Peficitinib Inhibits the Chemotactic Activity of Monocytes via Proinflammatory Cytokine Production in Rheumatoid Arthritis Fibroblast-Like Synoviocytes

Abstract

Background: This study was performed to examine the effects of the Janus kinase (JAK) inhibitor peficitinib on fibroblast-like synoviocytes (FLS) obtained from patients with rheumatoid arthritis (RA). Methods: To examine the expression of JAK1, JAK2, and JAK3 in RA synovial tissue (ST) and FLS, immunohistochemistry was performed. We investigated the effects of peficitinib on interleukin 6 and IL-6 receptor responses in RA FLS. Phosphorylation of STAT was determined by western blot. To examine the functional analysis of peficitinib, we performed a proliferation and chemotaxis assays with FLS using THP-1 and peripheral blood mononuclear cells (PBMC). The inflammatory mediator expression of FLS was estimated by enzyme-linked immunosorbent assay. Results: JAK1, JAK2, and JAK3 were expressed in RA STs and FLS. Phosphorylation of STAT1, STAT3, and STAT5 in RA FLS was suppressed by peficitinib in a concentration-dependent manner. Peficitinib-treated RA FLS-conditioned medium reduced THP-1 and PBMC migration (p < 0.05) and proliferation of RA FLS (p < 0.05). Peficitinib suppressed the secretion of MCP-1/CCL2 in the RA FLS supernatant (p < 0.05). Conclusion: Peficitinib suppressed the JAK-STAT pathway in RA FLS and also suppressed monocyte chemotaxis and proliferation of FLS through inhibition of inflammatory cytokines.

Keywords: fibroblast-like synoviocytes; monocyte chemotaxis; peficitinib; rheumatoid arthritis.

Figure 1

JAK1, JAK2, and JAK3 were expressed in rheumatoid arthritis (RA) synovial tissue (ST) and fibroblast-like synoviocytes (FLS). Frozen sections of RA ST and RA FLS isolated from ST were stained for JAK1, JAK2, or JAK3. (A) JAK1, JAK2, and JAK3 were expressed in RA ST. JAK1 and JAK3 were observed in the RA ST lining layers. JAK2 was expressed entirely in the RA ST cell nucleus. (B) JAK1, JAK2, and JAK3 were expressed in RA FLS (original magnification 200×).

Figure 2

IL-6 and IL-6R activate JAK-STAT pathway in RA FLS. The RA FLS were stimulated with IL-6 (100 ng/mL) and IL-6R (100 ng/mL) for 10 or 30 min. (A) Representative western blot showing phospho STAT1 (pSTAT), (B) phospho STAT3 (pSTAT3), and (C) phospho STAT5 (pSTAT5). (D) Expression of pSTAT1 band intensities was quantified and the data are expressed as the mean and SEM. pSTAT1, (E) pSTAT3, and (F) pSTAT5 were increased 10 min after stimulation with IL-6 and IL-6R. The data are expressed as the mean ± SEM (n = 3 patients). * p < 0.05 when unstimulated (0 min).

Figure 3

Effects of peficitinib on IL-6 and IL-6R responses in RA FLS. The RA FLS were stimulated with IL-6 (100 ng/mL) and IL-6R (100 ng/mL) after treating with peficitinib (0.1, 1, 5 μM) for 24 h. (A) Representative western blot images showed that peficitinib suppressed the phosphorylation of STAT1, (B) STAT3, and (C) STAT5 in RA FLS. (D) The expression of pSTAT1 band intensities was quantified and the data are expressed as the mean and SEM. pSTAT1, (E) pSTAT3, and (F) pSTAT5 were suppressed by peficitinib (0.1, 1, and 5 µM) in a concentration-dependent manner. The data are expressed as the mean ± SEM (n = 3 patients). * p < 0.05 vs. control.

Figure 4

Blocking JAK-STAT signaling with peficitinib in RA FLS reduced monocyte chemotaxis. The RA FLS were stimulated with IL-6 (100 ng/mL) and IL-6R (100 ng/mL) after treating with peficitinib (5 μM) for 24 h. (A) The peficitinib-treated RA FLS-conditioned medium reduced THP-1 migration as compared to the untreated RA FLS-conditioned medium (n, number of experiments; 12 patients). (B) Peficitinib-treated RA FLS-conditioned medium reduced PBMC migration as compared to the untreated RA FLS-conditioned medium (n, number of experiments; 12 patients). * p < 0.05 vs. control.

Figure 5

Peficitinib suppressed RA FLS proliferation. RA FLS were stimulated with IL-6 (100 ng/mL) and IL-6R (100 ng/mL) after treating with peficitinib (5 μM) for 24 h. The proliferation of peficitinib-treated RA FLS was reduced as compared to the untreated RA FLS (n, number of experiments; 4 patients). * p < 0.05 vs. control.

Figure 6

Peficitinib suppressed the secretion of inflammatory mediators in RA FLS. The amounts of RANTES/CCL5 (A), MCP-1/CCL2 (B), MMP-3 (C), fractalkine/CX3CL1 (D), ENA78/CXCL5 (E), and IL-8 (F) in IL-6 and IL-6R-stimulated peficitinib treated RA FLS-conditioned medium were determined and were compared to IL-6 and IL-6R-stimulated untreated RA FLS-conditioned medium. MCP-1/CCL2 in RA FLS supernatant was suppressed after adding peficitinib. (D) Fractalkine/CX3CL1 in IL-6 and IL-6R-stimulated peficitinib-treated RA FLS-conditioned medium was not different as compared to the untreated medium. (E) ENA-78 in IL-6 and IL-6R-stimulated peficitinib-treated RA FLS conditioned medium was increased. The data are expressed as the mean ± SEM (n = 4 patients). * p < 0.05 vs. control.