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. 2007 Feb 1;26(4):1129–1139. doi: 10.1038/sj.emboj.7601552

Figure 4.

Figure 4

HMGB1-mediated chemotaxis and transendothelial migration of leukocytes. (A) Immunofluorescence for Syk (green) or F-actin (red) was performed followed by confocal microscopy. Representative immunofluorescence of THP-1 cells that were incubated in the absence (−) or presence of HMGB1 (50 ng/ml) for 30 min is shown. Double-stained images were merged. HMGB1 induced the enrichment of F-actin and Syk staining at the leading edge of the cell. (B) Chemotaxis of wild-type mouse neutrophils towards no chemoattractant (open bar), 50 ng/ml MIP-2 (gray bar) or 50 ng/ml HMGB1 (filled bar) is shown. (C) Chemotaxis of wild-type, LFA-1−/−, Mac-1−/−, and RAGE−/− mouse neutrophils towards no chemoattractant (open bars), or 50 ng/ml HMGB1 (filled bars) is shown. (D) Chemotaxis of human neutrophils towards no chemoattractant (open bar), 50 ng/ml IL-8 (gray bar) or 50 ng/ml HMGB1 (filled bars) is shown in the absence (−) or presence of blocking mAb to Mac-1, mAb to LFA-1, soluble RAGE, or antibody to HMGB1 (each at 20 μg/ml). In (B), (C), and (D), chemotaxis data are shown as percent of control. In (B) and (C), chemotaxis of wild-type mouse neutrophils in the absence of stimuli or competitors represents the 100% control; in (D) chemotaxis of human neutrophils in the absence of stimuli or competitors represents the 100% control. *P<0.02; ns: not significant. (E) The transmigration of human neutrophils towards 50 ng/ml IL-8 (gray bars) or 50 ng/ml HMGB1 (filled bars) across HUVEC is shown in the absence (−) or presence of blocking mAb to Mac-1, mAb to LFA-1, soluble RAGE, or antibody to HMGB1 (each at 20 μg/ml). Transmigration is represented as percent of control. Transmigration through HUVEC in the absence of stimuli or competitors represents the 100% control. +P<0.01 as compared to transmigration towards IL-8 (gray bars) and in the absence of competitors (−); ns1: not significant as compared to transmigration towards IL-8 (gray bars) and in the absence of competitors (−); &P<0.01 as compared to transmigration towards HMGB1 (filled bars) and in the absence of competitors (−); ns2: not significant as compared to transmigration towards HMGB1 (filled bars) and in the absence of competitors (−). Data are mean±s.d. of three independent experiments each performed in triplicate.