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S. B. Douglas, E. J. Lee, S. R. Planck, J. T. Rosenbaum; Involvement of Beta1-integrin in Extravascular Leukocyte Migration in the Murine Cornea. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4310.
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Leukocytes establish adhesive contacts with the underlying tissue stroma to generate traction for migration. The integrin superfamily of adhesion molecules includes cell surface receptors that mediate binding to extracellular matrix molecules. Intravital microscopy was used to test the role of ß1-integrin in extravascular cell migration in the collagen-rich corneal stroma.
Mice expressing enhanced green fluorescent protein (eGFP) in the lysozyme M (lys) locus were used. Injury was induced in the central cornea with a silver nitrate applicator. At 5 h post-injury, corneas were imaged in vivo by time-lapse widefield fluorescence microscopy for 30 min at a rate of 3 frame/min. Immediately after imaging, animals were treated with AlexaFluor594-labeled antibodies (to verify and visualize distribution within the stroma) administered as a subconjunctival injection (1.25 ng /2.5 µl) and topical application (1.25 ng /2.5 µl). Eyes were treated in one of the following ways: 1) antibody to ß1-integrin, 2) isotype control antibody, or 3) not treated. At 2.5 h after treatment, corneas were imaged again in the same manner. Videos were stabilized and then randomly selected cells were tracked by masked observers. Acquired images were also used to determine eGFP+ cell density within the cornea and limbus.
Before treatment, eGFP+ cells (n=67) in the cornea (n=9) traveled at an average speed of 7.0±0.3 µm/min (avg±SEM). Treatment with anti ß1-integrin antibody resulted in increased speed to 9.5±0.3 µm/min (p<0.0001 vs. pre-treatment) compared to 7.8± 0.4µm/min with isotype control antibody (p=0.27 vs. pre-treatment), with no change in extent of randomness in migration pattern in either case, as determined by mean displacement plots. Antibody to ß1-integrin was detected in association with limbal blood vessels as well as with infiltrating cells.
Our results suggest that in the cornea, mobility of leukocytes is partially dependent upon ß1-integrin. They are also somewhat surprising, since blockade of ß1-integrin resulted in increased rather than decreased extravascular speed as has been demonstrated in other tissues.
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