Scandinavian Journal of Rheumatology
ISSN: 0300-9742 (Print) 1502-7732 (Online) Journal homepage: http://www.tandfonline.com/loi/irhe20
Dissimilar Biosynthesis of Interleukin4 by Different Areas of Synovial Membrane of Patients wiith Rheumatoid Arthitis and Osteoarthritis A. Falus, T. Lakatos & J. Smolen To cite this article: A. Falus, T. Lakatos & J. Smolen (1992) Dissimilar Biosynthesis of Interleukin4 by Different Areas of Synovial Membrane of Patients wiith Rheumatoid Arthitis and Osteoarthritis, Scandinavian Journal of Rheumatology, 21:3, 116-119 To link to this article: http://dx.doi.org/10.3109/03009749209095081
Published online: 12 Jul 2009.
Submit your article to this journal
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=irhe20 Download by: [University of California, San Diego]
Date: 22 January 2016, At: 11:35
Dissimilar Biosynthesis of Interleukin4 by Different Areas of Synovial Membrane of Patients wiith Rheumatoid Arthitis and Osteoarthritis A. Falus', T. Lakatos', and J. Smo1en2 'Departments of Molecular Biology and Orthopedic Surgery, National Institute of Rheumatology and Physiotherapy, Budapest, Hungary, *MA 17 Krankenhaus der Stadt, Dept. Med. 2., Wien-Lainz, Austria
Downloaded by [University of California, San Diego] at 11:35 22 January 2016
Falus A, Lakatos T, Smolen J. Dissimilar Biospthesis of Interleukin-6 by Different Areas of Synovial Membrane of Patients with Rheumatoid Arthritis and Osteoarthritis. Scand J Rheumatol. 1992; 21: 116-19.
In this study the IL6 production was studied by synovial cells isolated from patients with either rheumatoid arthritis (RA) or osteoarthritis (OA). The kinetics of spontaneous I L 6 production differs in both groups. Furthermore, the induction of I L 6 by bacterial lipopolysaccharide (LPS) in synovial cell cultures of RA is much more rapid than in those of OA patients. On the other hand, more PGE2 was detected in culture supernatants from synovial adherent cells of OA than in those of RA patients. We also compared the IL6 production and the amount of I L 6 mRNA in fragments derived from the areas of synovial tissue showing macroscopic signs of intensive inflammation (area A), with those from relatively intact (area B) synovial sites. In synovial fragments, but not in isolated adherent cells at area A the in vitro IG6 production starts earlier in RA than in OA. In the area A, significantlymore CD14+, CD43+ and HLA-DR+ cells were detected than in the other compartment less involved in local inflammatory events.
Key words: interleukin-6, synovial membrane, rheumatoid arthritis, lipopolysaccharide, PGE2
Dysregulation of local immune and metabolic events in the inflamed joints is closely associated with the abnormal production of some cytokines in rheumatoid synovium (1, 2, 3). Several studies suggest the important role of interleukin-6 (IG6) in inflammatory joint diseases, especially in rheumatoid arthritis (RA) (4, 5, 6). In this study we demonstrate that constitutive in vitro production of I L 6 by synovial lining cells, may be a common feature of RA and other types of synovitis. In another set of experiments we investigated whether the topology of local inflammation of the knee joints may be related to disproportionate production of I L 6 by cells in the corresponding areas of the joint. Materials and Methods
Surgical materials were obtained by synovectomy or elective orthopaedic surgery from 13 patients with definite or classical RA according to the ARA criteria (7) and 9 patients with classical osteoarthritis (OA). For multiple sampling the areas within a single joint were standardized as follows: Synovial fragments or cells from Part A correspond to the medial point of the patello-femoral surface of the Andr6s Falus, Dept. Molecular Biology, Natl. Inst. of Rheumatology and Physiotherapy, Budapest Frankel Leo u. 17/19. H-1027Budapest, Hungary Received 16 September 1991 Accepted 7 Febuary 1992
116
femur maximally involved in the joint destruction, based on a macroscopic evaluation. The specimens of Part B were obtained from distinct, suprapatellar areas of the knee joints - based on conventional macroscopic evaluation - and were not involved in the inflammatory process. The samples were taken from identical areas in surgical materials from both OA and RA patients. Culture of synovial adherent cells and synovial membrane tissue Synovial tissues were collected in TC Medium 199 containing 100 U/ml penicillin and 100 ug/ml streptomycin during surgery. All specimens were processed within 1 hour of collection. After removal of the blood clots, adipose tissues and fibrous material, the superficial layer. of synovium was minced into fragments of about 1-2 mm3 followed by washing three times in medium and culturing in tubes. About 100 mg tissue fragments were kept in 1 ml TC Medium 199 supplemented with 10% FCS and antibiotics as above. To obtain mononuclear cells, synovial membrane fragments were digested in serum-free medium containing 2 mg/ml collagenase (Sigma, 150 U/mg) and 0.15 mg/ml DNase (Serva, 30000 Dornase E/mg) for 4 hours at 37°C under frequent shaking. The digested fragments were filtered through two layers of nylon mesh, and the eluate was centrifuged at 1500 rpm for 15 minutes at room temperature followed by washing with medium twice. The cells were resuspended in medium (supplemented as above) at a concentration of 5 X 106 cells/ml, and incubated in petri
Dissimilar IL-6 within synovial membrane Tible 1. Interleukin-6production by
(A) Synovial Cells (ngllO5 cells). Incubation of adherent synovial cells for 1 day without
6 days with
without
LPS
with LPS
RA 16.9k 5.3 n=13
18.0f 9.5
23.3k10.7'
24.9k 11.4
OA n=9
16.9f11.4
17.8k 8.7
16.5k14.1
15.2f 8.7
Downloaded by [University of California, San Diego] at 11:35 22 January 2016
(6) by Synovial Membrane Fragments (ng/ 100 mg tissue). Incubation of synovial tissue fragments for 1 day without
6 days with
without
LPS
with LPS
RA 24.4512.0 n=13
31$1k10.1
31.8k 13.9
38.2f19.6
OA n=9
24.8f 9.3
25.7f11.2
40.2k10.9'L
23.9f18.4
Values given are mean k SD 'significantly higher (p=0.03) than the same values of OA patients 'significantly higher (p=0.02) than without LPS
dishes for 2 hours at 37°C in C0,-incubator. Removing the nonadherent cells by extensive washing with medium, the adherent cells were cultured for up to 7 days either without or with LPS (E.coli LPS, Sigma, 2 usfml) stimulation. The concentrations of I L 6 and prostaglandinE2 (PGE2) released into the culture supernatants were estimated by enzyme-immunoassay (ELISA) (8, 9) or by radioimmunoassay (Amersham), respectively. Immunofluorescent detection of cell markers CD 14 (for immature monocytes), CD 43 (for a mostly T cell activation marker) and HLA-DR were performed by commercially available (Beckton-Dickinson) mouse monoclonal antibodies. Total RNA was extracted by LiC1-urea procedure (10). Hybridization of RNA blots with 32P-labelled I L 6 cDNA probe (1 1) was performed as described elsewhere (12). For statistical analysis, the Students's t probe was used applying the Windows/ Statistics computer program.
the beginning of the culture of synovial cells regardless of whether LPS has been present or not (Table I.A.). However, the accumulation of IL-6 in 6 days culture of cells from R A patients significantly exceeds that of OA patients. In RA, but not in OA, the synovial fragments can be stimulated by LPS (2 ug/ml of LPS was used, found optimal in this system) to produce more IL-6 even after one day of culture. (Table I.B.). However, after a longer period of culture, synovial fragments from both patient groups are sensitive to LPS induction. The standard deviations of mean values manifest rather high individual variability in the response to LPS stimulation in both groups. These findings refer to dissimilar constitutive (baseline) and LPS-inducible I L 6 production in synovial adherent cells from RA and OA patients. The unresponsiveness of synovial adherent cells to LPS and in particular the effectiveness of LPS on synovial tissue, suggests a complex cellular interaction in tissue fragments of synovial membranes. Again, an immediate effect of LPS on synovial tissue from RA and a delayed one in the samples from OA patients shows obviously dissimilar cellular architecture. However, the comparisons between the inducibility of I L 6 in the synovial fragments from patients with RA and OA require precaution. Even when the surgical sampling is done based on a careful macroscopic evaluation, different values resulting from indifferent cellular composition and activation cannot be excluded. PGE2 production is increased in synovial adherent cells in both patients' group by LPS, but due to considerable individual variations the differences are not significant (Table 11.). Since the negative effect of PGE2 on cytokine production has been described (13, 14), a higher level of PGE2 (692 2 520 ng/105cells) in long term culture of adherent synovial cells from OA as compared to that of RA patients (327 370 ng/105 cells), might explain the reduced sensitivity to LPS
*
Table 11. PGE2 Production (ng1105 cells) by Synovial Cells Incubation of adherent synovial cells for
6 days
1 day without
with
without
LPS
Results and Discussion IL-6 and PGE2 production by adherent synovial
cells and in synovial membrane fragments No significant differences between RA and non RA group in the IL-6 production are observed at
RA
401f482
544k657
327k370
n=7
OA
388f352
368k293 n=10
439k370 n=8
with LPS
692k520
815f546 n= 5
Mean? SD
117
A . Falus et al.
Downloaded by [University of California, San Diego] at 11:35 22 January 2016
IL-6 production by synovial membrane fragments from site A and site 6
IL-6 production by different areas of synovial tissue In some R A cases, where multiple samples could be obtained from different synovial areas of a single joint, we could find relevant differences in the levels and kinetics of IL-6 released into the culture supernatants (Fig. l.A). At the center of inflammation of synovial tissue (part A) the peak of the IL-6 production (day 2.) precedes that of part B tissue (day 4.), sampled relatively far from the local inflammatory site. IL-6 mRNA values show parallel changes and, when compared to the “housekeeping” (unchanged) actin mRNA , parallel differences in the relative amount of IL-6 mRNA (Fig. l.B) were detected. A very detailed histopathological analysis as well as studies on local expression of cytokine genes and receptors by in situ hybridization are in progress in our laboratory. However, when adherent cells isolated from synovial fragments of part A or B were compared, we failed to detect analogous differences both in levels and kinetics of IL-6 production (Fig. 1.C). These results suggest that in addition to adherent synovial cells (presumably mostly macro-
IgG production by synovial fragments
Fig. I. Kinetics of in vitro production of IL-6 in fragments (A) (n=5) and adherent cells (C) (n=4) of synovial membrane obtained from different areas of the same knee joint of RA patients. IL-6 mRNA from synovial fragments and cells are also depicted. Relative 1L-6 mRNA values (B) (n=3) (compared to housekeeping actin mRNA) are given ,as well. For the sake of clarity of the figure only the means are given, the S.E.M. values are all under 10% of the mean. The standard sites of sampling from a single joint were marked with A and B (for details see Materials and Methods).
of the cells deriving from patients with O A (Table I. A). The high individual variability requires however further experimental data (e.g on the in vitro effect of indomethacine) to clarify the local regulatory processes more precisely.
118
Fig. 2. Levels of IgG (A) and IgM (B) in the media of synovial fragment cultures after 1 and 6 days incubation. (n=5).
Downloaded by [University of California, San Diego] at 11:35 22 January 2016
Dissimilar IL-6 within synovial membrane phages), other cells are definitely involved in the local network regulating the expression of the IL-6 gene. The measurement of TNF (alpha and beta), IL-1 (alpha and beta) and TGF-beta in the synovial fragmentkell culture will help our better recognition of the elements of local regulatory events. To elucidate the functional relevance of the differences in the level and kinetics of local IL-6 production, IgG and IgM concentrations were also measured in the cell culture supernatants. The data (Fig. 2.) show a dissimilar regulation of the two Ig isotypes, IgG (A) and IgM (B). Similar IgG levels which tended to decrease during the time period of the culture were detected in part A and B, while the amount of IgM secreted from day 1 during the culture was higher in part B than in part A-derived synovial cells. This difference is probably due to dissimilar maturation, activation and cytokine receptor expression of B lymphocytes in the two synovial compartments. Further studies using isotype (class, subclass)- specific serological and molecular reagents are needed to clarify the exact mechanism of isotype regulation in synovial tissue. The cellular compartments A and B were partially further characterized: The occurrence of putative LPS receptor CD14 positive monocytes (near 70%0),CD 43 positive cells(indicating activated T-cells) (about 35%) and HLA-DR-positive cells (85%) in part A , exceeded significantly those in part B (10, 7.7 and 25%, respectively). Again, the occurrence of plasma cells in part A and part B of synovial tissue was closely similar, corresponding to earlier observations (15). There was a moderate correlation between the amounts of IL-6 measured in the culture supernatants and plasma cell accumulation in the corresponding areas of the same joint (checked in four patients with RA, not shown). Similar results were shown recently by Field and coworkers (16). Our preliminary data demonstrate a significant local heterogeneity in the ability of synovial cells to produce IL-6. These findings, completed with studies focusing on the detection and localization of other inflammatory cytokines , cytokine inhibitors and soluble cytokine receptors in the rheumatoid joints, may provide a more precise idea on the local regulatory circuits in the inflamed synovia. Grants: Austrian-Hungarian Common Research Project A30DVR-175. Hungarian Ministry of Health and Welfare 7041 1988.
References 1. Husby G, Williams RC. Immunohistochemical studies of interleukin 2 and gamma interferon in rheumatoid arthritis. Arthritis Rheum 1985; 28: 174-81. 2. Leizer T, Clarris BJ, Ash PE, van Damme J, Saklatvala J, Hamilton JA. Interleukin-lp and interleukin-la stimulate the plasminogen activator activity and prostaglandin E2 levels of human synovial cells. Arthritis Rheum 1987; 30: 562-6. 3. Danis VA, March LM, Nelson DS, Brooks PM. Interleukin-1 secretion by peripheral blood monocytes and synovial macrophages from patients with rheumatoid arthritis J Rheumatol 1987; 14: 33-9. 4. Houssiau FA, Devogelaer J-P, van Damme J , Nagant de Deuxchaisnes C, van Snick J. Interleukin-6 in synovial fluid and serum of patients with rheumatoid arthritis and other inflammatory arthritides. Arthritis Rheum 1988; 31: 784-8. 5 . Swaak AJG, Rooyen A, van Nieuwenhuis E, Aarden LA. Interleukin-6 (IL-6) in synovial fluid and serum of patients with rheumatic diseases. Scand J Rheumatol 1988; 17: 469-74. 6. Hovdenes J, Kvien TK, Hovdenes AB. IL-6 in synovial fluids, plasma and supernatants from cultured cells with rheumatoid arthritis and other inflammatory arthritides Scand J Rheumatol 1990; 19: 177-82. 7. Ropes MW, Bennett GA, Cobb S, Jacox R, Jessar RA. 1958 revision of diagnostic criteria for rheumatoid arthritis. Bull Rheum Dis 1958; 9: 175-6. 8. Brozik M, Rosztoczy I, Walcz E et al. Comparison of ELISA and biological assay in determination of IL-6; application for IL-6 detection in synovial fluids and sera. Clin Exp Rheumatol 1990; 8: S-5, 106. 9. Brozik M, Rosztoczy I, Meretey K et al. Interleukin-6 levels in synovial fluids of patiens with different arthritides; correlation with local IgM-rheumatoid factor and systemic acute phase protein production. J Rheumatol. (in press). 10. Thomas PS. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci USA 1980; 77: 5201-5. 11. Hirano T, Yasukawa K, Harada H et al. Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin. Nature, 1986; 324: 73-6. 12. Falus A, Beuscher HU, Auerbach HS, Colten HR. Constitutive and IL-1 regulated murine complement gene expression is strain and tissue specific. J Imrnunol, 1987; 138: 856-60. 13. Rappaport RS , Dodge GR. Prostaglandin E inhibits the production of human interleukin 2. J Exp Med 1982; 155: 943-8. 14. Lehmmann V, Benninghoff B , Droge W. Tumor necrosis factor-induced activation of peritoneal macrophages is regulated by prostaglandin E2 and CAMP. J Immunol, 1988; 141: 587-91. 15. Chien HD, Walcz E, Lakatos T, Awai K, Br6zik M, MerCtey K. Immunoglobulins in synovial fragmenty cultures. Hung. Rheumatol. Suppl. 1989; 57-63. 16. Field M, Chu C, Feldmann M , Maini RN. Interleukin-6 localisation in the synovial membrane in rheumatoid arthritis. Rheumatol. Int. 1991; 11: 45-50.
119
ISSN: 0300-9742 (Print) 1502-7732 (Online) Journal homepage: http://www.tandfonline.com/loi/irhe20
Dissimilar Biosynthesis of Interleukin4 by Different Areas of Synovial Membrane of Patients wiith Rheumatoid Arthitis and Osteoarthritis A. Falus, T. Lakatos & J. Smolen To cite this article: A. Falus, T. Lakatos & J. Smolen (1992) Dissimilar Biosynthesis of Interleukin4 by Different Areas of Synovial Membrane of Patients wiith Rheumatoid Arthitis and Osteoarthritis, Scandinavian Journal of Rheumatology, 21:3, 116-119 To link to this article: http://dx.doi.org/10.3109/03009749209095081
Published online: 12 Jul 2009.
Submit your article to this journal
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=irhe20 Download by: [University of California, San Diego]
Date: 22 January 2016, At: 11:35
Dissimilar Biosynthesis of Interleukin4 by Different Areas of Synovial Membrane of Patients wiith Rheumatoid Arthitis and Osteoarthritis A. Falus', T. Lakatos', and J. Smo1en2 'Departments of Molecular Biology and Orthopedic Surgery, National Institute of Rheumatology and Physiotherapy, Budapest, Hungary, *MA 17 Krankenhaus der Stadt, Dept. Med. 2., Wien-Lainz, Austria
Downloaded by [University of California, San Diego] at 11:35 22 January 2016
Falus A, Lakatos T, Smolen J. Dissimilar Biospthesis of Interleukin-6 by Different Areas of Synovial Membrane of Patients with Rheumatoid Arthritis and Osteoarthritis. Scand J Rheumatol. 1992; 21: 116-19.
In this study the IL6 production was studied by synovial cells isolated from patients with either rheumatoid arthritis (RA) or osteoarthritis (OA). The kinetics of spontaneous I L 6 production differs in both groups. Furthermore, the induction of I L 6 by bacterial lipopolysaccharide (LPS) in synovial cell cultures of RA is much more rapid than in those of OA patients. On the other hand, more PGE2 was detected in culture supernatants from synovial adherent cells of OA than in those of RA patients. We also compared the IL6 production and the amount of I L 6 mRNA in fragments derived from the areas of synovial tissue showing macroscopic signs of intensive inflammation (area A), with those from relatively intact (area B) synovial sites. In synovial fragments, but not in isolated adherent cells at area A the in vitro IG6 production starts earlier in RA than in OA. In the area A, significantlymore CD14+, CD43+ and HLA-DR+ cells were detected than in the other compartment less involved in local inflammatory events.
Key words: interleukin-6, synovial membrane, rheumatoid arthritis, lipopolysaccharide, PGE2
Dysregulation of local immune and metabolic events in the inflamed joints is closely associated with the abnormal production of some cytokines in rheumatoid synovium (1, 2, 3). Several studies suggest the important role of interleukin-6 (IG6) in inflammatory joint diseases, especially in rheumatoid arthritis (RA) (4, 5, 6). In this study we demonstrate that constitutive in vitro production of I L 6 by synovial lining cells, may be a common feature of RA and other types of synovitis. In another set of experiments we investigated whether the topology of local inflammation of the knee joints may be related to disproportionate production of I L 6 by cells in the corresponding areas of the joint. Materials and Methods
Surgical materials were obtained by synovectomy or elective orthopaedic surgery from 13 patients with definite or classical RA according to the ARA criteria (7) and 9 patients with classical osteoarthritis (OA). For multiple sampling the areas within a single joint were standardized as follows: Synovial fragments or cells from Part A correspond to the medial point of the patello-femoral surface of the Andr6s Falus, Dept. Molecular Biology, Natl. Inst. of Rheumatology and Physiotherapy, Budapest Frankel Leo u. 17/19. H-1027Budapest, Hungary Received 16 September 1991 Accepted 7 Febuary 1992
116
femur maximally involved in the joint destruction, based on a macroscopic evaluation. The specimens of Part B were obtained from distinct, suprapatellar areas of the knee joints - based on conventional macroscopic evaluation - and were not involved in the inflammatory process. The samples were taken from identical areas in surgical materials from both OA and RA patients. Culture of synovial adherent cells and synovial membrane tissue Synovial tissues were collected in TC Medium 199 containing 100 U/ml penicillin and 100 ug/ml streptomycin during surgery. All specimens were processed within 1 hour of collection. After removal of the blood clots, adipose tissues and fibrous material, the superficial layer. of synovium was minced into fragments of about 1-2 mm3 followed by washing three times in medium and culturing in tubes. About 100 mg tissue fragments were kept in 1 ml TC Medium 199 supplemented with 10% FCS and antibiotics as above. To obtain mononuclear cells, synovial membrane fragments were digested in serum-free medium containing 2 mg/ml collagenase (Sigma, 150 U/mg) and 0.15 mg/ml DNase (Serva, 30000 Dornase E/mg) for 4 hours at 37°C under frequent shaking. The digested fragments were filtered through two layers of nylon mesh, and the eluate was centrifuged at 1500 rpm for 15 minutes at room temperature followed by washing with medium twice. The cells were resuspended in medium (supplemented as above) at a concentration of 5 X 106 cells/ml, and incubated in petri
Dissimilar IL-6 within synovial membrane Tible 1. Interleukin-6production by
(A) Synovial Cells (ngllO5 cells). Incubation of adherent synovial cells for 1 day without
6 days with
without
LPS
with LPS
RA 16.9k 5.3 n=13
18.0f 9.5
23.3k10.7'
24.9k 11.4
OA n=9
16.9f11.4
17.8k 8.7
16.5k14.1
15.2f 8.7
Downloaded by [University of California, San Diego] at 11:35 22 January 2016
(6) by Synovial Membrane Fragments (ng/ 100 mg tissue). Incubation of synovial tissue fragments for 1 day without
6 days with
without
LPS
with LPS
RA 24.4512.0 n=13
31$1k10.1
31.8k 13.9
38.2f19.6
OA n=9
24.8f 9.3
25.7f11.2
40.2k10.9'L
23.9f18.4
Values given are mean k SD 'significantly higher (p=0.03) than the same values of OA patients 'significantly higher (p=0.02) than without LPS
dishes for 2 hours at 37°C in C0,-incubator. Removing the nonadherent cells by extensive washing with medium, the adherent cells were cultured for up to 7 days either without or with LPS (E.coli LPS, Sigma, 2 usfml) stimulation. The concentrations of I L 6 and prostaglandinE2 (PGE2) released into the culture supernatants were estimated by enzyme-immunoassay (ELISA) (8, 9) or by radioimmunoassay (Amersham), respectively. Immunofluorescent detection of cell markers CD 14 (for immature monocytes), CD 43 (for a mostly T cell activation marker) and HLA-DR were performed by commercially available (Beckton-Dickinson) mouse monoclonal antibodies. Total RNA was extracted by LiC1-urea procedure (10). Hybridization of RNA blots with 32P-labelled I L 6 cDNA probe (1 1) was performed as described elsewhere (12). For statistical analysis, the Students's t probe was used applying the Windows/ Statistics computer program.
the beginning of the culture of synovial cells regardless of whether LPS has been present or not (Table I.A.). However, the accumulation of IL-6 in 6 days culture of cells from R A patients significantly exceeds that of OA patients. In RA, but not in OA, the synovial fragments can be stimulated by LPS (2 ug/ml of LPS was used, found optimal in this system) to produce more IL-6 even after one day of culture. (Table I.B.). However, after a longer period of culture, synovial fragments from both patient groups are sensitive to LPS induction. The standard deviations of mean values manifest rather high individual variability in the response to LPS stimulation in both groups. These findings refer to dissimilar constitutive (baseline) and LPS-inducible I L 6 production in synovial adherent cells from RA and OA patients. The unresponsiveness of synovial adherent cells to LPS and in particular the effectiveness of LPS on synovial tissue, suggests a complex cellular interaction in tissue fragments of synovial membranes. Again, an immediate effect of LPS on synovial tissue from RA and a delayed one in the samples from OA patients shows obviously dissimilar cellular architecture. However, the comparisons between the inducibility of I L 6 in the synovial fragments from patients with RA and OA require precaution. Even when the surgical sampling is done based on a careful macroscopic evaluation, different values resulting from indifferent cellular composition and activation cannot be excluded. PGE2 production is increased in synovial adherent cells in both patients' group by LPS, but due to considerable individual variations the differences are not significant (Table 11.). Since the negative effect of PGE2 on cytokine production has been described (13, 14), a higher level of PGE2 (692 2 520 ng/105cells) in long term culture of adherent synovial cells from OA as compared to that of RA patients (327 370 ng/105 cells), might explain the reduced sensitivity to LPS
*
Table 11. PGE2 Production (ng1105 cells) by Synovial Cells Incubation of adherent synovial cells for
6 days
1 day without
with
without
LPS
Results and Discussion IL-6 and PGE2 production by adherent synovial
cells and in synovial membrane fragments No significant differences between RA and non RA group in the IL-6 production are observed at
RA
401f482
544k657
327k370
n=7
OA
388f352
368k293 n=10
439k370 n=8
with LPS
692k520
815f546 n= 5
Mean? SD
117
A . Falus et al.
Downloaded by [University of California, San Diego] at 11:35 22 January 2016
IL-6 production by synovial membrane fragments from site A and site 6
IL-6 production by different areas of synovial tissue In some R A cases, where multiple samples could be obtained from different synovial areas of a single joint, we could find relevant differences in the levels and kinetics of IL-6 released into the culture supernatants (Fig. l.A). At the center of inflammation of synovial tissue (part A) the peak of the IL-6 production (day 2.) precedes that of part B tissue (day 4.), sampled relatively far from the local inflammatory site. IL-6 mRNA values show parallel changes and, when compared to the “housekeeping” (unchanged) actin mRNA , parallel differences in the relative amount of IL-6 mRNA (Fig. l.B) were detected. A very detailed histopathological analysis as well as studies on local expression of cytokine genes and receptors by in situ hybridization are in progress in our laboratory. However, when adherent cells isolated from synovial fragments of part A or B were compared, we failed to detect analogous differences both in levels and kinetics of IL-6 production (Fig. 1.C). These results suggest that in addition to adherent synovial cells (presumably mostly macro-
IgG production by synovial fragments
Fig. I. Kinetics of in vitro production of IL-6 in fragments (A) (n=5) and adherent cells (C) (n=4) of synovial membrane obtained from different areas of the same knee joint of RA patients. IL-6 mRNA from synovial fragments and cells are also depicted. Relative 1L-6 mRNA values (B) (n=3) (compared to housekeeping actin mRNA) are given ,as well. For the sake of clarity of the figure only the means are given, the S.E.M. values are all under 10% of the mean. The standard sites of sampling from a single joint were marked with A and B (for details see Materials and Methods).
of the cells deriving from patients with O A (Table I. A). The high individual variability requires however further experimental data (e.g on the in vitro effect of indomethacine) to clarify the local regulatory processes more precisely.
118
Fig. 2. Levels of IgG (A) and IgM (B) in the media of synovial fragment cultures after 1 and 6 days incubation. (n=5).
Downloaded by [University of California, San Diego] at 11:35 22 January 2016
Dissimilar IL-6 within synovial membrane phages), other cells are definitely involved in the local network regulating the expression of the IL-6 gene. The measurement of TNF (alpha and beta), IL-1 (alpha and beta) and TGF-beta in the synovial fragmentkell culture will help our better recognition of the elements of local regulatory events. To elucidate the functional relevance of the differences in the level and kinetics of local IL-6 production, IgG and IgM concentrations were also measured in the cell culture supernatants. The data (Fig. 2.) show a dissimilar regulation of the two Ig isotypes, IgG (A) and IgM (B). Similar IgG levels which tended to decrease during the time period of the culture were detected in part A and B, while the amount of IgM secreted from day 1 during the culture was higher in part B than in part A-derived synovial cells. This difference is probably due to dissimilar maturation, activation and cytokine receptor expression of B lymphocytes in the two synovial compartments. Further studies using isotype (class, subclass)- specific serological and molecular reagents are needed to clarify the exact mechanism of isotype regulation in synovial tissue. The cellular compartments A and B were partially further characterized: The occurrence of putative LPS receptor CD14 positive monocytes (near 70%0),CD 43 positive cells(indicating activated T-cells) (about 35%) and HLA-DR-positive cells (85%) in part A , exceeded significantly those in part B (10, 7.7 and 25%, respectively). Again, the occurrence of plasma cells in part A and part B of synovial tissue was closely similar, corresponding to earlier observations (15). There was a moderate correlation between the amounts of IL-6 measured in the culture supernatants and plasma cell accumulation in the corresponding areas of the same joint (checked in four patients with RA, not shown). Similar results were shown recently by Field and coworkers (16). Our preliminary data demonstrate a significant local heterogeneity in the ability of synovial cells to produce IL-6. These findings, completed with studies focusing on the detection and localization of other inflammatory cytokines , cytokine inhibitors and soluble cytokine receptors in the rheumatoid joints, may provide a more precise idea on the local regulatory circuits in the inflamed synovia. Grants: Austrian-Hungarian Common Research Project A30DVR-175. Hungarian Ministry of Health and Welfare 7041 1988.
References 1. Husby G, Williams RC. Immunohistochemical studies of interleukin 2 and gamma interferon in rheumatoid arthritis. Arthritis Rheum 1985; 28: 174-81. 2. Leizer T, Clarris BJ, Ash PE, van Damme J, Saklatvala J, Hamilton JA. Interleukin-lp and interleukin-la stimulate the plasminogen activator activity and prostaglandin E2 levels of human synovial cells. Arthritis Rheum 1987; 30: 562-6. 3. Danis VA, March LM, Nelson DS, Brooks PM. Interleukin-1 secretion by peripheral blood monocytes and synovial macrophages from patients with rheumatoid arthritis J Rheumatol 1987; 14: 33-9. 4. Houssiau FA, Devogelaer J-P, van Damme J , Nagant de Deuxchaisnes C, van Snick J. Interleukin-6 in synovial fluid and serum of patients with rheumatoid arthritis and other inflammatory arthritides. Arthritis Rheum 1988; 31: 784-8. 5 . Swaak AJG, Rooyen A, van Nieuwenhuis E, Aarden LA. Interleukin-6 (IL-6) in synovial fluid and serum of patients with rheumatic diseases. Scand J Rheumatol 1988; 17: 469-74. 6. Hovdenes J, Kvien TK, Hovdenes AB. IL-6 in synovial fluids, plasma and supernatants from cultured cells with rheumatoid arthritis and other inflammatory arthritides Scand J Rheumatol 1990; 19: 177-82. 7. Ropes MW, Bennett GA, Cobb S, Jacox R, Jessar RA. 1958 revision of diagnostic criteria for rheumatoid arthritis. Bull Rheum Dis 1958; 9: 175-6. 8. Brozik M, Rosztoczy I, Walcz E et al. Comparison of ELISA and biological assay in determination of IL-6; application for IL-6 detection in synovial fluids and sera. Clin Exp Rheumatol 1990; 8: S-5, 106. 9. Brozik M, Rosztoczy I, Meretey K et al. Interleukin-6 levels in synovial fluids of patiens with different arthritides; correlation with local IgM-rheumatoid factor and systemic acute phase protein production. J Rheumatol. (in press). 10. Thomas PS. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci USA 1980; 77: 5201-5. 11. Hirano T, Yasukawa K, Harada H et al. Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin. Nature, 1986; 324: 73-6. 12. Falus A, Beuscher HU, Auerbach HS, Colten HR. Constitutive and IL-1 regulated murine complement gene expression is strain and tissue specific. J Imrnunol, 1987; 138: 856-60. 13. Rappaport RS , Dodge GR. Prostaglandin E inhibits the production of human interleukin 2. J Exp Med 1982; 155: 943-8. 14. Lehmmann V, Benninghoff B , Droge W. Tumor necrosis factor-induced activation of peritoneal macrophages is regulated by prostaglandin E2 and CAMP. J Immunol, 1988; 141: 587-91. 15. Chien HD, Walcz E, Lakatos T, Awai K, Br6zik M, MerCtey K. Immunoglobulins in synovial fragmenty cultures. Hung. Rheumatol. Suppl. 1989; 57-63. 16. Field M, Chu C, Feldmann M , Maini RN. Interleukin-6 localisation in the synovial membrane in rheumatoid arthritis. Rheumatol. Int. 1991; 11: 45-50.
119
