CLASSICAL COMPLEMENT PATHWAY ACTIVATION ON NUCLEATED CELLS - ROLE OF FACTOR-H IN THE CONTROL OF DEPOSITED C3B

The restriction of alternative complement pathway activation in fluid phase or on nonactivator surfaces has been described as the major phys iologic function of the complement regulatory protein factor H. In thi s study, we provide evidence that factor H is also a restriction facto r of classical pathway activation on the surface of nucleated cells. W e found that C3b was rapidly converted to inactivated C3b (iC3b) on hu man SK-MEL-93-2 melanoma cells after classical pathway activation with the murine monoclonal IgG3 Ab R24 directed against the disialoganglio side surface Ag G(D3). The SK-h4EL-93-2 cells are nonactivators of the alternative pathway and express neither CR1 (CD35) nor the C3b-cleavi ng protease p65. The cells are further characterized by the expression of only moderate amounts of DAF (CD55) and approximately 5 x 10(3) MC P (CD46) molecules/cell. FAGS analysis and direct quantitation using [ (125)l]factor H revealed high level binding of factor H to the melanom a cells (5.6 x 10(6) molecules/cell) during classical pathway activati on. The binding of factor H could be inhibited under conditions that i nactivate the classical complement pathway (EGTA and heat treatment), but not by factor B depletion of the serum, demonstrating that classic al pathway activation was responsible for factor H binding. Treatment of factor B-depleted serum with neutralizing concentrations of polyclo nal anti-factor H resulted in the prolonged presence of intact C3b on the cells and a significantly reduced generation of iC3b. The increase d amount of C3b on these cells correlated with a 2.65-foId greater rat e of cell death. In contrast, the increase in cell death effected by n eutralizing concentrations of anti-CD46 or anti-CD55 Ab was only 0.13- or 0.35-fold, respectively. In addition, the supplementation of serum with purified factor H decreased the extent of lysis of the cells. Co llectively, these data provide experimental evidence that factor H, th rough its cofactor activity for C3b degradation, is involved in the re striction of the classical pathway of complement on the surface of nuc leated cells, a function that to date has been exclusively attributed to the membrane regulatory proteins CD35 and CD46.