Complement component C3 is required for recruitment and retention of Ly-6G⁺ cells in the splenic MZ after Ad injection.

<p>(A) Localization of Ly-6G⁺ cells in the MZ of <i>Il1a/b</i>^−/− mice 2 hours after Ad injection in relation to virus-containing MZ cells. Spleen sections of virus-injected <i>Il1a/b</i>^−/− mice were simultaneously stained with FITC anti-Ly-6G (green) and anti-Ad-hexon (red) antibodies and pictures were taken using a Leica fluorescent microscope. The regions of the spleen that are selected by rectangles A and B on the left panel are shown on the right panels at higher magnification. Scale bar is 25 µm. Anatomical borders of germinal centers are depicted by the dotted lines. Ly-6G⁺ cells that are in contact with virus-containing cells in the MZ are indicated by arrows. Representative spleen sections are shown. N = 5. (B) Distribution of Ly-6G⁺ leukocytes on sections of the spleens harvested from <i>WT</i> and complement component C3-deficient (<i>C3</i>^−/−) mice injected with Ad virus. Spleen sections were simultaneously stained with FITC anti-Ly-6G (green) and anti-Ad-hexon (red) antibodies and pictures were taken using a Leica fluorescent microscope. Representative spleen sections for each experimental setting are shown. N = 5. Scale bar is 100 µm. (C) Quantitative representation of Ly-6G⁺ (PMN) distribution on sections of the spleens of <i>WT</i> and <i>C3</i>^−/− mice injected with Ad 2 hours after the virus administration. The quantification of Ly-6G⁺ cell distribution was done as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004035#ppat-1004035-g003" target="_blank">Figure 3B</a>. N = 5. RP – red pulp; MZ – marginal zone. * - P<0.05, compared to corresponding mock groups. Inverted triangle – P<0.05, compared to Ly-6G⁺ cell distribution observed for corresponding compartments in <i>WT</i> mice. (D) Mouse cytokine array panel showing expression of the proinflammatory cytokines and chemokines in spleens of <i>WT</i> and <i>C3</i>^−/− mice injected with saline (Mock) or Ad5 (HAdv5) 1 hour after virus injection, determined by Proteome Profiler antibody array. Representative blots from three independent experiments are shown. Pos-C are dots that show the manufacturer's internal positive control samples on the membrane.