Cofactor regulation of C5a chemotactic activity in physiological fluids. Requirement for the vitamin D binding protein, thrombospondin-1 and its receptors

Abstract

Factors in physiological fluids that regulate the chemotactic activity of complement activation peptides C5a and C5a des Arg are not well understood. The vitamin D binding protein (DBP) has been shown to significantly enhance chemotaxis to C5a/C5a des Arg. More recently, platelet-derived thrombospondin-1 (TSP-1) has been shown to facilitate the augmentation of C5a-induced chemotaxis by DBP. The objective of this study was to better characterize these chemotactic cofactors and investigate the role that cell surface TSP-1 receptors CD36 and CD47 may play in this process. The chemotactic activity in C-activated normal serum, citrated plasma, DBP-depleted serum or C5 depleted serum was determined for both normal human neutrophils and U937 cell line transfected with the C5a receptor (U937-C5aR). In addition, levels of C5a des Arg, DBP and TSP-1 in these fluids were measured by RIA or ELISA. Results show that there is a clear hierarchy with C5a being the essential primary signal (DBP or TSP-1 will not function in the absence of C5a), DBP the necessary cofactor and TSP-1 a dependent tertiary factor, since it cannot function to enhance chemotaxis to C5a without DBP. Measurement of the C5a-induced intracellular calcium flux confirmed the same hierarchy observed with chemotaxis. Moreover, analysis of bronchoalveolar lavage fluid (BALF) from patients with the adult respiratory distress syndrome (ARDS) demonstrated that C5a-dependent chemotactic activity is significantly decreased after anti-DBP treatment. Finally, results show that TSP-1 utilizes cell surface receptors CD36 and CD47 to augment chemotaxis, but DBP does not bind to TSP-1, CD36 or CD47. The results clearly demonstrate that C5a/C5a des Arg needs both DBP and TSP-1 for maximal chemotactic activity and suggest that the regulation of C5a chemotactic activity in physiological fluids is more complex than previously thought.

Systemic alterations in plasma proteins from women with chronic widespread pain compared to healthy controls: a proteomic study

“…The cause of elevated levels of VDBP in the CWP group is unknown; one theory could be physiological, since the levels of vitamin D is increased, this could follow with increased levels of VDBP, due to elevated transportation of the protein is required. Another theory could be VDBP’s function as a cofactor, together with one of the by-products from the alternative and classical pathway of complement, c5a, which essentially needs VDBP to be able to work as chemoattractant for neutrophils 56. One could speculate that since several proteins from the complement system are upregulated in the CWP group, it could possibly lead to elevated levels of complement byproducts.…”

Circulating complexes of the vitamin D binding protein with G-actin induce lung inflammation by targeting endothelial cells

“…Measurement of cell movement of normal human neutrophils and differentiated HL-60 cells was performed using a 48 well microchemotaxis chamber as previously described in detail (Trujillo, et al, 2011). Human neutrophils were isolated from the venous blood of healthy medication-free volunteers who gave informed consent using a standard 3-step purification protocol (Trujillo, et al, 2011).…”

“…Human neutrophils were isolated from the venous blood of healthy medication-free volunteers who gave informed consent using a standard 3-step purification protocol (Trujillo, et al, 2011). The human promyelocytic cell line HL-60 was also used and differentiated for 4 days using 1.3% DMSO to obtain a neutrophil-like phenotype (Hauert, et al, 2002).…”

Behind the scenes of vitamin D binding protein: More than vitamin D binding

“…Although there is no evidence that TSP-1 directly interacts with either annexin A2 or CD44, indirect associations between TSP-1 and CD44 have been proposed. One or more of the multiple ligands of both players could bridge TSP-1 and CD44, facilitating the DBP binding site complex [99]. receptor, CYP27B1 and the 1,25(OH) 2 -vitamin D 3 -24-hydroxylase CYP24A1 of the cells locally involved in the immune response [110].…”