Human Cathelicidin LL-37 Is a Chemoattractant for Eosinophils and Neutrophils That Acts Via Formyl-Peptide Receptors
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Original Paper Int Arch Allergy Immunol 2006;140:103–112 Received: January 3, 2005 Accepted after revision: December 19, 2005 DOI: 10.1159/000092305 Published online: March 24, 2006 Human Cathelicidin LL-37 Is a Chemoattractant for Eosinophils and Neutrophils That Acts via Formyl-Peptide Receptors a a b G. Sandra Tjabringa Dennis K. Ninaber Jan Wouter Drijfhout a a Klaus F. Rabe Pieter S. Hiemstra a b Departments of Pulmonology, and Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden , The Netherlands Key Words ting using antibodies directed against phosphorylated Eosinophils Neutrophils Chemotaxis Antimicrobial ERK1/2. Results: Our results show that LL-37 chemoat- peptides Innate immunity Lung infl ammation tracts both eosinophils and neutrophils. The FPR antag- onistic peptide tBoc-MLP inhibited LL-37-induced che- motaxis. Whereas the FPR agonist fMLP activated ERK1/2 Abstract in neutrophils, LL-37 did not, indicating that fMLP and Background: Infl ammatory lung diseases such as asth- LL-37 deliver different signals through FPRs. Conclu- ma and chronic obstructive pulmonary disease (COPD) sions: LL-37 displays chemotactic activity for eosinophils are characterized by the presence of eosinophils and and neutrophils, and this activity is mediated via an FPR. neutrophils. However, the mechanisms that mediate the These results suggest that LL-37 may play a role in in- infl ux of these cells are incompletely understood. Neu- fl ammatory lung diseases such as asthma and COPD. trophil products, including neutrophil elastase and anti- Copyright © 2006 S. Karger AG, Basel microbial peptides such as neutrophil defensins and LL- 37, have been demonstrated to display chemotactic activity towards cells from both innate and adaptive im- Introduction munity. However, chemotactic activity of LL-37 towards eosinophils has not been reported. Therefore, the aim of Eosinophils and neutrophils play an important role in the present study was to investigate the chemotactic ac- infection and infl ammation, and increased numbers have tivity of LL-37 for eosinophils and to explore the mecha- been demonstrated in various infl ammatory diseases, in- nisms involved in LL-37-mediated attraction of neutro- cluding asthma and chronic obstructive pulmonary dis- phils and eosinophils. Methods: Neutrophils and eo- ease (COPD). At the site of infl ammation, these granulo- sinophils were obtained from venous blood of healthy cytes contribute to the infl ammatory process by releasing donors. Chemotaxis was studied using a modifi ed Boy- a range of mediators including proteinases, antimicrobial den chamber technique. Involvement of formyl-peptide peptides, lipid mediators and reactive oxygen intermedi- receptors (FPRs) was studied using the antagonistic pep- ates. In addition to causing tissue injury, granulocyte tide tBoc-MLP. Activation of the mitogen-activated pro- products may contribute to the recruitment of infl amma- tein kinase (MAPK) ERK1/2 was studied by Western blot- tory cells. This is illustrated by recent studies demonstrat- © 2006 S. Karger AG, Basel Correspondence to: Pieter S. Hiemstra 1018–2438/06/1402–0103$23.50/0 Department of Pulmonology, Leiden University Medical Center Fax +41 61 306 12 34 Building 1, C3-P, PO Box 9600 E-Mail [email protected] Accessible online at: NL–2300 RC Leiden (The Netherlands) www.karger.com www.karger.com/iaa Tel. +31 71 526 2950, Fax +31 71 526 6927, E-Mail [email protected] ing that neutrophil-derived antimicrobial peptides like mediate LL-37 induced processing and release of IL- , -defensins and LL-37 mediate chemotaxis of infl amma- but its involvement in chemotactic responses has not yet tory cells via activation of chemokine and formyl-peptide been reported. receptors (FPRs) [1, 2] . Therefore, the aim of the present study was to investi- LL-37 is the C-terminal active part of the only human gate whether LL-37 displays chemotactic activity towards cathelicidin that has been identifi ed to date, hCAP-18 [for human eosinophils, and to compare the mechanisms in- recent reviews, see 3, 4] . Cathelicidins are a family of an- volved in eosinophil and neutrophil chemotaxis by LL- timicrobial peptides that consist of a conserved N-termi- 37. Our results show that LL-37 chemoattracts both eo- nal cathelin domain and a variable C-terminal antimicro- sinophils and neutrophils via FPRs. In addition, we dem- bial domain, which can be cleaved off by proteinases [5, onstrated that formoterol, a 2 -adrenoceptor agonist 6] . Expression of hCAP-18/LL-37 has been demonstrated widely used in the treatment of infl ammatory lung dis- in neutrophils [7] , monocytes, T cells [8] , keratinocytes ease, inhibits LL-37-induced chemotaxis. Finally, activa- in infl amed skin [9] and in various squamous epithelia tion of the mitogen-activated protein kinase (MAPK) [10] . In the airways, hCAP-18/LL-37 expression was ERK1/2 by fMLP and LL-37 was studied. While both shown in surface epithelia of the conducting airways and fMLP and LL-37 were shown to chemoattract neutrophils in the submucosal glands [11] , and hCAP-18/LL-37 was and eosinophils, only fMLP induced activation of the demonstrated in broncheoalveolar lavage fl uid [12] . LL- MAPK ERK1/2. The results from this study suggest that 37 was originally identifi ed as an antimicrobial peptide LL-37 may play a role in infl ammatory lung diseases such [13] , and different studies have suggested that LL-37 as asthma and COPD. plays an important role in host defense [14, 15] . How- ever, additional functions have been described which sug- gest a regulatory role for LL-37 in infl ammatory respons- Materials and Methods es. These include LPS neutralization [16, 17] , activation LL-37 Synthesis of epithelial cells [18, 19] and chemotactic activity to- LL-37 (amino acid sequence LLGDFFRKSKEKIGKEFKR- wards neutrophils, monocytes, T cells and mast cells [2, IVQRIKDFLRNLVPRTES) was synthesized by solid-phase pep- 20] . Collectively, these results show that LL-37 may in- tide synthesis on a TentagelS AC (Rapp, Tübingen, Germany) using hibit infl ammatory responses to microbial stimuli, but 9-fl uorenylmethoxycarbonyl (Fmoc)/tBu chemistry, benzotriazole- may also act as a proinfl ammatory mediator. In addition, 1-yl-oxy-tris -pyrrolidino-phosphonium hexafl uorophosphate/N- methylmorpholine for activation and 20% piperidine in N-methyl- its ability to affect differentiation of dendritic cells [21] pyrrolidone for Fmoc removal. The peptide was cleaved from the and chemotaxis of monocytes and T cells [2] , its angio- resin, deprotected with trifl uoroacetic acid/water and purifi ed by genic activity [22] and its involvement in epithelial wound reverse-phase high-performance liquid chromatography on a Vy- healing [23] suggest that LL-37 may also be involved in dac C18 column. The molecular mass was confi rmed by Maldi-Tof adaptive immunity and wound repair. mass spectrometry. While LL-37 displays chemotactic activity towards Chemotaxis Assay different cell types from both innate and adaptive immu- Granulocytes were isolated from heparinized blood obtained nity, chemotactic activity towards eosinophils has not from healthy volunteers by density gradient centrifugation using been demonstrated yet. In addition, limited data are Percoll (density: 1.082 g/ml) as previously described [25] . Eosino- available on mechanisms mediating LL-37-induced che- phils were isolated from the mixed granulocyte population by neg- ative selection using anti-CD16 beads [26] . The neutrophil and motaxis of infl ammatory cells. Two receptors have been eosinophil suspensions were at least 95% pure. The chemotactic described that are involved in LL-37-mediated signaling activity of eosinophils and neutrophils was determined using the in leukocytes, the FPR-like 1 (FPRL1) [2] and the puri- modifi ed Boyden chamber technique [27] . Briefl y, 26 l of chemo- nergic P2X receptor [24] . Yang et al. [2] suggested that tactic stimuli in assay buffer was added to the lower compartment 7 ! 6 LL-37-induced chemotaxis is mediated via the FPRL1 and 50 l of cell suspension (2.5 10 cells/ml in assay buffer) was added to the upper compartment of a 48-well chamber (Neuro- based on experiments using FPRL1-transfected HEK293 probe, Cabin John, Md., USA). The assay buffer was composed 2+ cells and cross-desensitization of LL-37-induced Ca of 20 mM N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acid fl uxes in monocytes using an FPRL1-specifi c agonist. In (Hepes buffer; pH 7.4), 132 mM NaCl, 6 mM KCl, 1.2 mM KH2 PO4 , contrast, mast cell chemotaxis was suggested not to be 1 mM MgSO4 , 5.5 mM glucose, 0.1 mM CaCl2 and 0.5% human mediated via the FPRL1 [20] . Direct evidence for a role serum albumin (Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands) diluted of FPRL1 and related receptors in chemotaxis of granu- 1:1 with serum-free RPMI supplemented with 2 mM L -glutamine, locytes is missing. The P2X7 receptor has been shown to 20 U/ml penicillin and 20 g/ml streptomycin. The compartments 104 Int Arch Allergy Immunol 2006;140:103–112 Tjabringa/Ninaber /Drijfhout /Rabe / Hiemstra Eosinophils Neutrophils 100 125 ✽ ✽ 100 75 ✽ 75 ✽ 50 ✽ 50 Cells per 6 HPF Cells per 6 HPF 25 25 0 0 0 0.1 1 10 100 0 0.1 1 10 100 µ LL-37 (µM) fMLP LL-37 ( M) fMLP Fig. 1. Effect of LL-37 on migration of human eosinophils and neutrophils. Human eosinophils and neutrophils were isolated from peripheral blood, and chemotaxis induced by various concentrations of LL-37 and by fMLP (10 –8 M ) was determined using a modifi ed Boyden chamber technique. Cells in 6 high-power fi elds (HPF) were counted and expressed as mean 8 SEM of three individual experiments using cells from different donors. Data were analyzed for statistical differences by Student’s t test for paired samples.