Regulation of Neutrophil Migration in the Corneal Stroma by Chemokines and Proteoglycans in Endotoxin–Induced Keratitis

Abstract

Abstract: : Purpose: How the corneal stromal environments changes with respect to chemokine and proteoglycan expression during keratitis is currently unknown. The goal of this study is to determine the role of MIP–2 in inflammatory cell migration through the cornea in lipopolysaccharide–induced keratitis and the impact on keratocan expression. Methods: Keratitis was induced by intrastromal injection of 2 µg of lipopolysaccharide into the corneal stroma of mice. In order to determine the function of a chemokine, MIP–2, an in vivo migration assay was performed using EGFP–chimeric mice. A scratch was made in the temporal paracentral cornea at time –24 hrs to recruit inflammatory cells to a localized site. At time 0 hrs, fluorescent–labeled LPS was injected into the nasal paracentral cornea and a MIP–2 neutralizing antibody or an IgG control was injected into central cornea. In vivo fluorescent stereomicrographs were captured and quantitated at various time points to track the response of EGFP–positive inflammatory cells to the LPS stimulus in the presence or absence of MIP–2 neutralizing antibody. Corneas and enucleated eyes were harvested at 6, 24, 48, and 72 hrs following intrastromal injection of LPS and a keratocan western blot was performed. Inflammatory cells were quantitated by performing NIMP–R14 and F4–80 immunohistochemistry on enucleated eye frozen sections. Results: EGFP–positive bone marrow derived inflammatory cells migrate into the corneal stroma in response to LPS. MIP–2 neutralization blocked 47 % of inflammatory cell migration through the corneal stroma when compared to an IgG control in response to LPS. Keratocan expression decreased 75 % in 24 hrs following instrastromal injection of LPS, but increased 300 % between 24 and 48 hrs reaching naive corneal levels by 72 hrs. The number of neutrophils in the corneal stroma peaked at 24 hrs and began to subside 48 hrs following LPS intrastromal injection correlating with keratocan expression. Conclusions: Migration of neutrophils through the corneal stroma in LPS–induced keratitis is partially mediated by the chemokine MIP–2. The influx of neutrophils into the corneal stroma results in decreased keratocan levels which may facilitiate neutrophil migration by compromising the integrity of the highly–organized stromal matrix.

Keywords: inflammation • keratitis