LL-37 inhibits serum amyloid A-induced IL-8 production in human neutrophils

Abstract

Serum amyloid A (SAA) has been regarded as an important mediator of inflammatory responses. The effect of several formyl peptide receptor-like 1 (FPRL1) ligands on the production of IL-8 by SAA was investigated in human neutrophils. Among the ligands tested, LL-37 was found to specifically inhibit SAA-induced IL-8 production in transcriptional and post-transcriptional levels. Since SAA stimulated IL-8 production via ERK and p38 MAPK in human neutrophils, we tested the effect of LL-37 on SAA induction for these two MAPKs. LL-37 caused a dramatic inhibition of ERK and p38 MAPK activity, which is induced by SAA. LL-37 was also found to inhibit SAA-stimulated neutrophil chemotactic migration. Further, the LL-37-induced inhibitory effect was mediated by FPRL1. Our findings indicate that LL-37 is expected to be useful in the inhibition of SAA signaling and for the development of drugs against SAA-related inflammatory diseases.

Figure 1

Effect of several FPRL1 agonists on IL-8 production in human neutrophils. Freshly isolated human neutrophils stimulated by fMLF (1 µM), MMK-1 (1 µM), WKYMVm (1 µM), LL-37 (10 µM), F2L (20 µM), SAA (2 µM) or LPS (100 ng/ml) for 24 h (A). Human neutrophils stimulated by fMLF (1 µM), MMK-1 (1 µM), WKYMVm (1 µM), LL-37 (10 µM), F2L (20 µM), and subsequently stimulated with SAA (2 µM) for 24 h (B). The IL-8 levels secreted were determined by ELISA. The data represent the mean ± SE of three independent experiments performed in duplicate (A, B).

Figure 2

LL-37 specifically inhibits SAA-induced IL-8 production in human neutrophils. Human neutrophils were stimulated at various concentrations of LL-37 for 10 min prior to addition of SAA (2 µM) for 24 h (A). Human neutrophils were stimulated with LPS (100 ng/ml) for 24 h in the absence or presence of LL-37 (10 µM) (B). The data represent the mean ± SE of three independent experiments performed in duplicate (A, B). Human neutrophils, stimulated with LL-37 (10 µM), SAA (2 µM), LL-37 (10 µM) + SAA (2 µM), LPS (100 ng/ml), or LL-37 (10 µM) + LPS (100 ng/ml) for 6 h, were harvested for RNA preparation. A RT-PCR was performed using specific primers for human IL-8 and GAPDH. Next, the PCR products were electrophoresed in 2% agarose gel and stained with ethidium bromide. The data obtained from one representative experiment of experiments performed in quadruplicate is shown (C).

Figure 3

LL-37 blocks SAA-induced ERK and p38 MAPK activity in human neutrophils. Human neutrophils were preincubated with vehicle (DMSO), PD98059 (50 µM) for 60 min, or SB203580 (20 µM) for 15 min prior to treatment with SAA (2 µM) for 24 h (A). The amount of IL-8 secreted was measured by ELISA. The data represent the mean ± SE of three independent experiments performed in duplicate. Human neutrophils were stimulated with LL-37 (10 µM), SAA (2 µM), or LL-37 (10 µM) + SAA (2 µM) for 5 min. Samples (30 µg of protein) were subjected to 10% SDS-PAGE, and the phosphorylated ERK or phosphorylated p38 MAPK levels were determined by immunoblot analysis using anti-phospho-ERK antibody or anti-phospho-p38 MAPK antibody (B). The results shown are representative of at least three independent experiments (B).

Figure 4

Role of FPRL1 on the inhibitory effect of LL-37 against SAA-induced IL-8 production. FPRL1- (A) or vector- (B) expressing RBL-2H3 cells were stimulated with LL-37 (10 µM), SAA (2 µM), or LL-37 (10 µM) + SAA (2 µM) for 6 h and then harvested for RNA preparation. A RT-PCR was performed using specific primers for rat IL-8 and actin. The PCR products were electrophoresed in 2% agarose gel and stained with ethidium bromide. The data reported from one experiment is representative of the four (A, B).

Figure 5

Effect of LL-37 on SAA-induced chemotactic migration in human neutrophils. Isolated human neutrophils (1 × 10⁶ cells/ml of serum free RPMI 1640 medium) were added to the upper wells of a 96-well chemotaxis chamber. Next, the migration across the 3 µm pore size polycarbonate membrane was assessed after incubating at 37℃ for 1.5 h. Various concentrations of LL-37 or SAA were used for the chemotaxis (A). The isolated human neutrophils were incubated in the absence or presence of LL-37 (10 µM) for 10 min prior to the chemotaxis assay using SAA (1 µM or 2 µM) (B). The number of migrated cells was assessed by counting in 5 high power fields (400×). The data are expressed as the means ± SE of three independent experiments performed in duplicate (A, B).

Figure 6

LL-37 does not bind to SAA directly. The ligand (A: SAA, C: LL-37) was immobilized onto CM5 sensor chip. 1 µM of each analyte (B: LL-37 for SAA-immobilized chip, D: SAA for LL-37 immobilized chip) was passed over the chip at the flow rate of 30 µl/min. The binding response was monitored with BIAcore 2000. The results are representative of two independent experiments.