Differential survival of leucocyte subsets by synovial, bone marrow and skin fibroblasts: site-specific rescue of CD4 T cells versus activation-induced rescue of neutrophils

Background

Chronic inflammatory disease is characterised by predilection for certain sites, the molecular basis of which remains obscure. We investigated the role that fibroblasts play in governing the survival (persistence) as well as the type of infiltrate (predominantly neutrophil versus lymphocyte) using a leucocyte–fibroblast co-culture model. We used matched synovial, bone marrow and skin fibroblasts to test the hypothesis that a fibroblast-derived, stromal area post code identifies microenvironments that can support differential leucocyte accumulation.

Methods

Matched synovial (SFb), bone marrow (BMFb) and dermal fibroblast (DFb) lines were established from eight patients satisfying ACR criteria for rheumatoid arthritis undergoing total hip or knee arthroplasty. Activated peripheral blood CD4 T cells prepared by negative depletion and neutrophils prepared by density gradient centrifugation were co-cultured with fibroblasts. Survival, apoptosis and proliferation were assessed using total cell counts and active caspase 3 staining (T cells) or DiOC6 staining (neutrophils).

Results

Site-specific survival of CD4 T cells was observed in all eight sets of matched samples: at 3 days, DFb maintained better T-cell survival than culture in medium alone (40 ± 1.2% (mean ± standard error) compared with 25.4 ± 1.4% (P < 0.05)). However, both SFb and BMFb maintained even better T-cell survival compared with DFb: 58.8 ± 1.2% (P < 0.001 versus DFb) and 51.4 ± 2% (P < 0.05 versus DFb). Fibroblast pre-activation with a wide range of proinflammatory signals had no effect on differential survival. T-cell survival was partially reconstituted by medium from T cell:fibroblast co-cultures and reduced by 50% in the presence of transwells suggesting that T cell:fibroblast interactions are necessary for increased survival. Unlike the case for T cells, resting fibroblasts did not affect neutrophil survival. However pre-treatment of all fibroblasts with tumour necrosis factor alpha significantly increased neutrophil survival: SFb 51.2 ± 6.1%, BMFb 51.1 ± 5.5%, DFb 50.6 ± 4.7%, versus control 16.2 ± 3.3%, P < 0.001. Fibroblast conditioned media reconstituted the survival effect, indicating that a soluble survival factor(s) as opposed to cell–cell contact mechanism is involved in neutrophil survival.

Conclusions

Fibroblasts differentially regulate leucocyte subset survival in both a site-dependent and activation-dependent manner. Our results provide an alternative explanation for site-specific differences in the pattern of T-cell and neutrophil accumulation in different chronic inflammatory diseases.