Characterization of Combined Effects of Reactive Oxygen Metabolites, Complement System, and Antimicrobial Peptides In Vitro

Objective: Phagocytes activation results in the production of reactive oxygen metabolites exerting antimicrobial and host-damaging activity. Although the main pool of papers shows their potentiating action on a key humoral nexus of innate immunity, complement system, the data are controversial. Combined action of reac­tive oxygen metabolites with antimicrobial peptides of phagocytes also remains poorly characterized. Methods: We have investigated the influence of oxidative burst products on complement activation in various in vitro models and assessed the combined bactericidal action of hypochlorous acid with antimicrobial peptides. Results and Discussion: Hydrogen peroxide, including that in medium with Fe-EDTA did not affect parameters of complement activity in human blood serum. HOCl in millimolar concentrations stimulated production of C3a and C5a anaphylatoxins in 80% serum, the effect was inhibited by EDTA. We have identified bivalent ions-independent C5 cleavage in the presence of 16 mM HOCl. At the same time, HOCl served as an inhibitor of the alternative complement pathway in the model of membrane-associated activation in 5% serum in the presence of Mg-EGTA and rabbit erythrocytes. It inhibited production of C3a (IC₅₀ ~4 mM) and C5a as well as serum hemolytic activity (IC₅₀ ~0.2 mM). The inhibition of C5a generation was less pro­nounced in the presence of relatively high HOCl concentration (4–16 mM). Decrease in anaphylatoxins generation was also observed in the system with zymosan in 5% serum with Mg-EGTA. Under similar conditions but without activating surfaces, moderate HOCl concentrations enhanced C3a accumulation and C5a accumulation; EDTA inhibited this effect completely (C3a) or partially (C5a). Finally, in 70% serum, 16 mM HOCl enhanced the anaphylatoxins accumulation in the absence of zymosan but it inhibited this process almost completely under the conditions of the zymosan-triggered amplification loop. According to our hypothesis, HOCl can attack the thioester bond in C3 protein to form C3(HOCl) adduct which is capable of fluid-phase C3 and C5 convertases formation; however, the attack of the same group in C3b can prevent its covalent fixation on membranes and blocks the complement amplification loop. In addition, the transformation of C3 to C3(HOCl) which is not able to serve as a substrate of C3 convertases may also be responsible for complement inhibition. Besides, we have demonstrated the additive character of the combined action of HOCl with antimicrobial peptides (LL-37 cathelici­din and α-defensins HNPs) against Listeria monocytogenes and Escherichia coli. Conclusions: According to our results, hydrogen peroxide and hydroxyl radical do not participate in complement modulation. HOCl is an activator of fluid-phase and an inhibitor of surface complement activation. HOCl may also induce complement-independent C5 cleavage in serum. HOCl and antimicrobial peptides of phagocytes kill bacteria in an additive manner. The data obtained clarify the picture of the interaction between bactericidal factors of phagocytes and complement as key participants of the immune defense and host damage.