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 αMI-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.

The broad recognition specificity exhibited by integrin α_Mβ₂ (Mac-1, CD11b/CD18) has allowed this adhesion receptor to play innumerable roles in leukocyte biology, yet we know little about how and why α_Mβ₂ binds its multiple ligands. Within α_Mβ₂, the α_MI-domain is responsible for integrin’s multiligand binding properties. To identify its recognition motif, we screened peptide libraries spanning sequences of many known protein ligands for α_MI-domain binding and also selected the α_M I-domain recognition sequences by phage display. Analyses of >1400 binding and nonbinding peptides derived from peptide libraries showed that a key feature of the α_MI-domain recognition motif is a small core consisting of basic amino acids flanked by hydrophobic residues. Furthermore, the peptides selected by phage display conformed to a similar pattern. Identification of the recognition motif allowed the construction of an algorithm that reliably predicts the α_MI-domain binding sites in the α_Mβ₂ ligands. The recognition specificity of the α_MI-domain resembles that of some chaperones, which allows it to bind segments exposed in unfolded proteins. The disclosure of the α_Mβ₂ binding preferences allowed the prediction that cationic host defense peptides, which are strikingly enriched in the α_MI-domain recognition motifs, represent a new class of α_Mβ₂ ligands. This prediction has been tested by examining the interaction of α_Mβ₂ with the human cathelicidin peptide LL-37. LL-37 induced a potent α_Mβ₂-dependent cell migratory response and caused activation of α_Mβ₂ on neutrophils. The newly revealed recognition specificity of α_Mβ₂ toward unfolded protein segments and cationic proteins and peptides suggests that α_Mβ₂ may serve as a previously proposed “alarmin” receptor with important roles in innate host defense.

Background: Opioid peptides, including dynorphin A, besides their analgesic action in the nervous system, exert a broad spectrum of effects on cells of the immune system, including leukocyte migration, degranulation and cytokine production. The mechanisms whereby opioid peptides induce leukocyte responses are poorly understood. The integrin Mac-1 (alpha(M)beta(2), CD11b/CD18) is a multiligand receptor which mediates numerous reactions of neutrophils and monocyte/macrophages during the immune-inflammatory response. Our recent elucidation of the ligand recognition specificity of Mac-1 suggested that dynorphin A and dynorphin B contain Mac-1 recognition motifs and can potentially interact with this receptor.

Results: In this study, we have synthesized the peptide library spanning the sequence of dynorphin AB, containing dynorphin A and B, and showed that the peptides bound recombinant alpha I-M-domain, the ligand binding region of Mac-1. In addition, immobilized dynorphins A and B supported adhesion of the Mac-1-expressing cells. In binding to dynorphins A and B, Mac-1 cooperated with cell surface proteoglycans since both anti-Mac-1 function-blocking reagents and heparin were required to block adhesion. Further focusing on dynorphin A, we showed that its interaction with the alpha I-M-domain was activation independent as both the alpha 7 helix-truncated (active conformation) and helix-extended (nonactive conformation) alpha I-M-domains efficiently bound dynorphin A. Dynorphin A induced a potent migratory response of Mac-1-expressing, but not Mac-1-deficient leukocytes, and enhanced Mac-1-mediated phagocytosis of latex beads by murine IC-21 macrophages.

Conclusions: Together, the results identify dynorphins A and B as novel ligands for Mac-1 and suggest a role for the Dynorphin A-Mac-1 interactions in the induction of nonopiod receptor-dependent effects in leukocytes.

Over the last two decades, our knowledge concerning intracellular events that regulate integrin’s affinity to their soluble ligands has significantly improved. However, the mechanism of adhesion-induced integrin clustering and development of focal complexes, which could further mature to form focal adhesions, still remains under-investigated. Here we present a structural model of tandem IgC2 domains of skelemin in complex with the cytoplasmic tails of integrin α[subscript IIb]β[subscript 3]. The model of tertiary assembly is generated based upon NMR data and illuminates a potential link between the essential cell adhesion receptors and myosin filaments. This connection may serve as a basis for generating the mechanical forces necessary for cell migration and remodeling.