Identification of a transitional fibroblast function in very early rheumatoid arthritis

Abstract

Objectives: Synovial fibroblasts actively regulate the inflammatory infiltrate by communicating with neighbouring endothelial cells (EC). Surprisingly, little is known about how the development of rheumatoid arthritis (RA) alters these immunomodulatory properties. We examined the effects of phase of RA and disease outcome (resolving vs persistence) on fibroblast crosstalk with EC and regulation of lymphocyte recruitment.

Methods: Fibroblasts were isolated from patients without synovitis, with resolving arthritis, very early RA (VeRA; symptom ≤12 weeks) and established RA undergoing joint replacement (JRep) surgery. Endothelial-fibroblast cocultures were formed on opposite sides of porous filters. Lymphocyte adhesion from flow, secretion of soluble mediators and interleukin 6 (IL-6) signalling were assessed.

Results: Fibroblasts from non-inflamed and resolving arthritis were immunosuppressive, inhibiting lymphocyte recruitment to cytokine-treated endothelium. This effect was lost very early in the development of RA, such that fibroblasts no longer suppressed recruitment. Changes in IL-6 and transforming growth factor beta 1 (TGF-β₁) signalling appeared critical for the loss of the immunosuppressive phenotype. In the absence of exogenous cytokines, JRep, but not VeRA, fibroblasts activated endothelium to support lymphocyte.

Conclusions: In RA, fibroblasts undergo two distinct changes in function: first a loss of immunosuppressive responses early in disease development, followed by the later acquisition of a stimulatory phenotype. Fibroblasts exhibit a transitional functional phenotype during the first 3 months of symptoms that contributes to the accumulation of persistent infiltrates. Finally, the role of IL-6 and TGF-β₁ changes from immunosuppressive in resolving arthritis to stimulatory very early in the development of RA. Early interventions targeting 'pathogenic' fibroblasts may be required in order to restore protective regulatory processes.

Keywords: Fibroblasts; adhesion; endothelial cells; lymphocytes; rheumatoid arthritis.

Figure 1

Fibroblasts from patients with resolving and persistent arthritis differentially modulate lymphocyte recruitment from flow. Cocultures were established by culturing endothelial cells and fibroblasts on opposite sides of a porous insert, prior to treatment (A) without or (B) with TNFα+IFNγ for 24 hours. Endothelial monolayers without fibroblasts (none) were used as controls. Lymphocytes were perfused and their interactions with endothelial cells were assessed by digital microscopy. (C, D) Micrograph images showing lymphocyte adhesion to (i) endothelial cells cultured alone, with fibroblasts from (ii) resolving, (iii) VeRA or (iv) JRep patients (C) in the absence of cytokine treatment and (D) in response to TNFα+IFNγ treatment. White arrow indicates an adherent lymphocyte. In A and B, Kruskal-Wallis test shows a significant effect of fibroblasts on lymphocyte adhesion (p<0.01). Data are the mean±SEM for n experiments; each incorporated a different donor for all three cell types. *p<0.05 and **p<0.01 by Dunn post-test. EC, endothelial cells; IFNγ, interferon gamma; JRep, joint replacement; NI, non-inflamed; Res, resolving; TNFα, tumour necrosis factor alpha; VeRA, very early RA.

Figure 2

Resolving fibroblasts mediated immunosuppressive effect through IL-6 and TGF-β₁. Actions of IL-6 or TGF-β₁ were neutralised, alone or in combination, in TNFα+IFNγ-treated cocultures incorporating fibroblasts from patients with (A) resolving synovitis, (B) very early RA or (C) joint replacement RA (JRep). Dotted line (-----) represents adhesion to TNFα+IFNγ-treated endothelial monocultures for paired experiments. IgG represents cocultures incubated with isotype control antibodies. In A and B, ANOVA shows a significant effect of antibody treatment on lymphocyte adhesion (p<0.01). Data are the mean±SEM from three to five independent experiments each incorporating a different donor for all cell types. *p<0.05 compared with None (untreated cocultures) by Dunnett post-test. ANOVA, analysis of variance; IFNγ, interferon gamma; IL-6, interleukin 6; JRep, joint replacement; RA, rheumatoid arthritis; TGF-β, transforming growth factor beta; TNFα, tumour necrosis factor alpha.

Figure 3

Secretion and signalling of IL-6 in cocultures. (A) IL-6 release during TNFα+IFNγ-treated cocultures. ANOVA shows a significant effect of culture conditions on the secretion of IL-6 (p<0.001). (B) SOCS3 and (C) SOCS1 gene expression analysed by qPCR. Data are expressed as 2^−ΔCT relative to 18S expression. Data are the mean±SEM from three to five independent experiments each incorporating a different donor for all cell types. *p<0.05, **p<0.01 and ***p<0.001 compared with None (endothelial monoculture) by Dunnett post-test, unless otherwise indicated. ANOVA, analysis of variance; IFNγ, interferon gamma; IL-6, interleukin 6;  JRep, joint replacement; Res, resolving; SOCS, suppressor of cytokine signalling; TNFα, tumour necrosis factor alpha; VeRA, very early RA.

Figure 4

Secretome from resolving and very early RA cocultures. Conditioned media from resolving or very early RA fibroblast cocultures were measured by multiplex analysis. (A) CXCL10, (B) IL-8, (C) CXCL5, (D) CXCL1, (E) CCL5 and (F) CCL2 expression. Data are the mean±SEM from five to nine independent experiments each incorporating a different donor for all cell types. *p<0.05 by unpaired t-test. IL-8, interleukin 8, also known as CXCL8; Res, resolving; VeRA, very early RA.