Helene B. Juel

Chemokine Expression in Retinal Pigment Epithelial ARPE-19 Cells in Response to Coculture with Activated T Cells

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.

Retinal pigment epithelial cells upregulate expression of complement factors after co-culture with activated T cells.

In this study we examined the effect of T cell-derived cytokines on retinal pigment epithelial (RPE) cells with respect to expression of complement components. We used an in vitro co-culture system in which CD3/CD28-activated human T cells were separated from the human RPE cell line (ARPE-19) by a membrane. Differential gene expression in the RPE cells of complement factor genes was identified using gene arrays, and selected gene transcripts were validated by q-RT-PCR. Protein expression was determined by ELISA and immunoblotting. Co-culture with activated T cells increased RPE mRNA and/or protein expression of complement components C3, factors B, H, H-like 1, CD46, CD55, CD59, and clusterin, in a dose-dependent manner. Soluble factors derived from activated T cells are capable of increasing expression of complement components in RPE cells. This is important for the further understanding of inflammatory ocular diseases such as uveitis and age-related macular degeneration.

Inflammatory Cytokines Protect Retinal Pigment Epithelial Cells from Oxidative Stress-Induced Death

Purpose To investigate the effects of inflammatory factors and oxidative stress on cell survival of the human retinal pigment epithelial (RPE) cell line, ARPE-19. Methods Confluent RPE cells were treated with peripheral blood mononuclear cells-conditioned medium (PCM), H2O2, NaIO3, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, or combinations of these. Cell viability was determined by viability assays and by light microscopy. Effector molecules of cell death were investigated by immunofluorescence microscopy and flow cytometry. Microarrays were performed to screen for differential expression of anti-oxidative enzymes, and protein expression was validated by immunoblotting. Results Viability of RPE cells was reduced by exposure to inflammatory agents (PCM, IFNγ+/-TNFα) or to oxidative agents (H2O2 or NaIO3). Unexpectedly, cells treated with either H2O2 or NaIO3 were partially protected from cell death by the addition of PCM. This protection was conferred, at least in part, by IFNγ and TNFα. Cell death induced by H2O2 or NaIO3 was preceded by mitochondrial dysfunction and by p62 upregulation, both of which were attenuated by PCM and/or by IFNγ+TNFα. RPE cells co-cultured with activated T cells, or treated with cytokines showed increased expression of anti-oxidative genes, with upregulation of superoxide dismutase 2 protein following PCM treatment. Conclusion Oxidative stress-induced cell death was reduced by concomitant inflammatory stress. This is likely due to the cytokine-mediated induction of the anti-oxidative stress response, upregulating protective anti-oxidant pathway(s). These findings suggest caution for the clinical use of anti-inflammatory agents in the management of immune-associated eye diseases such as age-related macular degeneration.

Inflammation-induced chemokine expression in uveal melanoma cell lines stimulates monocyte chemotaxis.

PURPOSE Uveal melanoma (UM) is the most common primary intraocular tumor in adults and the presence of infiltrating leucocytes is associated with a poor prognosis. Little is known how infiltrating leucocytes influence the tumor cells. The purpose of this study was to investigate the effect of activated T cells on the expression of chemotactic cytokines in UM cells. Furthermore, we examined the ability of stimulated UM cells to attract monocytes. METHODS We used an in vitro coculture system in which UM cell lines and T cells were cultured together, but separated by a membrane. Uveal melanoma gene expression was quantified using a microarray. Protein expression in the supernatant was quantified with ELISA or cytometric bead array. For the monocyte migration assay, a transwell plate was used. RESULTS Gene-expression analysis of UM cell lines showed that coculture with activated T cells resulted in an upregulation of chemokines such as CXCL8, CXCL9, CXCL10, CXCL11, CCL2, CCL5, VEGF, intracellular adhesion molecule 1 (ICAM1), and granulocyte-macrophage colony-stimulating factor (GM-CSF). The upregulation of these molecules was confirmed at the protein level. This increase of chemokines coincided with an increased chemotactic capacity of the supernatant toward monocytes. CONCLUSIONS 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.

Early and exudative age-related macular degeneration is associated with increased plasma levels of soluble TNF receptor II

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.

Systemic and Ocular Long Pentraxin 3 in Patients with Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) has been associated with both systemic and ocular alterations of the immune system. In particular dysfunction of complement factor H (CFH), a soluble regulator of the alternative pathway of the complement system, has been implicated in AMD pathogenesis. One of the ligands for CFH is long pentraxin 3 (PTX3), which is produced locally in the retinal pigment epithelium (RPE). To test the hypothesis that PTX3 is relevant to retinal immunohomeostasis and may be associated with AMD pathogenesis, we measured plasma PTX3 protein concentration and analyzed the RPE/choroid PTX3 gene expression in patients with AMD. To measure the ability of RPE cells to secrete PTX3 in vitro, polarized ARPE-19 cells were treated with activated T cells or cytokines (interferon (IFN)-gamma and/or tumor necrosis factor (TNF)-alpha) from the basolateral side; then PTX3 protein concentration in supernatants and PTX3 gene expression in tissue lysates were quantified. Plasma levels of PTX3 were generally low and did not significantly differ between patients and controls (P=0.307). No statistically significant difference was observed between dry and exudative AMD nor was there any correlation with hsCRP or CFH genotype. The gene expression of PTX3 increased in RPE/choroid with age (P=0.0098 macular; P=0.003 extramacular), but did not differ between aged controls and AMD patients. In vitro, ARPE-19 cells increased expression of the PTX3 gene as well PTX3 apical secretions after stimulation with TNF-alpha or activated T cells (P<0.01). These findings indicate that PTX3 expressed in the eye cannot be detected systemically and systemic PTX3 may have little or no impact on disease progression, but our findings do not exclude that locally produced PTX3 produced in the posterior segment of the eye may be part of the AMD immunopathogenesis.

Astrocytoma cells upregulate expression of pro-inflammatory cytokines after co-culture with activated peripheral blood mononuclear cells

Jehs T, Faber C, Juel HB, Nissen MH. Astrocytoma cells upregulate expression of pro‐inflammatory cytokines after co‐culture with activated peripheral blood mononuclear cells, APMIS 2011; 119: 551–61.