiating bacterium, The observation that HLA-B27 is associated with ReA was though in more chronic cases cross reactivity with an first made over 20 years ago, shortly after the demon- autoantigen may play a part (discussed further in the stration of an association with ankylosing spondylitis (AS); section on T cells). For this hypothesis to be correct in the similar associations have since been shown for other case of ReA, several pieces of information are required; spondyloarthropathies (SpA). As HLA-B27 is now known firstly, can HLA-B27 present peptides derived from the to have nine subtypes, one critical question is whether all triggering bacteria, secondly, can bacteria specific HLA- http://ard.bmj.com/ predispose equally to SpA. Wordsworth (Oxford, United B27 restricted CD8+ T cells be found in patients, and Kingdom) reviewed previously published data from the thirdly, are such T cells pathogenic? Studies reviewed at Gambia suggesting that HLA-B2703 might not be the meeting examined the first two aspects. Bowness associated with SpA in the context of more recent work. (Oxford, United Kingdom) identified some 60 peptides, It has now been shown that a significant number of HLA- from chlamydial proteins whose sequence is known, which B27+ Gambian villagers carry the more usual HLA-B2705 would be predicted to bind to HLA-B27 from their subtype, which is clearly associated in Europeans with AS. sequences and which could be shown to do so in an in vitro on September 25, 2021 by guest. Protected copyright. Yet, in the Gambia neither those who are HLA-B2705 binding assay. So far, one of these peptides, derived from positive nor those carrying HLA-B2703 develop SpA, heat shock protein 70 has been shown to stimulate a T cell suggesting that the explanation for the absence of SpA does line derived from a patient with SpA. Marker-Hermann not lie with the HLA-B27 subtype but rather with some (Mainz, Germany) has previously identified bacteria other aspect of their lifestyle. A rare new HLA-B27 allele, specific, HLA-B27 restricted CD8+ T cell clones from the HLA-B2706, which is found in Thailand, has also not been synovial fluid of patients with enteric ReA and AS; unlike described in association with AS, but again it may be that CD4+ T cell clones from the same patients, these showed the population in which it occurs is not susceptible for other limited repertoire of their T cell receptors. The third reasons. Wordsworth also considered whether other genes question, of pathogenicity of specific T cells, is extremely may be involved in the pathogenesis ofSpA. No association difficult to address. In conclusion, there is some tantalising has been shown, in a number of studies, with proteasome, evidence in favour of the arthritogenic peptide hypothesis, transporter, or T cell receptor genes, but he suggested that but the difficulties encountered by the many groups trying there may be an association, at least in AS, with the class to confirm it suggest that other mechanisms may be II MHC gene, HLA-DR1, or with an extended B27-DR1 important. haplotype. Clearly, however, HLA-B27 remains the One way in which further information may be gained on strongest genetic risk factor so far identified. the association between HLA-B27 and SpA is by study of Numerous explanations for the association with HLA- the various HLA-B27 transgenic animal models currently B27 have been proposed, yet none has gained universal being developed. The most advanced of these is the model acceptance. Unlike AS, where virtually all patients with the developed by Taurog and Hammer (Dallas, Texas, USA). disease are HLA-B27 positive, in ReA a substantial In an update of this model Dr Hammer reminded us that proportion ofpatients do not carry this antigen, suggesting Lewis or Fisher rats, made transgenic for the human HLA- that it cannot be vital for pathogenesis. Leirisalo-Repo B27 and human P2 microglobulin genes, develop arthritis, (Helsinki, Finland) presented two reviews at the meeting, gut inflammation, epididymitis, and skin/nail lesion; some the first on the strength of the association with HLA-B27 of these features bear a strong resemblance to 566 Kingsley, Sieper manifestations seen in human SpA. A high copy number by bacteria specific, HLA-B27 restricted CD8 T cells of the HLA-B27 gene is required to develop these remains attractive, the situation is likely to be much more symptoms, which is hard to reconcile with the idea that complex than this and the only definite conclusion which Ann Rheum Dis: first published as 10.1136/ard.55.8.564 on 1 August 1996. Downloaded from HLA-B27 is simply acting as a restriction element for can be drawn is that both CD4+ and CD8+ cells are likely peptides. The condition does not develop in rats which are to play a part in pathogenesis. bred in pathogen free conditions, suggesting that bacteria Another critical question, discussed by Marker-Hermann have a role (though no specific bacterium has been singled (Mainz), Gaston (Birmingham), Sieper (Berlin), and out), nor can it be induced in nude mice, showing that T Campbell (London) is to determine which bacterial cells are required. Cell transfer experiments have, however, proteins and epitopes are recognised by the bacteria specific shown that the disease is best transferred to naive animals T cells in ReA. This is relevant for several reasons. Firstly, by HLA-B27 positive non-T cells from the bone marrow. the epitope repertoire may be different from that seen in Further examination provides a good chance of elucidating uncomplicated urethritis or enteritis, or epitope recognition the role of HLA-B27 in this model. A more difficult task may differ between acute and chronic ReA. Either of these will be to confirm its relevance to human SpA. would suggest that recognition of certain epitopes may be essential for rapid resolution or elimination of infectious organisms while, conversely, recognition of others may Pathogenesis of reactive arthritis: T cells, bacterial predispose to chronicity. Secondly, for chlamydia, the antigens, and autoantigens epitopes recognised can be compared with the genes Early studies of the immune response in ReA focused on expressed by the chlamydia in the joint. Thirdly, the humoral immune response, but more recent immuno- identification of bacterial epitopes may be of therapeutic logical and histological work has suggested that the cellular relevance as inducing tolerance to harmful epitopes could immune response is pathogenetically more relevant. It is be attempted. Initial approaches to the identification of now beyond doubt that a T cell proliferative response potential epitopes previously relied on the application of specific for the triggering bacterium is found in the ReA biochemical or immunological separation techniques to joint with little or no response in paired blood. Current bacterial preparations but have now been given a major studies are aimed at determining how such cells could boost by the increasing availability of recombinant bacterial induce synovitis. proteins whose identity and purity is much more certain. Because of the association of ReA with HLA-B27 and, Most of the work to date has been carried out on disease more specifically, the consequent arthritogenic peptide induced by yersinia or chlamydia. In yersinia, several hypothesis, it is of major importance to determine whether groups agree that the 19 kDa urease 3 subunit, which has the major response is found in the CD4+ or the CD8+ previously been shown to induce arthritis in mice, is of population. This question, which was addressed at the relevance. This is a cationic protein so will stick readily to meeting by Gaston and Young (Birmingham) and by cartilage, which may be of importance. The 14 kDa L23 Marker-Hermann (Mainz), is more difficult to answer than ribosomal protein also seems to be a relevant antigen. In it seems. Two approaches, immunohistological examin- chlamydial disease the 57 kDa heat shock protein, ation of sections from ReA joints and in vitro cell cycle analogous to the 60 kDa heat shock proteins found in studies, have been used. Immunohistological studies have almost all bacterial and mammalian cells, has been found shown that, as in the other inflammatory arthritides, most by two groups to be a major target antigen. Although at T cells in the joint are CD4+. Many T cells in the joint, first sight the recognition of such a widely expressed heat http://ard.bmj.com/ however, are not specific for the aetiological antigen but shock protein might suggest that cross reactivity with are "bystanders" migrating through the inflamed joint as human antigen was an important pathogenetic mechan- they would through any inflamed tissue; such cells may ism, in fact the epitope recognised has little similarity and make a contribution to perpetuating inflammation but are no cross reactivity with its human counterpart. Clones not the primary progenitors. In an attempt to refine the have also been derived which recognise the 18 kDa histone immunohistological approach, investigators have exam- protein, another cationic protein, and a 30 kDa antigen ined the presence of activation markers on T cells, which has not been fully identified. In contrast, despite on September 25, 2021 by guest. Protected copyright. suggesting that the most relevant subset would express intensive study by at least one group, no clones have been more activation markers. Unfortunately, the results found to recognise the chlamydial major outer membrane conflict both between different experimenters and between protein, which is a major component of the protective different activation markers so no firm conclusion can be immune response to chlamydia in normal individuals. drawn. Turning to the in vitro analyses, both in bulk cell Interestingly, chlamydia in the joint has been shown to cycle studies and in studies of T cell clone phenotype, the express only low levels of major outer membrane protein vast majority of cells which proliferate in response to the but continues to express the 57 kDa heat shock protein. triggering bacteria are CD4+. Again, this cannot be In further support of the idea that there may be different considered the definitive answer as culture conditions may recognition in patients with ReA compared with those who selectively allow CD4+ rather than CD8+ cells to grow. develop only uncomplicated urethritis, a 23 kDa There is certainly clear evidence in mice that chlamydia chlamydial antigen has been found that is recognised by and enteric bacteria can generate cytotoxic T cell re- patients with urethritis but not by those with ReA. sponses. Furthermore, as mentioned above, using specific To date, all the effort in ReA has gone into identifying culture conditions and antigen presenting cells, Marker- bacterially derived peptides that stimulate ReA synovial T Hermann derived bacteria specific CD8+ T cell clones cells. There has been no systematic search for autoantigens from the ReA joint and interestingly, these, unlike their in this condition, though at least in the more chronic or CD4+ counterparts, show a restricted T cell receptor persistent cases a transition from a purely infectious to a usage.To complicate the issue further, ithas become moreautoimmuneprocess seems plausible. Autoantigens, apparent that some CD8+ T cells in the ReA joint display such as collagen type II, proteoglycans, or HLA-B27, have MHC unrestricted but bacteria specific cytotoxicity; been suggested as stimulatory proteins in another although some of these are T cell receptora,B positive, spondyloarthropathy, ankylosing spondylitis. A presenta- others carryry5the T cell receptor. The epitope specificity tion by Wucherpfennig (Boston), suggesting that the T cell and role of these unusual cells remain to be established. expansion to myelin basic protein seen in multiple sclerosis Thus, although the idea that ReA synovitis may be induced might be initially virally driven, served to focus on the idea Third International Workshop on Reactive Arthritis 567 that the infection-autoimmune transition should be more treatment. Much of what we know about the clinical extensively investigated in ReA. aspects of ReA stems from the careful studies performed

in the past in Scandinavia, especially in Finland. Leirisalo- Ann Rheum Dis: first published as 10.1136/ard.55.8.564 on 1 August 1996. Downloaded from Repo (Helsinki, Finland) presented interesting results Pathogenesis ofreactive arthritis: studies of from a 20 year follow up epidemiologic study in Finland. cytokines There had clearly been some change in the organisms The role of cytokines in the pathogenesis ofReA and other triggering ReA. In sporadic ReA the incidence of forms of arthritis has been a recent topic of interest. As chlamydia induced disease remained constant while the alluded to above, although it is relatively easy to establish incidence of yersinia arthritis was decreasing, associated the phenotype of the bacteria specific T cells in the joint, with a general decrease in the incidence of all yersinia it is much more problematical to analyse the functional role infection. Conversely, the incidence of salmonella induced of such cells. In this respect, cytokines can be used as ReA was increasing in Finland. In general, the frequency surrogate markers. Suggesting that interferon -y was of cases of ReA induced by a particular organism mirrors important in the elimination of intracellular bacteria while interleukin-4 and interleukin-10 might encourage bacterial INITIATION PHASE persistence, Sieper (Berlin) investigated the presence of these molecules using a variety of methods, including immunohistology and polymerase chain reaction. Inter- estingly, most synovial tissue whether from patients with ReA or rheumatoid arthritis showed the presence of EXPOSURE El-> LOCAL INFECTION TO TRIGGERING I >- WITH OR WITHOUT interferon y, but interleukin-4 was found much more BACTERIUM SYMPTOMS commonly in ReA, whatever the stage of the disease. Clearly, there is considerable scope for further analysis here looking at the many other cytokines which may *Bacnrial load -Bacterial strain contribute to the immune response in the synovium. *Local host immunity -Systemic host immunity Cytokines

Pathogenesis ofreactive arthritis: a comprehensive hypothesis Based on the studies presented above, we have developed a hypothesis for the pathogenesis of ReA (figure). In this ACUTE REACTIVE ARTHRITIS hypothesis the pathogenesis is divided into three phases: the initiation phase, the acute ReA phase, and the chronic ReA phase. In the initiation phase the subject is exposed or to the triggering bacterium (chlamydia, yersinia, B27+ =:E > = salmonella, shigella, campylobacter) and develops a local REACTnVE SPONDYLOARTHROPATHIC primary infection (gastroenteritis or urethritis) which may ARTHRMS FEATURES or may not be symptomatic. This primary infection resolves in most subjects without sequelae but in a minority SYNOVITIS http://ard.bmj.com/ of people ReA (or another secondary consequence such as B 27- - pelvic inflammatory disease) ensues. The reason why this happens in some patients but not others is not known; 7()G37 0g however, both host specific and, possibly, bacterial strain specific factors-may be important. In the acute ReA phase all subjects develop a peripheral synovitis, but classical spondyloarthropathic features are primarly seen in HLA- on September 25, 2021 by guest. Protected copyright. B27+ subjects. This dichotomy may suggest that different mechanisms are responsible for arthritis and spondylo- CHRONIC REACTIVE ARTHRITIS arthropathic features. In acute arthritis the presentation of bacterial peptides to CD4+ T cells may be of primary importance, whereas other mechanisms more associated with B27-, such as presentation to CD8+ T cells or presentation of HLA-B27 derived peptides, may be relevant to the spondyloarthropathic manifestations. In the chronic ReA phase we postulate that the dichotomy between HLA-B27+ and HLA-B27- subjects continues, although those who are HLA-B27+ are more likely to develop persistent or recurrent disease. The factors which predispose to chronicity or recurrence are likely to include the host genetic and immune (especially cytokine) back- ground, persisting bacterial antigen, and possibly the evolution of responses to cross reactive autoantigens (Sieper J, Kingsley G. Recent advances in the pathogenesis of reactive arthritis. Immunol Today 1996; 17: 160-3.). * BACTERIUM @ BACTERIAL PEPTIDE ( Clinical aspects ofreactive arthritis: epidemiology, HLA-827 * HLA-827 DERIVED PEPTIDE clinical features, prognosis, and treatment '= HLA-DR 0 AUTOANTIGEN DERIVED PEPTIDE SACROIUITIS, ENTHESOPATHY, IRITIS The last part of the workshop concentrated on more clinical aspects of ReA, including diagnosis, prognosis, and Hypothetical schemefor the pathogenesis ofReA 568 Kingsley, Sieper the frequency of primary infection with that organism or refractory and to those who develop recurrent or chronic within the population. However, an exception is campylo- disease. The second line agents that are usually considered bacter, which is a relatively frequent cause ofgastroenteritis are sulphasalazine and methotrexate, though azathioprine Ann Rheum Dis: first published as 10.1136/ard.55.8.564 on 1 August 1996. Downloaded from and which rarely seems to progress to ReA. Although has also been used. Unfortunately, many studies of these similar changes are likely to have occurred in other drugs in ReA are short term and uncontrolled and countries, because the cause of ReA depends so closely on therefore firm treatment guidelines cannot be drawn up. the local prevalence of the organism, the incidence of the Nevertheless, Zeidler proposed the following policies: various forms of ReA will vary from one locale to another. sulphasalazine for a prolonged initial attack, methotrexate In epidemic disease the causative organisms again vary. In (or even azathioprine) if the disease became more severe developed countries epidemic ReA is mainly due to and refractory, and longer term sulphasalazine for patients salmonella, but shigella also plays a part. Although Shigella with recurrent disease. He also noted two particular points sonnei is the commonest strain in developed countries, its about methotrexate; firstly, it is especially efficacious in association with ReA is uncertain, and most ReA cases are mucocutaneous lesions and, secondly, it should be avoided probably associated with Shigella flexneri. World wide, in HIV positive patients. especially in less developed countries, Shigellaflexneri is, in The role of antimicrobial treatment in ReA is another any case, the major pathogen. area of major interest. There are two related questions. The study also provided some interesting information Firstly, can antibiotic treatment for the primary infection about prognosis. The 20 year prognosis was influenced by prevent subsequent ReA and, secondly, can antibiotic four major factors: the nature of the triggering infection, treatment in established disease shorten its course. In the presence of HLA-B27, the patient's gender, and the sexually acquired ReA it is clear that the use of appropriate presence of recurrent arthritis. When yersinia arthritis was antibiotics to treat the initiating urethritis can substantially compared with classical Reiter's syndrome (presumed but decrease the risk of subsequent ReA. Some studies have not proved to be primarily due to Chlamydia trachomatis also suggested that prolonged tetracycline treatment after non-specific urethritis) it was found that the patients with the onset of arthritis can decrease its duration and severity Reiter's syndrome had worse joint symptoms, more but this is far from being proved. In enteric ReA the chronic or recurrent disease, and more joint erosions. situation is still less clear: for most of the primary Interestingly, the incidence of other features, such as infections, antibiotic treatment is generally considered sacroiliitis, spondylitis, and iritis, did not differ between the inappropriate and such data as there are do not support two groups. As already alluded to above, HLA-B27 antibiotic use after the onset of ReA. A trial with cipro- patients had a worse prognosis. Recurrent arthritis, floxacin, however, which is ongoing in Berlin, and a pan- enthesopathy, sacroiliitis, and iritis, as well as progression European study of azithromycin, which is just starting, to a chronic condition including ankylosing spondylitis was should help to answer these questions. The more basic much commoner in these subjects. True spondyloarthro- studies examining whether the triggering organisms for pathic features, such as enthesopathy, sacroiliitis, and ReA persist in the joint or elsewhere in the body, and, if progression to anklosing spondylitis, were unknown in the so, in what state, will also help to design appropriate HLA-B27 negative subjects. Sacroiliitis and progression to antimicrobial treatment. ankylosing spondylitis, but not other features, were worse An alternative therapeutic modality which has become in men with yersinia arthritis than in women; there were popular in the treatment ofwvarious immune rheumatic too few women in the group with Reiter's disease to make diseases is oral tolerance. Van Eden (Utrecht, http://ard.bmj.com/ a meaningful comparison. Finally, as might seem self Netherlands) reviewed the evidence for its effectiveness in evident, patients who have disease recurrences get more rat adjuvant arthritis. He concluded that certain myco- chronic disease; interestingly, however, most still do not bacterial peptides, but not others, when given orally could get erosive disease. make the animals tolerant to adjuvant arthritis. Of interest, Mielants (Ghent, Belgium) presented an exhaustive the antirheumatic drug, Subreum', an Escherichia coli analysis of their series of studies of colonoscopic changes extract, which is also thought to be an oral tolerogen, also in patients with spondyloarthropathy. In brief, they suppressed arthritis in these rats, though less effectively on September 25, 2021 by guest. Protected copyright. showed that many patients with various spondylo- than the mycobacterial peptides. The mechanism of action arthropathies have inflammatory changes in the gut, usually of oral tolerance remains hotly disputed by the various not associated with clinical features of intestinal disease. In workers in the field, though one popular theory, supported some cases these lesions are histologically acute and by van Eden's studies, is that it induces Th2 (interleukin-4, disappear on repeat examination; other patients develop interleukin- 10, and transforming growth factor 3 pro- more chronic enteric disease (which appears to predispose ducing) T cells in the gut, which then migrate to the joint to chronicity ofthe accompanying locomotor disease). Such and suppress local immune activation. Sieper (Berlin, changes are rarely seen in the general population. These Germany) reviewed the use of oral tolerance in human data have normally been interpreted as showing that the gut rheumatoid arthritis where, once again, the data are has a causative role in many spondyloarthropathies. Recent conflicting, with some studies showing efficacy and others studies, however, have shown that even patients with not. He further made the point that in any case oral sexually acquired ReA have colonoscopic lesions in about tolerance might not be pertinent to ReA where, unlike 25% of cases, suggesting that in some cases these lesions rheumatoid arthritis, there appeared to be considerable represent yet another extra-articular manifestation of local interleukin-4 production in the joint. In addition, if spondyloarthropathy. ReA were truly due to persistent bacterial antigen in the Treatment of ReA, like that of most rheumatic diseases, joint, no benefit would be anticipated as interleukin-4 remains largely empirical. Most patients who have self would exacerbate bacterial persistence. limiting disease receive only symptomatic treatment with anti-inflammatory agents and, where appropriate, intra- articular steroid injections. Disease modifying agents are Clinical aspects ofreactive arthritis: clinical and increasingly prescribed, however, and Zeidler (Hannover, laboratory aids to diagnosis Germany) reviewed the rationale for their use. The In the final section of the workshop a series of consensus consensus appears to be that disease modifying agents groups were held in an attempt to develop diagnostic should be given to patients whose initial attack is prolonged guidelines of value for clinical practice. Third Intemnational Workshop on Reactive Arthritis 569

CONSENSUS ON DIAGNOSTIC CLINICAL FEATURES chlamydia. If chlamydia is cultured from a patient without Various diagnostic criteria have been developed for the recent genital symptoms it cannot be presumed with spondyloarthropathies, including ReA; those from the certainty to be related to their arthritis. Nevertheless, in the Ann Rheum Dis: first published as 10.1136/ard.55.8.564 on 1 August 1996. Downloaded from European Spondyloarthropathy Study Group have absence of a clear enteric trigger, urethral culture should probably been the most extensively validated. However, all be considered as treatment of genital chlamydia will avoid these criteria viewed ReA in the context of the spondylo- other consequences such as pelvic inflammatory disease. arthropathy disease group. As discussed above, most Heesemann (Wurzburg, Germany) pointed out the major patients with ReA, especially if they are HLA-B27 technical problems in detecting some organisms associated negative, may never develop spondyloarthropathic with ReA which are quite fastidious in their growth features. More recently the German Society of requirements. For yersinia, shigella, salmonella, and Rheumatology proposed that ReA should be diagnosed in campylobacter, stool culture should be used, but selective patients with an asymmetrical predominantly lower limb or enriched media may be required. Salmonella may oligoarthritis and a history or microbiological evidence of additionally be excreted in the urine. For chlamydia, preceding infection. These criteria, however, have not been urethral swab and culture is the traditional detection validated. method, but this organism is often difficult to grow. In At the workshop Zeidler (Hannover, Germany) and many laboratories, therefore, polymerase chain reaction for Toivanen (Turku, Finland) proposed that the diagnosis of chlamydial DNA is being used on urethral swabs or even ReA should require the presence of typical clinical features urine. The question of what cultures should be done in (lower limb or asymmetrical oligoarthritis ± enthesopathy patients with an arthritis typical of ReA, but no symptoms ± sacroiliitis) plus evidence ofinfection within the past four of primary infection, left the group divided as there was no weeks with typical organisms such as chlamydia or yersinia; agreement on the pick up rate. It was generally thought to exclusion criteria would be required for other known be quite high for chlamydia and lower for enteric rheumatic diseases, such as gout and other spondylo- organisms, but few hard data exist. arthropathies. The cut offpoint offour weeks for preceding If the interpretation of culture results can be difficult, infection is somewhat arbitrary as some patients may have serological tests, by comparison, are a veritable minefield. infection six or more weeks before arthritis; however, the Heesemann (Wurzburg) discussed the various antibody extension of the time beyond four weeks makes these tests available. For yersinia there is a Widal-type criteria less specific. They further suggested that in view agglutination reaction, but enzyme linked immunosorbent of the weakening association of HLA-B27 with ReA its assay (ELISA) or immunoblotting for the YOP protein is determination should play no part in diagnostic criteria. considered more accurate. Again for salmonella, the Widal Other members of the group were uncertain how much test remains in use but has been abandoned in other emphasis should be placed on the presence of centres as too unreliable; an ELISA for salmonella lipo- enthesopathic features because these features are only polysaccharide is available in some laboratories. Immuno- found in a minority of patients. The issue of whether ReA blotting is available for campylobacter, but for shigella should be diagnosed in a patient with enthesitis but no there is currently no serological test. How should the peripheral arthritis remained unresolved. In patients with finding of a significant titre of antibodies to an enteric peripheral arthritis it was agreed that additional features, organism be interpreted? Although there are few hard data whether of spondyloarthropathy or of Reiter's syndrome directly concerning patients with ReA, it is clear that a (conjunctivitis, skin lesions, and so on), should be correct interpretation requires some knowledge of the http://ard.bmj.com/ recorded but not required for diagnosis. prevalence of such antibodies in the local population. For example, yersinia infection is relatively unusual in Britain compared with Finland; appropriate local microbiological USEFULNESS OF POTENTIAL DIAGNOSTIC TESTS advice should be sought in cases of doubt. Serological tests The remainder ofthe consensus session was devoted to the are available for Chlamydia trachomatis, including micro- methods available for obtaining evidence of preceding immunofluorescence (which is time consuming and infection. In what is probably a minority of patients this difficult) and the more usual ELISA techniques. Vischer on September 25, 2021 by guest. Protected copyright. is a simple problem for they have a symptomatic preceding (Geneva, Switzerland) presented data from his studies on urethritis or gastroenteritis and culture of urethral or stool the diagnostic usefulness of detecting IgM, IgA, and IgG samples reveals appropriate organisms. In such patients no antibodies in the serum and synovial fluid of patients with further tests are needed. However, many patients, particu- oligoarthritis. In serum, even when all three antibody larly those with chlamydia infection, do not have any isotypes were detected, this gave a positive predictive value symptoms of their primary infection; this is probably the of only 58% because of the high prevalence of such norm in women. Although this is rare in most forms of antibodies in the general population and the existence of enteritis, it can occur; in yersinia infection, asymptomatic patients with chlamydia induced ReA who do not make a infection may be more usual. In addition, by the time humoral immune response against the organism. He patients present with arthritis the primary infection may concluded that serum antibodies were not really useful in have resolved so local culture is unsuccessful. In such daily clinical practice unless the patient came from an area cases, even if the patient had, for example, a urethritis, it where local antibody prevalence was unusually low. He will be unclear whether this was the initiating infection or also presented limited data suggesting that the detection merely a clinical manifestation of Reiter's syndrome. In of chlamydia specific IgA in the synovial fluid might be these circumstances, clinicians usually resort to serological more helpful, but further studies are required. testing; more recently, lymphocyte proliferation of synovial Regarding the more experimental diagnostic techniques T cells and detection of bacterial antigen in the joint by of bacterial detection by immunofluorescence, bacteria immunological or molecular methods have been used in specific synovial lymphocyte proliferation, and bacterial diagnosis. What is the true usefulness of such tests? DNA detection by polymerase chain reaction, it was Even the interpretation of stool and urethral cultures generally agreed that none was currently ready for use in may not be as clear cut as it seems. Although carriage of the clinic. Granfors (Turku, Finland), who has enormous enteric organisms associated with ReA is rare, a substantial experience of the use of bacterial immunofluorescence to minority of the population, possibly as high as 20% in detect salmonella, shigella, and yersinia, felt that it would sexually active young people, are asymptomatic carriers of probably never be suitable for routine use because it was 570 Kingsley, Sieper

Table 1 Diagnostic criteriafor reactive arthritis Table 2 Laboratory testsfor preceding infection Typical peripheral arthritis Stool culture

Predominantly lower limb, asymmetric oligoarthritis Helpful if positive; should be routine ifprevious diarrhoea; stool culture in Ann Rheum Dis: first published as 10.1136/ard.55.8.564 on 1 August 1996. Downloaded from absence of diarrhoea rarely positive plus Urethral culture Evidence ofpreceding infection Urethral culture often positive in absence of symptoms; need to interpret in (a) Where clear clinical diarrhoea or urethritis within preceding four weeks, light of local asymptomatic carriage rate laboratory confirmation is desirable but not essential (b) Where no clear clinical infection, laboratory confirmation of infection is Urine/urethral PCR* essential These tests can be used, where available, instead of urethral culture or urethral immunofluorescence for bacteria Exclusion criteria Patients with other known causes ofmono/oligoarthritis, such as other defined Serology spondyloarthropathies, septic arthritis, crystal arthritis, Lyme disease, and Chlamydial IgG of no value because high prevalence in community; rising titre streptococcal ReA, should be excluded or IgA antibodies useful in presence of non-specific urethritis history The diagnosis of ReA does not require the presence of HLA-B27 or extra- Yersinia, salmonella, and campylobacter antibodies by ELISA: IgG articular features of Reiter's syndrome (conjunctivitis, iritis, skin lesions, non- antibodies-a fourfold change in titre or a strongly raised titre (difficult to infectious urethritis, cardiac and neurological features) or typical specify; depends on local situation); IgA or IgM antibodies-may be more spondyloarthropathic features (inflammatory back pain, alternating buttock specific; useful if >2 standard deviations above control populations pain, enthesitis, iritis) but these, ifpresent, should be recorded Haemagglutination (Widal) tests for yersinia or salmonella are specific for recent infection but insensitive, especially for salmonella extremely technically demanding, very time consuming Shigella serology is of no use because of cross reactivity with Escherichia coli and relatively insensitive. Similar arguments were The following tests should currently be regarded as research tools: advanced by Braun (Berlin, Germany) against routine use Immunofluorescence for bacteria in synovium and proliferation of synovial lymphocytes of the synovial lymphocyte proliferation test. In this test These tests are labour intensive and technically difficult tests whose sensitivity lymphocytes are separated from fresh synovial samples and and specificity is uncertain. They are unlikely ever to be suitable for routine tested for their proliferation to a panel ofbacterial antigens. diagnostic use Apart from the demanding nature of the technique, the PCR for chlamydial DNA in the joint A potentially valuable test which could be practicable for routine use. Its requirement for some 10-20 ml of synovial fluid, and the sensitivity and specificity are being further investigated. This approach cannot inability to store the synovial fluid before separation, many currently be used for enteric ReA because it is uncertain whether DNA from samples respond to more than one bacterium, leading to enteric organisms reaches the joint significant difficulties in interpretation. In contrast, *PCR = polymerase chain reaction. Kingsley (London, United Kingdom) suggested that detection of chlamydial DNA in synovial fluid by the Financial help was also received from Bayer, Kabi-Pharmacia, Tosse, Lederle, polymerase chain reaction has great potential as a diag- Ciba-Geigy, Smith-Kline Beecham, and Henning. nostic technique. Only small volumes of fluid, or even better, small needle biopsy samples are required, which can United Medical and Dental Schools, GABRIELLE KINGSLEY be stored frozen until tested and many samples can be Guy's Campus, tested simultaneously. Polymerase chain reaction tech- Guy's Hopsital, London, niques are already in use to detect microbes in many and Lewisham Hospital, London routine microbiological laboratories. It remains, however, Klinikum Benjamin Franklin, JOCHEN SIEPER for the experimental systems currently used in research to Free University ofBerlin, and be converted into robust and reproducible systems for Deutsches Rheuma Forschungszentrum, Berlin, Germany http://ard.bmj.com/ routine use and for them then to be tested for sensitivity and specificity. So far, the polymerase chain reaction Key references cannot be applied to the enteric organisms as, with the BACKGROUND exception of a few samples positive for campylobacter 1 Kingsley G, Panayi G. Antigenic responses in reactive arthritis. Rheum Dis Clin North Am 1992; 18: 49-66. DNA their DNA has not been detected in the joint. 2 Sieper J, Braun J. Pathogenesis of spondylarthropathies. Persistent bacterial However, it is important to note that initial attempts to antigen, autoimmunity, or both? Arthritis Rheum 1995; 38: 1547-54. detect chlamydial DNA in the joint were negative, THE ROLE OF BACTERIA IN REACTIVE ARTHRITIS probably because of a combination of low levels of 3 Ward M E. The immunobiology and immunopathology of chlamydial on September 25, 2021 by guest. Protected copyright. infections. APMIS 1995; 103: 769-96. organism and the presence of inhibitors in synovial fluid. 4 Heesemann J, Gaede K, Autenrieth I B. Experimental Yersinia entercolitica Indeed, the later successes required the development of infection in rodents: a model for human yersiniosis. APMIS 1993; 101: 417-29. extremely sensitive techniques far removed from the 5 Bas S, Griffais R, Kvien T K, Glennas A, Melby K, Vischer T L. routine techniques capable of detecting chlamydia in urine Amplification of plasmed and chromosome Chlamydia DNA in synovial fluid of patients with reactive arthritis and undifferentiated seronegative or urethral swabs. Similar attempts will have to be made oligoarthropathies. Arthritis Rheum 1995; 38: 1005-13. for the enteric organisms before the search is abandoned. GENETICS OF REACTIVE ARTHRITIS A summary of the consensus groups is presented in the 6 Feltkamp T E W, Khan M A, Lopez de Castro J A. The pathogenetic role form of two tables, the first presenting diagnostic criteria of HLA-B27. Immunol Today 1996; 17: 5-7. for ReA and, the second, the usefulness of the available IMMUNOLOGY OF REACTIVE ARTHRITIS tests in diagnosing preceding infection. These criteria are 7 Gaston J S H, Deane K H 0, Jecock R M, Pearce J H. Identification of 2 chlamydia trachomatis antigens recognised by synovial T cells from not intended to compete with those already established for patients with chlamydia reduced ReA. JRheumatol 1996; 23: 130-6. the spondyloarthropathy group by the European 8 Mertz A, Daser A, Skumik M, et al. The evolutionary conserved ribosomal probein L23 and the cationic urease 13-subunit of yersinia enterocolitica Spondyloarthropathy Study Group but, rather, to take 0:3 belong the the immunodominant antigens in yersinia-triggered account of the fact that, particularly in the community, reactive arthritis: implications of autoimmunity. Molecular Medicine 1994; 1: 44-55. many patients with ReA do not show spondyloarthropathic 9 Hermann E, Yu D T Y, Meyer zum Buschenfelde K-H, Fleischer B. HLA- features and would therefore be excluded by previous B27 restricted CD8 T cells derived from synovial fluids of patients with reactive arthritis and ankylosing spondylitis. Lancet 1993; 342: 646-50. criteria. Two further points require emphasis; firstly, these 10 Simon A K, Seipelt E, Sieper J. Divergent T-cell cytokine patterns in criteria will require prospective validation and, secondly, inflammatory arthritis. Proc Natl Acad Sci USA 1994; 91: 8562-6. for the moment they take a narrow view of ReA, excluding CLINICAL ASPECTS OF REACTIVE ARTHRITIS virally induced diseases, Lyme disease, and streptococcal 11 Lauhio A, Leirisalo-Repo M, Lahdevirta J, Saikku P, Repo H. Double-blind placebo-controlled study of three-month treatment with lymecycline in arthritides, for example. reactive arthritis with special reference to chlamydia arthritis. Arthritis Rheum 1991; 34: 6-14. We would like gratefully to acknowledge the financial support we received from 12 Mielants H, Veys E M, Cuvelier C, et al. The evolution of spondylo- the Fritz Thyssen Stiftung and the European Concerted Action for arthropathies in relation to gut histology. II. Histological aspects. J Rheumatology without whose help the meeting would not have been possible. Rheumatol 1995; 22: 2273-8. Annals ofthe Rheumatic Diseases 1996; 55: 571-584 571

Abstracts ofinvited "primary" diseases may actually reflect Southampton General Hospital, S016 6YD, lectures immunopathologies triggered by unknown or United Kingdom unrecognised common or special viruses. Ann Rheum Dis: first published as 10.1136/ard.55.8.564 on 1 August 1996. Downloaded from Bas et al, using four different polymerase chain reactions (PCRs) for three different Interactions between the immune chlamydial gene targets, showed that system and infectious agents: Immune response to intraceliular Chlamydia trachomatis was present in the protection, immunopathology, bacteria joints of 22 of 71 patients with various automimunity, subversion Stefan H E Kaufmann arthropathies.' Other studies have shown the Rolf M Zinkemagel presence of chlamydiae in a layer of cells Department ofImmunology, University of Urm, beneath the synovium and have suggested Institute ofExperimental Immunology, Albert-Einstein-Allee 11, D-89070 Ulm and that the organism must be viable because University ofZurich, Switzerland Max-Planck-Institute for Infection Biology, primary chlamydial mRNA transcripts can be Monbijoustrasse 2, 10117 Berlin, Germany detected by reverse transcriptase PCR in Viruses have coevolved with vertebrate hosts synovial tissue from patients with arthritis. and their immune systems to reach Although all intracellular bacteria, by Chlamydiae, unlike yersiniae, are obligate evolutionarily balanced states; therefore definition, reside within host cells, their intracellular pathogens. Glycosamino- many of these virus-host arrangements show precise intracellular location may vary. glycans, abundant in joints, help mediate limiting facets of the immune system. For Mycobacterium bovis BCG remains in the chlamydial adhesion to target cells. Antibody example, immunological unresponsiveness to endosome, whereas, Listeria monocytogenes directed against neutralising epitopes on the viruses may be established by infection of lives in the cytosol. Mycobacterium tuberculosis major outer membrane protein (MOMP) the thymus, by sequestration of antigens primarily resides in the endosome, though it may block infection of host cells, though exclusively in epithelia or neurons where they has been shown that it can move out into these epitopes are subject to antigenic drift are ignored by T cells, or by exhaustive the cytosol. Protective immunity against (point mutations giving rise to serovariants) differentiation of T cells in the periphery. intracellular bacteria is mediated by T and antigenic shift (arising from recombina- Induction and activation of antiviral T cells lymphocytes. The T cell receptor (TCR) ota tion events). Once established in the host cell, depends on proper antigen presentation in expressing CD4 or CD8 T cells is considered chlamydiae will be protected from neutral- lymphoid organs where sufficient concentra- responsible for "endosomal" or "cytosolic" ising antibody, resisting oxidative stress by tion of cytokines prevails. Mechanisms of T pathogens, respectively. In addition, yb T elaborating heat shock proteins. One ofthese, cell mediated antiviral protection also vary cells seem to be required for efficient a 57 kilodalton protein, shares B and T cell with the virus. For cytopathic viruses either protection. We used gene disrupted mutant epitopes with human heat shock protein 60 CD8+ or CD4+ T cell dependent cytokines mice to assess the role of different T cell (hsp-60) and is thought to have a key role in or antibodies provide protection, dependent populations in immunity against intracellular chlamydial immunopathology. Some pep- upon the site of viral replication. Against bacteria. tides on chlamydial hsp-60 are able to bind non-cytopathic viruses cytotoxic perforin Effective protection against the intra- to HLA-B27. Once chlamydial infection dependent CD8+ T cells are mandatory for cellular bacteria, L monocytogenes, BCG, and becomes chronic, in vitro studies suggest that virus elimination. M tuberculosis, required all three subsets, production of interferon y by T helper 1 and B cell induction by viruses depends very though in varying degrees. Thus protection natural killer cells may lead to a state of much on antigen pattem and organisation; against the endosomal bacterium BCG not persistent infection, in which shedding of repetitive identical determinants with defined only involved CD4 but also CD8 T cells. infectious elementary bodies is curtailed but distances induce an IgM response in- Reciprocally, protection against the cytosolic aberrant chlamydiae are transferred at host dependent of T help, whereas B cell pathogen, L monocytogenes, depended on cell division.2 This may be accompanied by responses against monomeric antigens are CD4 T cells in addition to CD8 T cells. up regulation of immunopathological hsp-60 tightly controlled by T cells. These Although the contribution of yb T cells to and down regulation of the MOMP. Inter- characteristics of B cell responses may protection against BCG and L monocytogenes feron y probably has a protective and a http://ard.bmj.com/ explain some autoantibody responses. The seemed small, these cells were essential for pathological role, determined by the fragile key parameters of antivirally protective combat ofM tuberculosis. balance between the cell mediated immune antibody responses include high affinity and The Thl cytokine, interferon -y, is central responses and chlamydial replication. In apparent absence of so called affinity to protective immunity against intracellular ocular chlamydial infection (trachoma), maturation. bacteria. In contrast, Th2 cytokines seem to severe disease has been associated with Immunopathological consequences of exacerbate disease. Cytokines produced by anergic cell mediated responses to chlamy- protective antiviral immune responses are cells of the innate immune system directly diae. Chlamydial antigens, particularly lipo- seen whenever non- or influence the divergence into Th 1 or Th2 poorly cytopathic polysaccharide, persist for long periods in on September 25, 2021 by guest. Protected copyright. viruses spread too widely before they can be dominated immunity. Thus interleukin-12 macrophages. Chlamydial survival may be stopped by antiviral cytotoxic T cells, (IL-12) favours development of ThI cells, aided by a chlamydial homologue of the antibodies, or other mechanisms; this whereas IL-4 promotes that of Th2 cells. Legionellapneumophila macrophage inhibitory depends on many viral factors (for example, Although the source of early IL-4 production protein. Chlamydial antigens are processed replication kinetics, tropism, susceptibility to remains enigmatic, recent evidence points to by both the exogenous and endogenous interferons, etc) as well as on host factors CD4+ natural killer 1.1+ (NK1.1+) T cell pathways. Animal studies suggest T helper 1 (including kinetics and quality ofT and B cell receptor a,'t (TCRac'nt) lymphocytes. responses and interferon y are more responses). Because T cells recognise Infection with intracellular bacteria induced important for protection than cytotoxic T peptides bound by major histocompatibility the disappearance of these lymphocytes from lymphocyte responses, but the role of the molecules (HLA in humans) and because the the liver and, as a consequence, abolished latter in immunopathology deserves further T cell immune response (and not the non- IL-4 production. We conclude that down study, particularly in reactive arthritis. cytopathic virus) is pathogenic, susceptibility regulation of CD4+ NK1.1+ TCRot3'n to immunopathologically mediated disease lymphocytes by intracellular bacteria favours 1 Bas S, Griffais R, Kvien T K, Glennas A, triggered by seemingly harmless viruses may unconstrained antimicrobial protection. Melby K, Vischer T L. Amplification of with plasmid and chromosome chlamydia DNA in be associated HLA. Aggressive hepatitis synovial-fluid of patients with reactive B virus in man or Key papers by hepatitis lymphocytic Kaufmann S H E. Immunity to intracellular arthritis and undifferentiated seronegative choriomeningitis virus (LCMV) in mice, bacteria. Annu Rev Immunol 1993; 11: 129-63. oligoarthropathies. Arthritis Rheum 1995; 38: where specific CD8+ T cells destroy critical Kaufmnann S H E. Immunity to intracellular 1005-13. infected host cells, illustrates immunopatho- microbial pathogens. Immunol Today 1995; 16: 2 Beatty W L, Morrison R P, Byrne G I. Persistent 338-42. chlamydiae - from cell-culture to a paradigm genesis of such disease. Similarly, the for chlamydial pathogenesis. Microbiol Rev immunosuppression caused by LCMV, and 1994; 58: 686-99. possibly by HIV (particularly if HIV should turn out to be a non-cytopathic virus in vivo), Bacterial persistence in reactive may be caused by T cells destroying antigen arthritis: how do chlamydia evade the presenting cells infected by LCMV. These immune system and persist in the joint? examples may easily impress as autoimmune Michael E Ward Detection of Chlamydia pneumoniae disease if the infectious agents are not known and Chlamydia trachomatis in the and one can speculate that possibly many Molecular Microbiology Group, Mailpoint 814, joints ofpatients with arthritis autoimmune or chronic, degenerative, Southampton University Medical School, N Z Wilkinson*t, G Kingsley*, M E Wardt 572 Abstracts ofinvited lectures

*Rheumatology Unit, Hunts House, Guy's evidence exists to indicate different patho- reactive arthritis in humans. To study the Hospital, London Bridge, London, United genetic mechanisms for reactive arthritis pathogenesis of yersiniosis and its sequela

Kingdom; tMolecular Microbiology, triggered by different groups of bacteria. An appropriate animal models are required. Ann Rheum Dis: first published as 10.1136/ard.55.8.564 on 1 August 1996. Downloaded from Southampton University, Southampton General induction period ofone to three weeks occurs Fortunately, experimental yersiniosis of Hospital, Tremona Rd, Southampton, United always between the initial infection and rodents resembles that of humans and thus Kingdom development of arthritis. During this period offers suitable opportunities for studying the the host becomes immunologically sensitised sequential steps of the infectious process and Whether chlamydial DNA is present in the against the causative microbe. When the the host response.' To analyse the immune synovial fluid of patients with reactive microbial components then enter the joint response we used yersinia susceptible Balb/c arthritis is controversial. Present reports are tissue, a local delayed-type hypersensitivity mice and resistant C57 BL/6 mice.2 As major conflicting and it is unclear whether the reaction manifests as an acute reactive components of the protective immune whole organism or merely bacterial fragments arthritis. So far, synovial localisation of respone we identified antibodies directed are present. To clarify the discrepancies bacterial structures originating from the B27 against the virulence proteins YadA and V between results we developed polymerase independent species has been little studied. antigen.3 Results obtained from the mouse chain reactions (PCRs) to detect C infection model showed the protective role of pneumoniae and C trachomatis, targeting the Key papers T cells which produce interferon Innate Toivanen A, Toivanen P. Aetiopathogenesis of y.' Omp 1 gene and the cryptic plasmid reactive arthritis. Rheumatology in Europe 1995; 24: susceptibility of Balb/c mice to yersinia respectively. Subsequently, 85 synovial fluid 5-8. infection seemed to be associated with samples from patients with a range of Toivanen A, Toivanen P. Epidemiologic, clinical suppression or failure of interferon y rheumatic diseases, but predominantly re- and therapeutic aspects of reactive arthritis and ankylosing spondylitis. Curr Opin Rheumatol 1995; production of infected tissue (liver, spleen). active arthritis and undifferentiated oligo- 7: 279-83. Lewis rats develop reactive arthritis-like arthritis, were tested. disease after intravenous challenge with viru- Both PCRs could amplify about 10 fg of lent Yersinia enterocolitica, serotype 08. the relevant DNA, the equivalent of 10 Yersinia mutants impaired in full virulence organisms, and there was no cross reactivity. Shigelia as a paradigm for bacteria that expression are significantly less arthrito- Thirty patients were positive for C induce reactive arthritis genic.' Microbiological and immuno- trachomatis, which has been verified by Christoph Dehio*, Philippe Sansonettit histological examination of tarsal joints of sequencing, and no patients were positive for yersinia infected Lewis rats showed trans- C pneumoniae. It is likely that C trachomatis is *Max-Planck-Institutfuir Biologie, Abteilung location and persistence of cultivable yer- present in the joint of patients with reactive Infektionsbiologie, Spemannstra,8e 34, D-72076 siniae in synovial tissue accompanied by sig- arthritis and undifferentiated oligoarthritis. C Tiibingen, Germany; tInstitut Pasteur, Unite de nificant infiltration of ot4 T cells during the pneumoniae does not have a prominent role in Pathoginie Microbienne MolMculaire, INSERM prearthritic phase.' 6 7 Thereafter (arthritic reactive arthritis. U 389, 28 rue du Dr Roux, F-75724 Paris phase) the number of cultivable yersiniae Cedex 15, France drops. Surprisingly, treatment of rats with antibody against os4B T cell receptor did not Shigella species cause bacillary dysentery in suppress development of yersinia induced humans by invading the intestinal mucosa of arthritis, whereas treatment with cobra Comparisons between various infections the colon. reactive arthritis: After infection 1-4% of patients venom factor (depletion of complement) did triggering develop reactive arthritis. Compared with SO.6 7 implications for the pathogenesis reactive arthritis induced by other From these results we conclude that Paavo Toivanen intracellular bacteria, this postdysenteric reactive arthritis may be related (a) to an reactive arthritis is of special interest because ineffective immune response against arthrito- Department ofMedical Microbiology, Turku (a) the association with HIA-B27 is genic microorganisms, (b) to synovial tissue University, FIN-20520 Turku, Finland particularly strong, (b) shigella strains show susceptible for translocation of microbes and intra/interspecies variation in their ability to microbial antigen, and (c) to hyperreactivity http://ard.bmj.com/ Infections triggering reactive arthritis can be trigger reactive arthritis, and (c) post- of infected synovial tissue. grouped in two different ways. Firstly, most dysenteric reactive arthritis is the most of them are either urogenital, enteric, or common form of this arthritis in developing 1 Heesemann J, Gaede K, Autenrieth I B. respiratory tract infections; in addition, countries. Experimental Yersinia enterocolitica infection several other more or less generalised infec- in rodents: a model for human yersiniosis. It has been suggested that molecular APMIS 1993; 101: 417-29. tions may induce reactive arthritis, with the mimicry ofbacterial epitopes with epitopes of 2 AutenriethIB, BeerM, BohnE, KaufmannS HE, causative microbe identified or unknown. HLA-B27 may help to induce reactive Heesemann J. Immune responses to Yersinia Another, equally useful way to divide the enterocolitica in susceptible Balb/c and arthritis. This popular is on September 25, 2021 by guest. Protected copyright. microbes associated with reactive arthritis is hypothesis resistant C57 BI6 mice: an essential role for supported by several reports on cross gamma interferon. Infect Immun 1994; 62: by their dependence on the presence ofHLA- reactivity of monoclonal antibodies directed 2590-9. B27. A firm association between bacterial against HLA-B27 or shigella proteins, or 3 Vogel U, Autenrieth I B, Bemer R, HeesemannJ. genera and B27 has been established in only Role of plasmid-encoded antigens of Yersinia both. A particular mimetic epitope to HLA- enterocolitica in humoral immunity against six cases: campylobacter, chlamydia, clos- B27 was identified as part of a polypeptide secondary Y enterocolitica infection in mice. tridium, salmonella, shigella, and yersinia. sequence encoded by a 2 megadalton Microb Pathog 1993; 15: 23-6. The prevalence of B27 positive patients which was found to be associated 4 Bohn E, Heesemann J, Ehlers S, Autenrieth I B. for of these plasmid, Early gamma interferon mRNA expression is varies between 60 and 80% any with a number of arthritogenic Shigella associated with resistance of mice against triggers. For instance, of 19 B27 typed flexneri strains. This plasmid was not present, Yersinia enterocolitica. Infect Immun 1994; patients with Clostridium difficile induced however, in two non-arthritogenic Shigella 62: 3027-32. reactive arthritis reported on so far, 63% were 5 Gaede K I, Heesemann J. Arthritogenicity of sonnei strains. l genetically manipulated Yersinia B27 positive. On the other hand, because no enterocolitica serotype 08 for Lewis rats. clostridial structures have been demonstrated 1 Stieglitz H, Lipsky P. Association between Infect Immun 1995; 63: 714-9. in the synovium, it remains unclear whether reactive arthritis and antecedent infection 6 Gaede K I, Baumeister E, Heesemann J. the arthritis is due directly to C difficile or to with Shigella flexneri carrying a 2-Md Decomplementation by cobra venom factor plasmid and encoding an HLA-B27 mimetic suppresses yersinia-induced arthritis in rats. changes in the intestinal flora, parallel to that epitope. Arthritis Rheum 1993; 36: 1387-91. Infect Immun 1995; 63: 3697-701. occurring, for example, in the intestinal 7 Gaede K I, Nazet M, Bosse D, Hinig T, bypass syndrome. Heesemann J. Treatment of arthritis in Lewis rats by a monoclonal antibody against a,B T A remarkable characteristic of reactive cell receptor: differential sensitivity of arthritides triggered by species of the six B27 What can aniimal models tell us about yersinia-induced arthritis versus adjuvant dependent bacterial genera is their true human reactive arthritis? arthritis. Clin Immunol Immunopathol (in reactive nature-that is, these species do not J Heesemann, K Gaede, I Autenrieth press). usually induce bacterial arthritis. This is in contrast with those bacteria (borrelia, Institute ofHygiene and Microbiology, brucella, haemophilus, leptospira, mycobac- University of Wiirzburg, J7osef-Schneider-StraJ3e terium, neisseria, staphylococcus, strepto- 2, 97080 Wurzburg, Germany coccus, ureaplasma, and vibrio) which are Are CD8+ T cells really the major B27 independent and which may cause both Enteropathogenic bacteria, such as salmon- players in reactive arthritis? reactive and bacterial arthritis. However, no ellae, shigellae, and yersiniae, can induce Elisabeth Marker-Hermann Abstracts ofinvited lectures 573

First Department ofMedicine, University of SF macrophages) is also suggested by the results suggest that antigen recognition by yB Mainz, 55101 Mainz, Germany high "background" proliferative responses T cells is independent of classical MHC which are commonly seen in reactive arthritis molecules and their associated antigen One current theory trying to explain the SF T cells, and by the responses to processing pathways. Indeed, preliminary Ann Rheum Dis: first published as 10.1136/ard.55.8.564 on 1 August 1996. Downloaded from association of HLA-B27 with reactive autologous SF occasionally noted. Flow experiments suggest that at least one T cell arthritis is the "arthritogenic peptide model". cytometry of reactive arthritis SF T cells also clone can be triggered by intact Y This model is based on the physiological shows evidence of activation of CD4+ T enterocolitica. antigen presenting function of HLA class I cells, often to a greater extent than CD8+ Our study supports existing experiments in molecules. This theory proposes that cells. We have derived antigen specific T cells both humans and mice indicating that some "arthritogenic" bacteria produce a peptide from SM biopsy samples cultured in y8 T cells can recognise antigens in a distinct that primes a cytotoxic T lymphocyte interleukin-2, again suggesting activation manner from aot T cells. Experiments in mice response when presented by HLA-B27. (and interleukin-2 receptor expression) of suggest that -yb T cells can influence the Consequently, synovial fluid CD8+ rather CD4+ T cells in SM. Lastly, the antigenic pathology of bacterial infections. Moreover, than CD4+ T cells should have a prominent targets of synovial CD4+ T cells have in yB T cells have been proposed to act as a first pathogenetic role. The in vivo observation several cases proved to be highly cationic line of defence at the mucosal surface, the that severe reactive arthritis may occur in proteins (for example, chlamydial Hcl), a point of entry of bacteria associated with HIV infected individuals essentially devoid property associated with arthritogenicity in reactive arthritis. With our demonstration of functional CD4+ cells supports this experimental models of antigen induced that yS T cells with potent yersinia respon- hypothesis. The reactive arthritis synovitic arthritis. siveness can be found within the disease site, infiltrate contains CD8+ lymphocytes, some In view of these findings it is unlikely that we suggest that a role for yb T cells in the ofwhich are HLA-B27 restricted and specific CD4+ T cells are irrelevant to the patho- pathology of reactive arthritis should be for the triggering bacterium or for genesis of joint inflammation. HIA-B27, in considered. autoantigens. Furthermore, we have isolated conferring susceptibility to reactive arthritis, CD8+ cytotoxic T lymphocytes that kill may determine whether a response by CD4+ bacterial infected targets but are not T occurs in the joint (for example, by restricted by HLA molecules. T lymphocytes influencing whether antigen gets to this site). themselves can present bacterial antigens to such HLA unrestricted CD8+ cytotoxic T Key papers Antigenic targets for T cells in reactive lymphocytes and may thus contribute to the Gaston J S H, Deane K H 0, Jecock R M, Pearce arthritis-which enterobacterial J H. Identification of two Chlamydia trachomatis Is there a role for cytotoxic potential. Peptides derived from antigens recognized by synovial fluid T cells from antigens? bacterial proteins can be recognised by B27 chlamydia-induced reactive arthritis patients. J autoantigens? restricted cytotoxic T lymphocytes. Rheumatol (in press). Elisabeth Marker-Hermann, Rainer Gaston J S H, Pearce J H. Immune responses to Duchmann, Karl-Hermann Meyer zum We should consider the question whether heat shock proteins in reactive arthritis. In: van mimicry of B27 presented peptides is Eden, ed. Stress proteins in medicine. New York: Buschenfelde necessary to explain autoimmune cytotoxic T Marcel Dekker (in press). lymphocyte mediated phenomena and First Department ofMedicine, University of whether the number of T cells (either CD4+, Mainz, 55101 Mainz, Germany CD8+ or -yb+) of certain specificities or the demonstration of oligoclonal expansion of In enterogenic reactive arthritis the preva- subpopulations in reactive arthritis is really Are -yf+ T cells involved in the lence in the affected joints of T lymphocytes decisive for disease induction. pathogenesis ofreactive arthritis? specific for the triggering bacteria is high J L Young, J S H Gaston compared with the prevalence ofT cells from paired peripheral blood samples. However, CCRIS, University ofBirmingham, Edgbaston, recruitment of memory T cells of different Birmingham, United Kingdom specificities into the joint may accompany T cells and the pathogenesis ofreactive this response. There is increasing evidence http://ard.bmj.com/ arthritis: is the CD4+ T cell response A defective or inappropriate cellular immune that T cell reactivity to enterobacterial arthritogenic, regulatory-or response has been postulated to be antigens varies depending on the stage of irrelevant? responsible for reactive arthritis. However, disease and may spread to other antigens, J S H Gaston the precise disease mechanism remains to be that peripheral blood T cell responsiveness to elucidated. In this study we examined the in enterobacterial antigens is often reduced in CCRIS, The Medical School, University of vitro responses of synovial fluid and active arthritis, and that different T cell Birmingham, Birmingham B15 2TT, peripheral blood mononuclear cells stimu- subsets may recognise different dominant United Kingdom lated with autologous B lymphoblastoid cell antigens. on September 25, 2021 by guest. Protected copyright. lines infected with Yersinia enterocolitica, a 1 Specific T cell response to the triggering CD4+ T lymphocytes specific for organisms bacterium associated with triggering reactive enterobacterium: Synovial fluid T cells were which trigger reactive arthritis are readily arthritis. This procedure resulted in a pro- shown to recognise dominantly the entero- demonstrable in the synovial fluid (SF) and found expansion of yb+ T cells which bacterial protein antigens Yersinia entero- membrane (SM) ofaffected joints; these cells displayed major histocompatibility complex colitica urease ,B subunit and Yersinia 50 S have been cloned and their peptide major (MHC) independent cytotoxicity for yersinia ribosomal protein L23. The Escherichia coli histocompatibility complex (MHC) speci- infected target cells. In the absence of B GroEl is recognised in reactive arthritis and ficity determined. However, given the lymphoblastoid cell lines, bacteria fixed with other spondyloarthropathies. Cytotoxic T association between reactive arthritis and the formaldehyde, but not heat inactivated, also cells can also respond to peptides from some class I MHC antigen, HLA-B27, do these preferentially stimulated yB+ T cell pro- of these antigens. class II restricted CD4+ T cells have any liferation from mononuclear cell cultures. We 2 "Secondary" T cell responses to auto- pathogenic relevance? obtained T cell clones after such stimu- logous intestinal flora: Our studies indicated Infections associated with reactive arthritis lations-namely, CD2+, CD3+, CD4-, that in inflammatory bowel disease, laminar provoke a CD4+ T cell response, so their CD8or '°, T cell receptor -y8+. As with cell propria T lymphocytes from inflamed presence in SF and SM is not in itself lines, these clones killed both infected intestine respond to autologous intestinal surprising, as memory CD4+ T cells may be autologous and allogeneic cells. Infected cell flora and thus break the selective tolerance to preferentially recruited to the joint. If lines deficient in MHC class I or class II these bacteria. This mechanism may also be bacterial antigens are present in the joint, expression, or both, were also lysed, relevant in reactive arthritis, where T cells however, T cells will not only be recruited but including K562 (class I and II"), C 1R (HLA- with specificity for autologous gut flora could also activated. In these circumstances their A-, B-, C'"), and Daudi (,B2m-). Likewise, migrate to extraintestinal (joint?) tissues and activation is likely to be important in driving infected T2 cells, which have impaired contribute to inflammation. the inflammatory process. Evidence for the antigen processing for both MHC class I and 3 Autoantigens: Autoreactive CD4+, presence of antigen in the joint is increasing; class II presented peptides, were susceptible CD8+, and -yS+ T cells can be isolated from for Chlamydia trachomatis, organisms (in targets. Interestingly, a lower level of reactive arthritis synovial fluid. The signifi- small numbers) are present and may produce cytotoxicity was observed when autologous cance of these findings is still unclear; cross antigens, such as heat shock protein 60, rather than allogeneic targets were used. We reactivity to enterobacteria is not a pre- which we have recently shown to be a target have yet to determine if HLA-B27 is requisite to explain the induction and in vivo of the CD4+ response. Antigen in SF (or on implicated in this reduced cytotoxicity. Our expansion of such T cells. 574 Abstracts ofinvited lectures

Molecular mimicry in T cell mediated *Nuffield Department ofClinical Medicine, Prevalence ofHLA-B27 in patients with autoimmunity: relevance to Jrohn Radcliffe Hospital, Oxford OX3 9DU, reactive arthritis spondyloarthropathies United Kingdom; tRoyal National Hospitalfor Kai W Wucherpfennig Rheumatic Diseases, Bath, United Kingdom Patients collectedfrom: HLA-B27+ (V.) Ann Rheum Dis: first published as 10.1136/ard.55.8.564 on 1 August 1996. Downloaded from University clinic 77-88 Dana-Farber Cancer Institute and Harvard A genetic contribution of ankylosing spondy- Community referral cinic 45-83 con- Self referral 45-69 Medical School, Boston, MA, USA litis is clearly evident from the different Community outbreak 36 cordance rates in monozygotic (60%) and Single outbreak 0-69 Viral and bacterial infections may be dizygotic twins (12%). It has how been important in the initiation or progression of established beyond reasonable doubt that human T cell mediated autoimmune dis- HLA-B27 itself is an important part of this different sources.3 The incidence of joint eases. T cells specific for immunodominant genetic component, but it is apparent that symptoms varies from 2 to 30%, but the self peptides may be activated by viral/ other genes are also likely to play a part. prevalence of HLA-B27 in such patients is bacterial peptides with sufficient structural Analysis of the nine B27 subtypes currently considerably lower than previously reported similarity ("molecular mimicry").' recognised suggests positive associations with (table). Most such reports concern By using the knowledge of how an *01, *02, *04, *05, *07, but there is emerg- salmonella outbreaks, and calculations from immunodominant epitope of human myelin ing evidence that *03, *06, and *09 may not the reports show that an average of 29% of basic protein (MBP, residues 85-99) is bound be associated with the disease or only weakly the patients have HLA-B27. by HLA-DR2 and which of its amino acid so. The discrepancy between different reports side chains are important in contacting the T In a study of 100 nuclear families with at may be due to differences in the arthritogenic cell receptor, seven viral and one bacterial least two affected sibling pairs we found properties of the bacteria, in the antigenic peptide were identified that activate evidence of an extended DRI/B27 haplotype load in different outbreaks, different popu- MBP(85-99) specific T cell clones.2 Two overrepresented in patients compared with lations, or due to bias in patient selection. principles were important in the identifi- B27 controls (30% v 19%, p < 0 01), The most reasonable explanation would be a cation of these mimicry peptides: (a) the suggesting that another gene within the major bias in the patient selection. HLA-DR2 binding motif is degenerate as histocompatibility complex (MHC) could It is concluded that as shown before, HLA- structurally related amino acids can be also be involved. However, analysis of the B27 positive patients have more severe substituted for the two anchor residues ofthe TAP and LMP loci, in association studies, disease, with more complicated acute MBP(85-99) peptide, (b) the two primary T has excluded a significant role for these genes disease, and worse long term prognosis than cell receptor contact residues of the peptide which are involved in the class I pathway of HLA-B27 negative patients.4 Thus patients have to be conserved. The importance of antigen presentation. Analysis of patterns of treated in hospitals have more severe disease, these structural considerations is demon- HIA haplotype sharing among 100 affected with enrichment of HLA-B27 positive cases. strated by the fact that seven ofeight mimicry sibling pairs showed 6% sharing 0 HLA The mild cases are HI-A-B27 negative, peptides do not have obvious sequence haplotypes. This compares with predicted recover quickly, and have no need to be similarity with MBP(85-99) and would not sharing of 1-8% derived from estimates of referred to specialists. have been identified by sequence alignments. sibling recurrence risk (7% in 134 families) The fact that several different viral peptides and the population prevalence (0 5%). It 1 Keat A. Reiter's syndrome and reactive arthritis in perspective. N Engl J Med 1983; 309: can activate the same T cell clones suggests compares with expected sharing of 25% if 1606-15. that different viruses may trigger the same HLA was not linked with susceptibility to 2 Mattila L, Leirisalo-Repo M, Koskimies S, autoimmune syndrome. ankylosing spondylitis. These results there- Granfors K, Siitonen A. Reactive arthritis following an outbreak of salmonella infection This approach may also provide insights fore confirm linkage of ankylosing spondylitis in Finland. Br J7 Rheumatol 1994; 33: into the pathogenesis of rheumatic diseases, to HIA but suggest that a significant genetic 1136-41. in particular to the pathogenesis of rheuma- contribution comes from outside the MHC. 3 Inman R D, Johnston M E A, Hodge M, Falk J, toid arthritis, which is associated with certain Consequently, we have now embarked on a Helewa A. Postdysenteric reactive arthritis. A clinical and immunogenetic study following alleles of major histocompatibility complex genomic screen for susceptibility genes in an outbreak of salmonellosis. Arthritis Rheum http://ard.bmj.com/ (MHC) class II genes. A structural approach ankylosing spondylitis using a panel of 350 1988; 31: 1377-83. may also be useful for the identification of microsatellite DNA markers, which should 4 Leirisalo M, Skylv G, Kousa M, et al. Followup study on patients with Reiter's disease and peptides that mimic MHC class I bound self result in a rough linkage map of susceptibility reactive arthritis, with special reference to peptides. However, a key difference between loci within the next 18 months. HLA-B27. Arthritis Rheum 1982; 25: MHC class I and class II bound peptides will 249-59. have to be considered: for MHC class II bound peptides the conformation of the How strong is the association ofreactive

backbone is on September 25, 2021 by guest. Protected copyright. peptide largely sequence arthritis with HLA-B27? HLA-B27 restricted peptide independent as the peptide is fixed to the to T in site a number of bonds Marjatta Leirisalo-Repo*, Leena Mattilat, presentation cytotoxic cells binding by hydrogen Anja Siitonent, Saija Koskimiest, Kaisa reactive arthritis along the peptide backbone. In contrast, the Granfors§ Paul Bowness, Rachel Allen, Andrew J conformation ofMHC class I bound peptides McMichael is sequence dependent as the peptides are only at N and C terminuses to the *Department ofMedicine, University of fixed their Helsinki; tNational Public Health Institute; Molecular Immunology group, Institute of MHC class I binding site.3 Molecular Medicine, Nuffield Department of Consideration of the structural aspects of tFinnish Red Cross Blood Transfusion Service will in the Helsinki; §National Public Health Institute, Medicine, John Radcliffe Hospital, Oxford MHC-peptide interactions help Turku, Finland identification of viral and bacterial peptides In an attempt to identify HLA-B27 restricted that may be responsible for the loss of self T cells from the and blood of diseases. HLA-B27 is considered as the most cytotoxic joints tolerance in human autoimmune important risk factor for the development of patients with reactive arthritis we used a 1 Oldstone M B A. Molecular mimicry and reactive arthritis. It is present in 60-80% of "reverse immunogenetic" approach based autoimmune disease. Cell 1990; 50: 819-20. patients from different centres,' patients with upon knowledge of the unique peptide 2 Wucherpfennig K W, StromingerJ L. Molecular chlamydia as the triggering infection usually binding properties of HLA-B27s. One mimicry in T cell mediated autoimmunity: hundred and HLA-B27 viral peptides activate human T cell clones having slightly lower prevalence. Most of the twenty potential specific for myelin basic protein. Cell 1995; reports come from academic institutions or binding peptides of eight to 10 amino acids 80: 695-705. from rheumatological centres, which might length, identified by a database search of 3 Madden D R, Garboczi D N, Wiley D C. The cause bias in the patient selection, only the known chlamydial proteins, were synthe- antigenic identity of peptide-MHC com- to plexes: a comparison of the conformation of more severe cases being referred to the sised. Sixty two were found to bind five viral peptides presented by HLA-A2. Cell specialists. HLA-B*2705 in vitro. These peptides were 1993; 75: 693-70.8. Patient data from a single outbreak would used in pools to stimulate synovial fluid and be more informative. Although such epi- peripheral blood mononuclear cells from demiological data concern only a small patients with reactive arthritis, other HLA and non-HLA genes in the number of patients with reactive compli- HLA-B27 associated spondyloarthropathies, spondyloarthropathies cations, recent epidemiological studies on and healthy controls. CD8 positive P Wordsworth*, M Brown*, K Pile*, patients collected during salmonella out- HLA-B27 restricted cytotoxic T cell lines M Rudwaleit*, G Kennedyt, A Calint breaks in Finland2 confirm the data from were grown from blood and synovial fluid of Abstracts ofinvited lectures 575 two patients (one with reactive arthritis, one We noted that B27 and related class I alleles Most information on this topic has been with ankylosing spondylitis). In one case a that bind the Me-1 monoclonal antibody obtained from study of cytokine mRNAs and of invasion into in T cells, and long term in vitro culture was maintained and confer a relative inhibition cytokine proteins synovial of Ann Rheum Dis: first published as 10.1136/ard.55.8.564 on 1 August 1996. Downloaded from shown to be specific for a single nonamer target cells. Using Yersinia enterocolitica wild cytokines produced by peripheral blood peptide derived from chlamydial heat shock type and mutants deficient in expression of lymphocytes and T cells cloned out of protein 70. Cytotoxic T cell responses to this Yad A, inv, or ail genes, we determined that synovial fluid. So far these studies have dealt or other peptides were not detected in three the YadA protein is central to the B27 only with established disease. We hope to patients with other HLA-B27 associated modulation of yersinia invasion into target extend them to reactive arthritis, so as to arthropathies, one HLA-B27 negative patient cells. Examining this in vivo, we challenged encompass the initial triggering infection. A with reactive arthritis, or from the blood of the following mice intragastrically with Y prospective study ofthis sort could be carried one HLA-B*2705 positive healthy control. enterocolitica 0:8:B27 transgenic mice, non- out only in a population with a high level of expressing littermate controls, A2 transgenic the triggering infection, where a cohort of mice, and C57 BL/6 mice. infected individuals could be recruited that B27 transgenic mice showed persistence of would be large enough to generate a Control ofthe availability ofHLA-B27 yersinia at the level of the gut for prolonged reasonably large sample of overt arthritis. peptides at the levels ofthe periods compared with controls, and showed With colleagues in London, Paris, and Delhi proteasomes, TAP and HLA-B27 dissemination to liver and spleen when we plan to carry out a study in Dhaka at the Takashi Ikawa, Jens Kuipers, David Yu control mice had eliminated the pathogen. International Centre of Research in Diarrheal Serological studies showed higher IgA Diseases, Bangladesh. We have chosen to Rheumatology Division, Department of response, and IgGl response in the anti- examine only shigella infections owing to Los USA bodies to yersinia. their high prevalence in developing countries, Medicine, UCLA, Angeles, CA, These studies suggest that B27 may high B27 association, and lower likelihood of of HLA-B27 to present a modulate the handling of the organism at the encountering reactive arthritis driven directly The ability level of the gut, and influence the course of by bacteria in the joint. The indications are particular peptide to the immune system that Dhaka will have a European-type depends on several factors: the transcription the infection. of the prevalence of B27. Our aim is to determine of the HLA-B27 gene, the expression Key paper the factors in the infecting bacterium and in HLA-B27 protein, the generation of the Careless D J, Inman R D. Etiopathogenesis of which favour of peptide by the proteasome complex, the reactive arthritis and ankylosing spondylitis. Curr the host response triggering Opin Rheumatol 1995; 7: 290-8. reactive arthritis. translocation of the peptide into the endo- We believe that the major histocompat- plasmic reticulum, and the affinity of the ibility complex (MHC) associations provide peptide for the HLA-B27 molecules. a powerful tool for probing the causal role of We studied the effect of interferon -y on the Experimental spondyloarthropathy in these cytokines. Negative associations (pro- transcription of the genes of HLA-B27 as in RA LMP2 and LMP7 of the HLA-B27 transgenic rats tective effects) have been described well as of the Joel D Taurog*, Robert E Hammert and in early onset pauciarticular juvenile proteasome complex using RT-PCR; on the not as in molecules as rheumatoid arthritis, though yet expression of total HLA-B27 *Harold C Simmons Arthritis Research Center reactive arthritis. With Monika Brunner we well as those with certain specific peptides and Department ofInternal Medicine; tHoward have recently described a protective effect of using immunofluorescence with specific and the H2Ab allele in collagen induced athritis, monoclonal antibodies to HLA-B27. We also Hughes Medical Institute Department of which control Biochemistry, University of Texas Southwestern which parallels the immunosuppressive effect studied the residue specificities Medical Center, Dallas TX 75235, USA of this allele in other immunological the ability of a peptide to translocate into the responses.' In one of these parallel responses endoplasmic reticulum using a competition For five years we studied a spontaneous (to the antigen allo-4-hydroxy-phenyl- assay with a labelled reporter peptide, and the effect via which control the affinity of a peptide for the multisystem inflammatory disease in pyrovate-dioxygenase) operates with the of of an h) burst of http://ard.bmj.com/ HLA-B27 molecules using a stabilisation HLA-B27 transgenic rats, goal suppression early (>24 method. identifying the molecular role of HLA-B27 in interleukin-4 (IL-4) production,2 and we are its predisposition to inflammatory disease. currently testing whether a similar burst is The results showed that for a bacterial needed to establish chronic inflammatory peptide to become presented by HLA-B27 it Rats of the LEW, F344, and PVG strains, hurdles of bearing high copy numbers of transgenes for arthritis. When our data for IL-4 are has to overcome several control, HLA-B27 and human microglobulin, de- with those of Foumier et 4 and the factors of some of which can be analysed 12 compared al,3 of velop a multisystem disorder, many features with those of Rude et al for IL-12,s chronic with certain criteria algorithms. of which resemble aspects of the human inflammatory arthritis emerges as a disease in

1 Fukazawa T, WangJ, Huang F, et al. Testing the spondyloarthropathies, most dramatically which Th2 cells are required for initiation of on September 25, 2021 by guest. Protected copyright. importance of each residue in a HLA-B27 axial and peripheral arthropathy. The disease the disease, but become beneficial later in the binding peptide using monoclonal antibodies. is not seen in rats with comparable expression disease course. Our working hypothesis is j Immunol 1994; 152: 1190-6. 2 Huang F, Hermann E, Wang J, et al. Two of HLA-B7, and so is relatively specific for that polymorphism in the upstream peptide-dependent monoclonal antibodies as HLA-B27. It requires T cells and another regulatory regions of MHC class II genes is well as one cytotoxic T lymphocyte clone bone marow derived cell, as well as an intact conserved because it results in differential recognize HIA-B27-peptide complexes with flora. Characteristic in different of antigen low stringency for peptide sequences. Infect gut cytokine profiles expression types Immun (in press). distinguish the inflammatory infiltrates in the presenting cells (macrophages, dendritic gut and the joint, and the earliest T cell cells, B cells, activated T cells), and thus in infiltrates at these sites are polyclonal. Poly- prefential activation of either Thl or Th2 morphism at the Tap-2 peptide transporter cells. Sequencing of mouse upper regulatory locus has no significant effect on disease, regions by our colleagues R Lauster and M Influence ofHLA-B27 on host-microbial whereas the DA strain background abrogates Janitz demonstrates a high level of interactions disease. The molecular role of the B27 polymorphism, as occurs in man.6 R D Inman molecule in disease remains to be elucidated, Oral tolerance, as described elsewhere in but there is good reason to believe that it will this workshop by Sieper, is another way of The Toronto Hospital and the University of eventually yield to continued investigation of probing for T cell cytokine effects. We are Toronto, Toronto, Canada this animal model. impressed by the immunological impact of oral type II bovine collagen in the Berlin trial. The clinical features and immunological In the highest treatment group (10 mg/day) studies on reactive arthritis have raised the the level of antihuman type II collagen possibility that B27 may have a role in this dropped significantly among those patients disease other than the classical function of Role ofT ceil cytokines in iniiaion and with rheumatoid arthritis who showed a peptide presentation to CD8+ cytotoxic T maintenance of chronic inflammatory clinical response. cells. arthritis We examined direct microbe major histo- Jurgen Braun, Ulrike Gimsa, Avrion 1 Mitchison N A, Brunner M C. Association of compatibility complex interactions using an Mitchison, Joachim Sieper H2Ab with resistance to collagen-induced in vitro invasion assay, in which arthritogenic arthritis in H2-recombinant mouse strains: an allele associated with reduction of several Gram negative bacteria invade L cells Deutsches Rheuma Forschungszentrum, apparently unrelated responses. Immuno- transfected with various class I HLA alleles. Nordufer 20, 13353 Berlin, Germany genetics 1995; 41: 239-45. 576 Abstracts ofinvited lectures

2 Brunner M C, Larsen S, Sette A, Mitchison N A. Schumannstr. D-10098 Altered ThlI/Th2 balance associated with the 20-21, Berlin, spondyloarthropathies. However, at present immunosuppressive/protective effect of the Germany it is unknown if the same lymphocytes are

H-2Ab allele on the response to allo-HPPD. involved in the development of articular and Ann Rheum Dis: first published as 10.1136/ard.55.8.564 on 1 August 1996. Downloaded from Eur3JImmunol 1995; 25: 3285-9. Macrophages constitute a major part of the gut manifestations. Recent data provide 3 Boissier M C, Chiocchia G, Bessis N, et al. normal Biphasic effect of interferon-gamma in synovial lining. Their apparent role interesting new insights into the gut- murine collagen-induced arthritis. Eur J. appears to lie in scavenging debris originating synovium homing relation.' 2 Indeed, Immunol 1995; 25: 1184-90. from joint material as well as the elimination mucosal immunoblasts can bind (in vitro) to 4 Bessis N, Fradelizi D, Foumier C, Boissier M C. of microorganisms the In Treatment of inflammation with anti- entering joints. both mucosal and synovial high endothelial inflammatory cytokines: example in models of autoimmune diseases such as rheumatoid venules (HEV), but not to peripheral lymph auto-immune diseases. C R Seances Soc Biol arthritis, however, the monocyte-macrophage node HEV. Fil 1995; 189: 579-90. system seems to play a part in We the role of the 5 Germann T, Szeliga J, Hess H, et al. autoaggressive investigated integrin Administration of interleukin 12 in mechanisms. In infectious arthritides this o4AP7 in the proposed gut-synovium re- combination with type II collagen induces system has an important role in eliminating circulation pathway. ct4137 is expressed on severe arthritis in DBA/1 mice. Proc NatlAcad the inciting organisms, but monocytes- lymphocytes with specific homing affinity for Sci USA 1995; 92: 4823-7. also be involved in the 6 Louis P, Eliaou J F, Kerlan C S, Pinet V, macrophages may Peyer's patch HEV (the ligand being Vincent R, Clot J. Polymorphism in the subsequent pathogenesis of these diseases, MadCAM-1). In addition, this integrin has regulatory region of HLA-DRB genes possibly spreading the infectious organisms been reported to be overexpressed in correlating with haplotype evolution. or their antigenic material throughout the Immunogenetics 1993; 38: 21-6. synovium, compared with synovial fluid.3 body or providing a cytokine profile sup- The quantitative expression of cA4137 on porting the onset of joint inflammation, or interleukin-2 responsive T cell lines from both. This distinct local cytokine compo- ileum and colon in normal controls and Cytokine patterns in reactive arthritis sition may direct the subsequent T cell patients with inflammatory bowel disease J Sieper, J Braun, L Neure, P Wu, Z Yin response by leading to type I or II like (IBD) was assessed by flow cytometry, using reactions, and it may therefore be responsible the Act-I monoclonal antibody (a gift from Klinikum Benjamin Franklin and Deutsches for either antibody or cellularly dominated Dr A Lazarovits). T cell lines from patients Rheumaforschungs-Zentrum, Berlin, Germany immune reactions. wth IBD were significantly lower expressors One particular example of an infectious of (x4,87 than normal controls. These data Reactive arthritis is caused by obligate or arthritis is Lyme arthritis where, despite the may either suggest that the activated T cells facultative intracellular bacteria which have identification of Borrelia burgdorferi as the in IBD are low oaP47 expressors or that this been shown to persist inside the joint. For inciting organism, the pathogenesis is still integrin is down regulated by inflammatory other intracellular bacteria, like Myco- incompletely understood. A failure in the mediators in the IBD process. bacterium tuberculosis, it is known that Thl immune response to the infectious agent, an cells, mainly secreting interferon -y, are autoimmune mechanism triggered by 1 Salmi M, Andrew D P, Butcher E C,Jalkanen S. infection, microbial escape into protective Dual binding capacity of mucosal immuno- necessary to fight this kind of infection while blasts to mucosal and synovial endothelium in Th2 cells, secreting, among others, inter- tissue sites, and even intracellular persistence humans: dissection of the molecular leukin-4 (IL-4) inhibit an effective immune of B burgdorferi may be involved in this mechanisms. J7Exp Med 1995, 181: 137-49. response. Therefore the question was investi- process. 2 Salmi M, Granfors K, Leirisalo-Repo M, et al. To disclose a Selective endothelial binding of interleukin- gated whether elimination of bacteria might possible failure in the first line 2-dependent human T-cell lines derived from be hampered by a dominant Th2 response in of defence, human mononuclear phagocytes different tissues. Proc Natl Acad Sci USA were incubated with B burgdorferi. Light and 1992; 89: 11436-40. reactive arthritis. 3 A We have shown earlier by using polymerase electron microscopy showed that the Lazarovits I, Karsh J. Differential expression of the in rheumatoid synovium and synovial fluid of chain reaction and in situ hybridisation to presence spirochaetes induced distinct alpha 4 beta 7 integrin. A novel receptor for detect T cell cytokines in synovial membrane alterations in the phagocytes, including fibronectin and vascular cell adhesion (SM) that more IL-4 can be found in reactive coiling phagocytosis, the formation of leaky molecule-1. JImmunol 1993; 151: 6482-9. arthritis SM than in rheumatoid athritis SM. lysosomes, the invagination of large mem- http://ard.bmj.com/ We confirmed these results by investigating brane areas, the extralysosomal degradation more patients, applying in addition immuno- of intemalised B burgdorferi cells, and finally histochemical methods. We also showed that the formation of mononuclear syncytial cells IL-4 and homotypic cell clusters. Further analyses Epidemiology and prognostic features of positive cels are located in different reactive areas of the SM than interferon y positive showed distinct cytokine profiles with regard arthritis to interleukin-l (IL-1), tumour necrosis Marjatta Leirisalo-Repo*, Kirsi Laasila*, cells, and that both in very early reactive Petri Martti athritis (two patients with a disease duration factor cx, IL-10, and IL-12 released by Helenius*, NissilNt of less monocytes. In conclusion, as exemplified in on September 25, 2021 by guest. Protected copyright. than one week, one patient two weeks) *Department ofMedicine, University of and late reactive arthritis (one year) IL-4 is Lyme disease the monocyte-macrophage system may direct the subsequent Helsinki, Helsinki; tRheumatism Foundation present in the SM during active disease. specific Finland When synovial fluid T cells from patients immune system by (a) altered antigen Hospital, Heinola, processing and presentation, (b) changes of with reactive arthritis were stimulated, either Within the 20 Reiter's with phytohaemagglutinin or macrophage integritiy, and (c) a distinct past years syndrome, specific for which trachomatis is a bacterial antigen, more IL-4 was produced cytokine milieu. Chlamydia major cause, has remained as the most frequent than for patients with rheumatoid arthritis. In form of reactive one arthritis. In Scandinavia, patient IL-4 production was investigated reactive arthritis due to in serial yersinia infection has synovial fluid samples and a decrease been common, while reactive arthritis of IL-4 correlated with clinical improvement. Lymphocyte traffic in reactive arthritis induced salmonella In by infections has been conclusion, IL-4 is present either in the D Elewaut, F De Keyser, E M Veys uncommon.' 2 Within the same or greater amount as in past 10 years, interferon y however, there has been a worldwide increase joints of patients with reactive arthritis. The Department ofRheumatology, University in salmonella infections, which is reflected presence of Th2 cytokines might inhibit an Hospital, Ghent, Belgium also in the of effective immune frequency salmonella arthritis.3 response against bacteria At the same time there seems to be a decrease which trigger reactive arthritis. Spondyloarthropathies refer to a group of in yersinia infections and yersinia arthritis in disorders with overlapping manifestations, Scandinavia. characterised by common clinical and genetic We analysed the long term prognosis of features, of which reactive arthritis is an reactive arthritis to determine the role of the The monocyte-macrophage in infectious example. The presence of subclinical gut triggering infection, genetic factors, gender of diseases: simply a phagocyte or the inflammation (ileitis) in patients with the patients, and incidence of recurrent director ofthe immune response? spondyloarthropathies and its evolution in arthritis. Gerd R Burmester, Michael Rittig, Andreas some patients to clinically overt inflammatory The study included 50 patients with Krause bowel disease (Crohn's disease) strongly previous salmonella arthritis followed up for suggest a pivotal role of the gut immune a mean of 11 years, and 75 patients with Department ofMedicine III, Rheumatology and apparatus in the development of this disease yersinia arthritis and 75 patients with reactive Clinical Immunology, Charite University complex. T cells are believed to have an arthritis, both groups followed up for 20 Hospital, Humboldt University ofBerlin, important role in the pathogenesis of years. Abstracts ofinvited lectures 577

Long term prognosis of reactive arthritis campylobacter triggered), and 175 patients joint inflammatory disease in HIA-B27 transgenic were diagnosed as undifferentiated forms in rats. J Exp Med 1994; 180: 2359-64. Patients

which no triggering agent was found, but Ann Rheum Dis: first published as 10.1136/ard.55.8.564 on 1 August 1996. Downloaded from SA* YA* RS* presenting the same clinical picture. Inflammatory gut lesions were found in all Conventional disease modifying Mean follow up time (years) 11 20 20 but one patient with enterogenic reactive Chronic arthritis (%) 16 7 24 treatment for reactive arthritis: the role Ankylosing spondylitis (%) 9 16 23 arthritis (95%), most of them (69%) ofsulphasalazine and methotrexate Chronic iritis (0/o) 0 1 5 presenting the acute type of inflammation. In H Zeidler Radiology urogenital reactive arthritis, gut lesions were Erosions (%) ND 10 26 found in six patients (20%), all but one Sacroiliitis (22 grade) (c/o) 14 22 26 Division ofRheumatology, Medical Highschool featuring the acute type of inflammation. In Hannover, Germany *SA = salmonella arthritis; YA = yersinia arthritis; undifferentiated forms, gut lesions were RS = Reiter's syndrome. found in 65% of patients, with acute and Conventional disease modifying treatment chronic gut lesions equally distributed. This for reactive arthritis is hardly discussed in finding suggests that the gut is implicated in rheumatological textbooks. Only a few recent Patients with Reiter's syndrome had the the pathogenesis of most cases of reactive most severe prognosis with respect to the reviews have summarised the relevant litera- arthritis. ture in more detail.' 2 This review is based on development of chronic disease (table). They Seventy one of these original 224 patients also had a higher frequency of recurrent the literature search published by Creemers et were clinically reviewed four to nine years al' and has been updated by a recent search arthritis (found in 22% of patients with later. Forty three patients (60%) were in salmonella arthritis, in 28% of those with using Medline from 1993 to June 1995. clinical remission, presenting during at least Although no generally accepted criteria for yersinia arthritis, and in 53% of patients six months no clinical signs of arthritis, with Reiter's syndrome). HLA-B27 positive the indication of disease modifying treatment tendinitis, axial pain, complaining of no in reactive arthritis are available at present, patients more often had recurrent arthritis, morning stiffness, and most of them having enthesopathy, iritis, and they developed most authors consider this treatment in some normal inflammatory serum parameters. patients with severe early disease refractory to radiological sacroiliitis, chronic arthritis, and This finding confirms the good prognosis of ankylosing spondylitis more commonly than non-steroidal anti-inflammatory drugs but reactive arthritis, though most prospective more commonly in patients with prolonged, B27 negative patients. Most patients with studies describe an evolution to chronic joint Reiter's syndrome were male. The role of recurrent, or chronic disease.2 Most disease in only 20% of cases. Eleven patients experiences are based on uncontrolled gender was therefore analysed in patients (15%) developed a chronic or relapsing form with reactive arthritis triggered by enteric clinical observations: the beneficial effect in of pauciarticular arthritis. Fourteen patients placebo controlled trials is not always infections. In male patients the disease (20%) (one patient with urogenital reactive progressed more often to ankylosing spon- significant owing to the high placebo arthritis, four with enterogenic reactive arth- response, there is a lack of long term follow dylitis than in female patients. ritis, and nine with undifferentiated forms) It is concluded that triggering infection in up studies, and, additionally, outcome developed ankylosing spondylitis. variables are not standardised. Nevertheless, the urogenital tract, new attacks of arthritis, Four patients (6%) had developed a male gender, and HLA-B27 are markers of the efficacy ofsulphasalazine in the treatment proved Crohn's disease at review; three of of peripheral arthritis has established that it development of late sequels in reactive these patients had been previously diagnosed arthritis. should be the only drug to be accepted from as undifferentiated reactive arthritis, and one a scientific point of view.3 If further clinical as reactive arthritis. All four 1 Valtonen V V, Leirisalo M, Pentikainen P, et al. patient yersinia experience is included, based mainly on Triggering infections in reactive ardtritis. Ann patients had also developed ankylosing spon- observations in individual patients, case Rheum Dis 1985; 44: 399-405. dylitis and all initially presented histological reports, and uncontrolled cohort studies, 2 Leirisalo M, Skylv G, Kousa M, et al. Followup gut inflammation. study on patients with Reiter's disease and then methotrexate can also be recommended reactive arthritis, with special reference to In 19 of the 71 patients a secondary in cases where sulphasalazine fails or HLA-B27. Arthritis Rheum 1982; 25: ileocolonoscopy was performed at review. Of produces side effects. Especially in the case http://ard.bmj.com/ 249-59. the 12 patients who were in clinical remission of severe mucocutaneous lesions metho- 3 Kvien T K, Glennas A, Melby K, et al. Reactive at that none inflam- arthritis: incidence, triggering agents and moment, presented trexate may be advantageous in the treatment clinical presentation. J Rheumatol 1994; 21: matory gut lesions, whereas these lesions of severe refractory reactive arthritis. In 115-22. were found in six of the seven patients with chronic Reiter's syndrome a placebo persistent inflammatory joint symptoms. controlled crossover study showed that This finding confirms the close relation azathioprine reduce the joint score and non- Gut inflammation in different forms of between gut and joint inflammation in steroidal anti-inflammatory drug intake. reactive arthritis: prevalence and long reactive arthritis. All patients with active gut Bromocriptine could, in special cases, be the on September 25, 2021 by guest. Protected copyright. term evaluation inflammation at the second ileocolonoscopy last but experimental choice of treatment. H Mielants, E M Veys also presented lesions at the first inves- Finally, the most recent positive case reports tigation; they all showed chronic inflam- of treatment with the 5-aminosalicylic acid Department ofRheumatology, University matory gut lesions, three presenting the compounds, mesalazine and olsalazine, Hospital, Ghent, Belgium histological and clinical signs of Crohn's warrant further study to investigate the disease. possibility of using these drugs as an The concept of spondyloarthropathy gathers These findings substantiate the concept of alternative to sulphasalazine. together a group of chronic diseases with spondyloarthropathy as an interrelated group In conclusion, the treatment of reactive common clinical, biological, genetic, and of disorders in which one subdisease can arthritis with conventional disease modifying therapeutic characteristics. Ileocolonoscopic evolve into another. Gut inflammation seems drugs is poorly defined in view of the studies showed the presence of histological to have an important role in the pathogenesis indications, and only the efficacy of signs of gut inflammation in a relatively great and evolution of reactive arthritis, and the sulphasalazine has been sufficiently es- number of patients with different formins of absence of gut involvement seems to be a tablished in the treatment of the peripheral spondyloarthropathy (ankylosing spondylitis, good prognostic factor for the evolution of arthritis. The recommendations for other reactive arthritis, psoriatic arthritis, etc). The this disease. drugs are based on case reports and clinical histological gut lesions could be subdivided Key papers experience. There is an urgent need for into "acute" lesions, resembling acute bac- Mielants H, Veys E M, Goemaere S, Goethals K, controlled studies to be undertaken. terial enteritis, and "chronic" lesions, bearing Cuvelier C, De Vos M. Gut inflammation in the resemblance to the picture of inflammatory spondylarthropathies: clinical, radiologic, biologic 1 Creemers M, van Riel P, Franssen M, van der and genetic features in relation to the type of Putte L, Gribnau F. Second-line treatment in bowel disease, especially Crohn's disease. histology. A prospective study. J Rheumatol 1991; seronegative spondylarthropathies. Semin Ileocolonoscopy was performed in 224 18: 1542-51. Arthritis Rheum 1994; 24- 71-81. patients with spondyloarthropathy, in which Mielants H, Veys E M, Cuvelier C, et al. The 2 Amor B, Dougados M, Khan M A. Manage- evolution of spondylarthropathies in relation to gut ment of refractory ankylosing spondylitis and psoriatic arthritis, inflammatory bowel dis- histology. J Rheumatol (in press). related spondylarthropathies. Rheum Dis Clin ease, and ankylosing spondylitis were Leirisalo-Repo M, Turunen U, Stenman S, North Am 1995; 21: 117-28. excluded. Thirty patients had a form of uro- Helenius P, Seppala K. High frequency of silent 3 Dougados M, van der Linden S, Leirisalo-Repo genital reactive arthritis (mostly chlamydia inflammatory bowel disease in spondylarthropathy. M, et al. Sulfasalazine in the treatment of Arhtritis Rheum 1994; 37: 23-31. spondylarthropathy. A randomized, multi- triggered), 19 patients an enterogenic Taurog J D, Richardson J A, Crott J A. The center, double-blind, placebo-controlled reactive arthritis (yersinia, salmonella, or germfree state prevents development of gut and study. Arthritis Rheum 1995; 38: 618-27. 578 Abstracts ofinvited lectures

Can antimicrobial drugs really cure believed to be dominant. So far, three studies Studies in juvenile chronic arthritis have reactive arthritis?: evidence from for the treatment ofrheumatoid arthritis (one now suggested that similar mechanisms are studies ofciprofloxacin and other in Berlin, two in Boston) have been operative during disease remission of chronic antibiotics conducted with oral collagen type II, an arthritis. T cell responses to human hsp-60 Ann Rheum Dis: first published as 10.1136/ard.55.8.564 on 1 August 1996. Downloaded from Jurgen Braun, Ulrich Eggens, Jochen Sieper antigen abundant in the cartilage of the joint. were absent in patients with systemic and The results ofthese studies are promising but polyarticular forms of progressive disease, Rheumatology Subunit, Department of not yet conclusive. In reactive arthritis a whereas responses were present in patients Medicine, Klinikum Benjamin Franklin FU locally persistent bacterial antigen drives a T with oligoarticular remitting forms of the Berlin, Germany cell mediated response which seems to be disease. In longitudinal follow up it appeared responsible for the immunopathology. It can that remission was preceded by temporary Reactive arthritis can occur after preceding be speculated that the induction ofa Th2-like responsiveness to human hsp-60. bacterial infections, mainly in the urethra response by oral tolerance would hamper an We now have found that immunisation of owing to Chlamydia trachomatis and in the gut effective elimination ofbacteria and therefore rats with Subreum' elicited T cell immunity owing to Yersinia enterocolitica and others. might lead to a more chronic inflammation. directed at hsp-60 (Gro-EL) and hsp-70 Bacteria cannot be cultured from the joints, Therefore, oral tolerance is not likely to be a (DnaK) of Escherichia coli. Furthermore, but bacterial antigens and bacterial DNA therapeutic option in reactive arthritis. Subreum' was found to be protective against have been detected in the synovial membrane However, the possibility cannot be excluded adjuvant arthritis upon oral administration. and the synovial fluid of these patients. The that hypersensitivity against otherwise Possibly, the induction of active peripheral course ofreactive arthritis is generally benign: harmless bacteria in the joint, or even an tolerance (Th2?) for bacterial hsps is one of most cases are healed within one year. autoimmune response triggered by bacteria, the mechanism of action of Subreum' as an However, chronic courses have been reported is responsible for the local inflammation. In antirheumatic drug. in 20-30% of patients. There are no such a case oral tolerance might be established prognostic criteria for reactive favourable, with bacterially conserved pro- Key papers Anderton S M, van der Zee R, Noordzij A, van arthritis, but the presence of other features of teins such as heat shock proteins being likely Eden W. Differential mycobacterial 65-kDa heat spondyloarthropathy, such as enthesopathy candidates. shock protein T cell epitope recognition after and sacroiliitis and HLA-B27 positivity, has adjuvant arthritis-inducing or protective im- been reported to be associated with munization protocols. J7 Immunol 1994; 152: 3656-64. chronicity. Anderton S M, van der Zee R, Prakken B, Noordzij Antibiotic treatment in reactive arthritis is Restoration ofperipheral tolerance by A, van Eden W. Activation of T cells recognizing directed towards the eradication of bacteria self 60-kD heat shock protein can protect against immunisation with bacterial heat shock experimental arthritis. J Exp Med 1995; 181: persisting in the joints. Therapeutic aims are proteins: a possible mode ofaction of 943-52. effective prophylaxis, complete cure, and the antirheumatic drug Subreum® de Graeff-Meeder E R, van Eden W, Rijkers G T, shortening of arthritic symptoms. There is W van Eden, A B J Prakken, A Bloemendal, et al. Juvenile chronic arthritis: T cell reactivity to evidence that the short term treatment of human HSP60 in patients with a favorable course P J S van Kooten, R van der Zee, V P M G of arthritis. Clin Invest 1995; 95: 934-40. urethritis with an antibiotic appropriate to Rutten eradicate C trachomatis prevents the onset of arthritis in a significant percentage ofpatients Department ofInfectious Diseases and with chronic Reiter's syndrome. A three Immunology, Veterinary Faculty, Yalelaan 1, Chlamydia trachomatis antibody months' long term treatment with lyme- 3584CL Utrecht, The Netherlands isotypes in reactive arthritis: specificity cycline shortened the disease course in some and sensibility patients with chlamydia induced reactive The manipulation of peripheral tolerance by S Bas, T L Vischer arthritis but not in those with reactive relevant autoantigens may be used for the arthritis triggered by enterobacteria. In immunotherapy of certain autoimmune Division ofRheumatology, University Hospital contrast, there is no evidence that short or diseases. The proper definition of the Geneva, Switzerland long term treatment with antibiotics has any relevant antigen has, however, remained http://ard.bmj.com/ influence on the course of reactive arthritis elusive in many cases. Our most recent Results of studies on the clinical value of following enteritis. A placebo controlled studies have offered evidence that heat shock randomised in which a three serological methods in patients with reactive study, months' proteins (hsps) can be useful regulatory arthritis are equivocal as the prevalence of course of 1 g ciprofloxacin daily was given to antigens in rheumatic diseases. Chlamydia trachomatis infections (apparent 120 patients with reactive arthritis, is Synthesis ofhsps is enhanced in cells under and hidden) in general populations is currently being terminated in Berlin. A conditions ofcellular stress as is typically seen 1 relatively high. Few studies, however, have European study examining the effect of g at sites of inflammation. In chronic arthritis evaluated this formally in a large number of azithromycin given once weekly to patients this has been shown for hsp-60 in the well defined patients. We therefore de- on September 25, 2021 by guest. Protected copyright. with reactive arthritis has just been started. inflamed synovium of the arthritic joint, both termined isotype anti-chlamydia antibody So far, the effectiveness of long term in man and in experimental (rodent) models. concentrations in the serum of patients with treatment of reactive arthritis with antibiotics Recently, it has become clear that such various arthropathies. has not been established. overexpressed hsp-60 may be a potential Antibodies were detected by an enzyme target molecule for regulatory (or sup- linked immunosorbent assay (ELISA) using pressive) T cells. as antigen an elementary body enriched Oral tolerance in the treatment of Experimental arthritis can be prevented by fraction prepared from Verocells infected arthritis immunisation with mycobacterial hsp-60. with chlamydia. The same fraction prepared J Sieper, U Eggens, J Braun, N A Mitchison This was found to be caused by T cells from uninfected cells was used as control specific for highly conserved sequences in the antigen. An alkaline phosphatase labelled Klinikum Benjamin Franklin and Deutsches molecule. Cloned T cells responsive to such F(ab')2 fraction of goat antihuman Rheumaforschungs-Zentrum, Berlin, Germany conserved sequences were cross reactive with immunoglobulin isotype was used for the mammalian (rat and human) hsp-60 and detection. Normal values were obtained In animal models oral tolerance has been with stressed (heat shocked) cells in the through 100 serum samples from blood proved to be effective in preventing and absence of added antigen. Resistance was donors, and values at least three standard curing autoimmune diseases by feeding organ transferable with the latter cloned T cells and deviations higher than the mean antibody specific antigen. It is assumed that local not with T cells that recognised non- concentration of these samples were defined immunosuppression is mediated by secretion conserved (non-cross-reactive) hsp-60 epi- as positive. Patient groups included patients of Th2-like cytokines such as transforming topes. Immunisation with the conserved with sexually acquired reactive arthritis with growth factor [3 and interleukin-4 by T cells microbial hsp-60 peptide, induced protection proven C trachomatis origin, uroarthritis which have been primed by the orally fed against arthritis, both in the mycobacteria without proven C trachomatis origin, antigen in the Peyer's patches of the gut. and the non-microbially induced model. seronegative mono/oligo/monoarthritis, rheu- Therefore oral tolerance could be effective in Together, these findings indicate that T cell matoid arthritis, crystal induced arthritis and all diseases in which an inhibition of a recognition of conserved bacterial hsp-60 mechanical arthropathies. Sensitivity, specifi- deleterious Thl immune response by a Th2 epitopes contributes to protective immuno- city, negative and positive predictive values response is required. regulatory events, on the basis of cross were calculated using the results from In rheumatoid arthritis a T cell mediated recognition of self hsp-60 epitopes as ex- patients with C trachomatis proven sexually response to an unknown self antigen is pressed at the site of chronic inflammation. acquired reactive arthritis as expected Abstracts ofinvited lectures/Submitted abstracts 579 positives and the results from patients with Bacterial detection by Both ankylosing spondylitis and reactive rheumatoid arthritis, crystal induced arth- immunochemistry: is it practicable? arthritis are strongly associated with HLA- ritis, and mechanical arthropathies as ex- K Granfors B27, a class I product of the major pected negatives. histocompatibility complex. To explain this Ann Rheum Dis: first published as 10.1136/ard.55.8.564 on 1 August 1996. Downloaded from In all groups studied, positive values were National Public Health Institute, Department in association it has been suggested that the found for all three isotypes. Turku, FIN-20520 Turku, Finland unpaired cysteine residue at position 67 of If any of the three isotype determinations the heavy chain of B27 could form a was positive, sensitivity was 73% and Structures of causative microbes have been disulphide bond with thiol-containing agents, specificity 78%, with a positive predictive recently detected by immunochemical tech- causing a conformational change in B27, value of 58% and negative predictive value niques in inflamed joints in reactive arthritis: thereby triggering autoimmune cytotoxic T of 800/o. Thus isotype specific antibody in synovial fluid, synovial fluid cells, and lymphocyte (CTL) responses. determinations with the method used, have a synovial membrane. Structures from Here we report that homocysteine can limited value in clinical practice owing to the Chlamydia trachomatis, Yersinia enterocolitica modify B27 through disulphide bonding and high prevalence of antibodies to chlamydia 0:3, Salmonella typhimurium, Salmonella that modified cells can be specifically in the general population and to the absence enteritidis, and ShigeMla flexneri have been recognised by CTLs. The modification of antibodies in some of patients with demonstrated by immunofluorescence and in occurs in a pre-Golgi compartment in the well defined chlamydia induced reactive some cases also by enzyme linked immuno- cells, and we suggest that the altered B27 arthritis. sorbent assay (ELISA), electron microscopy, binds different peptides from native B27. We and immunoblotting techniques. Polyclonal have also identified homocysteine specific and monoclonal antibodies have been CTLs in a B27 negative patient with succesfully used, and specific bacterial ankylosing spondylitis. Possibly, B27 is more Diagnostic use ofspecific antibacterial structures detected are lipopolysaccharide, easily modified by homocysteine through lymphocyte proliferation heat shock protein, and outer membrane Cys67, whereas other HLA class I may also Jurgen Braun, Jochen Sieper proteins of these bacteria. Peripheral blood be modified through cysteine-containing cells from patients with salmonella and peptides bound to them. This suggests that Rheumatology Subunit, Department of yersinia triggered reactive arthritis have been presentation of homocysteine to CTLs may Medicine, Kinikum Benjamin Franklin FU studied also by immunofluorescence and be more relevant to the disease, as about 10% Berlin, Germany immunoblotting, and bacterial lipopoly- of patients with ankylosing spondylitis and saccharide and heat shock protein have been 30% with reactive arthritis are B27 negative. Reactive arthritis can occur after preceding detected. Homocysteine-CTL responses were found in bacterial infections, mainly in the urethra In reactive arthritis after intestinal 11/21 patients with ankylosing spondylitis or owing to Chlamydia trachomatis and in the gut infections bacterial structures in the joints reactive arthritis and in only 2/21 healthy owing to Yersinia enterocolitica. Bacteria and in peripheral blood are in highly controls. It is therefore proposed that these cannot be cultured from the joints, but degraded form; no live bacteria or bacterial CTLs could contribute to the pathogenesis of bacterial antigens have been detected in the DNA have been detected. This means that this form of arthritis. synovial membrane and the synovial fluid the structures existing in different tissues (SF) of these patients, particularly in early might not be easily detected by immuno- disease. Using lymphocyte proliferation chemical techniques as bacterial epitopes assays (LPAs) the immune response of SF recognised by antibodies might have Conserved bacterial proteins: and peripheral blood mononuclear cells can disappeared. implication for the pathogenesis of be measured in vitro. This phenomenon is Although published reports on microbial reactive arthritis found more markedly in SF than in structures in the inflamed joints in patients Sanja Ugrinovic*, Andreas Mertz*, Jurgen peripheral blood mononuclear cells, which is with reactive arthritis have shown that most Braun*, Joachim Sieper*t in accordance with the antigen specific T cell samples are positive, immunochemical frequency of 1/2000 in SF and 1/7500 in detection is difficult; especially owing to the *Klinikum Benjamin Franklin, Free University http://ard.bmj.com/ peripheral blood T cells. The local immune small amount of microbial material in tissues ofBerlin; tDeutsches response of T cells reacting to bacteria in the to be studied and to the highly processed Rheumaforschungszentrum Berlin, Germany joint is thought to be the major pathegenic form ofmicrobial structures. Antibodies used mechanism in reactive arthritis. so far have been prepared by different groups in the and no Reactive arthritis is triggered by infection Criteria for the definition of specific working field, systematic with obligate (chlamydia) or facultative responses to bacteria have been but distribution of them has been organised. proposed, to (yersinia, salmonella, shigella) intracellular sensitivity and specificity of LPAs could not Owing these problems bacterial detection bacteria. Examination of a of by immunochemistry cannot be recommen- group patients be determined because a standard" on September 25, 2021 by guest. Protected copyright. "gold ded as a test for reactive arthritis. with reactive arthritis shows that 60-70% of was lacking. However, DNA of non- diagnostic them are HLA-B27 positive. Yet the inter- enteropathogenic bacteria has recently been action of environmental and genetic factors detected in the synovial membrane and SF by in the pathogenesis of reactive arthritis is not polymerase chain reaction technology. No well understood. systematic comparison of polymerase chain We investigated which bacterial antigens reactions and LPAs has yet been performed, Submitted abstracts are immunodominant for CD4+ or CD8+ T but we report here, as a preliminary result, cells and whether they can be presented by that chlamydial DNA was detected in four SF HLA-B27. samples ofpatients with reactive arthritis with Homocysteine specific cytotoxic T Conserved ribosomal proteins L2, L23, an LPA response to the same microbe. The lymphocytes in patients with ankylosing Gro-EL, the 19 kilodalton urease subunit of probability of detecting a specific LPA spondylitis and reactive arthritis yersinia, the 18 kilodalton histone-like response in SF is highest in early disease, Xiao-Ming Gao*, Paul Wordswortht, protein, and the 57 kilodalton heat shock while 75% of SF samples taken in the first Andrew J McMichaelt, Martin Seifert§, protein of chlamydia are candidate antigens week after disease onset showed specific David Rees§, Joel D Taurog¶, Gordon for immunodominance in reactive arthritis. responses the frequency gradually decreased Dougan* Recombinant and native forms of these to 23% in more chronic stages of disease. proteins were first used in a proliferation Whether this correlates with the presence of *Department ofBiochemistry, Imperial College assay. In four patients with chlamydia bacterial antigen in the joint is not yet known. ofScience, Technology and Medicine, induced reactive arthritis, 57 kilodalton The specificity ofLPA results is influenced by Exhibition Road, London, SW7 2AZ, United protein was positive (SI: 5, 7, 50, 800), cross reactivity of enterobacteria, and the Kingdom; tNuffield Department ofClinical whereas in two patients with yersinia induced sensitivity is diminished by suppressor activity Medicine and tInstitute ofMolecular Medicine, reactive arthritis the 57 kilodalton protein of macrophages. Although the LPA provides J7ohn Radcliffe Hospital, Oxford, United was negative. The 18 kilodalton protein did additional information on triggering bacteria, Kingdom; §Department ofRheumatology, St not induce any proliferation. we do not recommend its use as a diagnostic Mary Hospital, London, United Kingdom; The nonamer peptides with HLA-B27 test. We think it important to identify T cell ¶Harold C Simmons Arthritis Research Centre binding motif were selected from these con- reactivity to bacteria in bulk cultures for and Department ofInternal Medicine, served proteins, tested in a standard HLA further analysis of epitopes and major University of Texas Southwestern Medical assembly assay, and used for evaluation of histocompatibility complex interaction. Center, DaUas, USA CD8 responses. 580 Submitted abstracts

We generated yersinia specific, synovial peptide(s) could lead to a better under- antibodies to investigate the T cell repertoire fluid derived cytotoxic T lymphocyte lines standing of the supposed role of the HLA- ofCD4 and CD8 positive cells in the synovial from three B27 positive patients and one B27 B27 antigen in bacterial infection and disease fluid and blood ofseven patients with reactive negative patient, using autologous macro- association. arthritis and one patient with peripheral Ann Rheum Dis: first published as 10.1136/ard.55.8.564 on 1 August 1996. Downloaded from phages infected with live yersinia as antigen oligoarthritis complicating ulcerative colitis. presenting cells. A line from one patient, used CD4/CD8 ratios and levels of -yb T cells were for further investigation, showed a specific also examined. cytotoxic response to L23 and Gro-EL (15% Response of synovial fluid derived T cell We identified several minor expansions in and 25% above background at an clones to recombinant Chlamydia the blood or synovial fluid, or both, of our effector:target ratio of 50:1 respectively), but trachomatis protein antigens patients by comparison with T cell reper- not to the 19 kilodalton derived peptides. F Campbell*, S Birkelundt, M E Wardt, toires of healthy individuals of similar age. Using Epstein-Barr virus transformed lines G S Panayi*, G H Kingsley* Different V gene segments identified expan- matched for B27, but mismatched for other sions in different patients, and expansions class I molecules as targets, indicates that *Department ofMedicine, Rheumatology Unit, were seen in both CD4 and CD8 positive these responses are B27 restricted. Guy's Hospital, London SEJ 9RT, United cells. One patient had V, 13 expansions in Therefore, epitopes from conserved proteins Kingdom; tMolecular Microbiology, both CD4 and CD8 cell subsets in synovial seem also to be relevant for a B27 restricted Southampton General Hospital, United fluid as well as VPB13 expansions in CD8 CDB response. Kingdom; tInstitute ofMedical Microbiology, positive peripheral blood lymphocytes. The ongoing investigation with chlamydia University ofAarhus, Denmark Another patient showed a 10-fold expansion specific cytotoxic T lymphocyte lines may tell of V,B12 in synovial fluid CD8 positive cells us if these CD8 epitopes are shared between We have previously reported the initial compared with CD8 positive peripheral different bacteria associated with reactive characterisation of a panel of T cell clones blood lymphocytes. There was no common arthritis. derived from an HLA-B27 negative patient pattern of expansions between patients and with chronic reactive arthritis. Subsequently, no major expansions were observed. we tested the potential of these clones to We aim to use V gene specific polymerase Induction ofcytotoxic T cells in proliferate against a panel of recombinant C chain reactions to determine the clonality of Chlamydia trachomatis infected trachomatis protein antigens. these expansions. Expanded T cell popu- HLA-B27,l2m homozygous transgenic T cell clones were made on two occasions lations may also be analysed for activation CBA mice from the synovial fluid of a 23 year old man markers using three colour flow cytometry. W Kuon*, R Laustert, U Bottcher*, with classical Reiter's syndrome by limiting No significant differences were detected in S Ugrinovic*, M Grolms*, J Braun*, dilution. Proliferation ofthe T cell clones was the levels of yb T cells between blood and A Koroknayt, M Ulbrechtt, E H Weisst, tested with C trachomatis, C pneumoniae, and synovial fluid, and there was no strong evi- J Sieper* a panel of C trachomatis recombinant pro- dence for the involvement of a superantigen teins, including the major outer membrane as suggested previously for rheumatoid *Klinikum Benjamin Franklin, Berlin; protein (MOMP), Gro-EL, Gro-ES, an 18 arthritis. kilodalton histone-like protein, and control tDeutsches RheumaforschungsZentrum Berlin; vectors. The HLA-DR restriction of the tInstitutfAnthropologie und Genetik, LM-Univ. Miinchen, Germany clones was also investigated using irradiated HLA typed antigen presenting cells. Clones Superposition ofhydropathy plots from Chlamydial species are intracellular living were either HLA-DR3 or 1301 restricted. T cell receptor ,1 chain CDR3 for bacteria that cause a variety of human We characterised fully eight T cell clones. All and three also detection ofconserved properties of diseases. It has been shown in mice that recognised C trachomatis corresponding antigenic peptides: a infected animals can a recognised C pneumoniae. One of the eight develop CD8+ C study in HLA-B27 restricted T cells cytotoxic T cell response against chlamydia. trachomatisiC pneumoniae reactive clones, E May, R Duchmann, B Ackermann, In this study we considered whether C recognised Gro-EL. None of the other seven K-H Meyer zum Biischenfelde, http://ard.bmj.com/ trachomatis can induce a cytotoxic T clones recognised any of the other E Marker-Hermann lymphocyte (CTL) response in HLA- recombinant antigens. No proliferation to the B*2705p2m homozygous transgenic mice. MOMP has been shown in 13 other partially First Department ofMedicine, University of The ability of C trachomatis specific immune characterised clones. Mainz, Germany cells to infected cells was It is concluded that the recognition of spleen lyse Gro-EL investigated in a standard five hour 51Cr is consistent with other reports of Bacterial and autologous peptides presented release assay. Our experiments with bulk responses to heat shock proteins and the up on HLA-B27 molecules may be crucial in the culture derived CTLs showed that CTLs can regulation of these proteins within the induction of CD8+ T lymphocyte (CTL) on September 25, 2021 by guest. Protected copyright. be raised in infected transgenic mice and that synovium. In addition, we extensively looked mediated inflammation in the spondylo- lysis was only observed for C trachomatis for MOMP responses as this is an immuno- arthropathies. infected targets. Preliminary blocking experi- dominant antigen, but so far unsuccessfully. The lack of In a previous study we showed the ments in vitro with monoclonal antibodies recognition, however, correlates presence of HLA-B27 restricted cytolytic T suggest that the bulk derived CTLs are with reports of the MOMP being down cells with specificity for bacterial or auto- restricted by both murine major histo- regulated in the synovium. logous antigens in the synovial fluid (SF) of compatibility complex class I antigen and HLA-B27+ patients with reactive arthritis.' the transgenic human HLA-B27 class I Seventeen HLA-B27 restricted CTL clones, molecule. four two-clonal lines, and 14 non-HLA-B27 At present we are in the process ofisolating Comparison ofT cell receptor restricted controls were grown from the site CD8+ T cell lines and clones, particularly repertoire in blood and synovial fluid of of inflammation of three patients or from those that are HLA-B27 restricted, to patients with reactive arthritis peripheral blood lymphocytes of a B27+ characterise in more detail the nature of the Rachel L Allen, Paul Bowness, Paul healthy donor. We analysed their T cell immune response. Moreover, in preliminary Wordsworth, Andrew J McMichael receptor (TCR) a chain rearrangement by experiments we investigated the role of the RT-PCR using primers for 24 different TCR chlamydial heat shock protein hsp-57, which Molecular Immunology Group, Institute of VP families and solid phase DNA sequencing has been claimed to have a crucial role Molecular Medicine, John Radcliffe Hospital, and showed limited V,B usage in HLA-B27 in CD4+ T cell mediated chlamydial in- Headington, Oxford, OX3 9DU, United restricted CTLs.2 Owing to the absence of flammatory processes in the guinea pig Kingdom information about TCR corresponding model. In our transgenic animal model antigenic peptides, special attention was paid chlamydial hsp-57 seemed not to be If reactive arthritis is a T cell mediated to the peptide contacting hypervariable responsible for evoking the observed CTL disease it may be possible to detect expanded CDR3. Formal amino acid sequence homol- response in C trachomatis infected animals. populations of T lymphocytes as sites of ogies in CDR3 of T cells with identical Taken together, our murine animal model inflammation. Such expansions may be specificity are often used to identify TCR offers the opportunity to isolate HLA-B27 identified by a bias in T cell receptor (TCR) sites that contact antigenic peptide epitopes. restricted lines and clones specific for V gene segment usage. However, it is known that identical peptide chlamydial antigen. Isolation and identifica- We used two colour flow cytometry with a epitopes can be recognised by formally non- tion of the so far unknown antigenic panel of 1 TCR V gene segment specific homologous CDR3 as this region arises from Submitted abstracts 581 random processes and is not genomically Immunofluorescence using polyvalent Interferon y induced growth inhibition encoded. anti-YadA was performed on colonic and of Chlamydia trachomatis in the human Nevertheless, peptide epitopes might synovial tissue. T cells were cloned by monocytic cell line THP-1 is not Ann Rheum Dis: first published as 10.1136/ard.55.8.564 on 1 August 1996. Downloaded from restrict the physicochemical variability of limiting dilution in an aspecific manner. mediated by tryptophan deficiency corresponding residues in the TCR CDR3 by Thirty seven clones from patient 1 and L Koehler*, C Bonk*, E Nettelnbreker*, charge-charge interaction or hydrophobic 53 clones from patient 2 were screened G Reisst, J Wollenhaupt*, H Zeidler* interaction and might thereby reflect their for proliferative responses to yersinia, Chla- own properties. We thus examined the mydia trachomatis, and Salmonella typhimu- *Division ofRheumatology and tDivision of distribution of polar and non-polar residues rium. Electron Microscopy, Medical School, of both CDR3 amino acid sequences and Yersinia antigens were demonstrated in Hannover, Germany known B27-binding peptides by generation colonic and synovial tissue of both patients. and superposition of hydrophobicity plots. Four CD4+ clones from patient 1 and six Recent data suggest that C trachomatis can Such hydrophobicity overlay plots from CD4+ clones from patient 2 showed a posi- persist in monocytes of the synovial fluid and control clones showed random distribution of tive response to Y enterocolitica. One clone membrane, and liberated chlamydial anti- hydrophilic and hydrophobic amino acids cross reacted with C trachomatis. gens may trigger the inflammatory process in over the entire segment. In clear contrast, It is concluded that the demonstration of the joint. In human fibroblasts persistence of hydrophobicity overlay plots from both types bacterial antigens in situ and the high C trachomatis has been attributed to a ofHLA-B27 restricted CTLs showed profiles percentage of Y enterocolitica reactive T cell cytokine mediated induction of indole- that were restricted and characteristically clones support the hypothesis that bacterial 2,3-diaminoxygenase, a tryptophan degrad- dissimilar from each other. None of the antigens are presented locally in the joint. ing enzyme. The mechanism of chlamydial CDR3 from 14 autoreactive clones used This may be one of the causes of chronic persistence in human monocytes, however, is charged amino acids at three distinct polyarthritis. not known. positions (5, 8, and 9 residues distant from Tissue plasminogen activator (TPA) a constant Cys), whereas such residues were differentiated THP-1 cells (human mono- present in yersinia specific CDR3. Here, in cytic cell line) and Hep-2 cells (human larynx tum, hydrophobic residues were absent from carcinoma) were preincubated with or with- P9, 10, and 12 positions which were Experimental reactive arthritis: effect of out interferon y (IFN-y) and/or tryptophan heterogeneic in CDR3 from autoreactive ciprofloxacin (200 ,ug/ml) for 24 hours and infected with CTLs. Accordingly, hydrophobicity overlay Yong Zhang, Christel Gripenberg-Lerche, C trachomatis. The number of inclusion plots of known HLA-B27 binding self- and Karl-Ove Soderstrom, Auli Toivanen, Paavo bodies was determined by immuno- bacterial nonapeptides closely mirrored the Toivanen fluorescence microscopy and the number of characteristic TCR CDR3 profiles of such infective chlamydiae by titration ofthe lysates TCRs that exhibited the corresponding Department ofMedical Microbiology, Turku of infected cells on Hep-2 cells. specificity. University, Finland Inoculation of TPA differentiated THP-1 Thus our data lead us to the conclusion cells with C trachomatis resulted in a low that this method of comparative graphical Significance of antiobiotic prophylaxis and productive infection. During the culture analysis might be useful for detecting treatment of reactive arthritis, following period of 10 days inclusion bodies were structural and functional concordances infections by a variety of micro-organisms, observed in 1-2% of the THP-l cells. In which would have remained cryptic using has remained an unsolved issue. As con- addition to the inclusion bodies, atypical are not to simple sequence alignment. trolled human studies easy perform chlamydiae were found in the monocytic we used a rat model. Yersinia entercolitica 0:8, phagosomes by electron microscopy. Pre- 1 Hermann E, Yu D T. HLA-B27-restricted CD8 when injected intravenously into Lewis rats, incubation with IFNy (25 U/ml) reduced the T cells derived from synovial fluids ofpatients causes in 70% ofthe animals a sterile arthritis number of inclusion bodies by 90% in with reactive arthritis and ankylosing closely resembling human reactive arthritis. spondylitis. Lancet 1993; 342: 646-50. THP-1 cells and productive infection was Arthritis in one to two weeks, in 2 Anonymous. 25th Meeting of the Society of develops converted into a non-infective state. After http://ard.bmj.com/ Immunology, Konstanz, Germany, Sep- some of the animals it remains chronic, and IFN-y treatment atypical chlamydiae were tember 21-24, 1994. Abstracts. Immuno- exacerbations occur. biology 1994; 191: 65-314. still seen in phagosomes of THP-1 cells. In With this model we studied the effect of a THP-1 exogenously added tryptophan (200 seven day treatment with ciprofloxacin, using ,ug/ml) did not reverse the antichlamydial two dosages (20 or 100 mg/kg/day) and four effect of in contrast with control enterocolitica: a cause of IFNy, Yersinia different timings. A seven day course with the experiments with Hep-2 cells where the chronic polyarthritis higher dosage, started on day 3 after the IFN-y mediated growth inhibition of C I M van der Heijden*, P C M Res*, bacterial inoculation, completely prevented

trachomatis was completely prevented by on September 25, 2021 by guest. Protected copyright. B Wilbrink, A Leow*, F C Breedveld*, development of reactive arthritis and elimi- J P P Tak* tryptophan. Heesemannt, nated yersinia during the 60 day experiment. These data show that (a) C trachomatis can If the lower dose was used, development of persist in human monocytic THP-1 cells and *Department ofRheumatology, Leiden acute arthritis was prevented, but some ofthe University Hospital, The Netherlands; (b) in contrast with fibroblasts, the persistent tInstitut animals had positive fecal cultures at the end chlamydial infection in THP-1 cells is not fPr Hygiene und Mikrobiologie der Universitat of experiment. If antibiotic treatment was Wurzburg, Germany mediated by tryptophan deficiency. Thus the started on day 5, the preventive effect was still persistence of C trachomatis is differentially observed but less pronounced. If treatment regulated in monocytes and fibroblasts. Yersinia enterocolitica is associated with was started at the peak of the arthritic reactive arthritis. Persistent polyarthritis development, no effect on arthritis was affects about 2% of patients with yersinia found. induced reactive arthritis. As cultures are The results suggest that patients with often negative after the acute phase, the cause infections known to induce reactive arthritis Amplification ofplasmid and may remain undetected. We attempted to should be considered for early antibiotic chromosome chlamydia DNA in demonstrate yersinia antigens in biopsy treatment. This applies particularly to those synovial fluid ofpatients with reactive specimens from two patients with chronic with chlamydia infections and, in the case of arthrits and undifferentiated polyarthritis, who had circulating antibodies enteric infections (yersinia, salmonella, seronegative oligoarthropathies to yersinia outer proteins. Responses of campylobacter), HLA-B27 positive indi- Sylvette Bas*, Remy Griffaist, synovial T cell clones against yersinia were viduals and patients with a history of reactive Tore K Kvient, Anne GlennAs4, also investigated. arthritis. Kjetil Melby§, Thomas L Vischer* Two patients were studied: (1) a 57 year old women with arthritis ofknees and elbows, Key papers *Divion ofRheumatology, University but no gastrointestinal complaints and (2) a Toivanen A, Yli-Kerttula T, Luukkainen R, Merilahti-Palo Granfors Hospital, Geneva, Switzerland; tLaboratoire 44 year old women with arthritis of knees, R, K, Seppdla J. Effect of antimicrobial treatment on chronic reactive des Rickettsiales et Chlamydiales, Institut ankles, and hands, and bouts of diarrhoea. arthritis. Clin Exp Rheumatol 1993; 11: 301-7. Pasteur, Paris, France; tOslo City Department Both patients were HLA-B27 negative. The Gripenberg-Lerche C, Skurnik M, Toivanen P. ofRheumatology, Norvegian Lutheran only lead was the demonstration of circu- Role ofYadA-mediated collagen binding in arthrito- genicity of Yersinia enterocolitica serotype 0:8; Hospital, Oslo, Norway; §Department of lating IgA and IgG antibodies to yersinia experimental studies with rats. Infect Immun 1995; Microbiology, Ullevdl University Hospital, outer proteins. 63: 3222-6. Oslo, Norway 582 Submitted abstracts

Oligoarticular synovitis of undetermined To test this hypothesis, we carried out Our results show that in our experimental origin can closely resemble an incomplete epitope mapping of 31 OspA-specific T cell setting dendritic cells phagocytose B burg- form ofreactive arthritis without history ofan lines and five T cell clones derived from the dorferi by two different uptake mechanisms. Ann Rheum Dis: first published as 10.1136/ard.55.8.564 on 1 August 1996. Downloaded from initial infectious event. Reactive arthritis can synovial fluid or peripheral blood of three These deliver the microbes into two different be related to an infectious cause, but an patients with treatment resistant Lyme cell compartments which may lead to both important question is whether whole micro- arthritis. Although each patient's T cell line major histocompatibility complex (MHC) organism is present in the joint or only recognises a broad array of OspA peptides class I and MHC class II associated antigen antigens. Different studies have been carried with different individual patterns, two presentation. The regulatory effects of IL-10 out with the aim of looking for chlamydia regions of OspA were dominantly recognised. and IL-12, especially on accessory dendritic nucleic acids, with variable results. Each patient's T cell line dominantly recog- cell functions and induction of T helper cell To clarify the discrepancies between these nised a C terminal epitope of OspA ranging type 1 and cytotoxic T lymphocyte responses, results we attempted to optimise the con- from amino acids 214-233 in one patient to are the subject of current investigations. ditions for DNA amplification with poly- 244-263 in another, and all three patients' T merase chain reaction, in synovial fluid. After cell lines dominantly recognised an epitope multiple trials to increase sensitivity and between amino acid 84 and 113. Thus the specificity we used four different amplifi- region of OspA between amino acid 84 and cation systems on samples from 71 patients 113 was the dominant T cell epitope shared with various arthropathies. Detection was by these three patients with treatment targeted both at the chlamydia cryptic resistant Lyme arthritis. If the T cell response Different interleukin-4 production in plasmid and at the chromosomal DNA. to OspA plays a part in the pathogenesis of the synovial fluid ofpatients with Specificity was improved in two ways: the use treatment resistant Lyme arthritis the epitope reactive arthritis and rheumatoid of nested primer sets that primed within the 84-113 is a candidate arthritogenic epitope. arthritis first sequence amplified, and the use of an Z Yin, J Braun, J Sieper internal labelled hybridisation probe to detect 1 Lengl-Janssen B, Strauss A F, Steere A C, Kamradt T. The T helper cell response in a specific fragment amplified by a polymerase Lyme arthritis: differential recognition of Deutsches Rheumaforschungszentrum Berlin chain reaction in a Southern blot. Borrelia burgdorferi outer surface protein A in and Department ofMedicine, Klinikum We amplified different sequences of patients with treatment-resistant or Benjamin Franklin Berlin, Germany chlamydia DNA from four patients with treatment-responsive Lyme arthritis. J Exp Chlamydia trachomatis sexually acquired Med 1994; 180: 2069-78. reactive athritis (CT-SARA), from six with Previously our group reported divergent T SARA, from 11 with undifferentiated sero- cell cytokine patterns in inflammatory negative mono/oligoarthritis, and from one arthritis.' It was shown in the report that in control patient. Although this method detects synovial tissue, rheumatoid arthritis samples small of the genome, different Phagocytosis ofBorrelia burgdorferi by only parts human dendritic cells express a Th 1-like pattern whereas in reactive DNA pieces were found in the samples. arthritis move towards a Th2 pattem. it is improbable that they corres- A Krause*, M Rittigt, F Steinbacht, Therefore, B Thiele*, G R Burmester* To analyse further the divergency of pond to residual genetic material from non- cytokine pattern, the interferon y (IFNy) and viable organisms. These results indicate that interleukin-4 (IL-4) production of peripheral of intra-articular viable *Department ofInternal Medicine III, Charite, the presence Berlin; tDepartment ofAnatomy I; University blood (PB) and synovial fluid (SF) mono- chlamydia in certain cases of SARA and nuclear cells from patients with reactive undifferentiated is ofErlangen, Erlangen; tInstitute of Virology, mono/oligoarthritis Free University, Berlin, Germany arthritis and rheumatoid arthritis stimulated probable. with optimal concentration of phyto- haemagglutinin were measured by enzyme

Although phagocytosis by dendritic cells has linked immunosorbent assay (ELISA). In PB http://ard.bmj.com/ been reported before, there still are contro- the IFN-y production from patients with versies about the phagocytic capacity of these rheumatoid arthritis and the healthy donor's Dominant recognition ofa Borrelia cells and the uptake mechanism used by control group was slightly lower than that in burgdorferi outer surface protein A them. This study aimed at investigating the the group with reactive arthritis, but for IL-4 peptide by T helper cells in patients with phagocytosis by dendritic cells of Borrelia production both groups showed similar treatment resistant Lyme arthritis burgdorferi, the causative agent of Lyme potential, though some rheumatoid arthritis Thomas Kamradt*, Beate Lengl-JanI3en, borreliosis, which appears to be particularly samples (three out of seven) produced less. However, in SF samples of patients with Ann F Strauss, Geetha Bansalt, difficult to internalise because of its length, on September 25, 2021 by guest. Protected copyright. Allen C Steere motility, and corkscrew-like morphology. reactive arthritis and rheumatoid arthritis Dendritic cells were generated from there was a difference of IL-4 production. Division ofRheumatology/Immunology, adherent peripheral blood mononuclear cells The IL-4 production from the group with RA Department ofMedicine, NEMCH, Tufts by stimulation with interleukin-4 (IL-4) and (1-8 (SD 2 6) pg/ml) was clearly lower than University School ofMedicine, Boston, MA granulocyte-macrophage colony stimulating that in reactive arthritis samples (16 3 (4 3) 02111. *Corresponding author, current address: factor for three to four days. A this time, cells pg/ml). Next, the same SF samples from Universitatsklinikum Charite, Medizinische had a dendritic morphology and exhibited patients with reactive arthritis were stimu- Klinik III, Rheumatologie und Klinische a CD1a+/CD14±/HLA-DR++ phenotype. lated with the triggering bacterium, yersinia Immunologie, D-10098 Berlin; tMedImmune, Dendritic cells were incubated with a 10-fold or chlamydia. All the samples showed similar Gaithersburg, MD excess of B burgdorferi (B burgdorferi sensu level of IFNy production (yersinia 1183 stricto strain LW2) for 24 to 48 hours. Phago- (217) pg/ml, chlamydia 1084 (270) pg/ml) cytosis of B burgdorferi was examined by compared with phytohaemagglutinin (1300 Lyme arthritis is a tick-borne infection electron microscopy. Culture supernatants (135) pg/ml). In contrast, only three out of caused by Borrelia burgdorferi sensu lato spiro- were harvested for the determination of the 14 samples produced a small amount ofIL-4. chaetes. Whereas most patients with Lyme immunoregulative cytokines IL-I 0 and IL- 12 Interestingly, one case included in this study, arthritis are cured with antibiotic treatment, by commerically available enzyme linked examined at three different times, showed a some 10% of the patients have persistent immunosorbent assay (ELISA). decrease of IL-4 production after stimulation arthritis after treatment. In an earlier study Electron microscopy studies showed that with phytohaemagglutinin which correlated we found that T cell lines from patients with dendritic cells readily phagocytose B burg- with improvement of disease. treatment resistant Lyme arthritis prefer- do?feri. As described for professional phago- Taken together, the Th2-type cytokines entially recognised B burgdorferi outer surface cytes before, dendritic cells used both may be the main reason for persisting protein A (OspA), but T cell lines from conventional and coiling phagocytosis simul- bacterium in the joints of patients with patients with treatment responsive arthritis taneously. Unlike conventional phagocytosis chronic reactive arthritis. These results only rarely recognised this protein.' which led to lysosomal degradation, the latter further confirm the data from the synovial Dominant T cell recognition of an arthrito- uptake mechanisms resulted in a cytosolic tissue mentioned above. genic OspA epitope is one possible way in disintegration of the engulfed microbes. stimulation with B dendritic which the immune response against OspA Upon burgdorferi 1 Simon A K, Seipelt E, Sieper J. Divergent T cell might be involved in the pathogenesis of cells produced high levels of IL-10 and low cytokine pattern in inflammatory arthritis. treatment resistant Lyme arthritis. amounts of IL- 12. Proc Natl Acad Sci USA 1994; 91: 8561-6. Submitted abstracts 583

Persistent knee synovitis in reactive HLA-B27 subtypes and ankylosing lymphocytotoxicity assay (LCA) and PCR. arthritis: clinical and histological spondylitis in the UK Amplified sequences (exon 2 and 3) of HLA- correlations M Brown*, M Rudwaleit*, A Calint, B27 positive patients were then tested with F Chieco-Bianchi*, L Cozzi*, C Rigon*, F Cornelist, K Pile*, P Wordsworth* eight sequence specific oligonucleotides to Ann Rheum Dis: first published as 10.1136/ard.55.8.564 on 1 August 1996. Downloaded from C Gallo*, M A Favaro*, A Fassinat, distinguish all HLA-B27 subtypes. G Espositot, A De Candia§, L Rubaltelli§, * The John Radcliffe, Oxford; tRNHRD Bath, In patients with rheumatic disease 61/398 U Fiocco*, S Todescot UK; tH6pital Lariboisiere, Paris patients tested HLA-B27 positive by micro- lymphocytotoxicity assay. When a PCR The association of HIA-B27 and ankylosing technique was used 78 patients tested HLA- *Division ofRheumatology; tInstitute of spondylitis is well known. It is less clear, B27 positive. Eighteen patients with a weak Pathology; tInstitute of Oncology, §Institute of however, whether the risk for the develop- signal or possible cross reaction (HLA-B7) in Radiology, University ofPadova, Italy ment of ankylosing spondylitis conferred by microlymphocytotoxicity testing gave a posi- HLA-B27 subtypes associated with anky- tive PCR result in 11 cases. Six patients with Knee joint involvement is one of the main losing spondylitis is the same for the a negative serological typing result for clinical manifestations in reactive arthritis, respective subtypes. Therefore we typed 284 HLA-B27 and a positive PCR result were and a common event in patients with chronic white patients with ankylosing spondylitis sequenced, and the positive PCR result was evolution of the disease. Measurement of and 1010 healthy white controls for B27 by confirmed. In patients with uveitis 23/97 synovial effusion and proliferation in the knee the polymerase chain reaction. 94% of the patients tested positive for HLA-B27 (LCA/ joint of patients with reactive arthritis can be patients with ankylosing spondylitis and PCR). Subtyping in 144 HLA-B27 positive useful for an objective assessment of the 9-4% of the controls were positive for B27 patients showed 24X B2702, 4X B2704, and severity and of changes in local disease (odds ratio (OR) 161, 95% confidence 116X B2705. Three patients tested positive activity. interval (CI) 113 to 230, p < 10A), confirm- for B2704 and B2705. Sequencing showed We conducted a prospective study to com- ing the previously reported strong association subtype B2705 in all three patients. No signi- pare the ultrasound pattern with the gross of B27 with ankylosing spondylitis. HLA- ficant association between any HLA-B27 appearance of knee synovitis and the B27 subtyping was performed on 172 of the subtype and a certain type of HLA-B27 histological picture. 268 HLA-B27 positive patients with associated disease could be found. At study entry synovial proliferation was ankylosing spondylitis and on 154 HLA-B27 The PCR technique is a reliable and evaluated (location, extent, and morpho- positive, healthy and ethnically matched reproducible method for HLA-B27 typing logic patterns) by a 10 MHz mechanical blood donors using single strand conforma- and can improve the common serological sectorial transducer and by scanning three tion polymorphism, a method which has also typing. Compared with LCA testing, the recesses of the knee (suprapatellar, lateral, the potential of detecting new subtypes. HL- PCR technique is able to classify uncertain and medial parapatellar). Findings were B*2705 was present in 169/172 patients with results (weak positive LCA results, cross then compared with arthroscopic visualisa- ankylosing spondylitis and in 147/154 reaction with HLA-B27). HLA-B27 sub- tion as the "gold standard" in six patients controls. HLA-B*2702 was found in 3/172 typing, so far, seems to have no clinical with persistent knee synovitis undergoing patients and 5/154 controls, and HIA- relevance for making a diagnosis. arthroscopic synovectomy. Moreover, to B*2708 was found in 2/154 controls only. monitor disease progression and response Overall, the frequencies of the B27 subtypes to treatment, ultrasound joint effusion were not significantly different in patients and synovial thickness were measured in and controls. No novel HIA-B27 subtypes four of these patients before and up to were detected in this study. Characterisation ofthe HLA-B27 linked six months after arthroscopic synovec- In addition, we typed all patients and mica gene tomy. controls for the B60 antigen by the poly- H Kellner*, M Schattenkirchner*, Ultrasound of localisation, morphology, merase chain reaction. Although no signifi- B Frankenbergert, E Benkhartj, E H Weisst and degree of synovial proliferation corres- cant difference in the overall B60 frequencies ponded with arthroscopic gross appearance was found between patients with ankylosing *Med. Poliklinik, D-80336, Miinchen, http://ard.bmj.com/ in every case. The predominant morpho- spondylitis and controls (32/268 v Germany; tInstitutfur Anthropologie und logical pattern on ulstrasound and subse- 122/1010), after stratification on B27 there Humangenetik, LMU, D-80333 Muanchen, quently confirmed by arthroscopic visual- was a significant increase of B60 in the Germany isation was villonodular (highly developed patients (11-9% v 6-2%, OR 2-0, 95% CI 1-4 hypertrophic villi) in four knees, and uniform to 3-1, p < 0-0005). The HLA-B27 antigen is known to be asso- thickening (uniform synovial pannus with ciated with ankylosing spondylitis and related tightly crowded villi) in two. The lympho- spondyloarthropathies. Several hypotheses cytic infiltrate, vascularity, and fibrosis of attempt to explain this association, based on September 25, 2021 by guest. Protected copyright. these two patterns also differed histologically. Analysis ofHLA-B27 subtype upon the function of the class I molecules of Immunohistological studies are now in frequencies in patients with rheumatic the major histocompatibility complex progress. disease: allelic typing by polymerase (MHC) as receptors for peptide ligands and Four male patients with reactive arthritis chain reaction and sequence specific as restriction elements for cytotoxic T cells. (three with urogenital forms and one with a oligonucleotides They propose a unique ligand presenting gastrointestinal form), with a mean age of H Kellner*, St Breitkopf*, function of the HLA-B27 antigen, stimu- 38-8 (SD 4-9) years and a synovitis duration B Frankenberger*, G Wildnert, S Thuraut, lating a local T cell response. Nevertheless, of 26-5 (12-6) months, received sulpha- M Schattenkirchner*, E Weisst the presence of linked or additional pre- salazine treatment for more than six months disposing genes within the HLA-B region and were then treated with arthroscopic *Med Poliklinik; tAugenklinik und; tInstitut cannot be ruled out. Novel polymorphic class synovectomy; a significant reduction in both furAnthropologie und Humangenetik, LMU I genes (MIC) within the human MHC have the global clinical index (p = 0 03) and the Munchen, Germany recently been identified which are mainly ultrasound index (p = 0 02) was seen at six transcribed in epithelial cell lines. months. This study aimed at investigating whether We analysed whether HLA-B27 haplotypes Although preliminary, these data seem to HLA-B27 typing by polymerase chain possess a unique MICA gene which is located confirm the persistence and severity of reaction (PCR) can provide better typing close to the HIA-B locus. We found that the proliferative knee joint synovitis process in results than those commonly obtained with MICA sequence of a homozygous lympho- reactive arthritis. Our ultrasound procedure serological typing (microlymphocytotoxicity blastoid B cell line differs from the eight enabled an accurate morphological and assay). Furthermore, all HLA-B27 positive MICA alleles characterised so far. We are quantitative definition of the synovial pro- patients with rheumatic disease or uveitis, or currently comparing MICA sequences from liferation, as confirmed by arthroscopic both, should be subtyped by sequence healthy and affected HLA-B27 positive visualisation. Therefore, in addition to histo- specific oligonucleotides to search for a individuals to find out whether the latter code logical evaluation, we suggest that ultrasound possible association between HIA-B27 sub- for a unique MICA allele. In addition, we are can be a useful, objective method for types and certain types of HLA-B27 asso- analysing the mRNA expression pattern of monitoring the progression of knee joint ciated disease. MICA in affected tissues. synovitis, and the response to treatment, with Three hundred and ninety eight consecu- In conclusion, the identification of a the advantage of non-invasiveness and low tive patients with rheumatic disease and 97 specific HLA class I gene in close proximity cost. patients with uveitis were HLA-B27 typed by to HLA-B27 might indicate the presence of 584 Submitted abstracts an alternative or additional predisposing gene cases (proband DRI allele frequency 30%, complex (MHC) class I antigen HLA-B27, for HLA-B27 associated diseases. relative risk (RR) 3-5, 95% confidence and the interaction between microbial anti- interval (CI) 2-6 to 4 7) and in sporadic cases gens and the HLA-B27 molecule is thought Ann Rheum Dis: first published as 10.1136/ard.55.8.564 on 1 August 1996. Downloaded from Supported by a grant from the Thyssen Stiftung. of ankylosing spondylitis (24% DR1+, RR to play an important part in the cause of the 2-5, 95% CI 1-7 to 3 7) compared with the disease. As MHC class II and non-HLA general population (11% DR1+). However, antigens also contribute to the immune this may be due to linkage disequilibrium response to chlamydia in patients with HLA class II associations ofankylosing between B27 and DRI as the frequency of reactive arthritis we studied the frequency of spondylitis DRI in our B27 positive controls was signifi- these MHC antigens in chlamydia induced M A Brown*, K D Pilet, J Klinke*, cantly higher than in the general population arthritis. K Gibson*, M Buncet, A Calin§, (19% v 11%, X2=15-8, p=7-2X 10-5-5). HLA class II antigens DRB1*, DQA1*, C Darke I1, J Davis¶, B P Wordsworth* When compared with our B27 positive DQB1*, and DPB1* and the MHC class III controls, the association between DR1 and antigens C2, Bf, C4A, and C4B were *Nuffield Department ofMedicine, J3ohn ankylosing spondylitis remained significant in determined by polymerase chain reaction- Radcliffe Hospital, Oxford; tQueen Elizabeth the familial cases of ankylosing spondylitis restriction fragment length polymorphism in Hospital, Adelaide, Australia; tOxford (RR 1-8, 95% CI 1-2 to 2 7) but not in the 30 patients with chlamydia induced arthritis Transplant Centre, Churchill Hospital, Oxford; group with sporadic ankylosing spondylitis (14 B27 positive and 16 B27 negative §Royal National Hospitalfor Rheumatic (RR 1-3, 95% CI 0-8 to 2-1). No other class patients). The diagnosis of chlamydia in- Diseases, Bath; II Regional Tissue Typing II allele was associated with ankylosing duced arthritis was based on either sympto- Laboratory, National Blood Transfusion Service spondylitis. DRI was not independently as- matic urogenital infection associated with (Wales), Rhydlafar, St. Fagans, Cardiff, sociated with ankylosing spondylitis in the raised IgG and IgA class serum antibodies to Wales; ¶Oxford Regional Transfusion Service, small subgroup of nine B27 negative patients chlamydia (n = 11) or chlamydia positive J7ohn Radcliffe Hospital, Oxford (DR1+22%, when compared with general urogenital smears (n = 19) in patients with population RR 2-3, 95% CI 0 77 to 6 8). otherwise unexplained arthritis. The fre- Ankylosing spondylitis is clearly associated Where haplotypes could be assigned in the quencies of MHC antigens in the patients with the HLA class I gene, B27, but class II familial cases, B27 and DR1 were mostly were compared with published antigen fre- associations have also been reported with inherited linked on the same haplotype (19 quencies among healthy white controls in DR1, DR2, and DR7. Many investigators linked, seven not linked, 11 indeterminate). Germany.' 2 have found no association between class II B60 was significantly associated with anky- Fourteen of 30 patients were HLA-DR4 alleles and ankylosing spondylitis, but in all losing spondylitis in both our familial and positive (47% v 13-2% controls; x2 = 30-887; these studies the number of patients and sporadic cases of ankylosing spondylitis p < 0-001). These patients had the following controls has been small. (familial ankylosing spondylitis 15% B60+, subtypes: DRB 1 *0401 (n = 5), DRB 1*0404 We studied the HLA class II allele fre- RR 79, 95% CI 2-6 to 23-8; sporadic anky- (n = 5), DRB1*0405 (n = 2), DRB1*0403 quencies in 110 cases of familial ankylosing losing spondylitis 9% B60+, RR 5.5, 95% CI and DRB1*0407 (one each). Nine of the 14 spondylitis (more than one case in the index 1-5 to 19 9). There was no difference in the DR4 positive patients were also HLA-B27 generation), 70 patients with sporadic DRI allele frequency between those who positive (9/30 (30%) v 2% controls; ankylosing spondylitis, and 147 B27 positive were B60 positive or negative. These results X2 = 88X398; p < 0 001). Normal frequencies controls (71 blood donors, 47 healthy raise the possibility of a second ankylosing were found for DQA1*, DQB1* and DPB1* individuals, 18 cadaveric organ donors, 11 spondylitis susceptibility gene on an antigens. Among MHC class III antigens, C2 patients with chronic lymphocytic leukae- extended B27/DR1 haplotype (probably not and Bfalleles were normally distributed while mia). Although these patients were not DR1), the effect ofwhich is seen most clearly C4A-QO was found with increased frequency known to have ankylosing spondylitis, as they in the familial cases. (14/30 (47%) patients v 2-9% controls; for this it had not been screened condition, X2= 88-017; p < 0 001). is possible that some cases would have been This study on a limited number of patients included in the control samples. This could http://ard.bmj.com/ Frequency ofHLA class II and non- suggests tFlat in addition to HLA-B27 the result in an underestimate of the relative risk HLA class II antigen DR4 and C4A-QO of any disease susceptibility gene. Class II HLA antigens in patients with chlarnydia induced reactive arthritis occur with increased frequency in chlamydia typing was carried out by polymerase chain induced arthritis. reaction (PCR)/sequence specific oligo- J Wollenhaupt*, H Zeidler*, U Voegelert, nucleotides in all patients with ankylosing B Kubenst, S Ferencikt, A Bialowona*, 1 Baur M P, et al. Population analysis on the basis spondylitis, whereas the controls were typed H Grosse-Wildet of deduced haplotypes from random families. In: Albert E D, Baur M P, Mayr W R, eds. by a mixture of serology and PCR/sequence *Division ofRheumatology, Hannover Medical Histocompatibility testing 1984. Berlin: specific primers. Familial cases of ankylosing School, Hannover, Germany; tInstitutefor Springer, 1984: 333-41. on September 25, 2021 by guest. Protected copyright. spondylitis were also typed for B27 and B60 2 Schendel D J, O'Neill G J, Wank R. MHC- Immunogenetics, University ofEssen, Germany linked class III genes. Analysis of C4 gene by PCR. frequencies complotypes and association with A significant association between DR1 and Chlamydia induced arthritis is strongly distinct haplotypes in German Caucasians. ankylosing spondylitis was found in familial associated with the major histocompatibility Immunogenetics 1984; 20: 23-31.