British Journal of Rheumatology 1992;31:59-61
BRIEF REPORT
T-CELL RECEPTOR GAMMA-DELTA POSITIVE LYMPHOCYTES IN SYNOVIAL MEMBRANE BY R. MELICONF 2, M. UGUCCIONP, A. D'ERRICO4, A. CASSISA4, L. FRIZZIERO5 AND A. FACCHINP ' Patologia Medica I, University of Bologna;2 Rheumatology Unit, Department of Medicine, UMDS, Guy's Hospital, London;j'Laboratorio di Immunologia e Genetica, Istituto di Ricerca Codivilla Putti, Bologna; ''Istituto di Anatomia Patologica, University of Bologna; 5Unita di Reumatologia, U.S.L. 27, Bologna, Italy
KEY WORDS:
Rheumatoid arthritis, Inflammatory arthritis, Degenerative joint disease, Synovium, Lymphocytes.
T-LYMPHOCYTES expressing
the y/5 (Ty/8) of the T-cell receptor (TCR) constitute a minority of CD3+ cells present in peripheral blood and in organized lymphoid tissues [1,2]. Although the precise function of Ty/5 is unknown, cytotoxic activity has been reported but unlike lymphocytes expressing TCRa/(3, Ty/5 cells mainly exhibit natural killer [3] or major histocompatibility complex (MHC) non-restricted cytotoxicity [4] even if class I dependent cytotoxicity has been demonstrated [5]. Great interest has been aroused by the recent finding that Ty/8 from normal subjects and rheumatoid arthritis (RA) patients are preferentially stimulated by mycobacteria extracts and by 65 kDa heat shock proteins (HSP) [4,6,7]. Since HSP are highly conserved molecules throughout various species (from bacteria to eukaryotes), these findings have led to the suggestion that immune response to HSP may provide a link between infection and autoimmunity [8]. Interest in Ty/8 in rheumatic disease has been further raised by conflicting data reported on Ty/8 cells in peripheral blood from patients with RA [9-11]. Furthermore preliminary data suggest increased numbers of Ty/6 in synovial fluid and membrane from patients with RA [9,10]. So far the presence ofTy/8 cells in noninflammatory arthritides has not been evaluated. In order to evaluate the presence and localization of Ty/5 cells in the synovial compartment, we have analysed by immunocytochemistry the distribution of Ty/8 positive T-cells in synovial membrane samples obtained from patients with inflammatory arthritis and from patients with non-inflammatory/degenerative arthritis. Ty/8 lymphocytes were mainly found in inflammatory arthritis.
arthroscopy from eight inflammatory arthritis: six classical RA patients [12], two spondyloarthritis (SA) (mean age 44.6 years, range 13-58, three males and five females) and from eight non-inflammatory arthritis patients: four osteoarthritis (OA) and four posttraumatic arthritis (PT) (mean age 39.5 years, range 17-59, six males and two females). At least two synovial membrane tissue specimens were obtained from each patient. The specimens were coated with OCT compound (Miles Laboratories, USA), snap frozen in liquid nitrogen and then stored at -70°C until used. Serial cryostatic sections, 4um thick, were air-dried, fixed in acetone at 4°C for 10 min and then in chloroform for 10 min in order to block endogenous peroxidase [13]. The sections were washed with Tris-HCl pH 7.6 and then incubated with mouse monoclonal antibodies (mAb). Ty/8 cells were identified using a m Ab directed against a common epitope of the 5 chain (TCR81, T-Cell Sciences, Cambridge, MA, USA). T-lymphocytes were stained with anti-CD3 mAb (OKT3, Ortho, Raritan, NJ, USA). MAbs were diluted in RPMI1640 medium supplemented with 10% FCS and the sections incubated for 45 min. After incubation the sections were washed and incubated with biotinylated horse anti-mouse IgG antibody for 30 min and then with a complex of biotinylated peroxidase and avidin DH (Vectastain ABC reagent) for 60 min. The slides were immersed in 3% diaminobenzidine in TrisHCl pH 7.6 solution with 30% hydrogen peroxide for 5 min and washed in Tris-HCl, contrasted with haematoxylin for 30 s, dehydrated in graded alcohols and xylene and permanently mounted with coverslips. Serial sections were examined (Axiophot Zeiss, Germany): the number of CD3+ and Ty/S+ cells were counted in the whole biopsy area and then Ty/5+ cells were expressed as percentage of CD3+ cells. The data were analysed using the chi-squared test with Yates' correction.
MATERIALS AND METHODS Samples of synovial membrane were obtained by Submitted 13 June; revised version accepted 20 October 1990. Correspondence to Dr R. Meliconi, I Patologia Medica, Policlinico S. Orsola, Via Massarenti 9, 40138 Bologna, Italy. 59
Downloaded from http://rheumatology.oxfordjournals.org/ at University of Exeter on July 17, 2015
SUMMARY The distribution of T-lymphocytes expressing CD3 and T-cell receptor gamma-delta (Ty/5) has been examined by immunocytochemistry in the synovial membrane of eight patients with inflammatory arthritis (six rheumatoid arthritis, two spondyloarthritis) and eight with non-inflammatory arthritis (four osteoarthritis, four post-traumatic arthritis). Ty/5 cells were present in eight out of eight inflammatory arthritis synovial membranes, but in only one out of eight noninflammatory membranes (F
FIG. 1.—Top: CD3 positive cells in a synovial biopsy specimen from a patient with rheumatoid arthritis (avidin-biotin-peroxidase/haematoxylin stain); bottom: T-cell receptor y/6 positive cells (Ty/6) in the same synovial biopsy specimen.
Downloaded from http://rheumatology.oxfordjournals.org/ at University of Exeter on July 17, 2015
RA RA RA
Treatment duration
Treatment
MELICONI ETAL.:
T-CELL GAMMA-DELTA POSITIVE LYMPHOCYTES
ACKNOWLEDGEMENTS
The authors wish to thank Prof. G. S. Panayi (London) for helpful comments and advice; Miss Patrizia Rappini for carefully typing this manuscript. This work was supported by grants from National Research Council (CNR), Italian Ministry of Education and Istituti Scientifici Rizzoli (Bologna). Dr Riccardo Meliconi is a recipient of a NATO-CNR Advanced Fellowship. REFERENCES
1. Bucy RP, Chen-Lo CH, Cooper MD. Tissue localization and CD8 accessory molecule expression of Ty/6 cells in humans. J Immunol 1989,142:3045-9. 2. Groh V, Porcelli S, Fabbi M, et al. Human lymphocytes bearing T-cell receptor y/5 are phenotipically diverse and evenly distributed throughout the lymphoid system. J Exp Med 1989;169:1277-94. 3. Bones V, Halvorsen R, Skaftadottir I, Gaudernack G, Thorsby E. Specificity of y/S receptor-bearing cytotoxic T-lymphocytes isolated from human peripheral blood. Scand J Immunol 1989;29:723-31.
4. Holoshitz J, Koning F, Coligan JE, De Bruyn J, Strober S. Isolation of CD4" CD8~ mycobacteriareactive T-lymphocyte clones from rheumatoid arthritis synovial fluid. Nature 1989;339:226-9. 5. Raulet DH. Antigens for y/6 T-cells. Nature 1989; 339:342-3. 6. Haregewoin A, Soman G, Hon RC, Finberg RW. Human y/S+ T-cells respond to mycobacterial heatshock protein. Nature 1989;340:309-12. 7. Born W, Hall L, Dallas A, et al., Recognition of a peptide antigen by heat shock-reactive y6 T-lymphocytes. Science 1990;249:67-9. 8. Lamb JR, Bal V, Mendez-Samperio P, et al. Stress proteins may provide a link between the immune response to infection and autoimmunity. Int Immunol 1989;l:191-6. 9. Brennan FM, Londei M, Jackson AM, et al. T-cells expressing y/5 chain receptors in rheumatoid arthritis. J Autoimmunity 1988;l:319-26. 10. Smith MD, Broker B, Moretta L, et al. Ty/5 cells in rheumatoid arthritis. Br J Rheumatol 1989;28:74. 11. Trujillo A, Andreu JL, Alonso JM, Mulero J, Martinez AC. Lymphocyte bearing gamma-delta T-cell receptor in rheumatoid synovial membrane. Br J Rheumatol 1989;28:74. 12. Ropes MW, Bennett GA, Cobb S, Jacox R, Jessar RA. Diagnostic criteria for rheumatoid arthritis: 1958 revision. Ann Rheum Dis 1959;18:49-53. 13. Tazzari PL, Gobbi M, Dinota A, et al. Normal and neoplastic plasma cell membrane phenotype: studies with new monoclonal antibodies. Clin Exp Immunol 1987;70:192-200. 14. De Maria A, Malnati M, Moretta A, et al. CD3 + 4-8WT31-(T-cell receptor y/6+) cells and other unusual phenotypes are frequently detected among spontaneously interleukin 2-responsive T lymphocytes present in the joint fluid in juvenile rheumatoid arthritis. A clonal analysis. Eur J Immunol 1987; 17:1815-19. 15. Reme T, Portier M, Frayssinoux F, et al. T cell receptor expression and activation of synovial lymphocytes subsets in patients with rheumatoid arthritis. Arthritis Rheum 1990;33:485-92. 16. Karlsson-Parra A, Soderstrom K, Ferm M, Ivanyi J, KiesslingR, KlareskogL. Presence of human 65 kD heat shock protein (HSP) in inflammed joints and subcutaneous nodules of RA patients. Scand J Immunol 1990;31:283-8. 17. Born W, Happ MP, Dallas A, et al. Recognition of heat shock proteins and y/6 cell function. Immunol Today 1990;ll:40-3.
Downloaded from http://rheumatology.oxfordjournals.org/ at University of Exeter on July 17, 2015
cytes which always constitute the majority of synovial infiltrating lymphocytes. The high percentage of synovial Ty/5 shown by one PT patient is an intriguing result. Unfortunately this patient was lost to follow-up and we do not know whether he developed an inflammatory arthritis after the biopsy was taken. The function of Ty/6 cells in synovium is still unclear. The two main functions so far described for Ty/5 are cytotoxicity and cytokine production. But the nature of the target(s) antigen(s) of their cytotoxic activity in the synovium remains elusive. Recent evidence suggests that Ty/5 are biased to proliferate in response to mycobacteria antigens including heat shock (stress) proteins [6]. In addition, HPSs have been detected by immunohistochemistry only in synovium from RA patients [16]. Thus do Ty/5 cells respond to synovial cells altered (stressed) by infection or other damaging insults? In this case, Ty/6 could be stimulated to produce a vast array of cytokines which, in turn, could exert several immunomodulatory activities on other inflammatory cells (Ta/|3 lymphocytes, macrophages, endothelial cells) [17]. At present we are attempting to elucidate the role of Ty/6 in synovium by studying the expression of different molecules (CD8, CD45RA, CD45RO, MHC class II, CD25) on their membrane.
61
BRIEF REPORT
T-CELL RECEPTOR GAMMA-DELTA POSITIVE LYMPHOCYTES IN SYNOVIAL MEMBRANE BY R. MELICONF 2, M. UGUCCIONP, A. D'ERRICO4, A. CASSISA4, L. FRIZZIERO5 AND A. FACCHINP ' Patologia Medica I, University of Bologna;2 Rheumatology Unit, Department of Medicine, UMDS, Guy's Hospital, London;j'Laboratorio di Immunologia e Genetica, Istituto di Ricerca Codivilla Putti, Bologna; ''Istituto di Anatomia Patologica, University of Bologna; 5Unita di Reumatologia, U.S.L. 27, Bologna, Italy
KEY WORDS:
Rheumatoid arthritis, Inflammatory arthritis, Degenerative joint disease, Synovium, Lymphocytes.
T-LYMPHOCYTES expressing
the y/5 (Ty/8) of the T-cell receptor (TCR) constitute a minority of CD3+ cells present in peripheral blood and in organized lymphoid tissues [1,2]. Although the precise function of Ty/5 is unknown, cytotoxic activity has been reported but unlike lymphocytes expressing TCRa/(3, Ty/5 cells mainly exhibit natural killer [3] or major histocompatibility complex (MHC) non-restricted cytotoxicity [4] even if class I dependent cytotoxicity has been demonstrated [5]. Great interest has been aroused by the recent finding that Ty/8 from normal subjects and rheumatoid arthritis (RA) patients are preferentially stimulated by mycobacteria extracts and by 65 kDa heat shock proteins (HSP) [4,6,7]. Since HSP are highly conserved molecules throughout various species (from bacteria to eukaryotes), these findings have led to the suggestion that immune response to HSP may provide a link between infection and autoimmunity [8]. Interest in Ty/8 in rheumatic disease has been further raised by conflicting data reported on Ty/8 cells in peripheral blood from patients with RA [9-11]. Furthermore preliminary data suggest increased numbers of Ty/6 in synovial fluid and membrane from patients with RA [9,10]. So far the presence ofTy/8 cells in noninflammatory arthritides has not been evaluated. In order to evaluate the presence and localization of Ty/5 cells in the synovial compartment, we have analysed by immunocytochemistry the distribution of Ty/8 positive T-cells in synovial membrane samples obtained from patients with inflammatory arthritis and from patients with non-inflammatory/degenerative arthritis. Ty/8 lymphocytes were mainly found in inflammatory arthritis.
arthroscopy from eight inflammatory arthritis: six classical RA patients [12], two spondyloarthritis (SA) (mean age 44.6 years, range 13-58, three males and five females) and from eight non-inflammatory arthritis patients: four osteoarthritis (OA) and four posttraumatic arthritis (PT) (mean age 39.5 years, range 17-59, six males and two females). At least two synovial membrane tissue specimens were obtained from each patient. The specimens were coated with OCT compound (Miles Laboratories, USA), snap frozen in liquid nitrogen and then stored at -70°C until used. Serial cryostatic sections, 4um thick, were air-dried, fixed in acetone at 4°C for 10 min and then in chloroform for 10 min in order to block endogenous peroxidase [13]. The sections were washed with Tris-HCl pH 7.6 and then incubated with mouse monoclonal antibodies (mAb). Ty/8 cells were identified using a m Ab directed against a common epitope of the 5 chain (TCR81, T-Cell Sciences, Cambridge, MA, USA). T-lymphocytes were stained with anti-CD3 mAb (OKT3, Ortho, Raritan, NJ, USA). MAbs were diluted in RPMI1640 medium supplemented with 10% FCS and the sections incubated for 45 min. After incubation the sections were washed and incubated with biotinylated horse anti-mouse IgG antibody for 30 min and then with a complex of biotinylated peroxidase and avidin DH (Vectastain ABC reagent) for 60 min. The slides were immersed in 3% diaminobenzidine in TrisHCl pH 7.6 solution with 30% hydrogen peroxide for 5 min and washed in Tris-HCl, contrasted with haematoxylin for 30 s, dehydrated in graded alcohols and xylene and permanently mounted with coverslips. Serial sections were examined (Axiophot Zeiss, Germany): the number of CD3+ and Ty/S+ cells were counted in the whole biopsy area and then Ty/5+ cells were expressed as percentage of CD3+ cells. The data were analysed using the chi-squared test with Yates' correction.
MATERIALS AND METHODS Samples of synovial membrane were obtained by Submitted 13 June; revised version accepted 20 October 1990. Correspondence to Dr R. Meliconi, I Patologia Medica, Policlinico S. Orsola, Via Massarenti 9, 40138 Bologna, Italy. 59
Downloaded from http://rheumatology.oxfordjournals.org/ at University of Exeter on July 17, 2015
SUMMARY The distribution of T-lymphocytes expressing CD3 and T-cell receptor gamma-delta (Ty/5) has been examined by immunocytochemistry in the synovial membrane of eight patients with inflammatory arthritis (six rheumatoid arthritis, two spondyloarthritis) and eight with non-inflammatory arthritis (four osteoarthritis, four post-traumatic arthritis). Ty/5 cells were present in eight out of eight inflammatory arthritis synovial membranes, but in only one out of eight noninflammatory membranes (F
FIG. 1.—Top: CD3 positive cells in a synovial biopsy specimen from a patient with rheumatoid arthritis (avidin-biotin-peroxidase/haematoxylin stain); bottom: T-cell receptor y/6 positive cells (Ty/6) in the same synovial biopsy specimen.
Downloaded from http://rheumatology.oxfordjournals.org/ at University of Exeter on July 17, 2015
RA RA RA
Treatment duration
Treatment
MELICONI ETAL.:
T-CELL GAMMA-DELTA POSITIVE LYMPHOCYTES
ACKNOWLEDGEMENTS
The authors wish to thank Prof. G. S. Panayi (London) for helpful comments and advice; Miss Patrizia Rappini for carefully typing this manuscript. This work was supported by grants from National Research Council (CNR), Italian Ministry of Education and Istituti Scientifici Rizzoli (Bologna). Dr Riccardo Meliconi is a recipient of a NATO-CNR Advanced Fellowship. REFERENCES
1. Bucy RP, Chen-Lo CH, Cooper MD. Tissue localization and CD8 accessory molecule expression of Ty/6 cells in humans. J Immunol 1989,142:3045-9. 2. Groh V, Porcelli S, Fabbi M, et al. Human lymphocytes bearing T-cell receptor y/5 are phenotipically diverse and evenly distributed throughout the lymphoid system. J Exp Med 1989;169:1277-94. 3. Bones V, Halvorsen R, Skaftadottir I, Gaudernack G, Thorsby E. Specificity of y/S receptor-bearing cytotoxic T-lymphocytes isolated from human peripheral blood. Scand J Immunol 1989;29:723-31.
4. Holoshitz J, Koning F, Coligan JE, De Bruyn J, Strober S. Isolation of CD4" CD8~ mycobacteriareactive T-lymphocyte clones from rheumatoid arthritis synovial fluid. Nature 1989;339:226-9. 5. Raulet DH. Antigens for y/6 T-cells. Nature 1989; 339:342-3. 6. Haregewoin A, Soman G, Hon RC, Finberg RW. Human y/S+ T-cells respond to mycobacterial heatshock protein. Nature 1989;340:309-12. 7. Born W, Hall L, Dallas A, et al., Recognition of a peptide antigen by heat shock-reactive y6 T-lymphocytes. Science 1990;249:67-9. 8. Lamb JR, Bal V, Mendez-Samperio P, et al. Stress proteins may provide a link between the immune response to infection and autoimmunity. Int Immunol 1989;l:191-6. 9. Brennan FM, Londei M, Jackson AM, et al. T-cells expressing y/5 chain receptors in rheumatoid arthritis. J Autoimmunity 1988;l:319-26. 10. Smith MD, Broker B, Moretta L, et al. Ty/5 cells in rheumatoid arthritis. Br J Rheumatol 1989;28:74. 11. Trujillo A, Andreu JL, Alonso JM, Mulero J, Martinez AC. Lymphocyte bearing gamma-delta T-cell receptor in rheumatoid synovial membrane. Br J Rheumatol 1989;28:74. 12. Ropes MW, Bennett GA, Cobb S, Jacox R, Jessar RA. Diagnostic criteria for rheumatoid arthritis: 1958 revision. Ann Rheum Dis 1959;18:49-53. 13. Tazzari PL, Gobbi M, Dinota A, et al. Normal and neoplastic plasma cell membrane phenotype: studies with new monoclonal antibodies. Clin Exp Immunol 1987;70:192-200. 14. De Maria A, Malnati M, Moretta A, et al. CD3 + 4-8WT31-(T-cell receptor y/6+) cells and other unusual phenotypes are frequently detected among spontaneously interleukin 2-responsive T lymphocytes present in the joint fluid in juvenile rheumatoid arthritis. A clonal analysis. Eur J Immunol 1987; 17:1815-19. 15. Reme T, Portier M, Frayssinoux F, et al. T cell receptor expression and activation of synovial lymphocytes subsets in patients with rheumatoid arthritis. Arthritis Rheum 1990;33:485-92. 16. Karlsson-Parra A, Soderstrom K, Ferm M, Ivanyi J, KiesslingR, KlareskogL. Presence of human 65 kD heat shock protein (HSP) in inflammed joints and subcutaneous nodules of RA patients. Scand J Immunol 1990;31:283-8. 17. Born W, Happ MP, Dallas A, et al. Recognition of heat shock proteins and y/6 cell function. Immunol Today 1990;ll:40-3.
Downloaded from http://rheumatology.oxfordjournals.org/ at University of Exeter on July 17, 2015
cytes which always constitute the majority of synovial infiltrating lymphocytes. The high percentage of synovial Ty/5 shown by one PT patient is an intriguing result. Unfortunately this patient was lost to follow-up and we do not know whether he developed an inflammatory arthritis after the biopsy was taken. The function of Ty/6 cells in synovium is still unclear. The two main functions so far described for Ty/5 are cytotoxicity and cytokine production. But the nature of the target(s) antigen(s) of their cytotoxic activity in the synovium remains elusive. Recent evidence suggests that Ty/5 are biased to proliferate in response to mycobacteria antigens including heat shock (stress) proteins [6]. In addition, HPSs have been detected by immunohistochemistry only in synovium from RA patients [16]. Thus do Ty/5 cells respond to synovial cells altered (stressed) by infection or other damaging insults? In this case, Ty/6 could be stimulated to produce a vast array of cytokines which, in turn, could exert several immunomodulatory activities on other inflammatory cells (Ta/|3 lymphocytes, macrophages, endothelial cells) [17]. At present we are attempting to elucidate the role of Ty/6 in synovium by studying the expression of different molecules (CD8, CD45RA, CD45RO, MHC class II, CD25) on their membrane.
61
