Chemokine expression by subchondral bone marrow stromal cells isolated from osteoarthritis (OA) and rheumatoid arthritis (RA) patients

Abstract

We analysed the spontaneous and cytokine-stimulated production and expression in vitro of IL-8, GROalpha, MCP-1, RANTES, MIP-1alpha, MIP-1beta, by subchondral bone marrow stromal cells (BMSC) isolated from RA, OA, post-traumatic (PT) patients and normal donors (ND). BMSC were cultured in vitro in the presence or absence of IL-1beta and tumour necrosis factor-alpha (TNF-alpha), and assessed for chemokine production, expression and immunolocalization. BMSC from different sources constitutively released MCP-1, GROalpha and IL-8, but not MIP-1alpha or MIP-1beta, while BMSC from ND constitutively released only IL-8 and MCP-1. IL-8, GROalpha and RANTES production in basal conditions was significantly higher in RA patients than in ND. RANTES production was also higher in OA and RA than in PT patients. The combination of TNF-alpha and IL-1beta synergistically increased the production of all chemokines tested except for RANTES. Reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated that all chemokines not detectable in the supernatants were expressed at the mRNA level. Chemokine immunostaining was localized around the nuclei. This work demonstrates that BMSC from subchondral bone produce chemokines and indicates that these cells could actively participate in the mechanisms directly or indirectly causing cartilage destruction and bone remodelling.

Fig. 1

Constitutive production in vitro of IL-8, GROα, MCP-1, RANTES from bone marrow stromal cells (BMSC) isolated from OA, RA post-traumatic (PT) patients and normal donors (ND), evaluated after 72 h of culture as described in Patients and Methods. Data are expressed as means ± s.e.m. of all patients analysed for each group. IL-8, GROα and RANTES production was higher in RA patients than in ND (P < 0.05 for each chemokine). RANTES production was also higher in RA patients than in PT (P < 0.005) and in OA patients than in PT (P < 0.05). *RA versus ND; **RA versus PT; ***OA versus PT.

Fig. 2

Chemokine mRNA expression in bone marrow stromal cells (BMSC) derived from a normal donor (lanes 1–4) and a RA patient (lanes 5–8), in basal and stimulated conditions, analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) as described in Patients and Methods. M, Molecular weight marker (123-bp ladder); N, negative control (no template); lanes 1 and 5, unstimulated BMSC; lanes 2 and 6, BMSC stimulated with IL-1β (10 ng/ml); lanes 3 and 7, BMSC stimulated with TNF-α (500 U/ml); lanes 4 and 8, BMSC stimulated with IL-1β+ TNF-α in combination.

Fig. 3

Immunostaining of MIP-1α in bone marrow stromal cells (BMSC) isolated from subchondral bone of unstimulated normal donor (ND) (a), IL-1β + TNF-α–activated ND (b), OA patient (c), RA patient (d), evaluated after 72 h of culture.