Abstract
LL-37 is the sole antimicrobial peptide of human cathelicidin comprising 37 amino acids, which is expressed mainly in epithelial cells and neutrophils, and activates mast cells. In the present study, in order to elucidate the mechanism of mast cell activation by LL-37, the associations between the internalization of LL-37 and Mas-related gene X2 (MrgX2)-mediated mast cell activation (degranulation) was investigated using the human mast cell line, LAD2. LL-37 was rapidly internalized into the cells, and induced degranulation, as assessed by the extracellular release of β-hexosaminidase. Pertussis toxin, a G-protein inhibitor, significantly suppressed the internalization of LL-37 and the degranulation of LAD2 cells. Furthermore, small interfering (si)-RNA-mediated knockdown of MrgX2, a putative G protein-coupled receptor for LL-37, inhibited the internalization of LL-37 and degranulation of LAD2 cells. Notably, LL-37 internalization was enhanced by the stable expression of MrgX2 in HMC-1 and 293 cells. In addition, the internalized LL-37 mainly colocalized with MrgX2 in the perinuclear region of LAD2 cells. Furthermore, neuraminidase treatment, which removes negatively charged sialic acid from the cell surface, markedly reduced the internalization of LL-37 and degranulation of LAD2 cells, and clathrin-mediated endocytosis inhibitors (dynasore and chlorpromazine) inhibited the internalization and degranulation of LAD2 cells. Taken together, these observations indicated that LL-37 may bind the negatively charged cell surface molecules, rapidly internalize into the cells via clathrin-mediated endocytosis and interact with MrgX2 to activate mast cells (LAD2 cells).
Highlights
Mammalian cells express a number of peptide antibiotics that function as effector components in innate host defense systems [1,2,3]
Mast cells are usually present in submucosal tissues and connective tissues, and play a pivotal role in innate immunity by releasing several mediators such as histamine, leukotrienes, and tryptase [14,15]
To investigate the relationship between the LL‐37 internalization and mast cell degranulation, LAD2 cells were incubated with or without LL‐37 (5 μM) for 40 min, and internalized LL‐37 was detected by anti‐LL‐37 rabbit polyclonal antibodies (pAbs) and Alexa Fluor 488‐conjugated secondary antibody
Summary
Mammalian cells express a number of peptide antibiotics that function as effector components in innate host defense systems [1,2,3]. LL‐37 is the sole antibacterial peptide of human cathelicidin comprising of 37 amino acids, which is expressed mainly in epithelial cells and neutrophils, and cleaved from the 18‐kDa human cationic antibacterial polypeptide [5]. LL‐37 has an α‐helical amphiphilic structure, and can disrupt the outer and inner membranes of bacteria. In addition its broad killing activity against bacteria, fungi, and certain viruses [6], LL‐37 has diverse immunomodulatory effects, including the regulation of pro‐ and anti‐inflammatory mediator production [7,8], wound healing [9], angiogenesis [10,11], and expression of nerve elongation factors [12]. It was reported that LL‐37 induces chemotaxis and histamine release by mast cells [13]
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