ABSTRACT
Purpose: To determine to which extent inflammatory cytokines affect chemokine secretion by primary human choroidal melanocytes (HCMs), their capacity to attract monocytes, and whether HCMs are able to influence the proliferation of activated T cells. Methods: Primary cultures of HCMs were established from eyes of 13 donors. Human choroidal melanocytes 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 by 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 transwell plate. Proliferation of activated T cells was assessed in a direct coculture 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 proinflammatory cytokines. Supernatants derived from unstimulated cultures of 10 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 after exposure to proinflammatory cytokines. Direct coculture of HCMs with T cells resulted in inhibition of T-cell proliferation. Conclusions: These results showed that normal and activated HCMs are immunologically active by secreting chemokines, and that HCMs are able to attract monocytes in addition to inhibiting T-cell proliferation.
Subject(s)
Chemokine CXCL9/metabolism , Choroid/pathology , Cytokines/metabolism , Melanocytes/pathology , Melanoma/pathology , Monocytes/pathology , Uveal Neoplasms/pathology , Adolescent , Adult , Aged , Cell Movement , Cell Proliferation , Choroid/metabolism , Culture Media, Conditioned , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Melanoma/metabolism , Middle Aged , Photomicrography , Tumor Cells, Cultured , Uveal Neoplasms/metabolism , Young AdultABSTRACT
PURPOSE: To investigate the effects of T-cell-derived cytokines on gene and protein expression of chemokines in a human RPE cell line (ARPE-19). METHODS: We used an in vitro coculture system in which the RPE and CD3/CD28-activated T-cells were separated by a membrane. RPE cell expression of chemokine genes was quantified using three different types of microarrays. Protein expression was determined by single and multiplex ELISA and immunoblotting. RESULTS: Coculture with activated T-cells increased RPE mRNA and protein expression of chemokines CCL2 (MCP-1); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (MCP-2); CXCL1 (GRO-α); IL8 (CXCL8); CXCL9 (MIG); CXCL10 (IP10); CXCL11 (ITAC); and CX3CL1 (fractalkine). CCL7, CXCL9, CXCL10, and CXCL11 were secreted significantly more in the apical direction. Using recombinant human cytokines and neutralizing antibodies, we identified IFNγ and TNFα as the two major T-cell-derived cytokines responsible for the RPE response. For CCL5, CXCL9, CXCL10, CXCL11, CXCL16, and CX3CL1, we observed a synergistic effect of IFNγ and TNFα in combination. CCL20, CXCL1, CXCL6, and IL8 were negatively regulated by IFNγ. CONCLUSIONS: RPE cells responded to exposure to T-cell-derived cytokines by upregulating expression of multiple chemokines related to microglial, T-cell, and monocyte chemotaxis and activation. This inflammatory stress response may have implications for immune homeostasis in the retina, and for the further understanding of inflammatory ocular diseases such as uveitis and AMD.
