Rheumatoid arthritis synovial fibroblasts produce a soluble form of the interleukin-7 receptor in response to pro-inflammatory cytokines

Abstract

We previously demonstrated that baseline synovial overexpression of the interleukin-7 receptor α-chain (IL-7R) is associated with poor response to tumour necrosis factor (TNF) blockade in rheumatoid arthritis (RA). We found that IL-7R gene expression is induced in fibroblast-like synovial cells (FLS) by the addition of TNF-α, IL-1β and combinations of TNF-α+ IL-1β or TNF-α+ IL-17, thereby suggesting that these cytokines play a role in the resistance to TNF blockade in RA. Because FLS and CD4 T cells also produce a soluble form of IL-7R (sIL-7R), resulting from an alternative splicing of the full-length transcript, we wondered whether expression of sIL-7R is similarly regulated by pro-inflammatory cytokines. We also investigated whether sIL-7R is detectable in the serum of RA patients and associated with response to TNF blockade. RA FLS were cultured in the presence of pro-inflammatory cytokines and sIL-7R concentrations were measured in culture supernatants. Similarly, sIL-7R titres were measured in sera obtained from healthy individuals, early untreated RA patients with active disease and disease-modifying anti-rheumatic drug (DMARD)-resistant RA patients prior to initiation of TNF-blockade. Baseline serum sIL-7R titres were correlated with validated clinical measurements of disease activity. We found that exposure of RA FLS to pro-inflammatory cytokines (TNF-α, IL-1β and combinations of TNF-α and IL-1β or TNF-α and IL-17) induces sIL-7R secretion. Activated CD4 T cells also produce sIL-7R. sIL-7R serum levels are higher in RA patients as compared to controls. In DMARD-resistant patients, high sIL-7R serum concentrations are strongly associated with poor response to TNF-blockade. In conclusion, sIL-7R is induced by pro-inflammatory cytokines in RA FLS. sIL-7R could qualify as a new biomarker of response to therapy in RA.

Fig 1

FLS and activated CD4 T cells produce two IL-7R α-chain isoforms. (A) IL-7R Western blots on protein extracts from FLS and PBMC, and IL-7R PCR on cDNA from duplicate TNF-α and IL-1β activated FLS (1), IL-2 and PHA activated CD4 T cells (2), antigen-activated CD8 T cell clones (3) and B-EBV cells (4). Arrows indicate the expected sizes of the membrane-bound IL-7R. (B) Sequencing of purified IL-7R PCR fragments indicates that FLS produce a full-length IL-7R α-chain and a truncated form of the IL-7R α-chain lacking exon 6 encoding the transmembrane domain, thereby resulting in a secreted form of the molecule.

Fig 2

Pro-inflammatory cytokines stimulate sIL-7R production by FLS. (A) IL-7R (open bars) and sIL-7R (closed bars) real-time qPCR studies carried out on FLS stimulated with the indicated cytokines. Results are expressed as mean fold changes in IL-7R and sIL-7R gene expression (±S.E.M.) over unstimulated FLS, obtained from two to five different experiments each. (B) Flow cytometric evaluation of IL-7R expression by PBMC and FLS. Cells were incubated with a PE-conjugated IL-7R antibody (red) or a PE-conjugated isotype control (blue). Autofluorescence of the cells is depicted in green. Graphs are representative of three different experiments. Similar results were obtained using FLS stimulated with pro-inflammatory cytokines, alone or in combination. (C) sIL-7R measurements were performed by sandwich-ELISA in supernatants of FLS cultures stimulated with the indicated cytokines. Results are expressed as mean optical density (O.D.) units ×1000 (after subtraction of the baseline O.D.) ±S.E.M. obtained from three different experiments. (D) Effect of IL-7 and sIL-7R-Fc fusion protein on proliferation of synovial CD4 T cells cultured in the presence of autologous FLS, autologous serum and IL-2. Results are expressed as mean cpm (±S.E.M.) obtained from two different experiments. *P < 0.05; **P < 0.005; ***P < 0.0005 versus unstimulated cells.

Fig 3

Serum sIL-7R titres are significantly higher in early and DMARD-resistant RA patients. sIL-7R titres measured by sandwich ELISA in duplicate serum samples from healthy individuals, early RA and DMARD-resistant RA patients. The horizontal bar depicts the median value in each group.

Fig 4

sIL-7R serum concentrations predict response to TNF blockade in RA. (A) sIL-7R titres measured by sandwich ELISA in duplicate baseline serum samples obtained in DMARD-resistant RA patients treated with 3 mg/kg infliximab. Patients were categorized in good-responders (circles), moderate- (triangles) and non-responders (squares) according to EULAR response criteria. The horizontal bar depicts the median value in each group. (B) Linear correlation between baseline sIL-7R serum levels and DAS-Score differences (follow-up minus baseline DAS28-CRP). (C) Receiving operating characteristic curve evaluating the value of baseline sIL-7R in predicting response to therapy. The curve was plotted by calculating sensitivity and specificity of the test at several cut-off values.