Abstract
Factor J (FJ) is a complement inhibitor that acts on the classical and the alternative pathways. We demonstrated FJ-cell interactions in fluid phase by flow cytometry experiments using the cell lines Jurkat, K562, JY, and peripheral blood lymphocytes. FJ bound to plastic plates was able to induce in vitro adhesion of these cells with potency equivalent to fibronectin. As evidence for the specificity of this reaction, the adhesion was blocked by MAJ2, an anti-FJ monoclonal antibody, and by soluble FJ. Attachment of the cells required active metabolism and cytoskeletal integrity. The glycosaminoglycans heparin, heparan sulfate, or chondroitin sulfates A, B, and C inhibited to varying degrees the binding of FJ to cells, as did treatment with chondroitinase ABC. In the search for a putative receptor, a protein of 110 kDa was isolated by affinity chromatography, and microsequence analysis identified this protein as nucleolin. Confocal microscopy evidenced the presence of nucleolin in cell membrane by immunofluorescence with monoclonal (D3) and polyclonal anti-nucleolin antibodies in Jurkat cells. The interaction FJ-nucleolin was evidenced by Western blot and enzyme-linked immunosorbent assay. Furthermore, purified nucleolin and D3 inhibited adhesion of Jurkat cells to immobilized FJ, suggesting that the interaction was specific and that nucleolin mediated the binding.
Highlights
Factor J (FJ) is a complement inhibitor that acts on the classical and the alternative pathways
Maximal attachment was observed with Vero, JY, and Jurkat cell lines, and the lowest value corresponded to U937 (3%)
Our results described the ability of FJ, a complement inhibitor, to induce cell adhesion
Summary
Factor J (FJ) is a complement inhibitor that acts on the classical and the alternative pathways. We demonstrated FJ-cell interactions in fluid phase by flow cytometry experiments using the cell lines Jurkat, K562, JY, and peripheral blood lymphocytes. Purified nucleolin and D3 inhibited adhesion of Jurkat cells to immobilized FJ, suggesting that the interaction was specific and that nucleolin mediated the binding. CD11b/CD18 (Mac1) binds several soluble ligands including the complement fragment iC3b [10]. Heparin-binding proteins constitute a diverse group that includes extracellular matrix molecules, growth factors, degradative and lipolytic enzymes, protease inhibitors, nuclear proteins, and lipoproteins. The aim of this work was to investigate whether FJ could interact with different cell lines and peripheral blood lymphocytes (PBL). We analyzed FJ-cell interactions by flow cytometry and in adhesion assays using FJ immobilized on plastic plates.
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