ABSTRACT.Purpose: We have recently identified homeostatic alterations in the circulating T cells of patients with age-related macular degeneration (AMD). In cultures of retinal pigment epithelial cells, we have demonstrated that T-cell-derived cytokines induced the upregulation of complement, chemokines and other proteins implicated in AMD pathogenesis. The purpose of this study was to test whether increased plasma levels of cytokines were present in patients with AMD. Methods: We conducted a case-control study. Age-related macular degeneration status was assessed using standardized multimodal imaging techniques. Plasma was isolated from freshly drawn peripheral venous blood samples and analysed for interleukin (IL)15, IL18, interferon (IFN)c, soluble tumour necrosis factor (TNF) receptor II (sTNFRII) and complement factor H (CFH) Y402H genotype. Results: We included 136 individuals with early or late forms of AMD and 74 controls. Significantly increased levels of sTNFRII were observed in patients with early or exudative AMD (p < 0.01). After adjusting for CFH Y402H genotype, age, sex and smoking history, the level of sTNFRII remained a significant predictor for prevalence of AMD with odds ratios at 3.0 in the middle and 3.6 in the highest tertiles. Levels of IL15, IL18 and IFNc were low and not associated with AMD. Conclusions: Increased plasma level of sTNFRII is found to be associated with AMD. The data supports the observations of low-grade, systemic inflammatory alterations in patients with AMD. However, it remains to be determined whether increased levels of TNFa can be found, which directly reflects an increased activity of macrophages and T cells.

Cytokines derived from activated T cells shifted the UM cell transcriptome toward a more inflammatory state, including upregulation of several chemokines, which led to an increased migration of monocytes. Therefore, UM cells might actively participate in generating a tumor-promoting inflammatory microenvironment.

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.

IMPORTANCE Among the characteristics of uveal melanoma that are associated with a poor prognosis are a large tumor size and the presence of increased numbers of lymphocytes and macrophages. In rapidly growing tumors, reduction in oxygen tension may occur with increased distance from blood vessels, which we hypothesize may lead to an inflammatory microenvironment, further stimulating tumor growth.OBJECTIVES To analyze whether hypoxia induces uveal melanoma cells to express proinflammatory cytokines and whether tumor supernatant (TSN) affects monocyte migration and differentiation. DESIGN AND SETTINGThe expression of proinflammatory genes in freshly cultured uveal melanoma samples was studied in an in vitro 24-hour hypoxic culture system using quantitative polymerase chain reaction. In addition, cell lines cultured under normoxic and hypoxic conditions were used. The effect of TSN on monocyte chemotaxis was tested using a transwell migration system and by analyzing monocyte differentiation. The levels of the cytokines CCL2, IL6, and PGE2 in TSN were determined by enzyme-linked immunosorbent assay.PARTICIPANTS Five cell lines (OCM8, 92.1, Mel270,Mel290 and OMM2.5) and 11 primary short-term cultures.RESULTS Exposure of freshly cultured uveal melanoma cells to hypoxia led to an increased expression of the proinflammatory cytokines PLGF (OMIM 601121), TGF␤ (OMIM 190180), END1 (OMIM +131240), and ICAM1 (OMIM 147840) and a lower expression of AIMP1 (OMIM 603605) (EMAP2), CCL2 (MCP-1) (OMIM +158105), and IL1b (OMIM *147720). The TSN from cultured melanoma cell lines induced chemotaxis of monocytes, but this was independent of the normoxic or hypoxic state. The TSN of 1 cell line and 2 primary uveal melanoma cultures inhibited the dendritic cell maturation and did not induce M2 macrophage polarization in vitro. CONCLUSIONS AND RELEVANCEOur results indicate that under hypoxic conditions, immune response genes are differentially expressed in cultured primary uveal melanoma cells. The TSN from uveal melanoma cell lines is capable of affecting the chemotactic response and maturation of monocytes in vitro, but this is irrespective of hypoxia. 31. Cools-Lartigue J, Marshall JC, Caissie AL, Saraiva VS, Burnier MN Jr. Secretion of interleukin-6 and prostaglandin E2 during uveal melanoma-monocyte in vitro interactions. Exp Eye Res. 2004;79(4):451-454. 32. Müller-Quernheim UC, Potthast L, Müller-Quernheim J, Zissel G. Tumor-cell co-culture induced alternative activation of macrophages is modulated by interferons in vitro.

In this study, we investigated the effect of CD3/CD28-activated peripheral blood mononuclear cells (PBMCs) on two human astrocytoma cell lines (1321N1 and CCF-STTG1), with respect to the expression of cytokines and chemokines. We used an in vitro co-culture system in which the astrocytoma cells and PBMCs were separated by a membrane in a transwell system. Proliferation of T cells was quantified by a [(3) H]-thymidine incorporation assay. Differential gene expression of astrocytoma cells cultured alone or with PBMCs was identified using gene arrays. Protein expression of CCL3, CCL5, CXCL8, CXCL9, CXCL10, IL6, and IL1β was determined by Luminex. Co-culture of activated T cells and astrocytoma cells resulted in inhibition of T cell proliferation. Moreover, astrocytoma cells upregulated a number of pro-inflammatory genes including CCL3, CCL5, CXCL8, CXCL9, CXCL10, IL6, and IL1β upon co-culture with activated PBMCs. These results were confirmed on the protein level. Astrocytoma cells inhibited the proliferation of T cells, indicating that astrocytes may play an important role in controlling T cell mediated immune responses in the brain. Contrary to this, soluble factors derived from CD3/CD28-activated PBMCs affected the transcriptome of astrocytoma cells, which then upregulated several pro-inflammatory genes and proteins. This could be part of neuroinflammatory events and may play a role in neurodegenerative diseases.