Identification of human cathelicidin peptide LL-37 as a ligand for macrophage integrin α_Mβ₂ (Mac-1, CD11b/CD18) that promotes phagocytosis by opsonizing bacteria

Abstract:

LL-37, a cationic antimicrobial peptide, has numerous immune-modulating effects. However, the identity of a receptor that mediates the responses in immune cells remains uncertain. We have recently demonstrated that LL-37 interacts with the α_MI-domain of integrin α_Mβ₂ (Mac-1), a major receptor on the surface of myeloid cells, and induces a migratory response in Mac-1-expressing monocyte/macrophages as well as activation of Mac-1 on neutrophils. Here, we show that LL-37 and its C-terminal derivative supported strong adhesion of various Mac-1-expressing cells, including human embryonic kidney cells stably transfected with Mac-1, human U937 monocytic cells, and murine IC-21 macrophages. The cell adhesion to LL-37 was partially inhibited by specific Mac-1 antagonists, including monoclonal antibody against the α_M integrin subunit and neutrophil inhibitory factor, and completely blocked when anti-Mac-1 antibodies were combined with heparin, suggesting that cell surface heparan sulfate proteoglycans act cooperatively with integrin Mac-1. Coating both gram-negative and gram-positive bacteria with LL-37 significantly potentiated their phagocytosis by macrophages, and this process was blocked by a combination of anti-Mac-1 monoclonal antibody and heparin. Furthermore, phagocytosis by wild-type murine peritoneal macrophages of LL-37-coated latex beads, a model of foreign surfaces, was several fold higher than that of untreated beads. In contrast, LL-37 failed to augment phagocytosis of beads by Mac-1-deficient macrophages. These results identify LL-37 as a novel ligand for integrin Mac-1 and demonstrate that the interaction between Mac-1 on macrophages and bacteria-bound LL-37 promotes phagocytosis.

Keywords::

• LL-37
• integrin α_Mβ₂
• Mac-1
• CD11b/CD18
• phagocytosis
• opsonin

Acknowledgments

The authors thank Yishin Shi, the School of Life Sciences, Arizona State University, for providing Salmonella bacterial strains and advice. This article was presented at the Annual Meeting of Experimental Biology, March 28–April 1, 2015, Boston, and published in the abstract form in FASEB J, 29:571.15. This work was supported by the NIH grant HL 63199.

Disclosure

The authors report no conflicts of interest in this work.

Supplementary material

Figure S1 Correlation between adhesion of Mac-1-expressing cells to immobilized LL-37-GY and the surface density of plastic-bound LL-37-GY. Various concentrations of ₁₂₅I-labeled LL-37-GY were immobilized on wells of 96-well microtiter plates under the same conditions used for adhesion assays.

Notes: The plates were washed and bound radioactivity was measured in a g-counter. Right ordinate shows cpm/well (o) and left ordinate shows fluorescence of cells adherent to wells (·) coated with LL-37-GY.