High CXCR3 expression in synovial mast cells associated with CXCL9 and CXCL10 expression in inflammatory synovial tissues of patients with rheumatoid arthritis

Abstract

To improve our knowledge on the pathophysiology of rheumatoid arthritis (RA), we investigated gene expression patterns in synovial tissue from RA and osteoarthritis (OA) patients. DNA oligonucleotide microarray analysis was employed to identify differentially expressed genes in synovial tissue from pathologically classified tissue samples from RA (n = 20) and OA patients (n = 10). From 7131 gene sets displayed on the microarray chip, 101 genes were found to be upregulated and 300 genes to be downregulated in RA as compared with OA. Semiquantitative reverse-transcription polymerase chain reaction, Western blotting and immunohistochemistry were used to validate microarray expression levels. These experiments revealed that Cys-X-Cys receptor (CXCR)1, CXCR2 and CXCR3 mRNAs, as well as Cys-X-Cys ligand (CXCL)9 (monokine induced by IFN-gamma) and CXCL10 (IFN-gamma inducible protein 10) mRNAs, were significantly upregulated in RA as compared with OA disease. Elevated protein levels in RA synovial tissue were detected for CXCR1 and CXCR3 by Western blotting. Using immunohistochemistry, CXCR3 protein was found to be preferentially expressed on mast cells within synovial tissue from RA patients. These findings suggest that substantial expression of CXCR3 protein on mast cells within synovial tissue from RA patients plays a significant role in the pathophysiology of RA, accompanied by elevated levels of the chemokines CXCL9 and CXCL10. Mature mast cells are likely to contribute to and sustain the inflamed state in arthritic lesions (e.g. by production of inflammatory mediators such as histamine, proteinases, arachidonic acid metabolites and cytokines). Thus, the mast cell could become a potential target in therapeutic intervention.

Figure 1

Analysis of IL-6 mRNA levels within synovial tissue from rheumatoid arthritis (RA) as compared with that from osteoarthritis (OA) patients. Upper panels: quality control of total RNA preparations. Aliquots (300 ng) of total RNA extracted from synovial tissue from RA and OA patients were plotted on a RNA 6000 Nano-LabChip. Quality of RNA was scanned using a 2100 bioanalyzer. RNA gel electropherograms show the presence of 28S and 18S ribosomal units, indicating intact RNA of the investigated samples. Lower panels: differential IL-6 mRNA levels were determined by semiquantitative reverse transcription polymerase chain reaction (PCR). The figure shows a representative analysis of eight cDNA samples derived from patients with RA and of eight cDNA samples from patients with OA. cDNA samples were adjusted to equal glyceraldehyde-3-phosphate dehydrogenase (G3PDH) levels, performed by competitive PCR using an internal standard (see Materials and methods). Numbered lanes correspond to individual patients within Table 1.

Figure 2

Analysis of mRNA levels of selected genes in synovial tissue from rheumatoid arthritis (RA) as compared to that from osteoarthritis (OA) patients by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Bars represent means ± SD of signal intensities after amplification of samples (see Materials and methods). The data from one representative experiment with one determination per patient sample are shown. Differences between RA and OA sample groups were statistically evaluated using the Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001). (a) RT-PCR analysis of 10 cDNA samples derived from patients with RA and of 10 cDNA samples from patients with OA. cDNA samples were adjusted to equal glyceraldehyde-3-phosphate dehydrogenase (G3PDH) levels, performed by competitive PCR using an internal standard (see Materials and methods). Numbered lanes correspond to individual patients within Table 1. (b) Quantitation of the expression of Cys–X–Cys receptor (CXCR)1, CXCR2, CXCR3, T-cell receptor (TCR)-ζ, Cys–X–Cys ligand (CXCL)9, and CXCL10 mRNAs in RA and OA synovial tissues. (c) CXCR/TCR-ζ mRNA ratios in RA versus OA synovial tissues.

Figure 3

Western blot analysis of Cys–X–Cys receptor (CXCR)1, CXCR2, and CXCR3 protein expression in selected rheumatoid arthritis (RA) and osteoarthritis (OA) synovial tissues. (a) Tissue extracts from RA (n = 8) and from OA patients (n = 4) were analyzed. Numbered lanes correspond to individual patients within Table 1. Staining of the indicated proteins on parallel blots is shown. Equal loading of tissue extracts was controlled by β-actin protein staining. MW indicates a protein from ECL molecular weight markers. (b) Western blot signals on Hyperfilm™ ECL™ after the chemiluminescence reactions were analyzed semiquantitatively using densitometric scanning. Expression is given in arbitrary units and the means ± SD of the RA and OA groups are plotted. Differences between RA and OA groups were assessed statistically using the Student's t-test (*P < 0.05, **P < 0.01).

Figure 4

Cellular distribution of Cys–X–Cys receptor (CXCR)3 protein in synovial tissue from rheumatoid arthritis (RA) patients. Localization of strong CXCR3 protein signals in mast cells within the sublining areas of rheumatoid synovial tissues was found. Sequential sections of paraffin-embedded tissue were stained for CXCR3 and mast cell tryptase proteins or using an IgG₁ isotype-matched control. Each arrow refers the same cell that was positively stained for CXCR3 and mast cell tryptase (original magnification: upper panel × 200; lower panel × 400).