Assessment of granulocyte and monocyte ratios in the peripheral leukocyte population of A.BY WT and <i>Dstn^corn1</i> mice.

<p>A flow cytometric analysis revealed that the ratios of granulocytes, which are comprised mostly of neutrophils, and monocytes, which are the precursor to macrophages, are not significantly different between A.BY WT and <i>Dstn^corn1</i> mice. Ratios are expressed as the percentage of cells compared to the total leukocyte number. Error bars represent SEM.</p

Inflammatory cell recruitment in the corneas of <i>Dstn</i> mutant mice.

<p>(A): Immunofluorescent staining for macrophages (F4/80, red) and neutrophils (myeloperoxidase, green) in WT, <i>Dstn^corn1</i>, and <i>Dstn^corn1-2J</i> cornea at P14. Neutrophils are recruited to the stromal area in <i>Dstn^corn1</i>, but not WT or <i>Dstn^corn1-2J</i> cornea. Sections are counterstained with DAPI (blue). The central region of the cornea is shown in all images. The scale bar corresponds to 10 µm. (B): Quantification of inflammatory cells in WT and <i>Dstn^corn1</i> mice. Accumulation of neutrophils is observed in <i>Dstn^corn1</i> cornea as compared to WT beginning at P14. Significantly higher numbers of macrophages are also present in <i>Dstn^corn1</i> cornea beginning at P14, and most significantly at P28. Error bars represent standard error of measurement (SEM). * denotes statistical significance by <i>t</i>-test. (C): Quantification of inflammatory cells in different regions of the cornea shows that the greatest numbers of neutrophils and macrophages accumulate in the peripheral region, the area adjacent to the limbal vasculature. Error bars represent SEM. * denotes statistical significance by <i>t</i>-test.</p

Neovascularization in the cornea of compound mutants for <i>Dstn</i> and <i>Il8rb</i>.

<p>(A): Representative images showing CD31⁺ (green) blood vessel growth from the limbus (Li) in P21 <i>Dstn^corn1/corn1 Il8rb^+/−</i> and <i>Dstn^corn1/corn1 Il8rb^−/−</i> cornea. The scale bar corresponds to 100 µm. (B): Quantification of vascularized area at P21 in whole mount cornea. A tendency for a greater vascularized area is observed in <i>Dstn^corn1/corn1 Il8rb^−/−</i> cornea, although not statistically significant as determined by Mann-Whitney test. Error bars represent SEM.</p

Inflammatory cell recruitment in the compound mutants for <i>Dstn</i> and <i>Il8rb</i>.

<p>(A): Representative images showing a lack of myeloperoxidase-positive neutrophils (green) in the cornea of <i>Dstn^corn1/corn1 Il8rb^−/−</i> mice at P21. The peripheral region of the cornea is shown. The scale bar corresponds to 10 µm. (B): Quantification of neutrophils and macrophages in <i>Dstn^corn1/corn1 Il8rb^+/−</i> and <i>Dstn^corn1/corn1 Il8rb^−/−</i> cornea. Neutrophil recruitment is almost completely inhibited in <i>Dstn^corn1/corn1 Il8rb^−/−</i> cornea. A tendency for greater macrophage recruitment, although not statistically significant, is observed. Error bars represent SEM. * denotes statistical significance by <i>t</i>-test.</p

Expression of CXCL5 in <i>Dstn</i> mutant mice.

<p>(A): Real-time PCR analysis for <i>Cxcl5</i>. The relative expression level of <i>Cxcl5</i> is expressed as the number molecules per 1000 <i>B2m</i> molecules, and is significantly higher in the <i>Dstn^corn1</i> cornea as compared to WT. Error bars represent SEM. * denotes statistical significance by <i>t</i>-test. (B): Immunofluorescent staining for CXCL5 in <i>Dstn</i> mutant mice. Positive immunoreactivity for CXCL5 (green) in the corneal epithelium of <i>Dstn^corn1</i> mice at P9, P14, and P21 and in some stromal cells (arrowhead). CXCL5 is not expressed in WT or <i>Dstn^corn1-2J</i> epithelium at any time points tested. The central region of the cornea is shown in all P9 and P21 images. The peripheral region is shown in all P14 images. (C): Immunofluorescent staining for CXCL5 (green) with the intracellular neutrophil marker, Myeloperoxidase (red). Single slice confocal images demonstrate that CXCL5 is localized to the cytoplasm of neutrophils invading the <i>Dstn^corn1</i> cornea. Sections are counterstained with the nuclear marker, DAPI (blue). The central region of the cornea is shown. All scale bars correspond to 10 µm.</p