Neutrophil Interstitial Migration

Purpose: Molecular events regulating neutrophil extravasation have been extensively researched and described. However, relatively little is known about extravascular interstitial migration of neutrophils and then, much of what we do know has come from in vitro 2-D or 3-D matrix models. These models are limited by their ability to duplicate the nuances of the physiological or physical native environment. Neutrophils often migrate a considerable distance from the site of extravasation through the avascular corneal stroma to reach the site of injury. This migration involves contact with extracellular matrix and resident keratocytes. Ultrastructural morphometric data suggest neutrophil contacts with keratocytes are mediated by the leukocyte β2 (CD18) integrins and ICAM-1(a β2 ligand). While β1 (CD29) and β3 (CD61) integrin families are also expressed on extravascular migrating neutrophils, in vitro studies have shown that locomotion of activated neutrophils is dependent on integrin binding on 2-D surfaces, but not in 3-D matrixes. The role of integrin binding during in vivo corneal stroma migration has yet to be clearly defined. A greater understanding of this migration holds the promise of a more effective means for modulating neutrophil activity to control inflammation and improve the outcome of wound healing. Additionally, it may elicit details of motility applicable to other types of cells. The purpose of this dissertation is to provide insights into the mechanisms of neutrophil migration through the corneal stroma. Specifically, it addresses the influence of the keratocyte network on migrating neutrophils and the relative contribution of β1 (CD29), β2 (CD18) and β3 (CD61) integrins to neutrophil locomotion in the inflamed mouse cornea. In vivo data obtained using Heidelberg Retinal Tomographer III with Rostock Corneal Module (HRT-RCM) time lapse sequences provided the means, for the first time, to quantify speed and directionality of cellular movement while observing neutrophil interaction with stromal keratocytes in the living eye.

Methods: Corneal inflammation was induced in female wild type C57BL/6 mice by mechanical removal of the epithelium using an Algerbrush. Eight hours after injury the corneas were imaged with the HRT-RCM. Scanning sequences provided the means to track individual cells for extended time periods to determine motility characteristics. The contribution of integrin binding to neutrophil migration was assessed by blocking antibody (anti-β1-, β2-, or β3-integrin) or IgG control antibody applied to the cornea at the time of epithelial injury. Image stabilization, cell tracking and movement analysis were accomplished with a custom MatLab program.

Results: Time-lapse imaging showed an unequivocal preference for neutrophils to follow the network of keratocytes. Neutrophils in control eyes moved with an average speed of 7.56±0.20 (SE) µm/minute. The average confinement ratio (CR) of the neutrophil population was 0.55±0.02, where a value of 1.0 indicates confinement to a perfectly straight path. Compared to the results from control eyes, anti- β1-integrin antibody resulted in a 31 % reduction in speed (p<0.05) and a 33% reduction in CR (p<0.05), while anti-β2- or β3- integrin antibodies had no significant effect on cell speed or CR.

Conclusions: Results clearly show that the keratocyte network is the preferred route for neutrophil migration within the corneal stroma. Contrary to expectations based on previously published histological and in vitro evidence, blockade of β2-integrin does not affect in vivo motility and the same is true for β3-integrin blockade. However, β1 blockade produced a significant, but not total, reduction in cell speed and resulted in migrating cells being less confined to a straight path. Therefore, neutrophil locomotion within the physically confined environment of the corneal stroma does not require integrin binding, though β1 binding facilitates the process.