MrgX2‑mediated internalization of LL‑37 and degranulation of human LAD2 mast cells

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).

Pharmacokinetics and Pharmacodynamics of Murepavadin in Neutropenic Mouse Models

“…For 7/15 of these strains, a 2-log reduction was not observed, but this could be due to organism-specific characteristics, as it does not seem to be related to the MIC. Strain 5 only reached a static effect, and this may be due to the high MIC toward this isolate (1 mg/liter); also, since murepavadin, like many peptide drugs, is less tolerated by mice (which is attributed to a nonimmunogenic histamine release from mast cells), the maximum dose was given and did not allow higher drug doses to be investigated (13,21). In addition, for this isolate, the homogenates of the lung were streaked on nonselective agar to determine the CFU load; the MICs of the subsequent colonies were tested, and no increase in MIC was observed, suggesting that resistance to murepavadin was not the reason for treatment failure.…”

“…Dose-fractionation studies were undertaken for 2 P. aeruginosa strains (clinical isolate 18 and ATCC 27853). A dose range of 0.25 to 32 mg/kg was used 1, 2, 4, or 8 times in 24 h. Dose-response experiments were performed for an additional 13 P. aeruginosa strains (5,6,9,11,12,15,16,19,21,22, X11045, ATCC BAA 2113, and NCTC 13437). For dose-response models, murepavadin was administered as single (q24h), b.i.d.…”

Amelioration of Compound 48/80-Mediated Itch and LL-37-Induced Inflammation by a Single-Stranded Oligonucleotide

“…Although our data clearly demonstrate that ssON has the potent capacity to inhibit C48/80-mediated activation of mast cells, the discovery of ssON's capacity to modulate LL-37 is of more biological significance. LL-37 is a multifaceted immunomodulatory compound involved in a plethora of processes, ranging from inflammation, wound healing, and angiogenesis ( 48 ), that can be secreted by certain immune cells including mast cells, which are a main producer, and neutrophils, as well as keratinocytes ( 1 ). Notably, LL-37 can induce the degranulation of mast cells, which can in turn release histamine, cytokines, and various inflammatory mediators and can thus create an inflammatory milieu in the dermis by additionally activating keratinocytes and supporting the migration of neutrophils to the epidermis ( 1 ).…”

Mas-related G protein–coupled receptor X2 and its activators in dermatologic allergies

“…After binding to MRGPRX2, LL-37 is internalized, where it is colocalized with MRGPRX2 in the perinuclear region. 87 AMPs are thought to be involved in the pathogenesis of AD, CSU, and other cutaneous allergic and inflammatory diseases by promoting inflammatory responses via the activation of MCs (Table III). AMP protein levels, but not mRNA, are increased in the lesional skin of patients with AD.…”