BioProject

Purpose: To determine to which extend inflammatory cytokines affect chemokine secretion by primary human choroidal melanocytes (HCMs), their capacity to attract monocytes and if HCMs are able to influence the proliferation of activated T cells. More...

Purpose: To determine to which extend inflammatory cytokines affect chemokine secretion by primary human choroidal melanocytes (HCMs), their capacity to attract monocytes and if HCMs are able to influence the proliferation of activated T cells. Methods: Primary cultures of HCMs were established from eyes of thirteen donors. HCMs were stimulated with IFN-γ and TNF-α or with supernatant from activated T-cells (T-cell conditioned media (TCM)). Gene expression analysis was performed using microarrays. Protein levels were quantified with ELISA or cytometric bead array. Supernatants of HCMs were assessed for the capability to attract monocytes in a trans-well plate. Proliferation of activated T-cells was assessed in a direct co-culture with HCMs by a [3H]-thymidine incorporation assay. Results: Stimulation of HCMs with TCM or IFN-γ and TNF-α resulted in increased expression and secretion of CXCL8, CXCL9, CXCL10, CXCL11, CCL2, CCL5 and intercellular adhesion molecule 1. Vascular endothelial growth factor and monocyte migration inhibitory factor were constitutively expressed without changes in response to pro-inflammatory cytokines. Supernatants derived from unstimulated cultures of ten HCM donors induced a high initial level of monocyte migration, which decreased upon stimulation with either TCM or IFN-γ and TNF-α. The supernatants from three HCM donors initially showed a low level of monocyte attraction which increased following exposure to pro-inflammatory cytokines. Direct co-culture of HCMs with T-cells resulted in inhibition of T-cell proliferation. Conclusion: These results show that normal and activated HCMs are immunologically active by secreting chemokines, and that HCMs are able to attract monocytes in addition to inhibit T-cell proliferation. Overall design: Choroidal melanocyte cultures from thirteen donors were analyzed before and after exposure to supernatants from activated T-cells and to 27 ng/ml recombinant IFN-γ and 10 ng/ml recombinant TNF-α. Less...