LL-37/hCAP-18 is the only human member of the cathelicidin family and plays an important role in killing various pathogens, as well as in immune modulation. In this study, we investigated the effect of LL-37 on bacterial phagocytosis by macrophages and demonstrate that LL-37 enhances phagocytosis of IgG-opsonized Gram-negative and Gram-positive bacteria in a dose- and time-dependent manner by dTHP-1 cells. In addition, LL-37 enhanced phagocytosis of nonopsonized Escherichia coli by human macrophages. Consistently, LL-37 elevated the expression of FcγRs on macrophages but not the complement receptors CD11b and -c. Further studies revealed that the expression of TLR4 and CD14 is also increased on LL-37-treated macrophages. Several lines of evidence indicated that the FPR2/ALX receptor mediated LL-37-induced phagocytosis. However, TLR4 signaling was also coupled to the phagocytic response, as a specific TLR4 antibody significantly suppressed phagocytosis of IgG-opsonized E. coli and nonopsonized E. coli by dTHP-1 cells. Finally, macrophages from Cnlp^−/− mice exhibited diminished bacterial phagocytosis compared with macrophages from their WT littermates. In conclusion, we demonstrate a novel, immune-modulatory mechanism of LL-37, which may contribute to bacterial clearance.

Bioactive peptide LL-37/hCAP18, the only human member of the cathelicidin family, plays important roles in killing various pathogens, as well as in immune modulation. We demonstrate that LL-37 is internalized by human macrophages in a time-, dose-, temperature-, and peptide sequence–dependent endocytotic process. Both clathrin- and caveolae/lipid raft–mediated endocytosis pathways are involved in LL-37 internalization. We find that the P2X₇ receptor (P2X₇R) plays an important role in LL-37 internalization by human macrophages because significantly less internalized LL-37 was detected in macrophages pretreated with P2X₇R antagonists or, more specifically, in differentiated THP-1 cells in which the P2X₇R gene had been silenced. Furthermore, this P2X₇R-mediated LL-37 internalization is primarily connected to the clathrin-mediated endocytosis pathway. In addition, our results demonstrate that internalized LL-37 traffics to endosomes and lysosomes and contributes to intracellular clearance of bacteria by human macrophages, coinciding with increased reactive oxygen species and lysosome formation. Finally, we show that human macrophages have the potential to import LL-37 released from activated human neutrophils. In conclusion, our study unveils a novel mechanism by which human macrophages internalize antimicrobial peptides to improve their intracellular pathogen clearance.

In humans, the antimicrobial peptide LL‐37 and leukotriene B ₄ (LTB ₄ ) are important proinflammatory mediators, whereas lipoxin A ₄ (LXA ₄ ) and resolvin E1 (RvE1) possess anti‐inflammatory, prore‐solving properties. Previously, we reported that LTB ₄ triggers LL‐37 release from human neutrophils (PMNs) and, conversely, that LL‐37 promotes LTB ₄ production from these cells. Here we show that this effect of LL‐37 is mediated via the GPCR FPR2/ALX. LL‐37 (5–30 µg/ml) induces intracellular calcium mobilization in a dose‐dependent manner, and the signal transduction leading to LTB ₄ release involves p38 MAP kinase and phosphorylation of cPLA ₂ . LXA ₄ , an endogenous lipid ligand of FPR2/ALX, and a stable LXA ₄ analog [benzo‐LXA ₄ ] were ineffective as stimuli at the concentrations of 0.1–10 nM for LTB ₄ release from PMNs. Likewise, the BLT1 ligand RvE1, a derivative of eicosapentaenoic acid, inhibited LTB ₄ ‐induced LL‐37 production from PMNs at 1–100 nM, whereas chemerin, a peptide ligand of the RvE1 receptor ChemR23, failed to block LTB ₄ ‐induced LL‐37 release at the same concentrations. Hence, in human neutrophils, binding of LL‐37 to FPR2/ALX promotes LTB ₄ production, which can bind to BLT1 and elicit further LL‐37 release. This proin‐flammatory circuit might be inhibited by LXA ₄ and RvE ₁ acting at FPR2/ALX and BLT1, respectively, leading to dampened mediator release.—Wan, M., Godson, C., Guiry, P. J., Agerberth, B., Haeggström, J. Z. Leukotriene B ₄ /antimicrobial peptide LL‐37 proinflammatory circuits are mediated by BLT1 and FPR2/ALX and are counterregulated by lipoxin A ₄ and resolvin E1. FASEB J. 25, 1697–1705 (2011). www.fasebj.org