Expression of chemokines CXCL4 and CXCL7 by synovial macrophages defines an early stage of rheumatoid arthritis

Abstract

Background and objectives: For our understanding of the pathogenesis of rheumatoid arthritis (RA), it is important to elucidate the mechanisms underlying early stages of synovitis. Here, synovial cytokine production was investigated in patients with very early arthritis.

Methods: Synovial biopsies were obtained from patients with at least one clinically swollen joint within 12 weeks of symptom onset. At an 18-month follow-up visit, patients who went on to develop RA, or whose arthritis spontaneously resolved, were identified. Biopsies were also obtained from patients with RA with longer symptom duration (>12 weeks) and individuals with no clinically apparent inflammation. Synovial mRNA expression of 117 cytokines was quantified using PCR techniques and analysed using standard and novel methods of data analysis. Synovial tissue sections were stained for CXCL4, CXCL7, CD41, CD68 and von Willebrand factor.

Results: A machine learning approach identified expression of mRNA for CXCL4 and CXCL7 as potentially important in the classification of early RA versus resolving arthritis. mRNA levels for these chemokines were significantly elevated in patients with early RA compared with uninflamed controls. Significantly increased CXCL4 and CXCL7 protein expression was observed in patients with early RA compared with those with resolving arthritis or longer established disease. CXCL4 and CXCL7 co-localised with blood vessels, platelets and CD68(+) macrophages. Extravascular CXCL7 expression was significantly higher in patients with very early RA compared with longer duration RA or resolving arthritis

Conclusions: Taken together, these observations suggest a transient increase in synovial CXCL4 and CXCL7 levels in early RA.

Keywords: Cytokines; Early Rheumatoid Arthritis; Inflammation; Rheumatoid Arthritis; Synovitis.

Figure 1

Cytokine and chemokine mRNA expression in synovial biopsies from uninflamed controls and patients with resolving arthritis, early rheumatoid arthritis (RA) and established RA. (A) Synovial tissue sections were assessed from uninflamed controls (n=10) and patients with resolving arthritis (n=9), early RA (n=17) and established RA (n=12). Data for genes for which the Kruskal–Wallis and Dunn's post-test showed significant difference between the four groups are shown. * p<0.05, ** p<0.01, ***p<0.001. (B) Cytokine and chemokine genes were ranked by difference in mRNA expression between resolving arthritis and early RA groups. Means of each group are represented in the heat map. Green represents low and red high relative expression (z-score of mean expression levels).

Figure 2

Generalised matrix relevance learning vector quantisation-based discrimination of subject groups (A) All 117 cytokine/chemokine genes used to classify uninflamed controls versus patients with established rheumatoid arthritis (RA). The 10 genes most informative in discriminating groups are indicated. (B) Receiver-operating characteristic (ROC) characteristics of the obtained classifiers for uninflamed controls and patients with established RA. (C) Classification cytokine mRNA signals in resolving arthritis versus patients with early RA. The 10 genes most informative in discriminating groups are indicated. (D) ROC characteristics of the obtained classifiers for patients with resolving arthritis and early RA. AUC, area under the curve.

Figure 3

Immunofluorescence staining of CXLC4 and CXCL7 in synovial tissue sections. (A) Synovial tissue staining of CXCL4 (red), CD68 (blue), CD41 (green) and von Willebrand factor (vWF) (orange). (B) Synovial tissue staining of CXCL7 (red), CD68 (blue), CD41 (green) and vWF (orange). Nuclear counterstain is shown. Images are representative of early rheumatoid arthritis (RA) synovium (n=10). No staining was observed using isotype and concentration-matched negative controls. Images were taken at ×40 magnification. (C) Quantification of CXCL4 and CXCL7 staining, calculated as the number of pixels per μm² over 6× 2×2 tile scans at ×40 magnification, in synovial tissue sections from patients with resolving arthritis (n=9), early RA (n=10) and established RA (n=11). Patients with early RA showed a significantly higher level of CXCL4 (p<0.05) and CXCL7 (p<0.05) compared with patients with resolving arthritis and established RA. Kruskal–Wallis and Dunn's post-test; * p<0.05, ** p<0.01.

Figure 4

Co-localisation of CXCL4 and CXCL7 with CD68-positive cells. (A) Synovial tissue staining of CXCL4 (red) and CD68 (blue) and (B) staining of CXCL7 (red) and CD68 (blue) showed co-localisation of both cytokines with macrophages. Image representative of rheumatoid arthritis synovium (n=10).

Figure 5

Expression of CXCL4 and CXCL7 in the synovium is predominantly found outside the vasculature. Staining of CXCL4 and CXCL7 in synovial tissue sections was quantified inside and outside of the vasculature, as assessed by co-staining with von Willebrand factor. Expression of both chemokines was significantly elevated outside the vasculature. RA, rheumatoid arthritis. Kruskal–Wallis test, Dunn's post-test; *p<0.05, ***p<0.001.