Abstract
The objective of this study was to determine the role of individual NFAT isoforms in TNFα-induced retinal leukostasis. To this end, human retinal microvascular endothelial cells (HRMEC) transfected with siRNA targeting individual NFAT isoforms were treated with TNFα, and qRT-PCR was used to examine the contribution of each isoform to the TNFα-induced upregulation of leukocyte adhesion proteins. This showed that NFATc1 siRNA increased ICAM1 expression, NFATc2 siRNA reduced CX3CL1, VCAM1, SELE, and ICAM1 expression, NFATc3 siRNA increased CX3CL1 and SELE expression, and NFATc4 siRNA reduced SELE expression. Transfected HRMEC monolayers were also treated with TNFα and assayed using a parallel plate flow chamber, and both NFATc2 and NFATc4 knockdown reduced TNFα-induced cell adhesion. The effect of isoform-specific knockdown on TNFα-induced cytokine production was also measured using protein ELISAs and conditioned cell culture medium, and showed that NFATc4 siRNA reduced CXCL10, CXCL11, and MCP-1 protein levels. Lastly, the CN/NFAT-signaling inhibitor INCA-6 was shown to reduce TNFα-induced retinal leukostasis in vivo. Together, these studies show a clear role for NFAT-signaling in TNFα-induced retinal leukostasis, and identify NFATc2 and NFATc4 as potentially valuable therapeutic targets for treating retinopathies in which TNFα plays a pathogenic role.
Highlights
Chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6), which have established roles in leukocyte recruitment[8,23,24,25,26]
We have recently demonstrated that TNFα is a strong inducer of these gene products in human retinal microvascular endothelial cells (HRMEC), and that nuclear factor of activated T-cell (NFAT) family transcription factors regulate a subset of these genes related to leukocyte cell adhesion and recruitment[23]
TNFα -stimulation (1 ng/ml for 4 hrs) of HRMEC resulted in increased expression of the leukocyte adhesion proteins CX3CL1, VCAM1, E-Selectin, and ICAM1 (Table 1)
Summary
Chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6), which have established roles in leukocyte recruitment[8,23,24,25,26]. We have recently demonstrated that TNFα is a strong inducer of these gene products in human retinal microvascular endothelial cells (HRMEC), and that nuclear factor of activated T-cell (NFAT) family transcription factors regulate a subset of these genes related to leukocyte cell adhesion and recruitment[23]. The small molecule Inhibitor of NFAT-Calcineurin Association-6 (INCA-6) effectively blocks CN activation of the four CN-dependent isoforms, and this has been shown to reduce TNFα -induced expression of CX3CL1, VCAM1, CXCL10, and CXCL11 in HRMEC29,30. Taken together, this evidence suggests that NFAT-signaling may play an important role in TNFα -induced retinal leukostasis.
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