Modulation of the complement system by human beta-defensin 2

Abstract

Objective: Human beta-defensin (HBD) and the complement system are two important innate immune mechanisms active against a broad range of burn and wound pathogens. However, excessive or uncontrolled complement activation, following thermal injury, contributes to tissue damage. Previous studies from our laboratory suggested a decreased expression of HBD-2 in burn wounds and its absence in burn blister fluid. Prior studies have demonstrated that human neutrophil peptide can bind to the C1q component of the complement system and prevent complement activation. The objective of this study was to determine whether HBD-1 and HBD-2 can also bind to the C1q component and modulate complement activity.

Methods: The binding efficiency of HBD-1 and HBD-2 to the C1q component was determined by utilizing dot blot hybridization. The effect of HBD-2 on the activation of the complement system by the classical and alternative pathways was determined by CH50 and AP50 assays. In addition, the ability of HBD-1 and HBD-2 to inhibit C1q activity was predicted by a comparison with known C1q inhibitor peptide 2J in a DNAStar computer modeling study.

Results: C1q binding to HBD-2 was strong, whereas C1q binding to HBD-1 was weak. HBD-2 inhibits the classical pathway significantly without affecting the alternative pathway. In addition, a computer modeling study also revealed structural homology of HBD-2 with known C1q inhibitory sequences of HBD-2.

Conclusion: HBD-2 inhibits the classical pathway. The replacement of missing defensin, a natural inhibitor of the complement system, may have a dual protective role not only as an antimicrobial agent but also in providing protection against uncontrolled activation of the complement system.

Figure 1

The complement system consists of about 30 proteins that can be activated by 3 different cascades. The classical pathway is initiated when antigen-antibody complexes bind to the C1q component of C1, resulting in release of C1s and C1r, which act as protease to cleave C2 and C4. The mannin-binding lectin (MBL) pathway is triggered by activation of MBL-associated serine proteinases (MASP-1 and MASP-2) following the binding of polysaccharides on microbes to MBL. MASP-1 and MASP-2 convert C2 and C4. The alternative pathway is triggered by the deposition of spontaneously generated C3b on microbes, leading to the generation of alterative C3 convertase. The common final step is the activation of C5 by C5 convertases, which leads to the formation of the membrane attack complex that finally inserts into target cell membranes and causes cell lysis. The peptides C3a, C4a, and C5a mediate several reactions in the inflammatory response that could cause tissue injury when complement activation becomes uncontrolled.

Figure 2

The dot blot hybridizations were carried out as described in the “Material and Methods” section. The quantified Phosphor Imager counts were plotted against different peptide concentrations. *Significance (P < .05).

Figure 3

The effect of HBD-2 on hemolytic activity (CH50 and AP50) was determined as described in the “Material and Methods” section. The percentage of lysis was determined relative to a reagent blank and 100% lysis, expressed as units/mL (Z) and converted to percentage inhibition. *Significance (P < .05).

Figure 4

Alfa helix display of consensus sequence between peptide 2J and HBD-2 and their overlap using Laser Gene software.