Chemokine Expression in Retinal Pigment Epithelial ARPE-19 Cells in Response to Coculture with Activated T Cells
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Chemokine Expression in Retinal Pigment Epithelial ARPE-19 Cells in Response to Coculture with Activated T Cells. / Juel, Helene Bæk; Faber, Carsten; Udsen, Maja Søberg; Folkersen, Lasse ; Nissen, Mogens Holst.
In: Investigative Ophthalmology & Visual Science, Vol. 53, No. 13, 12.2012, p. 8472-8480.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Chemokine Expression in Retinal Pigment Epithelial ARPE-19 Cells in Response to Coculture with Activated T Cells
AU - Juel, Helene Bæk
AU - Faber, Carsten
AU - Udsen, Maja Søberg
AU - Folkersen, Lasse
AU - Nissen, Mogens Holst
PY - 2012/12
Y1 - 2012/12
N2 - 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.
AB - 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.
U2 - 10.1167/iovs.12-9963
DO - 10.1167/iovs.12-9963
M3 - Journal article
VL - 53
SP - 8472
EP - 8480
JO - Investigative Ophthalmology & Visual Science
JF - Investigative Ophthalmology & Visual Science
SN - 0146-0404
IS - 13
ER -
ID: 38304787