The *C5a chemotactic cofactor function of the vitamin D binding protein is regulated by thrombospondin 1 and CD36

The chemotactic activity of C5a and C5a des Arg can be enhanced significantly by the vitamin D binding protein (DBP), also known as Gc-globulin. DBP is a multifunctional 56--58 kDa plasma protein that can bind and transport several diverse ligands. Neutrophils have been utilized as the primary cell type to investigate the cochemotactic mechanisms of DBP. Since neutrophils are very complex cells that will display chemotactic movement to a myriad of stimuli, we sought to identify a defined cell culture model to study the chemotactic cofactor function of DBP. Previously, we generated undifferentiated U937 cells transfected with the C5a receptor (U937-C5aR cells). The objective of this dissertation was to determine if this transfected cell line could be utilized to investigate the mechanism by which DBP functions as the C5a chemotactic cofactor. The results demonstrate that U937-C5aR cells function as a de facto mutant, since they show C5a chemotactic enhancement only to DBP in serum but, unlike neutrophils, this cell line cannot respond to purified DBP or DBP in plasma. Pretreating U937-C5aR cells with either serum or activated platelet releasate supplements a missing factor and permits purified DBP to function as a chemotactic cofactor for C5a. These results clearly indicate that the immature undifferentiated U937-C5aR cells lack an essential cell surface molecule that permits DBP to function as a chemotactic cofactor for C5a. We have identified the molecule as thrombospondin 1 (TSP1), a homotrimeric glycoprotein released by activated platelets and neutrophils, and propose that TSP1 regulates the co-chemotactic function of DBP in neutrophils and in U937-C5aR via its cell surface receptor, CD36.