Scandinavian Journal of Rheumatology
ISSN: 0300-9742 (Print) 1502-7732 (Online) Journal homepage: http://www.tandfonline.com/loi/irhe20
Connective Tissue Activation by Synovial Fluids and Synovial-Tissue Extracts of Rheumatoid Arthritis Patients I. Takala, P. Mäkisara & E. Kulonen To cite this article: I. Takala, P. Mäkisara & E. Kulonen (1977) Connective Tissue Activation by Synovial Fluids and Synovial-Tissue Extracts of Rheumatoid Arthritis Patients, Scandinavian Journal of Rheumatology, 6:3, 172-176, DOI: 10.3109/03009747709095444 To link to this article: http://dx.doi.org/10.3109/03009747709095444
Published online: 12 Jul 2009.
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Article views: 1
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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=irhe20 Download by: [University of California Santa Barbara]
Date: 22 March 2016, At: 18:15
Scand J Rheumatology 6: 172-176, 1977
CONNECTIVE TISSUE ACTIVATION BY SYNOVIAL FLUIDS AND SYNOVIAL-TISSUE EXTRACTS O F RHEUMATOID ARTHRITIS PATIENTS I. Takala, P. Makisara and E. Kulonen
Downloaded by [University of California Santa Barbara] at 18:15 22 March 2016
From the Department of Medical Chemistry, University of Turku, Turku, Finland, and Rheumatism Foundation Hospital, Heinola, Finland
ABSTRACT. Prompted by Castor's investigations (4) on a connective-tissue-activatingpeptide (CTAP) we investigated the stimulative effect of synodal fluids and synodal-tissue extracts on the synthesis of collagen by incubated embryonicehick tendon cells. The stimulative effect was greater with synovial-tissueextracts from more severe cases than with samples from patients having milder forms of the disease. There was no correlation between the stimulation by synovial fluids and synovial-tissueextracts from the same patient. The stimulative activity was lost at dialysis. A slight stimulation in the incorporation of glucosamine was also observed. Treatment of the patients with gold, chloroquine or steroids decreased the stimulating capacity. These effects seem to depend on factors different from those described by Castor. The differences in the stimulant activity of samples from various groups cannot be entirely due to glutamine, which is one of the limiting nutrients of the embryonic-chick tendon cells.
This work was prompted by the series of papers on the CTAP (connective-tissue-activating peptide) separated from spleen and lymphocytes by Castor and co-workers (4) and by our own work on the connective-tissue stimulation which is observed in silicosis (2) and certain fatty liver disorders (15). The rheumatoid synovial tissue is a form of granulation tissue with a high content of hyaluronate. One of our original purposes was to relate the stimulative activity of the synovial fluid to the clinical picture. In most cases, parallel samples from the solid synovial tissue, from which extracts could be prepared, were also available for study. MATERIAL AND METHODS Patients The 18 synovial-tissue samples and the corresponding synovial fluids were collected during therapeutic Scand J Rheumatology 6
synovectomies of the knee joints carried out at the Rheumatism Foundation Hospital, Heinola, Finland. Ten additional synovial-fluid samples were taken by needle puncture without synovectomy. All the patients had classic rheumatoid arthritis according to the A.R.A. criteria (14). The radiologic grades of the joints were determined according to Larsen (13), and the joint indices according to Lansbury (12). Preparation of synovial tissue samples One g wet weight of synovial tissue was homogenized with an Ultra-Turrax (Janke & Kunkel) homogenizer in 10 ml of 0.15 M NaCl in an ice bath. The homogenate was centrifuged first at 2 500 g for 30 min and the supernatant was used as the synovial-tissue extract in the preliminary experiments. This extract was then recentrifuged at 25 OOO g for 30 min, and the supernatant was used in the final experiments. The synovial fluid was centrifuged at 1 OOOg for 15 min to remove the cells. The dialyses were performed against a 10000-fold volume of 0.15 M NaCI. Preparation and incubation of embryonicchick tendon cells Tendon cells from 17-day-old chick embryos were prepared by the method of Dehm & Prockop ( 5 ) as modified by Ronnemaa et al. (15). After isolation, the fibroblasts were suspended in the modified Krebs-Ringer medium containing 0.1 mM ascorbate, 14.5 p M L-proline and 0.15 gll bovine serum albumin (Sigma A4503). Aliquots containing about I@ cells in 3 ml of medium were used and 0.1 ml of synovial fluid or synovial-tissue extract was added to each. After a 15-min preincubation at 37°C. 5 pCi LG-[3H]proline (TRA 82, The Radiochemical Centre, Amersham) was added in 0.1 ml of 0.15 M NaCI. The incubation was continued for 120 min with air as the gas phase and terminated by adding 270 pg of cycioheximide (Sigma) and 400 pg of a,a'-dipyridyl (Fluka) in 0.2 ml of the medium. Cell viability was ascertained with the trypan blue test. Cells were separated from the medium by centrifugation (350g, 12 min). To disrupt the cells, 2 ml of water was added and the samples were sonicated for 30 sec. All samples were dialysed against cold running tap water for 24 h with L-proline (0.005 mglsample) as the carrier.
Connective tissue activation
173
Table I. Effpcts of centrifugation and dialysis on the activity of the synovial-tissue extract on protein synthesis in embryonic-chick tendon cells Fraction analysed Effect of centrifugation (n =4)
Total protein Collagen Effect of dial.vsis (n =6)
Downloaded by [University of California Santa Barbara] at 18:15 22 March 2016
Total protein Collagen
Synthesis expressed as percentage of control (100.0)
Distribution of synthesized protein in medium/cells
2 500g 128.8f5.8 137.7f5.8
25 OOOg 155.lf6.5 139.7f8.6
2 500g 1.18f0.30 1.71f0.66
25 OOOg 1.69f0.17* 2.39f0.39
Before 125.9f 9.6 125.2f 12.4
After
Before 1.54f0.27 2.05f 0.60
After 1.30f0. I5 1.53f0.25
101.4f 6.3) *** 98.6f 12.9
The averages fS.E.M. aregiven; *P 120 X-ray changes 1-3 X-ray changes 4-6 SCAT 64 Age 45 years Duration 7 years Treatments Gold: Steroids: Chloroquine: Salicylate:
No. of cases
Total synthesis (cells+medium)
Distribution mediumlcells
18
132.5f 7.4 126.9f 8.8 152.3f 8.6 124.5f 8.0 142.5f 13.2 132.6f 10.7 132.4f 11 .O 133.8f 8.9 129.8f 14.8 129.5f 10.8 134.9f 10.7 122.df 8.4 145.lf 12. I 136.3f 11.0 128.7f10.5
127.8f 8.6 124.2f10.5 140.3f 12.7 131.2f 10.8 123.5f 14.7 126.8f11.8 128.8f 13.3 127.9f 12.1 128.3f 10.8 119.9f 15.8 134.lf 9.4 127.9f 14.6 127.6f 8.0 134.7f 15.3 119.0f 9.3
14 4 10
8 9 9 12 6 8 10 10
8 9 9
Yes no Yes
no yes no Yes no
8 10
3 I5 9 9 12 6
125.1f10.6 138.4f 10.5 120.3f26.9 134.9f 7.6 145.9f 7.9 * 119.1+11.3) 138.0f 8.6 121.5f14.3
139.4f 17.2
118.5f 6.9 133.0f29.3 326.7f 9.1 124.9f 11.5 130.7f 6.4 324.4f 6.7 134.5k23.4
The data are expressed as percentages fS.E.M. of the respective controls (100.0); * P
ISSN: 0300-9742 (Print) 1502-7732 (Online) Journal homepage: http://www.tandfonline.com/loi/irhe20
Connective Tissue Activation by Synovial Fluids and Synovial-Tissue Extracts of Rheumatoid Arthritis Patients I. Takala, P. Mäkisara & E. Kulonen To cite this article: I. Takala, P. Mäkisara & E. Kulonen (1977) Connective Tissue Activation by Synovial Fluids and Synovial-Tissue Extracts of Rheumatoid Arthritis Patients, Scandinavian Journal of Rheumatology, 6:3, 172-176, DOI: 10.3109/03009747709095444 To link to this article: http://dx.doi.org/10.3109/03009747709095444
Published online: 12 Jul 2009.
Submit your article to this journal
Article views: 1
View related 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 Santa Barbara]
Date: 22 March 2016, At: 18:15
Scand J Rheumatology 6: 172-176, 1977
CONNECTIVE TISSUE ACTIVATION BY SYNOVIAL FLUIDS AND SYNOVIAL-TISSUE EXTRACTS O F RHEUMATOID ARTHRITIS PATIENTS I. Takala, P. Makisara and E. Kulonen
Downloaded by [University of California Santa Barbara] at 18:15 22 March 2016
From the Department of Medical Chemistry, University of Turku, Turku, Finland, and Rheumatism Foundation Hospital, Heinola, Finland
ABSTRACT. Prompted by Castor's investigations (4) on a connective-tissue-activatingpeptide (CTAP) we investigated the stimulative effect of synodal fluids and synodal-tissue extracts on the synthesis of collagen by incubated embryonicehick tendon cells. The stimulative effect was greater with synovial-tissueextracts from more severe cases than with samples from patients having milder forms of the disease. There was no correlation between the stimulation by synovial fluids and synovial-tissueextracts from the same patient. The stimulative activity was lost at dialysis. A slight stimulation in the incorporation of glucosamine was also observed. Treatment of the patients with gold, chloroquine or steroids decreased the stimulating capacity. These effects seem to depend on factors different from those described by Castor. The differences in the stimulant activity of samples from various groups cannot be entirely due to glutamine, which is one of the limiting nutrients of the embryonic-chick tendon cells.
This work was prompted by the series of papers on the CTAP (connective-tissue-activating peptide) separated from spleen and lymphocytes by Castor and co-workers (4) and by our own work on the connective-tissue stimulation which is observed in silicosis (2) and certain fatty liver disorders (15). The rheumatoid synovial tissue is a form of granulation tissue with a high content of hyaluronate. One of our original purposes was to relate the stimulative activity of the synovial fluid to the clinical picture. In most cases, parallel samples from the solid synovial tissue, from which extracts could be prepared, were also available for study. MATERIAL AND METHODS Patients The 18 synovial-tissue samples and the corresponding synovial fluids were collected during therapeutic Scand J Rheumatology 6
synovectomies of the knee joints carried out at the Rheumatism Foundation Hospital, Heinola, Finland. Ten additional synovial-fluid samples were taken by needle puncture without synovectomy. All the patients had classic rheumatoid arthritis according to the A.R.A. criteria (14). The radiologic grades of the joints were determined according to Larsen (13), and the joint indices according to Lansbury (12). Preparation of synovial tissue samples One g wet weight of synovial tissue was homogenized with an Ultra-Turrax (Janke & Kunkel) homogenizer in 10 ml of 0.15 M NaCl in an ice bath. The homogenate was centrifuged first at 2 500 g for 30 min and the supernatant was used as the synovial-tissue extract in the preliminary experiments. This extract was then recentrifuged at 25 OOO g for 30 min, and the supernatant was used in the final experiments. The synovial fluid was centrifuged at 1 OOOg for 15 min to remove the cells. The dialyses were performed against a 10000-fold volume of 0.15 M NaCI. Preparation and incubation of embryonicchick tendon cells Tendon cells from 17-day-old chick embryos were prepared by the method of Dehm & Prockop ( 5 ) as modified by Ronnemaa et al. (15). After isolation, the fibroblasts were suspended in the modified Krebs-Ringer medium containing 0.1 mM ascorbate, 14.5 p M L-proline and 0.15 gll bovine serum albumin (Sigma A4503). Aliquots containing about I@ cells in 3 ml of medium were used and 0.1 ml of synovial fluid or synovial-tissue extract was added to each. After a 15-min preincubation at 37°C. 5 pCi LG-[3H]proline (TRA 82, The Radiochemical Centre, Amersham) was added in 0.1 ml of 0.15 M NaCI. The incubation was continued for 120 min with air as the gas phase and terminated by adding 270 pg of cycioheximide (Sigma) and 400 pg of a,a'-dipyridyl (Fluka) in 0.2 ml of the medium. Cell viability was ascertained with the trypan blue test. Cells were separated from the medium by centrifugation (350g, 12 min). To disrupt the cells, 2 ml of water was added and the samples were sonicated for 30 sec. All samples were dialysed against cold running tap water for 24 h with L-proline (0.005 mglsample) as the carrier.
Connective tissue activation
173
Table I. Effpcts of centrifugation and dialysis on the activity of the synovial-tissue extract on protein synthesis in embryonic-chick tendon cells Fraction analysed Effect of centrifugation (n =4)
Total protein Collagen Effect of dial.vsis (n =6)
Downloaded by [University of California Santa Barbara] at 18:15 22 March 2016
Total protein Collagen
Synthesis expressed as percentage of control (100.0)
Distribution of synthesized protein in medium/cells
2 500g 128.8f5.8 137.7f5.8
25 OOOg 155.lf6.5 139.7f8.6
2 500g 1.18f0.30 1.71f0.66
25 OOOg 1.69f0.17* 2.39f0.39
Before 125.9f 9.6 125.2f 12.4
After
Before 1.54f0.27 2.05f 0.60
After 1.30f0. I5 1.53f0.25
101.4f 6.3) *** 98.6f 12.9
The averages fS.E.M. aregiven; *P 120 X-ray changes 1-3 X-ray changes 4-6 SCAT 64 Age 45 years Duration 7 years Treatments Gold: Steroids: Chloroquine: Salicylate:
No. of cases
Total synthesis (cells+medium)
Distribution mediumlcells
18
132.5f 7.4 126.9f 8.8 152.3f 8.6 124.5f 8.0 142.5f 13.2 132.6f 10.7 132.4f 11 .O 133.8f 8.9 129.8f 14.8 129.5f 10.8 134.9f 10.7 122.df 8.4 145.lf 12. I 136.3f 11.0 128.7f10.5
127.8f 8.6 124.2f10.5 140.3f 12.7 131.2f 10.8 123.5f 14.7 126.8f11.8 128.8f 13.3 127.9f 12.1 128.3f 10.8 119.9f 15.8 134.lf 9.4 127.9f 14.6 127.6f 8.0 134.7f 15.3 119.0f 9.3
14 4 10
8 9 9 12 6 8 10 10
8 9 9
Yes no Yes
no yes no Yes no
8 10
3 I5 9 9 12 6
125.1f10.6 138.4f 10.5 120.3f26.9 134.9f 7.6 145.9f 7.9 * 119.1+11.3) 138.0f 8.6 121.5f14.3
139.4f 17.2
118.5f 6.9 133.0f29.3 326.7f 9.1 124.9f 11.5 130.7f 6.4 324.4f 6.7 134.5k23.4
The data are expressed as percentages fS.E.M. of the respective controls (100.0); * P
