685
10. Lauritsen K, Laursen LS, Bukhave K, Rask-Madsen J. Effects of topical 5-aminosalicylic acid and prednisolone on prostaglandin E2 and leukotriene B4 levels determined by equilibrium in vivo dialysis of rectum in relapsing ulcerative colitis. Gastroenterology 1986; 91: 837-14. 11. Wallace JL, MacNaughton WK, Morris GP, Beck PL. Inhibition of leukotriene synthesis markedly accelerates healing in a rat model of inflammatory bowel disease. Gastroenterology 1989; 96: 29-36. 12. Stenson WF, Lobos E. Sulfasalazine inhibits the synthesis of chemotactic lipids by neutrophils. J Clin Invest 1982; 69: 494-97. 13. Lauritsen K, Laursen LS, Bukhave K, Rask-MadsenJ. In vivo effects of
orally administered prednisolone on prostaglandin and leucotriene production in ulcerative colitis. Gut 1987; 28: 1095-99. 14. Lauritsen K, Laursen LS, Bukhave K, Rask-Madsen J. In vivo profiles of eicosanoids in ulcerative colitis, Crohn’s colitis, and Clostridium difficile colitis. Gastroenterology 1988; 95: 11-17. 15. Bukhave K, Gréen K, Rask-Madsen J. Comparison of radioimmunological determinations with gas chromatography mass spectrometry dosage. A study of PGE2 and PGF2&agr; concentrations in gastrointestinal fluids. Biomed Mass Spectrom 1983; 10: 265-68.
K, Laursen LS, Bukhave K, Rask-Madsen J. Inflammatory intermediaries in inflammatory bowel disease. Int J Colorect Dis 1989; 4: 75-90. 17. Blackwell GJ, Carnuccio R, DiRosa M, Flower RJ, Parente L, Persico P. Macrocortin: a polypeptide causing the anti-phospholipase effect of glucocorticoids. Nature 1980; 287: 147-49. 18. Sircar JC, Schwender CF, Carethers ME. Inhibition of soybean lipoxygenase by sulfasalazine and 5-aminosalicylic acid: a possible mode of action in ulcerative colitis. Biochem Pharmacol 1983; 32: 170-72. 19. Nielsen OH, Bukhave K, Elmgreen J, Ahnfelt-Rønne I. Inhibition of 5-lipoxygenase pathway of arachidonic acid metabolism in human neutrophils by sulfasalazine and 5-aminosalicylic acid. Dig Dis Sci 16. Lauritsen
1987; 32: 577-82. 20. Marcus 21.
AJ. Eicosanoids as bioregulators in clinical medicine. Am J Med 1985; 78: 805-10. Goetzl EJ, Pickett WC. The human PMN leukocyte chemotactic activity of complex hydroxy-eicosatetraenoic acids (HETEs). J Immunol 1980; 125: 1789-91.
Salmonella
lipopolysaccharide in synovial cells from patients with reactive arthritis
Synovial cells from nine patients with reactive arthritis following Salmonella enteritidis or Salmonella typhimurium infection were examined for salmonella antigens. Extensive bacterial cultures of the synovial fluid were negative. Eight synovial-fluid cell samples stained positively on immunofluorescence with rabbit antisera against heat-killed S enteritidis or S typhimurium or with monoclonal antibodies specific for the causative salmonella lipopolysaccharide (LPS). Synovial tissue from the ninth patient stained positively in the avidin-biotin-peroxidase complex method with the monoclonal antibody. Control samples (synovial-fluid cells from thirteen patients with other rheumatic diseases and synovial tissue from two) were negative. Synovial cells from eight patients and five controls were studied by western blotting with the same monoclonal antibodies. Four of the eight patients but no controls had blots indicating salmonella LPS in the synovial cells. The presence of bacterial LPS in the joint is a common and pathogenetically important feature of reactive arthritis.
Introduction Reactive arthritis certain infections,
develops as a late complication after including those by species of Yersinia, Chlamydia, Salmonella, Shigella, and Campylobacter,
especially in susceptible people positive for HLA B27. Several theories have been suggested to explain how inflammation in the joint may be stimulated by microbial antigens at a remote site in the body, and all include the idea that the interaction between host and microbe is somehow abnormal and inefficient in HLA-B27-positive subjects in whom reactive arthritis develops. There is direct evidence that microbial antigens enter the joints in reactive arthritis following Chlamydia trachomatis urogenital infection 2-5 and Yersinia enterocolitica gastrointestinal infection.6,7 In addition, enhanced in-vitro responses to microbial agents of synovial-fluid mononuclear cells from patients with reactive arthritis suggest the presence of microbial antigens in the joints.8-10 In this study we sought bacterial lipopolysaccharide (LPS) in synovialfluid cells or synovial tissue from patients with salmonellatriggered reactive arthritis.
ADDRESSES: Departments of Medical Microbiology and Medicine, Turku University, Finland (K Granfors, PhD, S. Jalkanen, MD, O. Mäki-Ikola, MD, R Lahesmaa-Rantala, MD, R Saario, MD, Prof A. Toivanen, MD); Karolinska Institute, Department of Clinical Bacteriology, Huddinge Hospital, Sweden (Prof A. A. Lindberg, MD), Rheumatism Foundation Hospital, Heinola, Finland (R. von Essen, MD, Prof H. Isomaki, MD); and Division of Medicine, Lutheran General Hospital, Park Ridge, Illinois, USA (W. J Arnold, MD). Correspondence to Dr K Granfors, Department of Medical Microbiology, Turku University, SF-20520 Turku, Finland.
686
Patients and methods We studied nine adult patients with salmonella-triggered reactive arthritis, diagnosed on the basis of bacteriological, serological (enzyme immunoassayll), and clinical fmdings. S enteritidis was isolated from the stools of five patients and S typhimurium from three
(see table).
In
eight patients studied (including patient 8,
whose stool culture was negative) the serum antibody responses to the causative microorganism exceeded the mean response in a hundred healthy blood donors by six standard deviations (table). All but two patients had high IgM antibody levels, suggesting recent infection. Typical clinical signs of acute salmonella infection (fever, diarrhoea, or gastrointestinal pain) and of acute reactive arthritis occurred in all nine patients.
DETECTION OF BACTERIAL ANTIGENS IN JOINTS
+ + + ≥6 SD, + + ->4SD, *ABC method
on
+ 2 SD above
mean in
100 normal controls
synovial tissue
Fig 1-immunofluorescence of synovial-fluid cells. panels show positive staining with rabbit antiserum, ’B’ panels show positive staining with corresponding LPS-specific monoclonal antibody in patients 3 (S enteritidis) and 6 (S typhimurium) Panels 3A and 3B x 1000, panels 6A and 6B x 630 ’A’
687
Synovial-fluid leucocytes were obtained from eight patients. Peripheral-blood leucocytes were obtained at the same time as synovial fluid from patients 1, 2, and 3, and 4 weeks later from patient 5. Synovial tissue was obtained from one patient (patient 9; table) as part of an arthroscopic synovectomy 2 years after the first symptoms of gastroenteritis. The sample was immediately frozen in liquid nitrogen and stored at - 70°C. Control material from twelve patients meeting the American
8 8 8 8 8
3
8
II II Ii illl il if
Rheumatism Association’s criteria for rheumatoid arthritis was also studied (synovial-fluid cells from ten and synovial tissue from two). None had a history of recent gastrointestinal symptoms. Synovialfluid cells from three HLA-B27-positive patients with reactive arthritis triggered by Y enterocolitica 0:3 were also studied as controls. Synovial fluid aspirated from the knee was mixed with heparin (50 IU/ml) and centrifuged at 200 g for 10 min. Part of the cell deposit in the residual fluid was stored at - 70°C. Another part was suspended in Hanks’ balanced salt solution, cytocentrifuged onto slides
per slide), fixed in 70°C. The slides were used for indirect immunofluorescence testing and the solubilised cells for western blotting. For synovial samples from patients 1, 2, and 3 and for peripheral-blood samples from patients 1, 2, 3, and 5 ’FicollIsopaque’ gradient centrifugation was applied to separate mononuclear and polymorphonuclear cells. Part of each cell fraction was cytocentrifuged onto a slide and fixed as above, and another part was stored in liquid nitrogen. Synovial-fluid samples from all nine patients were cultured to isolate the causative micro-organism as described for samples from patients with yersinia-triggered reactive arthritis.’ Samples from three patients (1-3) were studied with special care. They were inoculated into three different media in an attempt to detect even minute amounts of bacteria, including L-forms.7 No viable organisms were recovered. Peripheral-blood lymphocytes were typed for HLA B27 antigen by a two-stage microlymphocytotoxicity test (’Histognost-B27’, Behringwerke AG, Marburg, Federal Republic of Germany). All eight patients tested were positive. Antisera against S enteritidis and S typhimurium were raised by immunisation of rabbits with heat-killed bacteria. The rabbits were injected intravenously once a week for 5 weeks with increasing amounts of bacteria (0-25, 05, 1-0, 1-5, and 2-0 ml bacterial suspension containing approximately 109 bacteria per ml phosphate- buffered saline). The animals were bled 1 week after the final injection. As controls, we used the same antisera, absorbed extensively with the immunising bacteria. Mouse monoclonal antibodies specific for the 0-polysaccharide chain of LPS of S enteritidis (MASE 09) and S typhimurium (MAST 04) were also used (Carlin et al, unpublished)." Subclass-matched monoclonal antibody (3G6) specific for chicken T cells was used as a negative were in control. These antibodies used indirect immunofluorescence testing of synovial-fluid and peripheral-blood cells,’ the avidin-biotin-peroxidase complex (ABC) method on synovial tissue (with monoclonal antibody MAST 04, and the ABC kit ‘Vectastain’, Vector Laboratories, Burlingame, California, USA),13 and western blotting (monoclonal antibodies).7 The reactivity of the antibodies in these techniques was ascertained as we described for yersinia-specific antibodies.7 The controls consisted of bacterial pellets and bacteria-fed mononuclear cells. at
200 g for
5 min
(200 000 cells
ethanol/acetic acid at - 20°C, and stored at
-
Results Salmonella antigens were detected by immunofluorescence in the synovial-fluid cells of all eight patients with rabbit antiserum or monoclonal antibody against the causative salmonella species (table). Staining patterns varied (fig 1). Fine-granular staining of cytoplasm of mononuclear phagocytes was seen in all but one patient (no 6), in whom large-granular staining alone was observed mostly in polymorphonuclear cells, but a few mononuclear phagocytes were also positive. The percentage of positively
Snt SFC PBC Patient 1
SFC P8C
S im Sm
Patient 9
patient 3
Fig 2-Western blot of synovial cells (SFC) and peripheral-blood cells (PBC) from three patients. S ent and membrane.
S tm=controls with
nauve
bacteria.
SM=synovial
stained cells also varied; it was below 10% for two patients (4 and 6) but 50% or more for the others. Samples of peripheral blood cells from four patients all showed positive
staining. No salmonella antigens were detected by immunofluorescence in any of the synovial-fluid cell samples from the thirteen control patients, including three patients with yersinia reactive arthritis, and none of the controls were positive for serum antibodies to salmonella. Synovial-fluid cells from three patients with salmonellatriggered arthritis (patients 4, 6, 8) were negative on immunofluorescence with yersinia-specific antibodies.’ ABC-staining was applied to synovial tissue from patient 9. Staining with the monoclonal antibody specific for S typhimurium LPS was positive, revealing several mononuclear cells containing salmonella LPS. The two control samples studied were negative. Three of five synovial-fluid cell samples and two of four peripheral-blood cell samples from patients with reactive arthritis after infection with S enteritidis were positive on western blots with monoclonal antibody specific for S enteritidis LPS (table). Broad bands with molecular weights of 50-90 kD were visualised in all samples but one; synovial-fluid cells from patient 3 had a narrow band of molecular weight 85 kD (fig 2). A synovial tissue sample from a patient with reactive arthritis triggered by S typhimurium was positive on western blot with monoclonal antibody MAST 04; a broad band with a molecular weight of 67-80 kD was visualised (fig 2). Western blots of synovial-fluid cells from S typhimurium patients were negative, as were cells from five patients with rheumatoid arthritis. On western blots the reactivity patterns of the salmonellaLPS-specific monoclonal antibodies were much narrower against patient samples than against S typhimurium or S enteritidis, indicating degradation of LPS within the patients’ cells. The patterns varied to some extent among patients, probably owing to differing degrees of degradation.
688
Discussion
study we found salmonella antigens in joint material from patients with salmonella-triggered reactive arthritis, by immunostaining in all nine patients studied and by western blotting in four of eight patients studied. Bacterial cultures from samples of synovial-fluid were undertaken with special care, but all were negative. As in yersinia-triggered reactive arthritis, there is extensive processing of bacteria inside the phagocytic cells in salmonella-triggered reactive arthritis. Granular staining of the cytoplasm was seen in mononuclear and polymorphonuclear phagocytes. The sample from patient 3 showed a clearly different pattern from the others; almost all the mononuclear phagocytes on the slides were positive in In this
immunofluorescence with both rabbit and monoclonal antibodies and, as well as the fine granular material seen in the cytoplasm with both antibodies, staining of cell surfaces was obvious with monoclonal antibody. This finding was confirmed with the same antibody in flow cytometry (data not shown). It is noteworthy that this patient remained a carrier of salmonella bacteria after the acute phase of infection; the unusual expression of the bacterial 0polysaccharide on synovial-fluid macrophages may reflect disturbed processing of the bacteria. The presence of 0-polysaccharide of bacterial LPS in synovial-fluid and peripheral-blood cells was evident both by immunofluorescence and by western blot analysis. In western blot analysis all positive samples showed a band in the same region as the lower molecular weight units of the native LPS. No evidence was obtained for the presence of other bacterial degradation products but it cannot be excluded, since western blotting used LPS-specific monoclonal antibodies. On the other hand, in similar experiments on yersinia reactive arthritis, only LPS was observed when a rabbit polyclonal antiserum recognising a wide spectrum of yersinia antigens was applied.’ The role of bacterial LPS in the pathogenesis of human and animal arthritis has long stimulated speculation.13 Polysaccharides have been demonstrated in synovial fluid and in synovial-fluid leucocytes of patients with rheumatoid arthritis.14 In an animal model, the lipid A part of LPS is the active moiety in arthritis induction. 15 Interestingly, all bacterial species associated with the development of reactive arthritis possess LPS, which, moreover, shows crossreactivity among species.16 In Reiter’s syndrome following Chlamydia trachomatis infection, the synovial vascular injury is characteristic of that caused by LPS.4 In gastrointestinal infection and inflammation microbial antigens enter the body. How they are transported to the joints (eg, as immune complexes or within cells) is not known. Immune complexes containing yersinia antigens are found in the circulation and in synovial fluid of patients with yersinia-triggered reactive arthritiS.6,111 Likewise, the intracellular demonstration of degraded LPS both in peripheral blood and in synovial fluid accords with the idea that bacterial structures are transported within cells to the
joint. Processed LPS is
especially effective at stimulating lymphocytes,’9 leading to production of various cytokines active in inflammation. It may also enhance lymphocyte entry to the joint at the level of vascular endothelium."2’ We conclude, on the basis of this and previous studies’ that the presence of bacterial LPS in the synovium is an important factor in the pathogenesis of reactive arthritis. This conclusion is supported by the strong IgA response directed
particularly against LPS in patients with yersinia-triggered reactive arthritis.22 We thank Prof Paavo Toivanen for critical review of the manuscript and Ms Nina Vainio and Ms Raya Raulimo for technical assistance. This study was supported by grants from the Yrjo Jahnsson Foundation, the Sigrid Juselius Foundation, the Academy of Finland, Swedish Medical Research Council (grant no 16X-656), Gastroenterology Research Foundation, and Astra Research Foundation.
REFERENCES 1. Granfors K, Vuento R, Toivanen A. Host-microbe interaction in reactive arthritis. In: Toivanen A, Toivanen P, eds. Reactive arthritis. Boca Raton: CRC Press, 1988: 15-49. 2. Ishikawa H, Ohno O, Yamasaki K, Ikuta S, Hirohata K. Arthritis presumably caused by Chlamydia in Reiter syndrome. J Bone Joint Surg 1986; 68-A: 777-79. 3. Keat A, Dixey J, Sonnex C, Thomas B, Osbom M, Taylor-Robinson D. Chlamydia trachomatis and reactive arthritis: the missing link. Lancet 1987; i: 72-74. 4. Schumacher HR Jr, Magge S, Cherian PV, et al. Light and electron microscopic studies on the synovial membrane in Reiter’s syndrome. Immunocytochemical identification of chlamydial antigen m patients with early disease. Arthritis Rheum 1988; 31: 937-46. 5. Taylor-Robinson D, Thomas BJ, Dixey J, Osborn MF, Furr PM, Keat AC. Evidence tha Chlamydia trachomatis causes seronegative arthritis in women. Ann Rheum Dis 1988; 47: 295-99. 6. Lahesmaa-Rantala R, Granfors K, Isomaki H, Toivanen A. Yersiniaspecific immune complexes in the synovial-fluid of patients with Yersinia-triggered reactive arthritis. Ann Rheum Dis 1987; 46: 510-14. 7. Granfors K, Jalkanen S, von Essen R, et al. Yersinia antigens in synovial-fluid cells from patients with reactive arthritis. N Engl J Med 1989; 320: 216-21. 8. Ford DK, da Roza DM, Schulzer M. Lymphocytes from the site of disease but not blood lymphocytes indicate the cause of arthritis. Ann Rheum Dis 1985; 44: 701-10. 9. Gaston JSH, Life PF, Granfors K, et al. Synovial T lymphocyte recognition of organisms which trigger reactive arthritis. Clin Exp Immunol 1989; 76: 348-53. 10. van den Broek MF, van den Putte LBA, van den Berg WB. Crohn’s disease associated with arthritis. a possible role for cross-reactivity between gut bacteria and cartilage in the pathogenesis of arthritis. Arthritis Rheum 1988; 31: 1077-79. 11. Isomaki O, Vuento R, Granfors K. Serological diagnosis of Salmonella infections by enzyme immunoassay. Lancet 1989; i: 1411-14. 12. Carlin NIA, Svenson SB, Lindberg AA. Role of monoclonal O-antigen antibody epitope specificity and isotype in protection against experimental mouse typhoid. Microb Pathogen 1987; 2: 171-83. 13. Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. JHistochem Cytochem 1981; 29: 577-80. 14. Hollingsworth JW, Atkins E. Synovial inflammatory response to bacterial endotoxin. Yale J Biol Med 1965; 38: 241-56. 15. Bartholomew LE, Bartholomew FN. Antigenic bacterial polysaccharide in rheumatoid synovial effusions. Arthritis Rheum 1979; 22: 969-77. 16. Stimson SA, Esser EE, Carter PB, Sartor RB, Cromartie WJ, Schwab JH. Lipopolysaccharide induces recurrence of arthritis in rat joints previously injured by peptidoglycan-polysaccharide. J Exp Med 1987; 165: 1688-702. 17. Nurminen M, Lemonen M, Saikku P, Makela PH. The genus-specific antigen of Chlamydia: resemblance to the lipopolysaccharide of enteric bacteria. Science 1983; 220: 1279-81. 18. Lahesmaa-Rantala R, Granfors K, Kekomaki R, Toivanen A. Circulating yersinia specific immune complexes after acute yersiniosis: a follow up study of patients with and without reactive arthritis. Ann Rheum Dis 1987; 46: 121-26. 19. Unanue ER, Allen PM. The basis for the immunoregulatory role of macrophages and other accessory cells. Science 1987; 236: 551-57. 20. Jalkanen S, Steere AC, Fox RI, Butcher EC. A distinct endothelial cell recognition system that controls lymphocyte traffic into inflamed synovium. Science 1986; 233: 556-58. 21. Jalkanen S. Leukocyte-endothelial cell interaction and the control of leukocyte migration into inflamed synovium. Springer Semin Immunopathol 1989; 11: 187-98. 22. Gransfors K, Ogasawara M, Hill JL, Lahesmaa-Rantala R, Toivanen A, Yu DTY. Analysis of IgA anti-lipopolysaccharide antibodies in Yersinia-triggered reactive arthritis. J Infect Dis 1989; 159: 1142-47.
10. Lauritsen K, Laursen LS, Bukhave K, Rask-Madsen J. Effects of topical 5-aminosalicylic acid and prednisolone on prostaglandin E2 and leukotriene B4 levels determined by equilibrium in vivo dialysis of rectum in relapsing ulcerative colitis. Gastroenterology 1986; 91: 837-14. 11. Wallace JL, MacNaughton WK, Morris GP, Beck PL. Inhibition of leukotriene synthesis markedly accelerates healing in a rat model of inflammatory bowel disease. Gastroenterology 1989; 96: 29-36. 12. Stenson WF, Lobos E. Sulfasalazine inhibits the synthesis of chemotactic lipids by neutrophils. J Clin Invest 1982; 69: 494-97. 13. Lauritsen K, Laursen LS, Bukhave K, Rask-MadsenJ. In vivo effects of
orally administered prednisolone on prostaglandin and leucotriene production in ulcerative colitis. Gut 1987; 28: 1095-99. 14. Lauritsen K, Laursen LS, Bukhave K, Rask-Madsen J. In vivo profiles of eicosanoids in ulcerative colitis, Crohn’s colitis, and Clostridium difficile colitis. Gastroenterology 1988; 95: 11-17. 15. Bukhave K, Gréen K, Rask-Madsen J. Comparison of radioimmunological determinations with gas chromatography mass spectrometry dosage. A study of PGE2 and PGF2&agr; concentrations in gastrointestinal fluids. Biomed Mass Spectrom 1983; 10: 265-68.
K, Laursen LS, Bukhave K, Rask-Madsen J. Inflammatory intermediaries in inflammatory bowel disease. Int J Colorect Dis 1989; 4: 75-90. 17. Blackwell GJ, Carnuccio R, DiRosa M, Flower RJ, Parente L, Persico P. Macrocortin: a polypeptide causing the anti-phospholipase effect of glucocorticoids. Nature 1980; 287: 147-49. 18. Sircar JC, Schwender CF, Carethers ME. Inhibition of soybean lipoxygenase by sulfasalazine and 5-aminosalicylic acid: a possible mode of action in ulcerative colitis. Biochem Pharmacol 1983; 32: 170-72. 19. Nielsen OH, Bukhave K, Elmgreen J, Ahnfelt-Rønne I. Inhibition of 5-lipoxygenase pathway of arachidonic acid metabolism in human neutrophils by sulfasalazine and 5-aminosalicylic acid. Dig Dis Sci 16. Lauritsen
1987; 32: 577-82. 20. Marcus 21.
AJ. Eicosanoids as bioregulators in clinical medicine. Am J Med 1985; 78: 805-10. Goetzl EJ, Pickett WC. The human PMN leukocyte chemotactic activity of complex hydroxy-eicosatetraenoic acids (HETEs). J Immunol 1980; 125: 1789-91.
Salmonella
lipopolysaccharide in synovial cells from patients with reactive arthritis
Synovial cells from nine patients with reactive arthritis following Salmonella enteritidis or Salmonella typhimurium infection were examined for salmonella antigens. Extensive bacterial cultures of the synovial fluid were negative. Eight synovial-fluid cell samples stained positively on immunofluorescence with rabbit antisera against heat-killed S enteritidis or S typhimurium or with monoclonal antibodies specific for the causative salmonella lipopolysaccharide (LPS). Synovial tissue from the ninth patient stained positively in the avidin-biotin-peroxidase complex method with the monoclonal antibody. Control samples (synovial-fluid cells from thirteen patients with other rheumatic diseases and synovial tissue from two) were negative. Synovial cells from eight patients and five controls were studied by western blotting with the same monoclonal antibodies. Four of the eight patients but no controls had blots indicating salmonella LPS in the synovial cells. The presence of bacterial LPS in the joint is a common and pathogenetically important feature of reactive arthritis.
Introduction Reactive arthritis certain infections,
develops as a late complication after including those by species of Yersinia, Chlamydia, Salmonella, Shigella, and Campylobacter,
especially in susceptible people positive for HLA B27. Several theories have been suggested to explain how inflammation in the joint may be stimulated by microbial antigens at a remote site in the body, and all include the idea that the interaction between host and microbe is somehow abnormal and inefficient in HLA-B27-positive subjects in whom reactive arthritis develops. There is direct evidence that microbial antigens enter the joints in reactive arthritis following Chlamydia trachomatis urogenital infection 2-5 and Yersinia enterocolitica gastrointestinal infection.6,7 In addition, enhanced in-vitro responses to microbial agents of synovial-fluid mononuclear cells from patients with reactive arthritis suggest the presence of microbial antigens in the joints.8-10 In this study we sought bacterial lipopolysaccharide (LPS) in synovialfluid cells or synovial tissue from patients with salmonellatriggered reactive arthritis.
ADDRESSES: Departments of Medical Microbiology and Medicine, Turku University, Finland (K Granfors, PhD, S. Jalkanen, MD, O. Mäki-Ikola, MD, R Lahesmaa-Rantala, MD, R Saario, MD, Prof A. Toivanen, MD); Karolinska Institute, Department of Clinical Bacteriology, Huddinge Hospital, Sweden (Prof A. A. Lindberg, MD), Rheumatism Foundation Hospital, Heinola, Finland (R. von Essen, MD, Prof H. Isomaki, MD); and Division of Medicine, Lutheran General Hospital, Park Ridge, Illinois, USA (W. J Arnold, MD). Correspondence to Dr K Granfors, Department of Medical Microbiology, Turku University, SF-20520 Turku, Finland.
686
Patients and methods We studied nine adult patients with salmonella-triggered reactive arthritis, diagnosed on the basis of bacteriological, serological (enzyme immunoassayll), and clinical fmdings. S enteritidis was isolated from the stools of five patients and S typhimurium from three
(see table).
In
eight patients studied (including patient 8,
whose stool culture was negative) the serum antibody responses to the causative microorganism exceeded the mean response in a hundred healthy blood donors by six standard deviations (table). All but two patients had high IgM antibody levels, suggesting recent infection. Typical clinical signs of acute salmonella infection (fever, diarrhoea, or gastrointestinal pain) and of acute reactive arthritis occurred in all nine patients.
DETECTION OF BACTERIAL ANTIGENS IN JOINTS
+ + + ≥6 SD, + + ->4SD, *ABC method
on
+ 2 SD above
mean in
100 normal controls
synovial tissue
Fig 1-immunofluorescence of synovial-fluid cells. panels show positive staining with rabbit antiserum, ’B’ panels show positive staining with corresponding LPS-specific monoclonal antibody in patients 3 (S enteritidis) and 6 (S typhimurium) Panels 3A and 3B x 1000, panels 6A and 6B x 630 ’A’
687
Synovial-fluid leucocytes were obtained from eight patients. Peripheral-blood leucocytes were obtained at the same time as synovial fluid from patients 1, 2, and 3, and 4 weeks later from patient 5. Synovial tissue was obtained from one patient (patient 9; table) as part of an arthroscopic synovectomy 2 years after the first symptoms of gastroenteritis. The sample was immediately frozen in liquid nitrogen and stored at - 70°C. Control material from twelve patients meeting the American
8 8 8 8 8
3
8
II II Ii illl il if
Rheumatism Association’s criteria for rheumatoid arthritis was also studied (synovial-fluid cells from ten and synovial tissue from two). None had a history of recent gastrointestinal symptoms. Synovialfluid cells from three HLA-B27-positive patients with reactive arthritis triggered by Y enterocolitica 0:3 were also studied as controls. Synovial fluid aspirated from the knee was mixed with heparin (50 IU/ml) and centrifuged at 200 g for 10 min. Part of the cell deposit in the residual fluid was stored at - 70°C. Another part was suspended in Hanks’ balanced salt solution, cytocentrifuged onto slides
per slide), fixed in 70°C. The slides were used for indirect immunofluorescence testing and the solubilised cells for western blotting. For synovial samples from patients 1, 2, and 3 and for peripheral-blood samples from patients 1, 2, 3, and 5 ’FicollIsopaque’ gradient centrifugation was applied to separate mononuclear and polymorphonuclear cells. Part of each cell fraction was cytocentrifuged onto a slide and fixed as above, and another part was stored in liquid nitrogen. Synovial-fluid samples from all nine patients were cultured to isolate the causative micro-organism as described for samples from patients with yersinia-triggered reactive arthritis.’ Samples from three patients (1-3) were studied with special care. They were inoculated into three different media in an attempt to detect even minute amounts of bacteria, including L-forms.7 No viable organisms were recovered. Peripheral-blood lymphocytes were typed for HLA B27 antigen by a two-stage microlymphocytotoxicity test (’Histognost-B27’, Behringwerke AG, Marburg, Federal Republic of Germany). All eight patients tested were positive. Antisera against S enteritidis and S typhimurium were raised by immunisation of rabbits with heat-killed bacteria. The rabbits were injected intravenously once a week for 5 weeks with increasing amounts of bacteria (0-25, 05, 1-0, 1-5, and 2-0 ml bacterial suspension containing approximately 109 bacteria per ml phosphate- buffered saline). The animals were bled 1 week after the final injection. As controls, we used the same antisera, absorbed extensively with the immunising bacteria. Mouse monoclonal antibodies specific for the 0-polysaccharide chain of LPS of S enteritidis (MASE 09) and S typhimurium (MAST 04) were also used (Carlin et al, unpublished)." Subclass-matched monoclonal antibody (3G6) specific for chicken T cells was used as a negative were in control. These antibodies used indirect immunofluorescence testing of synovial-fluid and peripheral-blood cells,’ the avidin-biotin-peroxidase complex (ABC) method on synovial tissue (with monoclonal antibody MAST 04, and the ABC kit ‘Vectastain’, Vector Laboratories, Burlingame, California, USA),13 and western blotting (monoclonal antibodies).7 The reactivity of the antibodies in these techniques was ascertained as we described for yersinia-specific antibodies.7 The controls consisted of bacterial pellets and bacteria-fed mononuclear cells. at
200 g for
5 min
(200 000 cells
ethanol/acetic acid at - 20°C, and stored at
-
Results Salmonella antigens were detected by immunofluorescence in the synovial-fluid cells of all eight patients with rabbit antiserum or monoclonal antibody against the causative salmonella species (table). Staining patterns varied (fig 1). Fine-granular staining of cytoplasm of mononuclear phagocytes was seen in all but one patient (no 6), in whom large-granular staining alone was observed mostly in polymorphonuclear cells, but a few mononuclear phagocytes were also positive. The percentage of positively
Snt SFC PBC Patient 1
SFC P8C
S im Sm
Patient 9
patient 3
Fig 2-Western blot of synovial cells (SFC) and peripheral-blood cells (PBC) from three patients. S ent and membrane.
S tm=controls with
nauve
bacteria.
SM=synovial
stained cells also varied; it was below 10% for two patients (4 and 6) but 50% or more for the others. Samples of peripheral blood cells from four patients all showed positive
staining. No salmonella antigens were detected by immunofluorescence in any of the synovial-fluid cell samples from the thirteen control patients, including three patients with yersinia reactive arthritis, and none of the controls were positive for serum antibodies to salmonella. Synovial-fluid cells from three patients with salmonellatriggered arthritis (patients 4, 6, 8) were negative on immunofluorescence with yersinia-specific antibodies.’ ABC-staining was applied to synovial tissue from patient 9. Staining with the monoclonal antibody specific for S typhimurium LPS was positive, revealing several mononuclear cells containing salmonella LPS. The two control samples studied were negative. Three of five synovial-fluid cell samples and two of four peripheral-blood cell samples from patients with reactive arthritis after infection with S enteritidis were positive on western blots with monoclonal antibody specific for S enteritidis LPS (table). Broad bands with molecular weights of 50-90 kD were visualised in all samples but one; synovial-fluid cells from patient 3 had a narrow band of molecular weight 85 kD (fig 2). A synovial tissue sample from a patient with reactive arthritis triggered by S typhimurium was positive on western blot with monoclonal antibody MAST 04; a broad band with a molecular weight of 67-80 kD was visualised (fig 2). Western blots of synovial-fluid cells from S typhimurium patients were negative, as were cells from five patients with rheumatoid arthritis. On western blots the reactivity patterns of the salmonellaLPS-specific monoclonal antibodies were much narrower against patient samples than against S typhimurium or S enteritidis, indicating degradation of LPS within the patients’ cells. The patterns varied to some extent among patients, probably owing to differing degrees of degradation.
688
Discussion
study we found salmonella antigens in joint material from patients with salmonella-triggered reactive arthritis, by immunostaining in all nine patients studied and by western blotting in four of eight patients studied. Bacterial cultures from samples of synovial-fluid were undertaken with special care, but all were negative. As in yersinia-triggered reactive arthritis, there is extensive processing of bacteria inside the phagocytic cells in salmonella-triggered reactive arthritis. Granular staining of the cytoplasm was seen in mononuclear and polymorphonuclear phagocytes. The sample from patient 3 showed a clearly different pattern from the others; almost all the mononuclear phagocytes on the slides were positive in In this
immunofluorescence with both rabbit and monoclonal antibodies and, as well as the fine granular material seen in the cytoplasm with both antibodies, staining of cell surfaces was obvious with monoclonal antibody. This finding was confirmed with the same antibody in flow cytometry (data not shown). It is noteworthy that this patient remained a carrier of salmonella bacteria after the acute phase of infection; the unusual expression of the bacterial 0polysaccharide on synovial-fluid macrophages may reflect disturbed processing of the bacteria. The presence of 0-polysaccharide of bacterial LPS in synovial-fluid and peripheral-blood cells was evident both by immunofluorescence and by western blot analysis. In western blot analysis all positive samples showed a band in the same region as the lower molecular weight units of the native LPS. No evidence was obtained for the presence of other bacterial degradation products but it cannot be excluded, since western blotting used LPS-specific monoclonal antibodies. On the other hand, in similar experiments on yersinia reactive arthritis, only LPS was observed when a rabbit polyclonal antiserum recognising a wide spectrum of yersinia antigens was applied.’ The role of bacterial LPS in the pathogenesis of human and animal arthritis has long stimulated speculation.13 Polysaccharides have been demonstrated in synovial fluid and in synovial-fluid leucocytes of patients with rheumatoid arthritis.14 In an animal model, the lipid A part of LPS is the active moiety in arthritis induction. 15 Interestingly, all bacterial species associated with the development of reactive arthritis possess LPS, which, moreover, shows crossreactivity among species.16 In Reiter’s syndrome following Chlamydia trachomatis infection, the synovial vascular injury is characteristic of that caused by LPS.4 In gastrointestinal infection and inflammation microbial antigens enter the body. How they are transported to the joints (eg, as immune complexes or within cells) is not known. Immune complexes containing yersinia antigens are found in the circulation and in synovial fluid of patients with yersinia-triggered reactive arthritiS.6,111 Likewise, the intracellular demonstration of degraded LPS both in peripheral blood and in synovial fluid accords with the idea that bacterial structures are transported within cells to the
joint. Processed LPS is
especially effective at stimulating lymphocytes,’9 leading to production of various cytokines active in inflammation. It may also enhance lymphocyte entry to the joint at the level of vascular endothelium."2’ We conclude, on the basis of this and previous studies’ that the presence of bacterial LPS in the synovium is an important factor in the pathogenesis of reactive arthritis. This conclusion is supported by the strong IgA response directed
particularly against LPS in patients with yersinia-triggered reactive arthritis.22 We thank Prof Paavo Toivanen for critical review of the manuscript and Ms Nina Vainio and Ms Raya Raulimo for technical assistance. This study was supported by grants from the Yrjo Jahnsson Foundation, the Sigrid Juselius Foundation, the Academy of Finland, Swedish Medical Research Council (grant no 16X-656), Gastroenterology Research Foundation, and Astra Research Foundation.
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