LL-37, the neutrophil granule- and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (FPRL1) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and T cells

Abstract

We have previously shown that antimicrobial peptides like defensins have the capacity to mobilize leukocytes in host defense. LL-37 is the cleaved antimicrobial 37-residue, COOH-terminal peptide of hCAP18 (human cationic antimicrobial protein with a molecular size of 18 kD), the only identified member in humans of a family of proteins called cathelicidins. LL-37/hCAP18 is produced by neutrophils and various epithelial cells. Here we report that LL-37 is chemotactic for, and can induce Ca(2+) mobilization in, human monocytes and formyl peptide receptor-like 1 (FPRL1)-transfected human embryonic kidney 293 cells. LL-37-induced Ca(2+) mobilization in monocytes can also be cross-desensitized by an FPRL1-specific agonist. Furthermore, LL-37 is also chemotactic for human neutrophils and T lymphocytes that are known to express FPRL1. Our results suggest that, in addition to its microbicidal activity, LL-37 may contribute to innate and adaptive immunity by recruiting neutrophils, monocytes, and T cells to sites of microbial invasion by interacting with FPRL1.

Figure 1

Induction by LL-37 of migration of (A) and Ca²⁺ flux in (B) human monocytes. (A) The migration of monocytes (10⁶ cells/ml) was assessed by chemotaxis assay using 5-μm uncoated membranes. Spontaneous cell migration (without LL-37) was ∼30–50 cells per HPF. The average C.I. (mean ± SD) of triplicate wells is shown. *P < 0.05 when compared with chemotaxis medium alone (open bar). (B) Arrow indicates the time point at which LL-37 was applied to the cells.

Figure 2

Effect of PTX (A) and serum (B) on LL-37–induced chemotaxis of monocytes. (A) Monocytes were incubated with (closed bar) or without (hatched bar) PTX at a final concentration of 200 ng/ml for 30 min at 37°C before performing chemotaxis assay. To show that the spontaneous cell migration (C.M.) was not affected by PTX pretreatment, the results are presented as no./HPF. (B) Chemotaxis assay was performed in the absence (hatched bar) or presence (closed bar) of 10% human AB serum, which can completely block the antimicrobial activity of LL-37 at 10⁻⁵ M.

Figure 3

LL-37 uses FPRL1 as its receptor. (A) Selective induction of FPRL1/293 cell chemotaxis by LL-37. The migration of parental HEK293 (open bars), FPRL1/293 (dotted bars), or ETFR (closed bars) cells was assessed by chemotaxis assay with the use of collagen-coated 10-μm membranes. Cells were used at a concentration of 10⁶ cells/ml. The average C.I. (mean ± SD) of triplicate wells is shown. Spontaneous cell migration (without LL-37) was ∼10–20 cells per HPF. *P < 0.05 when compared with chemotaxis medium alone. (B) LL-37–induced Ca²⁺ flux in FPRL1/293 cells. Arrow indicates the time point at which LL-37 was applied to the cells. (C) Cross-desensitization of LL-37–induced Ca²⁺ flux in monocytes by FPRL1-specific agonistic ligand. Arrows indicate the time points at which LL-37 and Su peptides were applied to the cells.

Figure 4

Chemotaxis of human neutrophils (A) and CD4 T lymphocytes (B) in response to LL-37. The cell migration was assessed by chemotaxis assay with the use of uncoated (A) or fibronectin-coated (B) 5-μm membranes. The results are presented as the average C.I. (mean ± SD) of triplicate wells. *P < 0.05 when compared with spontaneous cell migration (chemotactic medium alone). Neutrophils and CD4 T cells were used at a concentration of 10⁶ cells/ml and 5 × 10⁶ cells/ml, respectively. Spontaneous neutrophil and T cell migration (without LL-37) was ∼30–50 and ∼30–40 cells per HPF, respectively.