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S100A8 Triggers Oxidation-sensitive Repulsion of Neutrophils

¹ Department of Oral Medicine and Diagnostic Sciences, University of Illinois at Chicago, College of Dentistry (M/C 838), 801 S. Paulina St., Room 556, Chicago, IL 60612-7213, USA;
² Department of Medicine,
³ Howard Hughes Medical Institute, and
⁴ Department of Oral and Maxillofacial Surgery, University of California at San Francisco, San Francisco, CA, USA

View larger version (21K): [in a new window]   Figure 1. Transwell migration of peripheral neutrophils. (A) The data are the mean of a representative experiment conducted in triplicate (± SD) with cells isolated from different healthy adult volunteers. Il-8 was introduced in the lower chamber. S100A8 was introduced in the lower or upper chamber of the Transwells. Analysis of the data indicated a dose-dependent increase in neutrophil migration to the lower chambers of the Transwells when IL-8 and S100A8 were added to the lower and upper chambers, respectively. The addition of S100A8 to the lower wells did not have any measurable effect at the concentrations and conditions tested (#p < 0.05; *p < 0.01 when compared with control with no compound added). (B) The data represent the average of 4 experiments performed in duplicate (± SD) with cells isolated from different adult volunteers. S100A8 was introduced in the lower, upper, or both chambers of the Transwells at a concentration of 10–9 M. Analysis of the data indicated that the increased migration of neutrophils observed when S100A8 was added to the upper well was inhibited by the addition of S100A8 at an equimolar concentration in the lower well (*p < 0.01 when compared with control with no compound added). (C) The data represent the average of 2 experiments performed in duplicate (± SD) with cells isolated from different adult volunteers. S100A8 was added to the upper chamber of the Transwell at a concentration of 10–9 M. Il-8-mediated chemotaxis served as a positive control for the effect of pertussis toxin and was added to the lower chamber of the Transwell at a concentration of 10–9 M. The cells were incubated with different concentrations of pertussis toxin for 30 min at room temperature before the beginning of the assay. Analysis of the data indicated that the fugetactic effect of S100A8 was pertussis-toxin-sensitive. Il-8-mediated chemotaxis displayed a similar sensitivity (*p < 0.01 when compared with the migration ratio with no pertussis toxin for IL-8 and S100A8 separately).

View larger version (28K): [in a new window]   Figure 2. Non-reducing gel electrophoresis and chemotaxis assays of recombinant wild-type (WT) S100A8 and ala42S100A8 exposed to 10–5M hypochlorite (OCl). (A) Coomassie-blue-stained gel (1) WT S100A8 and (2) ala42S100A8. ala42S100A8 did not form covalently bound dimers. (B) Western blot analysis of WT S100A8 and mutated S100A8 with monoclonal antibodies to S100A8. (1) WT S100A8; (2) ala42S100A8. The antibodies recognized both WT S100A8 and ala42S100A8. (C) The data represent the average of 3 experiments performed in duplicate (± SD) with cells isolated from different adult volunteers. Transwell fugetaxis assays of neutrophils with WT S100A8 and ala42S100A8 oxidized by 10–5 M OCl on ice for 30 min. The fugetactic effect of ala42S100A8 was resistant to oxidation, whereas the WT S100A8 protein’s fugetactic effect was inhibited by oxidation (#p < 0.05; *p < 0.01 when compared with control with no compound added). We calculated the migration ratio by dividing the number of cells that migrated to the lower well in each experiment by the number of cells in the lower well in the untreated control wells.

View larger version (12K): [in a new window]   Figure 3. Effect of ala42S100A8 on lipopolysaccharide (LPS)-induced recruitment of neutrophils in a rat air-pouch model. The data represent a mean (± SD). (A) Air-pouches were injected with 300 µL of a PBS control, 30 ng LPS in PBS, and 30 ng LPS in PBS containing 10–5 M of ala42S100A8. Co-injection of ala42S100A8 inhibited the inflammatory response induced by LPS, as indicated by the number of neutrophils recruited by LPS into the air-pouch (*p < 0.01 when compared with PBS-only control). (B) Dose response of the inhibitory effect of ala42S100A8 on the recruitment of neutrophils induced by LPS in the air-pouch model. All the air-pouches were injected with 30 ng LPS in 300 µL PBS containing different concentrations of ala42S100A8. ala42S100A8 displayed dose-dependent inhibition of the recruitment of neutrophils (#p < 0.05; *p < 0.01 when compared with LPS-only control; n indicates the number of rats in each treatment group). PMN, polymorphonuclear leukocyte.