Abstract
Complement activation is associated with a variety of inflammatory conditions including atherosclerosis, but the mechanism of complement activation in these settings is poorly understood. Endothelial cells (EC) play an important role in vascular pathology and express a variety of complement receptors, including gC1qR/p33, recognizing the globular domain of the complement component C1q. In preliminary studies, purified recombinant gC1qR/p33 was found to support C1q-dependent C4 activation in vitro, comprising 19.5% ± 8.3% (mean ± S.D., n=5) of that produced by aggregated IgG. In contrast, a truncated form of gC1qR/p33, lacking the C1q binding domain, failed to support C4 activation. Additional studies were performed with immortalized bone marrow microvascular EC to investigate classical complement pathway activation and deposition. EC were exposed to anticoagulated (0.32 % sodium citrate) human plasma, diluted (1/10) in 0.01 M HEPES buffered modified Tyrode’s solution, pH 7.5, containing 2 mM Mg Cl2 and 1 mM CaCl2, for 60 min, 37°C. A solid phase ELISA approach was used to detect EC-associated C1q and C4 activation (C4d). Statistically significant deposition of C4d (0.72 ± 0.3. OD units (ODU), n=4)(p=0.04) and C1q (0.57 ± 0.19. ODU, n=4) (p=0.002) was observed on EC that had been immobilized on poly-L-lysine coated microtiter wells. Consistent with classical complement pathway activation, C4d deposition remained at baseline (0.23 ± 0.13, ODU, n=4) in the presence of 10 mM EDTA, but C1q deposition was unaffected. Moreover, no significant C1q or C4d deposition occurred when endothelial cells were exposed to C1q depleted serum. Similar studies were performed using EC grown to confluence on Type I collagen to examine the effect of shear stress (12 dynes/cm2 for 1 hour in a cone-and-plate shearing device), simulating flow conditions in coronary arteries, on classical complement pathway activation and deposition. Compared to static conditions, shear stress resulted in an approximately 50% increase in C1q and C4d deposition on EC. This was accompanied by an approximately 2-fold increase in EC binding of a monoclonal antibody, 60.11, recognizing the N-terminal C1q binding domain of gC1qR/p33. Taken together, these data present evidence for a potential paradigm shift, illustrating immune complex independent classical complement pathway activation by gC1qR/p33, and deposition of activated classical complement components on EC. The generation and deposition of active complement components on EC is likely to contribute directly to vascular inflammation and atherosclerotic changes.
Role of immunoglobulin G in killing of Borrelia burgdorferi by the classical complement pathway.