Selective recruitment of CCR6-expressing cells by increased production of MIP-3 alpha in rheumatoid arthritis

Abstract

Infiltration of various types of leucocytes has been shown to play a crucial role in the pathogenesis of rheumatoid arthritis (RA). Macrophage inflammatory protein-3 alpha (MIP-3 alpha) is a recently identified chemokine which is a selective chemoattractant for leucocytes such as memory T cells, naïve B cells and immature dendritic cells. In this study, we investigated the expression of MIP-3 alpha and its specific receptor CCR6 in the inflamed joints of patients with RA. Increased amounts of MIP-3 alpha were found by ELISA in synovial fluids (SF) of patients with RA. MIP-3 alpha was apparently detected in all synovial tissue specimens of RA patients (n = 6), but it could not be detected in that of osteoarthritis (OA) patients (n = 4). Expression of MIP-3 alpha was detected especially in the sublining layer, and infiltrating mononuclear cells in RA synovial tissue. Gene expression of MIP-3 alpha was also found in six out of 11 RA-synovial fluid cells by RT-PCR. Cultured synovial fibroblasts derived from either RA or OA patients were capable of producing MIP-3 alpha in response to IL-1 beta and TNFalpha in vitro. Furthermore, expression of CCR6 was found in infiltrating mononuclear cells in the cellular clusters and around the vessels of RA synovial tissue. These findings indicate that increased production of MIP-3 alpha may contribute to the selective recruitment of CCR6-expressing cells in RA.

Fig. 1

MIP-3α in synovial fluids. Concentration of MIP-3α in SF of RA patients (n = 20) and OA patients (n = 18) was determined by ELISA. Bars show the mean ±s.d. The mean concentration of MIP-3α in RA-SF is significantly higher than that of OA-SF (P = 0·0027).

Fig. 2

Expression of MIP-3α and CCR6 in RA. Expression of MIP-3α and CCR6 was determined by immunostaining and in situ hybridization of frozen synovial tissue from RA and OA patients, as described in Materials and methods. (a) Immunostaining of MIP-3α in RA synovial tissue (original magnification, ×100). (b) Immunostaining of MIP-3α in OA synovial tissue (original magnification, ×100). (c) Higher magnification of Fig. 2a (original magnification, ×200). (d) In situ hybridization of MIP-3α mRNA in RA synovial tissue (original magnification, ×200). (e) Immunostaining of CCR6-expressing cells in RA synovial tissue (original magnification, ×200). (f) In situ hybridization of CCR6 mRNA in RA synovial tissue (original magnification: ×200). Results were reproducible in the synovial tissue specimens obtained from six RA patients and four OA patients.

Fig. 3

Expression of MIP-3α by infiltrating cells from RA-SF. Gene expression of MIP-3α and the internal control (β2-microglobulin) was determined in SF cells from 11 RA patients by RT-PCR and Southern hybridization.

Fig. 4

Induction of MIP-3. gene expression in synovial fibroblasts. (a) RA synovial fibroblasts were incubated in the presence or absence of cytokines such as IL-1β (1 ng/ml), TNF-α (1 ng/ml) and TGF-β (1 ng/ml) for 1 h. (b) RA synovial fibroblasts were incubated for 1 h with IL-1β (1 ng/ml), or IL-1β (1 ng/ml) and TNF-α (1 ng/ml), or IL-1β (1 ng/ml) and TGFβ (1 ng/ml). (c) Synovial fibroblasts derived from RA patient (RA SyF) and OA patient (OA SyF) were incubated with IL-1β (1 ng/ml) for the indicated periods. Gene expression of MIP-3α and β₂-m was determined by RT-PCR and Southern hybridization.

Fig. 5

MIP-3α production of cultured synovial fibroblasts by IL-1β. Synovial fibroblasts were stimulated with various concentrations of IL-1β for 24 h and MIP-3α in the culture supernatant fluid was determined by specific ELISA. Data represent the mean ±s.d. of triplicate determinations.