Chemokine receptors in the rheumatoid synovium: upregulation of CXCR5

Abstract

In patients with rheumatoid arthritis (RA), chemokine and chemokine receptor interactions play a central role in the recruitment of leukocytes into inflamed joints. This study was undertaken to characterize the expression of chemokine receptors in the synovial tissue of RA and non-RA patients. RA synovia (n = 8) were obtained from knee joint replacement operations and control non-RA synovia (n = 9) were obtained from arthroscopic knee biopsies sampled from patients with recent meniscal or articular cartilage damage or degeneration. The mRNA expression of chemokine receptors and their ligands was determined using gene microarrays and PCR. The protein expression of these genes was demonstrated by single-label and double-label immunohistochemistry. Microarray analysis showed the mRNA for CXCR5 to be more abundant in RA than non-RA synovial tissue, and of the chemokine receptors studied CXCR5 showed the greatest upregulation. PCR experiments confirmed the differential expression of CXCR5. By immunohistochemistry we were able to detect CXCR5 in all RA and non-RA samples. In the RA samples the presence of CXCR5 was observed on B cells and T cells in the infiltrates but also on macrophages and endothelial cells. In the non-RA samples the presence of CXCR5 was limited to macrophages and endothelial cells. CXCR5 expression in synovial fluid macrophages and peripheral blood monocytes from RA patients was confirmed by PCR. The present study shows that CXCR5 is upregulated in RA synovial tissue and is expressed in a variety of cell types. This receptor may be involved in the recruitment and positioning of B cells, T cells and monocytes/macrophages in the RA synovium. More importantly, the increased level of CXCR5, a homeostatic chemokine receptor, in the RA synovium suggests that non-inflammatory receptor-ligand pairs might play an important role in the pathogenesis of RA.

Figure 1

Microarray analysis of chemokine and chemokine receptor expression in the rheumatoid arthritis (RA) and non-RA synovia. A pair of human cytokine array membranes were hybridized to ³³P-labelled cDNA probes prepared from pools of (a) RA mRNA (n = 8) and (b) non-RA mRNA (n = 9). The membranes were washed and autoradiographed. (c) The position of the 33 chemokines (C), the 16 chemokine receptors (CR), the nine positive control 'housekeeping genes' (HKG) and the six negative controls (NC). Each gene was printed in duplicate. The star indicates the position of the genes CXCR1, CXCR2, CXCR4 and CXCR5 (reading top to bottom) and shows their differential expression in RA and non-RA synovia. Exposure time was 7 days and 14 days for (a) and (b), respectively.

Figure 2

RT-PCR on rheumatoid arthritis and non-rheumatoid arthritis synovial tissue. CXCR5 RT-PCR products were separated on 0.8% agarose gels and stained with ethidium bromide. The reactions were performed on the individual RNA samples that were applied to the microarray membranes. The ribosomal RNA L27 was employed to normalize the amount of RNA used in each reaction.

Figure 3

Immunohistochemistry of CXCR5 in lymphoid cell aggregates of rheumatoid arthritis synovia. Sections of rheumatoid synovium were treated with (a) CXCR5 antibody or (b) isotype control. Serial sections were treated with (c) anti-CD20 as a marker of B lymphocytes or (d) isotype control. Arrows indicate B lymphocytes expressing CXCR5. (e) Rheumatoid synovium treated with the T-cell marker anti-CD3 followed by alkaline phosphatase and Vector red substrate (methyl green counterstain). T cells stain a light red colour. (f) Serial section from the same synovial sample as (e) treated with anti-CD3, alkaline phosphatase and Vector red followed by anti-CXCR5, peroxidase and 3,3'-diaminobenzidine (DAB)-Nickel substrate (no counterstain used). T cells that express CXCR5 are stained dark red whereas cells expressing CXCR5 alone are grey–black in colour. (g) Control for (e), in which isotype-matched rabbit immunoglobulin (Ig) was used instead of anti-CD3. (h) Control for (f), in which isotype-matched rabbit and mouse Ig were applied instead of CD3 and CXCR5 antibodies (no counterstain used). Unless stated otherwise, DAB substrate was used. (a), (c) and (e)–(h) Original magnification, 420 ×; isotype controls (b) and (d) original magnification, 280 ×.

Figure 4

Immunohistochemistry of CXCR5 in the intima and postcapillary venules in rheumatoid arthritis synovia. (a) CD68⁺ cells in the intima. (b) Serial section to (a) stained for CXCR5. Note the colocalization of CXCR5 and CD68 to the same group of cells. (c) and (d) Sections from the same region as (a) and (b), treated with isotype-matched control immunoglobulin instead of CD68 and CXCR5 antibodies, respectively. (e) Postcapillary venule positive for CXCR5 within a lymphoid aggregate. Labelling was revealed using 3,3'-diaminobenzidine substrate. (f) Isotype control for (e). (a), (b), (e) and (f) Original magnification, 420 ×; (c) and (d) original magnification, 350 ×.

Figure 5

Immunohistochemistry of CXCR5 in non-rheumatoid arthritis synovia. (a) CD68 staining in the intimal layer. (b) Serial section to (a) treated with anti-CXCR5, showing that CXCR5⁺ cells in the intimal layer included those also positive for CD68. (c) and (d) Sections from the same region as (a) and (b), treated with isotype-matched control immunoglobulin instead of CD68 and CXCR5 antibodies, respectively. (e) Subintimal postcapillary venule stains for CXCR5 expression (arrow). (f) Isotype-matched control for (e). Labelling was revealed using 3,3'-diaminobenzidine substrate. (a), (b), (e) and (f) Original magnification, 420 ×; (c) and (d) original magnification, 350 ×.

Figure 6

RT-PCR on monocytes/macrophages from peripheral blood (PB) and synovial fluid (SF). CXCR5 RT-PCR products were separated on 0.8% agarose gels and stained with ethidium bromide. The reactions were performed on four additional rheumatoid arthritis patients. The ribosomal RNA L27 was employed to normalize the amount of RNA used in each reaction.