British Journal of Rheumatology 1991;30:265-271
ROLE OF TNFa, IN RELATION TO IL-1 AND IL-6 IN THE PROTEOGLYCAN TURNOVER OF HUMAN ARTICULAR CARTILAGE BY B. WILBRINK, J. J. NIETFELD, W. DEN OTTER*, J. L. A. M. VAN ROY, J. W. J. BIJLSMA AND O. HUBER-BRUNING Departments of Rheumatology and * Pathology, University Hospital Utrecht, The Netherlands
KEY WORDS:
Explant culture, Cytokines, Bioassay.
induced IL-6 was a prerequisite for the effect of TNFa on the PG synthesis of the cartilage. Finally we investigated a possible synergism between TNFa and IL-1.
THE synovial fluid in rheumatoid arthritis (RA) can induce proteoglycan (PG) loss and inhibition of PG synthesis in cultured normal human articular cartilage [1] and bovine nasal cartilage [2]. This is presumably due to the activities of cytokines. The presence of tumour necrosis factor a (TNFa) [3,4], interleukin-1 (IL-1) [5-7] and interleukin-6 (IL-6) [8,9] has been demonstrated in RA synovial fluid. TNFa [10, 11] and IL-1 [12-14] have the potential to influence PG metabolism of articular cartilage in vitro and in vivo [15], but their combined influence on cartilage degradation in vivo is not fully understood. TNFa induces IL-1 in different cell types that participate in joint structures, such as macrophages [16], synoviocytes [17] and endothelial cells [18]. IL-1 can induce IL-6 production, not only in these and othercells [19-25], but also in human articular cartilage in explant culture [26-28] as well as in chondrocytes in vitro [29]. An hypothetical cytokine cascade for cartilage is that TNFa induces IL-1 production, which in turn induces IL-6 production. Such a cascade might play a role in the mechanism of cartilage degradation in RA. The produced IL-6 could ultimately determine the biological response of these cytokines in cartilage, since we have found that the IL-1-induced IL-6 is required for the inhibition of PG synthesis by IL-1 in human cartilage [27, 28]. We investigated the effects of recombinant human TNFa on the PG metabolism of human articular cartilage of different ages in explant culture since we have found a difference in sensitivity for IL-1 in human cartilage obtained from young and old donors [14]. In addition, we studied whether TNFa had the capacity to induce IL-1 and/or IL-6 production in these explants. Since we found a direct induction of IL-6 by TNFa we studied whether the presence of the Submitted 30 July; revised version accepted 23 November 1990. Correspondence to Dr B. Wilbrink, University Hospital, Department of Rheumatology, F.02.223, P. O. Box 85500, 3508 GA Utrecht, The Netherlands.
MATERIALS AND METHODS Microscopic normal human articular cartilage was obtained from the humeral head of donor bodies by sterile techniques within 18 h of death. Exclusion criteria were septicaemia, connective tissue disease, known hereditary defect or recent treatment with corticosteroids or cytostatic drugs. Slices of cartilage were removed aseptically from the articular surface, cut into square pieces, weighed (range 5-10 mg) and cultured individually at random in quintuplicate per group in a 96-well roundbottomed microtitre plate in 180ul medium consisting of 90% DMEM plus 10% heat-inactivated pooled human male AB serum, supplemented with glutamine (2 m\i), penicillin (100 IU), streptomycin sulphate (100ng/ml) and ascorbic acid (150 M-g/ml). Thefluidwas brought up to 200 ul by addition of 20 uJ PBS with 1 mg/ml bovine serum albumin (BSA) or 20 ul of PBS/BSA containing various concentrations of TNFa or IL-1. Then the microtitre plate was placed in a moist CO2 incubator (5% CO2 in air) at 37°C. To measure the effect of TNFa and IL-1 on the PG synthesis after 4 days, the cultures were supplied with 148 kBq Na235SO4 (carrier free, 0.37GBq/ml) in 10 ul per well for the last 4 h of the culture. To assess the induction of cytokines, culture media were collected after 4 days and stored at -30°C until determination. To measure the effect of TNFa on the PG content and the PG release the plates were cultured for 4 or 8 days, in the latter case with a change of the media and additions at the fourth day. After culture the explants and media were collected separately. To measure the reversibility of the effects of TNFa, explants cultured for 4 days in the presence or absence of TNFa were amply washed with medium of 37°C on day 4. Explants were cultured without TNFa from day 265
Downloaded from http://rheumatology.oxfordjournals.org/ at New York University on May 9, 2015
SUMMARY In both young and old human articular cartilage explants, TNFa induced a concentration-dependent, reversible suppression of the proteoglycan (PG) synthesis. Young cartilage was more sensitive to TNFa than old cartilage: 50% suppression of 3PG synthesis was reached at a TNFa concentration of 5 U/ml for young and 30U/ml for old cartilage, whereas at 10 U/ml the PG synthesis of young cartilage was blocked and that of old cartilage suppressed by 80%. These inhibition levels of PG synthesis resulted in 25% PG depletion of the explants after 8 days of culture. The release of cartilage PG was not enhanced. TNFa induced no detectable amounts of IL-1 (
ROLE OF TNFa, IN RELATION TO IL-1 AND IL-6 IN THE PROTEOGLYCAN TURNOVER OF HUMAN ARTICULAR CARTILAGE BY B. WILBRINK, J. J. NIETFELD, W. DEN OTTER*, J. L. A. M. VAN ROY, J. W. J. BIJLSMA AND O. HUBER-BRUNING Departments of Rheumatology and * Pathology, University Hospital Utrecht, The Netherlands
KEY WORDS:
Explant culture, Cytokines, Bioassay.
induced IL-6 was a prerequisite for the effect of TNFa on the PG synthesis of the cartilage. Finally we investigated a possible synergism between TNFa and IL-1.
THE synovial fluid in rheumatoid arthritis (RA) can induce proteoglycan (PG) loss and inhibition of PG synthesis in cultured normal human articular cartilage [1] and bovine nasal cartilage [2]. This is presumably due to the activities of cytokines. The presence of tumour necrosis factor a (TNFa) [3,4], interleukin-1 (IL-1) [5-7] and interleukin-6 (IL-6) [8,9] has been demonstrated in RA synovial fluid. TNFa [10, 11] and IL-1 [12-14] have the potential to influence PG metabolism of articular cartilage in vitro and in vivo [15], but their combined influence on cartilage degradation in vivo is not fully understood. TNFa induces IL-1 in different cell types that participate in joint structures, such as macrophages [16], synoviocytes [17] and endothelial cells [18]. IL-1 can induce IL-6 production, not only in these and othercells [19-25], but also in human articular cartilage in explant culture [26-28] as well as in chondrocytes in vitro [29]. An hypothetical cytokine cascade for cartilage is that TNFa induces IL-1 production, which in turn induces IL-6 production. Such a cascade might play a role in the mechanism of cartilage degradation in RA. The produced IL-6 could ultimately determine the biological response of these cytokines in cartilage, since we have found that the IL-1-induced IL-6 is required for the inhibition of PG synthesis by IL-1 in human cartilage [27, 28]. We investigated the effects of recombinant human TNFa on the PG metabolism of human articular cartilage of different ages in explant culture since we have found a difference in sensitivity for IL-1 in human cartilage obtained from young and old donors [14]. In addition, we studied whether TNFa had the capacity to induce IL-1 and/or IL-6 production in these explants. Since we found a direct induction of IL-6 by TNFa we studied whether the presence of the Submitted 30 July; revised version accepted 23 November 1990. Correspondence to Dr B. Wilbrink, University Hospital, Department of Rheumatology, F.02.223, P. O. Box 85500, 3508 GA Utrecht, The Netherlands.
MATERIALS AND METHODS Microscopic normal human articular cartilage was obtained from the humeral head of donor bodies by sterile techniques within 18 h of death. Exclusion criteria were septicaemia, connective tissue disease, known hereditary defect or recent treatment with corticosteroids or cytostatic drugs. Slices of cartilage were removed aseptically from the articular surface, cut into square pieces, weighed (range 5-10 mg) and cultured individually at random in quintuplicate per group in a 96-well roundbottomed microtitre plate in 180ul medium consisting of 90% DMEM plus 10% heat-inactivated pooled human male AB serum, supplemented with glutamine (2 m\i), penicillin (100 IU), streptomycin sulphate (100ng/ml) and ascorbic acid (150 M-g/ml). Thefluidwas brought up to 200 ul by addition of 20 uJ PBS with 1 mg/ml bovine serum albumin (BSA) or 20 ul of PBS/BSA containing various concentrations of TNFa or IL-1. Then the microtitre plate was placed in a moist CO2 incubator (5% CO2 in air) at 37°C. To measure the effect of TNFa and IL-1 on the PG synthesis after 4 days, the cultures were supplied with 148 kBq Na235SO4 (carrier free, 0.37GBq/ml) in 10 ul per well for the last 4 h of the culture. To assess the induction of cytokines, culture media were collected after 4 days and stored at -30°C until determination. To measure the effect of TNFa on the PG content and the PG release the plates were cultured for 4 or 8 days, in the latter case with a change of the media and additions at the fourth day. After culture the explants and media were collected separately. To measure the reversibility of the effects of TNFa, explants cultured for 4 days in the presence or absence of TNFa were amply washed with medium of 37°C on day 4. Explants were cultured without TNFa from day 265
Downloaded from http://rheumatology.oxfordjournals.org/ at New York University on May 9, 2015
SUMMARY In both young and old human articular cartilage explants, TNFa induced a concentration-dependent, reversible suppression of the proteoglycan (PG) synthesis. Young cartilage was more sensitive to TNFa than old cartilage: 50% suppression of 3PG synthesis was reached at a TNFa concentration of 5 U/ml for young and 30U/ml for old cartilage, whereas at 10 U/ml the PG synthesis of young cartilage was blocked and that of old cartilage suppressed by 80%. These inhibition levels of PG synthesis resulted in 25% PG depletion of the explants after 8 days of culture. The release of cartilage PG was not enhanced. TNFa induced no detectable amounts of IL-1 (
