Pro-inflammatory IL-1beta and/or TNF-alpha up-regulate matrix metalloproteases-1 and -3 mRNA in chondrocyte subpopulations potentially pathogenic in osteoarthritis: in situ hybridization studies on a single cell level

Abstract

Aim: In osteoarthritis chondrocytes, matrix metalloproteases (MMPs) and their inhibitors are induced by interleukin (IL)-1beta or tumor necrosis factor (TNF)-alpha and balanced by inhibitors, but their messenger RNA (mRNA) expression has not been studied in individual cells.

Methods: Normal articular chondrocytes (10 donors; age 50 ± 6 years, mean ± SEM) were stimulated in a monolayer for 24 h with IL-1beta, TNF-alpha, or transforming growth factor (TGF)-beta1 (10 ng/mL each), alone or in combination. mRNA expression for MMP-1, MMP-3 and tissue inhibitor of metalloproteinase (TIMP)-1 was studied by in situ hybridization ((35) S-cRNA) and quantitative reverse transcription polymerase chain reaction (RT-PCR) (n ≥ 3 each).

Results: Whereas < 5% chondrocytes constitutively expressed MMP-1, a higher percentage expressed MMP-3 and TIMP-1 (31.1 ± 1.8%; 36.7 ± 2.8%, respectively). Upon stimulation with IL-1beta, TNF-alpha or IL-1beta/TNF-alpha, the percentage of cells positive for MMP-1, MMP-3 and TIMP-1 rose significantly (IL-1beta: 31.5%, 54.5% and 60.2%, respectively; TNF-alpha: 35.4%, 56.6%, 50.9%; IL-1beta/TNF-alpha: 38.8%, 45.2%, 52.1%). In bulk population (RT-PCR), mRNA for MMP-1 and MMP-3 was also induced by IL-1beta (11.9-fold, 1.2-fold, respectively), TNF-alpha (4.8-fold, 1.0-fold) or IL-1beta/TNF-alpha (14.7-fold, 1.4-fold), an effect attenuated by TGF-beta1. TIMP-1 mRNA, in contrast, was down-regulated by IL-1beta, TNF-alpha or IL-1beta/TNF-alpha, an effect again partially reverted by TGF-beta1. Finally, collagen type II mRNA was down-regulated by IL-1beta, TNF-alpha or IL-1beta/TNF-alpha (by 90%, 50% and 98%, respectively) and that of collagen type I was up-regulated (5.7-fold, 3.0-fold, 3.7-fold).

Conclusions: Up-regulation of MMP-1/MMP-3 by IL-1beta and/or TNF-alpha in a fraction of chondrocytes in vitro suggests that a subpopulation of catabolic cells may also exist in osteoarthritis. These cells may undergo considerable dedifferentiation, as indicated by a decreased collagen-II/collagen-I ratio.

Keywords: IL-1beta; MMP; TGF-beta1; TIMP; TNF-alpha; chondrocyte subpopulations; in situ hybridization.