zed from abstract) Chemokines are small molecular weight inflammatory mediators responsible for chemoattracting neutrophils, monocytes and lymphocytes into sites of inflammation. The PI has discovered that IL-1a stimulated human corneal epithelial cells (HCEC) synthesize the a-chemokine IL-8 but not the b-chemokine MCP-1. The goal for this project is to determine if combinatorial interaction between NF-kB and C/EBP transcriptional factors accounts for the differential expression of the two chemokines in HCEC and to determine how the amounts of IL-8 produced by HCEC is regulated. RT-PCR, immunoblotting and electrophoretic mobility shift assays will be used to determine which members of the NF-kB and C/EBP family of transcriptional activators are expressed and activated in HCEC. To determine if transcriptional factors activated in IL-1a stimulated cells stimulate IL-8 transcription without stimulating MCP-1 transcription, reporter plasmids will be constructed in which NF-kB and C/EBP binding elements of the IL-8 and MCP-1 promoters are used to drive expression of the luciferase gene. The expression of reporter plasmids transfected into HCEC will then be monitored following IL-1a stimulation. RT-PCR and ELISA assays will be used to test the hypothesis that the same combination of transcriptional factors that activate IL-8 expression will also activate expression of ENA-78, a a-chemokine whose promoter possesses both sequence homology and structure similarity to the IL-8 promoter. The role of IL-1 receptor antagonist (IL-1RA) and soluble IL-1 type 2 receptor (IL-1RII) proteins in dampening HCEC responses to IL-1a will be tested by neutralizing these naturally produced epithelial cell inhibitors with anti-IL-1RA, and anti-IL-1RII antibodies during IL-1a stimulation. This study will increase our understanding of the molecular events which precipitate inflammatory responses on the eye surface.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY012713-03
Application #
6384837
Study Section
Visual Sciences A Study Section (VISA)
Program Officer
Fisher, Richard S
Project Start
1999-09-01
Project End
2004-08-31
Budget Start
2001-09-01
Budget End
2002-08-31
Support Year
3
Fiscal Year
2001
Total Cost
$274,235
Indirect Cost
Name
University of South Alabama
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
City
Mobile
State
AL
Country
United States
Zip Code
36688
McInnis, Karla A; Britain, Andrea; Lausch, Robert N et al. (2007) Human corneal epithelial cells synthesize ELR(-)alpha-chemokines in response to proinflammatory mediators. Ocul Immunol Inflamm 15:295-302
McInnis, Karla A; Britain, Andrea; Lausch, Robert N et al. (2005) Synthesis of alpha-chemokines IP-10, I-TAC, and MIG are differentially regulated in human corneal keratocytes. Invest Ophthalmol Vis Sci 46:1668-74
Ritchie, Mary H; Fillmore, Rebecca A; Lausch, Robert N et al. (2004) A role for NF-kappa B binding motifs in the differential induction of chemokine gene expression in human corneal epithelial cells. Invest Ophthalmol Vis Sci 45:2299-305
Tran, M T; Lausch, R N; Oakes, J E (2000) Substance P differentially stimulates IL-8 synthesis in human corneal epithelial cells. Invest Ophthalmol Vis Sci 41:3871-7