Synthesis of alpha-chemokines IP-10, I-TAC, and MIG are differentially regulated in human corneal keratocytes.
OBJECTIVE
Chemokines responsible for recruiting lymphocytes such as activated T cells into the cornea have not been clearly defined. IP-10, I-TAC, and MIG are chemoattractants for these lymphocytes. The goal of this study was to determine whether human corneal keratocyte (HCKs) in culture synthesize these chemokines in response to proinflammatory mediators.


METHODS
HCKs grown in vitro were stimulated with IL-1alpha, TNF-alpha, or IFN-gamma. Induction of alpha-chemokine gene expression was quantitated by real-time PCR and ELISA. Activation of the transcriptional activator STAT1 by IFN-gamma receptors expressed on HCKs was determined by Western blot analysis.


RESULTS
HCKs incubated with TNF-alpha, IL-1alpha, or IFN-gamma resulted in a >2000-fold increase in IP-10 protein secretion by 36 hours after stimulation. In contrast, stimulation with TNF-alpha, IL-1alpha, or IFN-gamma induced levels of MIG and I-TAC that were not significantly greater than constitutive levels. Treatment of HCKs with IFN-gamma activated STAT1 and, in combination with either TNF-alpha or IL-1alpha, enhanced MIG and I-TAC synthesis >20-fold.


CONCLUSIONS
IP-10 synthesis is induced in HCKs by IL-1alpha, TNF-alpha, and IFN-gamma. In contrast, induction of I-TAC and MIG synthesis in HCKs requires costimulation with IFN-gamma and either IL-1alpha or TNF-alpha. The results suggest therefore, that the upregulation of I-TAC and MIG gene expression at sites of corneal inflammation are more tightly regulated than that of IP-10. A role for differential induction of the three alpha-chemokine genes in corneal inflammatory processes at the eye surface is discussed.
McInnis
Karla A
KA
Department of Microbiology and Immunology, College of Medicine, University of South Alabama, 307 University Boulevard, Mobile, AL 36688, USA.
Britain
Andrea
A
Lausch
Robert N
RN
Oakes
John E
JE
D015153:Blotting, Western; D002478:Cells, Cultured; D054357:Chemokine CXCL10; D054371:Chemokine CXCL11; D054370:Chemokine CXCL9; D019743:Chemokines, CXC / Q000096:biosynthesis* / Q000235:genetics; D003315:Cornea / Q000187:drug effects / Q000378:metabolism*; D016207:Cytokines / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050794:STAT1 Transcription Factor; D015534:Trans-Activators / Q000378:metabolism; D015854:Up-Regulation
C114753:CXCL10 protein, human; C515202:CXCL11 protein, human; C080810:CXCL9 protein, human; D054357:Chemokine CXCL10; D054371:Chemokine CXCL11; D054370:Chemokine CXCL9; D019743:Chemokines, CXC; D016207:Cytokines; D004268:DNA-Binding Proteins; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D015534:Trans-Activators