Effects of Polymeric C3, C3b and iC3b on Neutrophil Expression of CD11b and CD18

Complement C3 C3b iC3b Neutrophil adhesion Correspondence to: Dr. M. Michael Glovsky, I luntington Memorial Hospital, Asthma and Allergy Center, 39 Congress Street, Suite 301, Pasadena, CA 91105 (USA), Tel. (818) 397 3383, Fax (818) 795 0982 Introduction Complement activation in vivo in rats is known to induce neutrophil (PMN) activation and endothelial damage in the lung and other organs [1]. The mechanisms for this injury are thought to involve C3, PMN-CDllb/CD18 selectin and endothelial adhesion molecules 1-CAM 1 and Pselectin [2, 3]. To clarify these events in vitro, we have purified C3, C3b and iC3b. The effect of C3 and its products were tested in human PMN expression of the B2-selectin, CDllb/CD18. Results Since the CD 1 lb/CD 18 complex is known to possess binding sites for iC3b, we explored the role of C3, C3b and iC3b on CDllb/CD18 expression with human PMNs. C3, C3b and iC3b were purified from human plasma by precipitation with polyethylene glycol, anion exchange chromatography and gel filtration [4]. C3 was further purified to remove traces of C5, IgE and factor 11. These procedures yielded C3 which was more than 98% pure. C3b was prepared from C3 by treatment with 1 μg trypsin/mg C3. The reaction was stopped with soybean trypsin inhibitor. iC3b was prepared from C3b by treatment with 9 μg factor H and 5 μg factor I/mg C3b. Both C3b and iC3b were further purified by gel filtration in Biogel A 0.5 m (BioRad, Richmond, Calif, USA). Purified C3, C3b and iC3b were cross-linked with dimethyl superimidate [5]. The effects of native (non-cross-linked) and cross-linked C3, C3b and iC3b were studied by cytofluorimetry on uptake of C3b as well as expression of CDllb/CD18. Cross-linked C3b bound to PMNs with five times greater fluorescent intensity than native C3b. To study CDllb/CD18 upregulation, PMNs were stimulated with FMLP (10”5 M) and showed a Ill% increase of both CD lib and CD18. Nonstimulated PMNs were incubated with native and cross-linked (1.25, 2.5 and 5.0 μg) C3, C3b and iC3b. Dose-response increases of CDlδ > CDllb were found with cross-linked C3b and iC3b. Cross-linked iC3b increased CD18 expression by 8,