Biochemical aspects of infection in rheumatoid arthritis and ankylosing spondylitis

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Biochemical aspects of infection in rheumatoid arthritis and ankylosing spondylitis VERA NEUMANN

Several arthritides have a well-documented relation to infection. The role of certain streptococcal infections in rheumatic fever is well established. Reactive arthritis (including Reiter's disease) may follow a variety of enteritic infections. These include salmonella (Berglof, 1963; Vartiainen and Hurri, 1964), shigella (Paronen, 1948; Noer, 1966), yersinia (Aho et al, 1974), Clostridium difficile (Bolton et al, 1981) and Campylobacter ]ejuni (Berden et al, 1979). Reactive arthritis following non-specific urethritis probably due to chlamydial infections has also been described, as has gonococcal arthritis in which the organism is sometimes but not always detectable within the joint. Lyme arthritis is also now recognized as developing in subjects infected by the spirochaete Borrelia burgdorperi via a tick bite through the skin (Steere et al, 1983). In many of the above examples, epidemiological data have provided the first clue to aetiopathogenesis. For example, in Lyme arthritis a patient's observation of seasonal variation in her own and her family's symptoms focused the attention of researchers on insect vectors. Biochemical and immunological data have been used mainly as supportive evidence. However, in two of the numerically most important arthritides, ankylosing spondylitis and rheumatoid arthritis, infection is widely believed to play a role in disease pathogenesis but epidemiological studies have so far yielded few clues as to the nature of any such infectious agent. Investigators have, therefore, been forced to look for other clues using a variety of microbiological, immunological and biochemical methods. The contribution of biochemistry to research into the pathogenesis of rheumatoid arthritis and ankylosing spondylitis is the subject of this chapter. ANKYLOSING SPONDYLITIS Uncomplicated ankylosing spondylitis, the arthritides associated with inflammatory bowel disease, and Reiter's disease show some striking clinical similarities; all can present as a peripheral mono- or poly-arthritis and can also manifest as sacroiliitis with or without spondylitis. Eye involvement

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(uveitis or conjunctivitis) is common. Subcutaneous rheumatoid nodules are not seen and there is no association with serum rheumatoid factor. There are other less obvious shared features; in particular familial aggregation of the conditions and association with a histocompatibility antigen, HLA B27. Recognition of these shared features led Moll et al (1974) to group them under the general heading--'the seronegative spondarthritides'. Thus, clinical, epidemiological and genetic data have provided evidence that uncomplicated ankylosing spondylitis is linked with other arthritides, several of which have an overt association with bowel inflammation or infection. Of the various spondarthritides, ankylosing spondylitis bears most clinical resemblance to Reiter's disease. Also the association with HLA B27 is strongest in these two disorders, implying a closer genetic similarity. Since Reiter's disease can follow certain types of bacterial gastroenteritis, it is not surprising that researchers into the aetiopathogenesis of ankylosing spondylitis have sought evidence of bowel inflammation or bacterial infection in the bowel. Several different experimental approaches have been used. Patients with uncomplicated ankylosing spondylitis have undergone investigations to look for hidden bowel inflammation. Faecal samples from patients with ankylosing spond.ylitis have been analysed for evidence of bowel infection with p articular orgamsms. Immunological techniques have been used to look for relationships between immune responses to certain gut-related organisms and ankylosing spondylitis. Finally, the function of HLA antigens has also been examined in much greater detail in the hope of ascertaining how HLA type may influence susceptibility. Evidence of bowel inflammation

Attempts to identify silent bowel pathology in patients with ankylosing spondylitis by direct methods have been largely unsuccessful. Jayson et al (1970) studied 47 patients with ankylosing spondylitis using barium enema, proctosigmoidoscopy and rectal biopsy but revealed only two cases of unsuspected bowel inflammation. More recently, Mielants et al (1985), using the more advanced technique of fibre-optic ileocolonscopy, detected previously unsuspected cases of inflammatory bowel disease in patients with ankylosing spondylitis. However, affected patients tended to have peripheral arthritis or to be B27 negative. Histological evidence for inflammatory changes in the bowel in patients with axial seronegative arthritis is, therefore, only weak. Investigators have turned to less direct approaches and revealed some evidence of abnormal bowel permeability using isotope markers and other methods (Smith et al, 1985). Various groups (Veys and Van Laere, 1973; Kinsella et al, 1975; Cowling et al, 1980) have reported that elevated serum IgA concentrations are detectable in cases of ankylosing spondylitis. Cowling and colleagues (1980) also reported that serum IgA concentrations were increased during active phases of the joint disease. In interpreting these findings they stated that serum IgA is 'produced mainly within the mucosal associated lymphoid tissues of the gastrointestinal tract'. They

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claimed that their result indicated an inflammatory process within the bowel and was therefore consistent with the hypothesis that microbial infection in the gut was triggering ankylosing spondylitis (Trull et al, 1983). Others also reported elevated serum IgA in association with active ankylosing spondylitis. Increased salivary IgA was noted in patients with the active disease (Calguneri et al, 1981). This seemed to lend support to the view that gastrointestinal mucosal inflammation was a feature of ankylosing spondylitis. However, in our own more recent study (Wright et al, 1985), whilst we were able to confirm that serum and salivary IgA are elevated in ankylosing spondylitis, we found no such changes in serum and salivary IgA in a group of 43 patients studied during and after bacterial gastroenteritis. Similar observations have been made in shigellosis (Reed and Williams, 1971), cholera (Waldman et al, 1971, 1972) and salmonella enteritis (La Brooy et al, 1980). Ulcerative colitis and Crohn's disease are by definition associated with bowel inflammation, yet some authors have found normal serum immunoglobulin in both disorders (El Khatib et al, 1978). Others (Hodgson and Jewell, 1978) have reported that, whilst all three immunoglobulin classes are elevated in ulcerative colitis, only IgM is elevated in the serum in Crohn's disease. It follows from the above observations that elevations in the serum and salivary IgA in ankylosing spondylitis do not necessarily indicate bowel inflammation. IgA may instead be produced in increased quantities from other sources, such as the bone marrow or the synovium. The search for specific microorganisms Earlier reports suggested cross-reactivity between B27-positive lymphocytes and several Gram-negative bacteria (Ebringer et al, 1977) and the search for putative microbial triggers in ankylosing spondylitis has concentrated on this group of organisms. Subsequently, Ebringer et al (1977, 1978) reported that Klebsiella pneumoniae was found more frequently in the faeces of patients with active ankylosing spondylitis than in patients with inactive disease or controls. Sequential sampling also suggested that acquisition of klebsiella was frequently followed by an increase in disease activity (Ebringer et al, 1978). These reports provoked both interest and criticism. The inclusion of 18 patients without definite sacroiliitis (and therefore not fulfilling Rome criteria for ankylosing spondylitis) was criticized, as was the use of hospital-based controls. It is also important to note that the yield of klebsiella in the ankylosing spondylitis group as a whole in this study was not significantly different from the yield in the control group. Therefore, the paper hinges on the division of patients into active, probably active and inactive disease categories. Ebringer et al (1978) defined active disease as those patients who had evidence of peripheral synovitis, acute anterior uveitis or a recent exacerbation of spinal pain or stiffness. However, this clinical definition is not widely accepted, particularly as acute anterior uveitis and peripheral synovitis may both exacerbate independently of spinal disease, or may even precede the onset of spinal

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symptoms by several years (Hart and MacLagan, 1955; Wilkinson and Bywaters, 1958). Subsequent attempts by other groups (Warren et al, 1980; Eastmond et al, 1980, 1982; Shinebaum et al, 1984) have yielded conflicting results. All these studies were hampered by the lack of well-defined objective measures of disease activity in ankylosing spondylitis. The use of patient diaries to record pain, analgesic requirements and morning stiffness is limited by their subjective nature. Objective clinical measurements of joint mobility, such as those described by Wright and Moll (1976), are undoubtedly reliable but not sufficiently sensitive to detect the minor fluctuations that occur in patients with ankylosing spondylitis over a period of months. The use of biochemical tests to assess disease activity is not yet fully established but may well help future research in this field. Although the erythrocyte sedimentation rate (ESR) has been used by some groups (Ebringer et al, 1978) their justification for the use of an ESR above 15 mm/h as a definition of active disease is not at all clear. This group and others (Dixon et at, 1981) have advocated the use of the C-reactive protein (CRP). Plasma viscosity has also been recommended (Dixon et al, 1981). Both these tests appear to have a higher sensitivity than ESR. Thus, in an initial study of 31 patients with ankylosing spondylitis judged on clinical grounds to have active disease, only 55% of cases had an elevated ESR, whereas CRP and plasma viscosity were abnormal in 83 and 90% respectively. Subsequent research by the same group (Sitton et al, 1987) produced similar findings: CRP was elevated in 88% of patients with ankylosing spondylitis and plasma viscosity in 87%, although this study was hampered by the use of an ESR greater than 30 mm/h as one of three selection criteria. Elevated total serum IgA concentrations are also claimed by some groups to correlate with active spondylitis (Cowling et al, 1980; Calguneri et al, 1981). Other groups, including ourselves (Wright et al, 1985), have been unable to confirm an association between elevated IgA and active disease. Further research to identify biochemical markers of active ankylosing spondylitis is dearly needed. The role of HLA B27: evidence for interaction with Enterobacteriaciae

Immunodiffusion experiments on the relationship between B27 lymphocytes and klebsiella have indicated reciprocal cross-reactivity between the two; antibody precipitation could be detected both when B27 lymphocytes were tested against rabbit anti-klebsiella sera and when klebsiella were tested against rabbit anti-B27 sera (Ebringer et al, 1978). Cross-reactivity of this type was, however, not confined to klebsiella, but was also demonstrated in certain strains of Enterobacter aerogenes, Yersinia enterocolitica, and Shigella sonnei. Anti-B27 sera were also shown, using radio-active labelling, to have increased binding affinity for K. aerogenes (Avakian et al, 1980; Welsh et al, 1980). Thus, partial cross-reactivity has been demonstrated between one or more antigens on human B27 lymphocytes, possibly the B27 molecule itself and an antigenic component found in several Gram-negative bacteria

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including klebsiellas and enterobacteria. Ebringer et al (1978) proposed that such cross-reactivity may result in 'inadequate or delayed immune response by a B27 individual against infecting microorganisms which carry similar antigens, because these organisms would be to some extent recognized as self'. This hypothesis has been called the molecular mimicry or the crossreactivity theory (Damian, 1964; Snell, 1968; Ebringer and Davis, 1973). The observations of Geczy's group in Australia also implied an interaction between Gram-negative bacteria and B27 but differed in certain fundamental aspects. They found that the cells of B27-positive patients with ankylosing spondylitis (B27+ AS+), but not those of B27-positive normal individuals (B27+ A S - ) , carry on their cell surface an antigenic complex which is cross-reactive with a wide range of enteric bacteria (Seagar et al, 1979; Geczy et al, 1983; Prendergast et al, 1983). Since this cell surface determinant was not serologically detectable in a SlCr release assay on the cells of B27- A S - patients or of B27+ A S - normal people, Geczy's group proposed that B27 either formed part of the cross-reactive complex or was in some way required for its expression (Geczy et al, 1983). Molecular mimicry between B27 and enterobacteria cannot explain these observations, since one would expect cross-reactivity to be detectable in all B27+ individuals, irrespective of whether they had ankylosing spondylitis. An 'altered self' hypothesis (Doherty and Zinkernagel, 1975), in which the HLA antigen or a closely associated structure is modified by encountering the foreign antigen, provides a better fit for these observations. Subsequent immunochemical research by these two groups has failed to resolve the debate between the proponents of 'altered self' and 'molecular mimicry'. Nevertheless, this immunochemical research has taught us much about the function of HLA antigens and their role in immunomodulation.

THE ROLE OF MICROBES IN THE PATHOGENESIS OF RHEUMATOID ARTHRITIS

The idea that rheumatoid arthritis is triggered by microbial infection is an attractive one which gains support from several sources. Clearly genetic factors are involved in disease pathogenesis. Rheumatoid arthritis shows an increased prevalence amongst relatives of probands with the disease. Furthermore, association between this disease and the histocompatibility antigen DR4 has now been shown by several groups. However, environmental factors must also be involved. The nature of such factors has intrigued researchers for the last 60 to 70 years. The earlier studies relied on direct microbiological methods. Tests resulted in sporadic reports of isolation of numerous different microorganisms including streptococci (Cecil et al, 1929), diphtheroids (Duthie et al, 1967) and, more recently, mycoplasma (Stewart et al, 1974) and viruses (Norval et al, 1979) from the tissues of patients with rheumatoid arthritis. However, such experiments could seldom be confirmed in other laboratories, and researchers were obliged to turn to other methods of investigation.

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The ability of a particular organism to transmit disease is normally confirmed by the demonstration of organisms upon examination of histological sections of animals that develop that disease. Unfortunately this method of sorting out relevant and irrelevant organisms is unavailable in rheumatoid arthritis because of the lack of good animal models. Certainly, some animals can get chronic arthritic synovitis; for example, Erysipelothrix insidiosa is a naturally occurring cause of chronic proliferative synovitis in swine (Sikes et al, 1969). However, full-blown nodular rheumatoid disease has not been documented. One group (Warren et al, 1969) claimed to have transmitted rheumatoid arthritis to other animals including mice, by injection of human synovial membrane extracts but attempts to perform similar experiments in animals phylogenetically closer to man, such as baboons, have failed (Mackay, 1983). Owing to these difficulties, biochemical and immunological methods have been used increasingly to provide evidence for particular pathogens. Unfortunately, this has resulted in a new set of difficulties mainly due tothe failure to recognize the limitations of these indirect methods of demonstrating infection. As an early example of this problem, consider the demonstration that rheumatoid sera have the ability to agglutinate certain strains of streptococci (Dawson and Boots, 1933). This observation 'caused a furore in rheumatism circles at the time' (Ragan, 1961) and was interpreted as strong evidence for an aetiological role of streptococcus in rheumatoid arthritis. Subsequent work has shown that other bacterial strains or even latex particles coated with y-globulin can be agglutinated by rheumatoid arthritis sera, the responsible agent being rheumatoid factor (immunoglobulin directed against the patients' own IgG) in the patients' sera (LamontHavers, 1954). This example should have taught us caution in subsequent data interpretation but this has not always been apparent. Thus, as the ELISA technique for detecting antibodies and antigens has become widely available, this technique has been applied to rheumatoid arthritis and resulted in numerous reports of specific antibodies in serum. Two Scandinavian laboratories (Larsen, 1980; Gripenberg, I981) have reported that antibodies to Yersinia enterocolitica, a bacteria known to be associated with both enteritis and reactive arthritis, can be detected not only in the yersina arthritis but also in a high proportion of patients with rheumatoid arthritis. Both authors have claimed that this is evidence of an aetiological role for Y. enterocolitica in rheumatoid arthritis. It should be remembered that whereas in some parts of Scandinavia yersiniosis is the most common cause of gastroenteritis (Larsen, 1980), in Britain and in warmer climates this organism is rarely detected. Epidemiological data indicating a worldwide distribution of rheumatoid arthritis does not support a link between this disease and yersinia. Using similar ELISA techniques, as well as Western immunoblotting and indirect bacterial agglutination, Ebringer and colleagues have more recently reported raised antibodies to Proteus mirabilis in the serum of patients with rheumatoid arthritis compared with patients with ankylosing spondylitis (Ebringer et al, 1987). These authors have suggested that rheumatoid arthritis 'may be the end stage of repeated episodes of proteus reactive

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arthritis'. Unlike Y. enterocolitica, the Proteus species is ubiquitous and hence a more suitable candidate for the role of trigger organism in rheumatoid arthritis. However, such immunochemical data alone are insufficient to prove this role. Similar techniques applied to the study of viruses in rheumatoid arthritis have shown that antibodies to both cytomegalovirus (CMV) and EpsteinBarr virus (EBV) are elevated in early disease (Male et al, 1982). Subsequent research has revealed that antibodies to a nuclear antigen (now named RANA) are detectable in some 70 to 90% of patients with rheumatoid arthritis but less than 20% of various control groups. Furthermore, RANA seems to be identical (or at least very similar) to the nuclear antigen produced by EBV (Alspaugh et al, 1978; Ferrell et al, 1981; Hazelton et al, 1987). Whilst the sophistication of such reports is impressive, it is disappointing that anti-RANA antibody titres show little or no correlation with clinical features of rheumatoid arthritis. The data implicating EBV and perhaps CMV, rubella and other candidate viruses could be interpreted as indicating altered handling of viruses by patients with rheumatoid arthritis as a consequence of the disease--just as the early serological studies of streptococcus in rheumatoid arthritis, which were thought to imply streptococcal infection, instead reflect a disease-related immunological abnormality. The limitations of certain biochemical methods of investigating the pathogenesis of rheumatoid arthritis have been discussed. These methods are valuable but need to be supplemented with data obtained by other experimental approaches. One approach has been to study the mode of action of sulphasalazine (SASP) in rheumatoid arthritis. This drug is capable of suppressing clinical and biochemical evidence of disease activity (Neumann et al, 1983; Pullar et al, 1983). It appears similar in efficacyto penicillamine (Neumann et al, 1983). Like chloroquine and dapsone, but unlike other established second-line agents in rheumatoid arthritis (such as gold and penicillamine), SASP is an antibiotic. Its antibiotic activity resides in its sulphonamide moiety, sulphapyridine (SP). Sulphasalazine provides an example of research where a combination of microbiological and biochemical methods has provided more information than the use of either approach alone. Bacteriological methods have been used to study faecal flora in rheumatoid arthritis and have demonstrated higher counts and carriage rates of the bacteria Clostridium perfringens in patients with rheumatoid arthritis than in non-rheumatic controls (Shinebaum et al, 1987). Moreover, counts of Clostridium perfringens and E. coil fall in parallel with clinical and biochemical improvement during treatment of rheumatoid arthritis with SASP (Neumann et al, 1986). Previous pharmacokinetic studies (Azad Khan et al, 1980) have demonstrated that much of SASP is unabsorbed until cleaved by the action of colonic bacteria into its two major constituents, SP and 5-aminosalicylic acid (5-ASA). SP is well absorbed systemically, whereas 5-ASA achieves much lower serum concentrations. Although administering 5-ASA alone to patients with rheumatoid arthritis can achieve similar or increased serum concentrations of this moiety compared with administering the parent drug

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(Astbury et al, personal communication), 5-ASA shows no second-line action (Neumann et al, 1986). In contrast, SP administered alone in doses equivalent to those supplied by the parent drug is comparable in efficacy to SASP (Neumann et al, 1986). Thus biochemical and bacteriological data support the hypothesis that certain gut flora (Cl. perfringens or perhaps E. coli) may be relevant to disease pathogenesis in rheumatoid arthritis. However, as well as being antibiotics, SASP and SP in common with other sulphonamides possess immunomodulating properties which may instead determine SASP's action in rheumatoid arthritis. Further research is now needed to elucidate this problem. The studies of poorly absorbed sulphonamides may help to establish whether the site of action of SASP is within the bowel. It is worth noting that the efficacy of SASP is not dependent on acetylator status (Pullar et al, 1985). Since the acetylator status would influence serum concentrations of the drug and its metabolites, this pharmacokinetic evidence points to a site of action for SASP other than in the serum and hence supports the bacteriological evidence of gut involvement cited above. Studies of structurally unrelated antibiotics which share SASP's antibiotic spectrum but lack its immunological properties, are also likely to be helpful. This chapter reviews the contribution of biochemical and immunochemical methods to research into the pathogenesis of rheumatoid arthritis and ankylosing spondylitis. The 'cause' of both these diseases remains a mystery despite intensive research. One can only conclude that the hypothesis that both diseases are triggered by silent bowel infections remains attractive. Perhaps the main lesson to be learnt is that no single methodology is likely to provide a firm ~answer. Close research co-operation between biochemists, pharmacologists, microbiologists and immunologists provides the best chance of establishing the cause of these diseases.

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Snell GD (1968) The H-2 locus of the mouse: observations and speculations concerning its comparative genetics and its polymorphism. Folia Biologica (Praha) 14: 335-358. Steere AC, Grodzicki RL, Kornblatt AN et al (1983) The spirochetal etiology of Lyme disease. New England Journal of Medicine 303: 733-740. Stewart SM, Duthie J JR, Mackay JMK, Marmion BP & Alexander WRM (1974) Mycoplasmas and rheumatoid arthritis. Annals of the Rheumatic Diseases 33: 346-352. Trull AK, Ebringer R, Panayi GS, Colthorpe D, James DCO & Ebringer A (1983) IgA antibodies to KlebsieUa pneumoniae in ankylosing spondylitis. Scandinavian Journal of Rheumatology 12: 249-253. Vartiainen J & Hurri L (1964) Arthritis due to Salmonella typhimurium. Acta Medica Scandinavica 175: 771-776. Veys EM & Van Laere M (1973) Serum IgA IgM IgA levels in AS. Annals of the Rheumatic Diseases 32: 49~496. Waldman RH, Bencic R, Sakazaki R et al (1971) Cholera immunology. 1. Immunoglobinlevels in serum, fluid from small intestine and faeces from patients with cholera and noncholeraic diarrhoea during illness and convalescence. Journal of Infectious Diseases 12: 579-586. Waldman RH, Bencic Z, Sinha R et al (1972) Cholera immunology. 2. Serum and intestinal secretion antibody response after naturally occurring cholera. Journal of Infectious Diseases 126: 401--407. Warren SL, Marmor L, Liebes DM & Hollins R (1969) An active agent from human rheumatoid arthritis which is transmissible in mice. Archives of Internal Medicine 124: 629-634. Warren SL, Marmor L, Warren RE & Brewerton DA (1980) Faecal carriage of KlebsieUa by patients with AS and RA. Annals of the Rheumatic Diseases 39: 37-44. Welsh J, Avakian H, Cowling P et al (1980) Ankylosing spondylitis, HLA B27 and Klebsiella. 1. Cross-reactivity studies with rabbit antisera. British Journal of Experimental Pathology 61: 85-91. Wilkinson M & Bywaters EGL (1958) Clinical features and course of AS. Annals of the Rheumatic Diseases 15: 209. Wright V & Moll JMH (eds) (1976) Seronegative Polyarthritis. Amsterdam: North Holland Publishing Company. Wright V & Watkinson G (1965) The arthritis of ulcerative colitis. British Medical Journal 2" 670-680. Wright V, Neumann VC, Shinebaum R & Dixon JS (1985) Seronegative spondylarthritis: clinical bacteriological and biochemical aspects. Advances in Inflammation Research 9: 249-259.