Mediators of inflammation are down-regulated while apoptosis is up-regulated in rheumatoid arthritis synovial tissue by polymerized collagen

Abstract

The aim of the study was to determine whether collagen-polyvinylpyrrolidone (collagen-PVP) modifies some proinflammatory responses in synovium cultures from rheumatoid arthritis (RA) patients. Synovium from 10 RA patients were cultured with or without 1% collagen-PVP. Tissues on the 3rd, 5th and 7th culture day were sectioned and stained by the Herovici technique. Total collagen and type I/III collagen ratios were evaluated by the Woessner micromethod and by interrupted gel electrophoresis, respectively. Collagenolytic activity was assessed by degradation of [3H]-collagen in supernatants. TIMP-1, IL-1beta and TNF-alpha were determined in supernatants by ELISA, and the results were normalized by DNA concentration. IL-1beta, TNF-alpha, IL-6, IL-8, MMP-1, TIMP-1, Cox-1, VCAM-1, ICAM-1 and Fas/APO95 expression was evaluated by immunohistochemistry. Apoptosis was detected by TUNEL technique. The histological analysis and electrophoresis revealed a 1.7-fold increase of type III collagen in a time-dependent fashion in collagen-PVP-treated cultures. Proinflammatory cytokines (IL-1beta: 58 +/- 9 versus 22 +/- 10; TNF-alpha: 41 +/- 6 versus 11 +/- 3; IL-8: 59 +/- 12 versus 29 +/- 9; treated versus untreated), adhesion molecule (ICAM-1: 57 +/- 11 versus 29 +/- 15; VCAM-1: 49 +/- 7 versus 21 +/- 13; treated versus untreated) as well as Cox-1 (59 +/- 10 versus 20 +/- 3) expression was down-regulated in RA synovium treated. Meanwhile, TIMP-1 (36 +/- 7 versus 57 +/- 11) and Fas expression (20 +/- 10 versus 55 +/- 13) and apoptosis (14 +/- 3 versus 55 +/- 5) were up-regulated in treated cultures compared with controls. In supernatants, the collagenolytic activity, as well as IL-1beta and TNF-alpha, levels were all down-regulated in treated cultures (two, three, fourfold, respectively). The addition of collagen-PVP to synovium-induced down-modulation of some inflammatory parameters and an increase in apoptosis of synovial cells. Perhaps this mechanism could contribute to inhibit outgrowth of pannus formation and to down-regulate inflammation of joints in patients with RA.

Fig. 1

Collagen-PVP effect on tissue architecture of synovium in culture. Photomicrographs of synovial tissue cultures with or without 1% collagen-PVP, stained with the Herovici technique. (a) Non-RA synovial control cultures on the 0, 3rd and 7th culture days. (b) Non-RA synovium collagen-PVP-treated cultures during days 3 and 7. (c) RA synovial tissue incubated without treatment during days 0, 3 and 7, respectively. The predominant extracellular matrix component is type I collagen (fibres in red). (d) RA synovium treated with collagen-PVP during days 3 and 7, respectively. There is an increase of type III collagen (fibres in blue). Scale bar: 100 µm.

Fig. 2

Collagen-PVP effect on types I and III collagen content in synovial tissue cultures. (a) SDS-PAGE of types I and III collagen of representative RA synovial tissue cultures with (+) or without (−) collagen-PVP on the 3rd, 5th and 7th culture days, respectively. MW: Type I collagen standard. (b) Type III collagen relative percentage in non-RA synovial tissue cultures. (c) Type III collagen relative percentage in RA synovial tissue cultures. Statistical differences between control and collagen-PVP-treated groups were obtained on the 5th (*P = 0·009) and the 7th culture days (*P = 0·00007). Data are the mean ± s.e.m. of synovial tissue cultures from five non-RA and 10 RA patients, each performed in duplicate. , Type III collagen in control group; ▪, Type III collagen in collagen-PVP group.

Fig. 3

Collagen-PVP effect on collagenase activity and TIMP-1 expression in synovial tissue cultures. (a) Total collagenolytic activity. Data were statistically significant with *P = 0·008, *P = 0·055 and *P = 0·05 for the 3rd, 5th and 7th culture days, respectively. (b) Calcium-dependent collagenolytic activity. (c) Calcium-independent collagenolytic activity. Collagen-PVP-treated groups were compared with untreated cultures and the differences were statistically significant with *P = 0·008, *P = 0·006 and *P = 0·002 for the 3rd, 5th and 7th culture days, respectively. Collagenase activity was expressed as cpm/24 h/µg of DNA at 35°C. Data are mean ± s.e.m. of synovial tissue cultures from 10 RA patients, each performed in duplicate. (d) MMP-1 immunoreactive cells on non-RA synovial tissue. (e) MMP-1 immunoreactive cells on RA synovial tissue. (f) TIMP-1 immunoreactive cells on non-RA synovial tissue. (g) TIMP-1 immunoreactive cells on RA synovial tissue. Data are mean ± s.e.m. of synovial tissue cultures from five non-RA and 10 RA patients, each performed in duplicate, where of each tissue at least two sections were evaluated. (h) TIMP-1 concentration in synovial tissue supernatant with or without collagen-PVP treatment (*P = 0·04 on the 7th culture day). Data are the mean ± s.e.m. of synovial tissue cultures from 10 RA patients, each performed in duplicate. □, Initial tissue; , control; ▪, collagen-PVP.

Fig. 4

Effect of collagen-PVP on proinflammatory cytokine expression in synovial tissue cultures. (a) IL-8 immunoreactive cells on blood vessels and stromal cells from non-RA patients. (b) IL-8 immunoreactive cells in synovial tissue from RA patients (*P < 0·05). (c) TNF-α immunoreactive cells on blood vessels and stromal cells from non-RA patients. (d) TNF-α immunoreactive cells on blood vessels and stromal cells from RA patients (*P < 0·05). Data are mean ± s.e.m. of synovial tissue cultures from five non-RA and 10 RA patients, each performed in duplicate, where of each tissue at least two sections were evaluated. (e) TNF-α production (*P = 0·03) on the 7th day. Data are the mean ± s.e.m. of synovial tissue cultures from 10 RA patients, each performed in duplicate. (f) IL-1β immunoreactive cells on blood vessels and stromal cells from RA patients (*P < 0·05). Data are mean ± s.e.m. of synovial tissue cultures from five non-RA and 10 RA patients, each performed in duplicate, where of each tissue at least two sections were evaluated. (g) IL-1β concentration in supernatants from RA synovium cultures (*P = 0·05). Data are the mean ± s.e.m. of synovial tissue cultures from 10 RA patients, each performed in duplicate. □, Initial tissue; , control; ▪, collagen-PVP.