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. 1999 Mar;58(3):175–181. doi: 10.1136/ard.58.3.175

Complement C1s activation in degenerating articular cartilage of rheumatoid arthritis patients: immunohistochemical studies with an active form specific antibody

K Nakagawa 1, H Sakiyama 1, T Tsuchida 1, K Yamaguchi 1, T Toyoguchi 1, R Masuda 1, H Moriya 1
PMCID: PMC1752845  PMID: 10364916

Abstract

OBJECTIVE—The first complement component C1s was reported to have novel functions to degrade matrix components, besides its activities in the classic complement pathway. This study explores participation of C1s in articular cartilage degradation in rheumatoid arthritis (RA).
METHODS—Normal articular cartilage (n=6) and cartilage obtained from joints with RA (n=15) and osteoarthritis (OA, n=10) were immunostained using anti-C1s monoclonal antibodies PG11, which recognises both active and inactive C1s, and M241, which is specifically reactive to activated C1s. The effects of inflammatory cytokines on C1s production by human articular chondrocytes were also examined by sandwich ELISA.
RESULTS—In normal articular cartilage, C1s was negative in staining with both PG11 and M241. In contrast, degenerating cartilage of RA was stained with PG11 (14 of 15 cases), and in most of the cases (13 of 15 cases) C1s was activated as revealed by M241 staining. In OA, C1s staining was restricted in severely degrading part of cartilage (5 of 10 cases), and even in that part C1s was not activated. In addition, C1s production by chondrocytes in vitro was increased by an inflammatory cytokine, tumour necrosis factor α.
CONCLUSION—These results suggest that C1s activated in degenerative cartilage matrix of RA but not in that of OA. C1s is thought to participate in the pathogenesis of RA through its collagenolytic activity in addition to the role in the classic cascade.

 Keywords: complement C1s; articular cartilage; rheumatoid arthritis

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Figure 1  .

Figure 1  

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Immunostaining of C1s in normal and OA articular cartilage. Articular cartilage obtained from knee joints of a normal person (A, B: 18 year old man) and a patient with OA (C, D, E, F: 75 year old woman) was fixed with 4% paraformaldehyde as described in the text. Frozen sections were immunostained with PG11 (A, C), M241 (D), and NG7 (B, E, for control staining). Parallel serial sections were stained with toluidine blue (F). In normal articular cartilage, neither chondrocytes nor matrix were stained with PG11 (A). In the degrading part of OA cartilage, superficial cartilage matrix (arrowheads) and chondrocytes (large arrows) but not chondrocytes in the deep zone (small arrow) was reactive to PG11 (C). However, active form C1s was not detected in the cartilage by immunostaining with M241 (D). Bar: 100 µm.

Figure 2  .

Figure 2  

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Differential reactivities of PG11 and M241 in RA articular cartilage. Articular cartilage of RA patients (A, C, E: 36 year old woman, B, D, F, G: 51 year old woman) was fixed and frozen sectioned as described in the text. The samples were immunostained with PG11 (A, B), M241 (C, D), and NG7 (E, F, for control staining) and stained with toluidine blue (G). Degenerative cartilage matrix was equally reactive to both PG11 (A, B arrowheads) and M241 (C, D arrowheads). Whereas, chondrocytes were stained with PG11 (A, B arrows) but not with M241 (C, D). Bar: 100 µm.

Figure 3  .

Figure 3  

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Immunolocalisation and activation of C1s at the site of pannus formation. Articular cartilage with pannus was fixed, and serial frozen sections were prepared as described in the text. They were stained with haematoxlin and eosin (C), toluidine blue (F) and immunostained with PG11 (A), M241 (D), and NG7 (B). Tartarate resistant acid phosphatase (TRAP) staining was also performed (E). Invasion of synovial cells and TRAP positive multinuclear cells (E arrows) was observed in the cartilage matrix. Invading synovia and degrading cartilage matrix were intensely stained with PG11 (A). They also stained with M241 (D), indicating that C1s was activated there. Bar: 50 µm.

Figure 4  .

Figure 4  

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Effects of IL1 and TNFα on C1s production by human articular chondrocytes. (A) Serum free culture medium of human articular chondrocytes was analysed on SDS-PAGE (10% gel) under non-reducing condition and immunoblotted with M81. Lane 1; culture medium, lane 2; moleculer marker, arrow; C1s. (B) Chondrocytes were seeded at a density of 3 × 104 cells/well in 96 well plates and grown in DMEM+F12 supplemented with 20% FCS. After they reached confluency (four days), the culture medium was switched to serum free medium, DMEM+F-12 supplemented with human transferrin (10 µg/ml), bovine insulin (10 µg/ml), and hydrocortisone (10-8 M), containing various concentration of IL1α or TNFα, (control: no addition). After a 48 hour incubation period, C1s in the medium was quantified by sandwich ELISA and the amounts were normalised to cell number. Values are the average (SD) of four determinations (bars). * p<0.05; ** p<0.01 v control.

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