SEARCH

SEARCH BY CITATION

Keywords:

  • asthma;
  • cathelicidins;
  • eosinophil cationic proteins;
  • human primary eosinophils;
  • leukotrienes

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflicts of interest
  8. References
  9. Supporting Information

Background

Eosinophils and their products, including leukotrienes and eosinophil cationic protein (ECP), are well-known mediators of inflammation and tissue damage in asthma. The antimicrobial peptide LL-37 exhibits a variety of immunomodulatory activities. However, the role of LL-37 in asthma has not been fully addressed. Here, we aim to investigate the effect of LL-37 on inducing inflammatory mediators in human eosinophils, probe the underlying mechanisms, and search for a clinical correlate.

Methods

Primary eosinophils were isolated from peripheral blood. Leukotriene and ECP levels were measured using EIAs or ELISAs. Activation of leukotriene-synthesizing enzymes and signaling kinases was analyzed by Western blot or immunofluorescent imaging. LL-37/its proform hCAP18 expression was analyzed by Western blot.

Results

LL-37, via formyl peptide receptor-2 (FPR-2), triggered the release of cysteinyl leukotrienes (cys-LTs) from eosinophils. The release was more prominent in cells primed with the eosinophilopoietic cytokine GM-CSF or IL-5 or cells from asthmatic patients. LL-37 stimulates lipid body formation and activates cys-LT-synthesizing enzymes by multiple mechanisms: enhancing cPLA2 activity by pERK1/2-mediated phosphorylation and inducing intracellular translocation and assembly of 5-LO and LTC4S at perinuclear locations and lipid bodies. In addition to cys-LTs, LL-37 enhances ECP release from eosinophils via pERK1/2. The expression of hCAP18 and its release following leukotriene stimulation are significantly higher in eosinophils from asthmatics.

Conclusions

This study identifies LL-37 as an eosinophil-activating peptide that triggers release of inflammatory mediators. The clinical correlation suggests that LL-37/hCAP18 and its signaling pathway represent potential therapeutic targets for this disease.

Abbreviations
cys-LTs

cysteinyl leukotrienes

LTB4

leukotriene B4

LTD4

leukotriene D4

ADRP

adipocyte differentiation-related protein

ECP

eosinophil cationic protein

FPR-2

formyl peptide receptor-2

cPLA2

cytosolic phospholipase A2

5-LO

5-lipoxygenase

LTC4S

leukotriene C4 synthase

Eosinophils are key effector cells of airway inflammation, hyper-responsiveness, and tissue damages in asthma. Activated eosinophils contribute to these processes by releasing numerous mediators including leukotrienes and granular eosinophil cationic protein (ECP) [1, 2].

Cysteinyl leukotrienes (Cys-LTs; LTC4, LTD4, and LTE4), are the most prominent lipid mediators generated by eosinophils and potent signaling molecules in airway inflammation, bronchoconstriction, and remodeling in asthmatic patients [3, 4]. Leukotriene biosynthetic pathways start with phospholipase (PL)A2-mediated release of arachidonic acid (AA). Subsequent oxygenation of AA by 5-lipoxygenase (5-LO) produces LTA4, which is rapidly converted to cys-LTs or LTB4 by the action of LTC4 synthase (LTC4S) or LTA4 hydrolase, respectively [5]. Eosinophils contain all essential leukotriene-synthesizing enzymes, the activities of which are subject to regulation in a stimulus-specific manner [6]. Eosinophil cationic protein is a granule-derived cytotoxic protein released by activated eosinophils and a marker of eosinophilic inflammation. High levels of ECP have been associated with an active-state asthma [7].

Antimicrobial peptides are important molecules of host innate immunity against invading microbes. The mammalian antimicrobial peptides are mainly cathelicidins and defensins. LL-37/hCAP18 is the only human cathelicidin found primarily in epithelial cells and immune cells such as neutrophils. They are stored predominantly as the unprocessed proform hCAP18 and processed to the active form LL-37 by serine proteases upon cell activation/degranulation and release [8]. LL-37/hCAP18 modulates a variety of inflammatory/immunological processes [8]. LL-37 levels are dysregulated in asthmatic patients [9]. However, the role of LL-37/hCAP18 in asthma is largely unknown, except for indirect evidence showing that the peptide has chemotactic effects on effector cells in asthma including eosinophils [10], neutrophils [11], and mast cells [1, 12]. To address its potential implications in asthma, we investigated the regulatory effects of LL-37 on leukotriene and cytotoxic protein production from eosinophils, on the synthesizing and feedback mechanisms, and on clinical correlation of LL-37/hCAP18 expression in eosinophils in a group of subjects with documented asthma.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflicts of interest
  8. References
  9. Supporting Information

Subjects

Fifteen patients with mild/moderate allergic asthma [five women and 10 men, mean age 34 (23–50) years] and 15 nonallergic, gender-matched healthy control subjects were recruited for this study. For inclusion, patients were required to display a positive methacholine challenge test, and/or reversibility in forced expiratory volume in 1-s (FEV1) according to GINA criteria (Global Strategy for Asthma Management and Prevention 2011), and at least one positive skin prick test to common allergens. All subjects signed informed consent, and the study was approved by the ethical committee at Karolinska Institutet, Stockholm, Sweden (Dnr 2011/1045-32/4).

Materials

Synthetic LL-37 peptide was purchased from Innovagen AB (Lund, Sweden). Monoclonal LL-37 antibody was generated as described [13].

Methods

Isolation of human peripheral blood eosinophils

Except for comparison of LL-37/hCAP18 expression between healthy and asthmatic subjects, primary eosinophils were obtained from peripheral blood of nonallergic subjects by density gradient centrifugation followed by an Eosinophil isolation kit (Milteny Biotech, Bergisch Gladbach, Germany). Cell viability and purity were determined by trypan blue exclusion staining and CD16 staining by FACS analysis, respectively. This preparation routinely yields eosinophils of more than 96% viability and 98% purity.

Polypeptide concentration and Western blot analysis of LL-37/hCAP18

After treatment of the eosinophils, supernatants were collected and cells were lysed with 60% acetonitrile in 1% trifluoroacetic acid (TFA) overnight at 4°C for optimal LL-37/hCAP18 recovery. Polypeptides were enriched from acidified supernatants or cell lysates using reverse-phase chromatography (OASIS cartridge; Waters®, Milford, MA, USA). Subsequent Western blot analysis of LL-37/hCAP18 was performed as previously described [14].

Western blot analysis for cytosolic PLA2 (cPLA2), phospho-cPLA2, extracellular signal-regulated kinase (ERK)1/2, and phospho-ERK1/2 (pERK1/2)

At the end of the designated treatment, cells were lysed with supplemented radioimmunoprecipitation assay lysis buffer (RIPA) (Sigma Chemical Co., St Louis, MO, USA). Protein concentrations were determined using a NanoDrop Spectrophotometer (Thermo Scientific, Newark, DE, USA). Western blot analysis was performed as previously described [14]. All antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA) except for the housekeeping protein GAPDH obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Enzyme immunoassays (EIAs) of leukotrienes

Concentrations of leukotrienes in the cell supernatants were determined with specific EIA kits (Cayman Chemical, Ann Arbor, MI, USA) following the manufacturer's instructions.

Immunofluorescence and confocal microscopy

Eosinophils adhering to polylysine-coated glass slides were fixed in 3.7% formaldehyde, permeabilized, and blocked with 0.1% Triton X-100 and 10% normal goat sera. The cells were then treated with rabbit anti-5-LO antibody or anti-LTC4S antibody followed by a Cy3-labeled anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA). Lipid bodies were localized with an anti-adipocyte differentiation-related protein (ADRP) antibody followed by FITC-labeled anti-guinea-pig IgG antibody. The slides were visualized with a Zeiss LSM700 confocal microscope (Oberkochen, Germany) equipped with plan-Apochromat 63X/1.4 and plan-Neofluor 40X/1.3 oil immersion lenses and LSM 3D image acquisition software.

Statistical analysis

Data are expressed as means ± SD. Statistical analyses were performed by independent t-test or, when multiple comparisons were made, by one-way anova using GraphPad Prism version 5 (GraphPad Software Inc., San Diego, CA, USA). A P value of <0.05 was considered a statistically significant difference.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflicts of interest
  8. References
  9. Supporting Information

LL-37 induces release of leukotrienes from eosinophils

Purified eosinophils were stimulated with 10, 15, or 30 μg/ml LL-37 for up to 30 min. Based on the quantitative analyses, cys-LTs are among the most abundant lipid mediators secreted from eosinophils (Fig. 1A). The increase in cys-LT release became significant 15 min after stimulation and decreased with prolonged incubation. An eosinophil activator and serpentine receptor agonist, viz. platelet-activating factor (PAF, 1 μM), triggered release of cys-LTs of comparable levels as LL-37 after 15 min and persisted until 30 min (Fig. 1A).

image

Figure 1. LL-37 via FPR-2 receptor triggers cys-LT release from human eosinophils. (A) Time and dose dependence of LL-37-induced cys-LT secretion (n = 4). PAF, as an eosinophil-activating agent, is a positive control. (B) Eosinophils isolated from healthy subjects were primed with GM-CSF or IL-5 for 15 min before LL-37 stimulation (n = 4). (C) Eosinophils isolated from asthmatic subjects were compared with those from healthy subjects (n = 3). (D) Eosinophils isolated from healthy subjects were preincubated with PTx, WRW4, or suramin for 10 min before LL-37 stimulation (n = 4). Results are the means ± SD from at least three independent experiments, each in triplicate measurements. *P < 0.05, **P < 0.01, vs Ctrl. &P < 0.05, &&P < 0.01, vs Veh (A) or untreated healthy (C). #P < 0.05, ##P < 0.01, vs LL-37.

Download figure to PowerPoint

Although eosinophils are not a major cellular source of LTB4, this compound was found in cell media after LL-37 stimulation, with a similar dose–response pattern as for cys-LTs (Suppl. Fig. S1).

Primed eosinophils or eosinophils from asthmatic patients exhibit enhanced cys-LT release in response to LL-37 stimulation

Cells were primed with 15 ng/ml GM-CSF or 10 ng/ml of IL-5 for 15 min before addition of LL-37. Priming itself did not affect basal leukotriene levels but significantly enhanced LL-37-induced cys-LT formation (at doses of 15 and 30 μg/ml). Notably, primed cells responded to 15 μg/ml of LL-37, a concentration to which unprimed cells did not respond significantly (Fig. 1B).

Eosinophils from asthmatic patients were more sensitive to LL-37 stimulation: they responded to a lower dose of LL-37, 15 μg/ml, and released comparably higher cys-LT amounts than cells from healthy control subjects (Fig. 1C).

LL-37-mediated cys-LT release from eosinophils is dependent on the FPR-2 receptor

Purified eosinophils were preincubated with 200 ng/ml pertussis toxin (PTx), a G inhibitor, an FPR-2 receptor antagonist WRW4 (1 or 5 μM), or a broadspectrum P2X receptor antagonist suramin (3 or 30 μM) for 10 min before LL-37 stimulation. The inhibitor or antagonist alone did not affect the basal levels of cys-LTs. PTx pretreatment significantly attenuated LL-37-induced cys-LT release (Fig. 1D). Preincubation of eosinophils with WRW4 (1 or 5 μM) essentially abolished the response, pointing to the role of FPR-2 in signal transduction. In contrast, the purinergic P2 receptor antagonist suramin had no effect on LL-37-triggered cys-LT release (Fig. 1D).

WKYMVm, a potent FPR-2 agonist, triggered cys-LT release from eosinophils, with the maximal effect observed at 1 μM of WKYMVm after 30 min of stimulation. The effect was abolished when cells were pretreated with PTx or WRW4 (Suppl. Fig. S2).

LL-37 stimulates phosphorylation of cPLA2, intracellular trafficking of 5-LO and LTC4S, and colocalization with lipid bodies

As demonstrated by Western blot analysis, LL-37 induced a rapid phosphorylation of cPLA2 in eosinophils, which reached maximum at 5 min. In contrast, total cPLA2, 5-LO, and LTC4S expression did not change (Fig. 2A, Suppl. Fig. 3A,B).

image

Figure 2. LL-37 activates cys-LT-synthesizing enzymes via different mechanisms in human eosinophils (n = 3). (A) LL-37 stimulates phosphorylation of cPLA2. Eosinophils were treated with 30 μg/ml LL-37 for up to 30 min. (B) LL-37 triggers intracellular translocation of 5-LO and LTC4S. Eosinophils were stimulated with LL-37 for 15 min. Upper two panels, anti-5-LO and anti-ADRP staining, Ctrl vs LL-37-treated. Lower two panels, anti-LTC4S and anti-ADRP staining, Ctrl vs LL-37-treated. Original magnification ×63. Representative blots/graphs from three independent experiments are shown.

Download figure to PowerPoint

image

Figure 3. LL-37 activates ERK1/2 in human eosinophils via FPR-2 (n = 4, A, B). The pERK1/2 are involved in LL-37-induced cPLA2 activation and cys-LT production (n = 4, C, D). (A) Cells were treated with 30 μg/ml LL-37 for up to 30 min. (B) Cells were pretreated with 200 ng/ml PTx or 5 μM WRW4 for 10 min before exposure to LL-37 for 5 min. (C) Cells were pretreated with 10 μM PD-98059 for 10 min before LL-37 stimulation for 5 min. Representative blots from four separate experiments were shown. (D) Cells were pretreated with PD-98059 before LL-37 stimulation for 15 min. Results are the means ± SD from four independent experiments, each in triplicate measurements. **P < 0.01, vs Ctrl. #P < 0.05, vs LL-37.

Download figure to PowerPoint

Immunofluorescent staining was then performed to determine whether LL-37 induces intracellular translocation of 5-LO and LTC4S. Lipid bodies were localized with an antibody directed against ADRP protein and identified as intense dots spread in the cytoplasm [15]. Staining of 5-LO was diffuse and faint throughout the cytoplasm prior to LL-37 stimulation. LL-37 treatment triggered a focal distribution of 5-LO around perinuclear region and colocalization of 5-LO with lipid body staining (Fig. 2B). Similar to 5-LO, LTC4S staining was colocalized with lipid bodies in LL-37-stimulated cells (Fig. 2B).

LL-37 activates ERK1/2 and pERK1/2 mediate LL-37-induced cys-LT synthesis

LL-37 induced a rapid, time-dependent, phosphorylation and activation of ERK1/2 in eosinophils (Fig. 3A, Suppl. Fig. S4A,B). Pretreatment of cells with 200 ng/ml PTx or 5 μM WRW4 diminished LL-37-induced pERK1/2 upregulation (Fig. 3B, Suppl. Fig. S4C,D).

Blocking ERK1/2 activation by pretreating eosinophils with a specific inhibitor ERK1/2, PD-98059, suppressed LL-37-induced phosphorylation of cPLA2 (Fig. 3C, Suppl. Fig. S5) and subsequent cys-LT release (Fig. 3D). Preliminary data indicated that treatment of eosinophils with PD-98059 alone did not change the basal expression of cPLA2 or p-cPLA2 (data not shown).

LL-37 induces ECP release from human eosinophils via pERK1/2

A significant increase in ECP release was observed after challenge with 15 μg/ml LL-37 after 30 min. At 30 μg/ml of LL-37, a similar effect was observed after 15 and 30 min of stimulation (Suppl. Fig. S6). The stimulated release of ECP was partially inhibited with PD-98059 pretreatment (10 μM) (Suppl. Fig. S6).

Enhanced hCAP18 expression and release from eosinophils from asthmatic patients

Eosinophils isolated from 12 asthma patients and 12 matched healthy subjects were compared for LL-37/hCAP18 expression. Eosinophils from asthmatics expressed significantly higher hCAP18 protein compared with those from healthy subjects (Fig. 4).

image

Figure 4. Eosinophils from asthma patients express higher levels of hCAP18 compared with those from healthy subjects (n = 12). A representative blot from three separate experiments is shown. Densitometry data and the histogram of hCAP18 expression were generated from a total of 12 samples after normalizing to GAPDH. ***P < 0.001, vs 12 nonasthmatic.

Download figure to PowerPoint

Purified eosinophils were pretreated with 1 μM cytochalasin B before stimulation with LTD4, (a cys-LT) or LTB4. Cytochalasin B, a cell-permeable mycotoxin that changes cytoskeleton integrity, is routinely added to amplify leukotriene-induced transmembrane responses. Cytochalasin B itself did not trigger significant release of hCAP18, whereas pretreatment with cytochalasin B prior to LT stimulation allowed rapid hCAP18 release. The maximal release was observed 5 min after stimulation (Fig. 5A, Suppl. Fig. S7A,B). Cells isolated from asthmatics released more hCAP18 upon leukotriene stimulation than did cells from healthy subjects (Fig. 5B, Suppl. Fig. S7C,D).

image

Figure 5. Leukotriene stimulation triggered release of hCAP18 from human eosinophils. Eosinophils isolated from nonasthmatic subjects (n = 3, A, B) or asthmatic patients (n = 3, C, D) were pretreated with 1 μM cytochalasin B before stimulation with LTD4 or LTB4. Cell media were collected and enriched for polypeptide content as described in the 'Materials and methods'. Protein concentrations were then determined and normalized before Western blot analysis of hCAP18 and GAPDH levels. Shown are representative Western blots from three separate experiments.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflicts of interest
  8. References
  9. Supporting Information

LL-37 triggers release of proinflammatory cys-LTs from eosinophils

Despite various immunomodulatory activities reported for LL-37, its role in eosinophil function and in asthma has not been clearly described.

Here, we demonstrate that LL-37 induces release of leukotrienes, importantly cys-LTs, from human eosinophils (Fig. 1A). Cys-LTs are potent proinflammatory lipid mediators characteristically increased in the airways of asthmatic patients [16]. The dose equipotent to 1 μM PAF to effectively trigger the response, that is, 30 μg/ml, is a pathophysiologically relevant concentration of LL-37 (Fig. 1A). Concentrations up to 50 μg/ml of LL-37 have been reported for its bioactions such as antimicrobial activity, release of inflammatory mediators, and chemotaxis [8].

In vitro priming of blood eosinophils with IL-5 family cytokines allows them to acquire a hyper-responsive phenotype and prolonged survival as the airway eosinophils [17]. Indeed, primed blood eosinophils are sensitized to LL-37 stimulation, which is similarly observed in cells isolated from asthmatic patients (Fig. 1B,C).

Receptors reported for LL-37 to transduce differential cellular responses include FPR-2 [10, 11], P2X7 receptor [18], epidermal growth factor receptor (EGFR) [19], Toll-like receptors (TLRs) [20], and CXCR2 [21]. Using PTx, which inhibits Giα protein–mediated signaling and WRW4, an FPR-2 receptor antagonist, we identify that LL-37-induced cys-LT release is mediated by FPR-2 (Fig. 1D). FPR-2 receptors on human eosinophils are also important in allergen-triggered cell activation [22] and LL-37-induced chemotaxis [10].

LL-37 induces activation and assembly of lipid-synthesizing enzymes at perinuclear locations and lipid bodies

Key enzymes including cPLA2, 5-LO, and LTC4S act sequentially to produce cys-LTs [5]. The observed increase in cys-LT production could be the result of either enhanced expression or activities of the enzymes, by mechanisms such as phosphorylation and/or intracellular translocation. The latter often elicits faster responses. Here, we observed several mechanisms in LL-37-induced cys-LT synthesis: phosphorylation of cPLA2 (Fig. 2A) and intracellular translocation of 5-LO and LTC4S to perinuclear localizations and lipid bodies (Fig. 2B). LL-37 stimulation of eosinophils was also accompanied by lipid body formation (Fig. 2B). Our findings are supported by earlier studies showing that cys-LT synthesis depends on intracellular localization of properly assembled enzymatic complexes [23, 24]. Lipid bodies, characteristically increased during eosinophil activation, are predominant sites for leukotriene synthesis [25, 26]. In eosinophils, 5-LO translocation is associated with leukotriene synthesis and differentially regulated by various stimuli [27].

LL-37-activated pERK1/2 are involved in lipid-synthesizing pathways

MAPK cascades are involved in mediating cellular responses elicited by human antimicrobial peptides [28, 29]. In eosinophils, we observe that LL-37 via FPR-2 induces ERK1/2 phosphorylation (Fig. 3A,B), and pERK1/2 are important in the regulation of cPLA2 phosphorylation and subsequent cys-LT release (Fig. 3C,D). These findings are in accordance with earlier evidence suggesting that cPLA2 and ERK1/2 share common signaling pathways [30, 31].

A positive feedback regulation between leukotrienes and LL-37/hCAP18

We observe that either LTD4 or LTB4 triggers release of hCAP18 from eosinophils. In vivo, hCAP18 may be processed by proteinase-3 from infiltrating neutrophils to generate active LL-37 [32, 33]. Thus, a positive feedback mechanism forms between leukotrienes and LL-37/hCAP18. Intriguingly, hCAP18 is the predominant form not only in eosinophils but also in epithelial cells [8]. As evidenced by altered expression and secretion in disease conditions, hCAP18 is likely to possess intrinsic biological functions yet to be discovered.

Of note, this proinflammatory circuit between LTs and LL-37 in eosinophils may be undesirable for the host during an antimicrobial process. Cys-LTs are proinflammatory and spasmogenic molecules that may cause asthma exacerbations. Furthermore, as both products of this feedback loop, LL-37 (Suppl. Fig. S6) and cys-LTs [34], stimulate ECP release, it may also enhance ECP-mediated cytotoxicity, tissue damage, and mucus production during asthma. It remains to be evaluated the contribution of eosinophils and ECP to LL-37-mediated antimicrobial host defense.

LL-37/hCAP18 a correlative marker for asthma

Earlier, the role of LL-37 in asthma seems controversial. LL-37 by itself chemo-attracts eosinophils, neutrophils, and T cells via FPR-2 [11] and triggers release of histamine and PGD2 from mast cells [12], pathogenetic mediators for allergic asthma. LL-37 also activates neutrophils and promotes release of LTB4 [14], which is a potent chemoattractant for many effector cells in the development of asthma [2]. On the other hand, deficiency of vitamin D, a transcriptional inducer of the CAMP (cathelicidin antimicrobial peptide) gene encoding LL-37, was causally associated with asthma [35], which must be cautiously interpreted as vitamin D is a pluripotent antioxidant that affects multiple gene products. Furthermore, Xiao et al. [9] found decreased levels of cathelicidin in induced sputum of asthmatic patients, as opposed to increased levels in sputum from patients with cystic fibrosis and chronic obstructive pulmonary disease. One may speculate that the low levels of LL-37 in asthma sputum reflects a different inflammatory state and cellular composition of the upper respiratory tract or perhaps effects of pharmacological agents. To better understand the potential role of LL-37 in asthma, further work is required with analysis of different tissues and body fluids from patients at various stages of disease and pharmacological treatments.

Here, our findings directly point to a ‘proasthmatic’ role of LL-37 by triggering release of pathogenetic mediators. In lungs, LL-37 could be generated from epithelial cells or infiltrating neutrophils and contribute to amplification of the allergic response by recruiting and activating eosinophils followed by triggering release of inflammatory mediators. A model for LL-37-initiated inflammatory signaling in eosinophils is depicted in Fig. 6. Of note, comparing samples from 12 asthmatic patients and 12 healthy controls, hCAP18 expression (Fig. 4) was significantly higher in the asthmatic patients.

image

Figure 6. A model for LL-37-mediated eosinophil recruitment and cellular signaling leading to cys-LT and ECP release. In the lung, LL-37 could be generated from epithelial cells and neutrophils. LL-37, in turn, can recruit additional leukocytes including eosinophils and amplify the inflammatory responses by triggering cys-LT and ECP production from human eosinophils. Via FPR-2, LL-37 activates cys-LT synthesis pathway at perinuclear regions and cytoplasmic lipid bodies and can also trigger ECP release from granules. Cys-LTs and ECP may contribute to airway inflammation, tissue damage, and remodeling during asthma. The figure was made with tools from www.proteinlounge.com.

Download figure to PowerPoint

Collectively, our study identifies LL-37 as an eosinophil activator that triggers intracellular trafficking and activation of cys-LT-synthesizing enzymes and subsequent release of proinflammatory mediators of importance in asthma. Eosinophils isolated from asthmatics expressed and released significantly higher levels of LL-37/hCAP18, suggesting that the peptide and its signaling pathway represent potential therapeutic targets for this disease.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflicts of interest
  8. References
  9. Supporting Information

The authors wish to thank Eva Ohlson and Monica Lindh for technical support, Olof Rådmark for 5-LO antisera, Anders Wetterholm for LTC4S antisera, Marianne Lundblad Eduards and Agneta Lindeberg for recruiting asthmatic subjects. The project was financially supported by the Swedish Research Council (20854, Linneus grant CERIC, 10350, 1217-01-06, 20368-04-3), the Swedish Strategic Foundation (SSF), the Swedish Cancer Society, Thorsten & Ragnar Söderberg's Foundation, the Swedish Heart Lung Foundation, the Stockholm County Council (20090136), and Karolinska Institutet.

Conflicts of interest

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflicts of interest
  8. References
  9. Supporting Information

The authors declare that there are no conflicts of interest relevant to this manuscript.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflicts of interest
  8. References
  9. Supporting Information
  • 1
    Costa JJ, Weller PF, Galli SJ. The cells of the allergic response: mast cells, basophils, and eosinophils. JAMA 1997;278:18151822.
  • 2
    Hallstrand TS, Henderson WR. An update on the role of leukotrienes in asthma. Curr Opin Allergy Clin Immunol 2010;10:6066.
  • 3
    Holgate ST, Peters-Golden M, Panettieri RA, Henderson WR. Roles of cysteinyl leukotrienes in airway inflammation, smooth muscle function, and remodeling. J Allergy Clin Immunol 2003;111(Suppl 1):S18S34; discussion S34-16.
  • 4
    Weller PF. Human eosinophils. J Allergy Clin Immunol 1997;100:283287.
  • 5
    Haeggström JZ, Funk CD. Lipoxygenase and leukotriene pathways: biochemistry, biology, and roles in disease. Chem Rev 2011;111:58665898.
  • 6
    Lai Y, Oslund RC, Bollinger JG, Henderson WR, Santana LF, Altemeier WA et al. Eosinophil cysteinyl leukotriene synthesis mediated by exogenous secreted phospholipase A2 group X. J Biol Chem 2010;285:4149141500.
  • 7
    Vatrella A, Ponticiello A, Parrella R, Romano L, Zofra S, DiLeva A et al. Serum eosinophil cationic protein (ECP) as a marker of disease activity and treatment efficacy in seasonal asthma. Allergy 1996;51:547555.
  • 8
    Kai-Larsen Y, Agerberth B. The role of the multifunctional peptide LL-37 in host defense. Front Biosci 2008;13:37603767.
  • 9
    Xiao W, Hsu YP, Ishizaka A, Kirikae T, Moss RB. Sputum cathelicidin, urokinase plasminogen activation system components, and cytokines discriminate cystic fibrosis, COPD, and asthma inflammation. Chest 2005;128:23162326.
  • 10
    Tjabringa GS, Ninaber DK, Drijfhout JW, Rabe KF, Hiemstra PS. Human cathelicidin LL-37 is a chemoattractant for eosinophils and neutrophils that acts via formyl-peptide receptors. Int Arch Allergy Immunol 2006;140:103112.
  • 11
    Yang D, Chen Q, Schmidt AP, Anderson GM, Wang JM, Wooters J et al. 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. J Exp Med 2000;192:10691074.
  • 12
    Niyonsaba F, Someya A, Hirata M, Ogawa H, Nagaoka I. Evaluation of the effects of peptide antibiotics human beta-defensins-1/-2 and LL-37 on histamine release and prostaglandin D(2) production from mast cells. Eur J Immunol 2001;31:10661075.
  • 13
    Yoshio H, Tollin M, Gudmundsson GH, Lagercrantz H, Jornvall H, Marchini G et al. Antimicrobial polypeptides of human vernix caseosa and amniotic fluid: implications for newborn innate defense. Pediatr Res 2003;53:211216.
  • 14
    Wan M, Sabirsh A, Wetterholm A, Agerberth B, Haeggström JZ. Leukotriene B4 triggers release of the cathelicidin LL-37 from human neutrophils: novel lipid-peptide interactions in innate immune responses. FASEB J 2007;21:28972905.
  • 15
    Mesquita-Santos FP, Vieira-de-Abreu A, Calheiros AS, Figueiredo IH, Castro-Faria-Neto HC, Weller PF et al. Cutting edge: prostaglandin D2 enhances leukotriene C4 synthesis by eosinophils during allergic inflammation: synergistic in vivo role of endogenous eotaxin. J Immunol 2006;176:13261330.
  • 16
    Thomas LH, Warner JA. The eosinophil and its role in asthma. Gen Pharmacol 1996;27:593597.
  • 17
    Zhu Y, Bertics PJ. Chemoattractant-induced signaling via the Ras-ERK and PI3K-Akt networks, along with leukotriene C4 release, is dependent on the tyrosine kinase Lyn in IL-5- and IL-3-primed human blood eosinophils. J Immunol 2011;186:516526.
  • 18
    Nagaoka I, Tamura H, Hirata M. An antimicrobial cathelicidin peptide, human CAP18/LL-37, suppresses neutrophil apoptosis via the activation of formyl-peptide receptor-like 1 and P2X7. J Immunol 2006;176:30443052.
  • 19
    Tokumaru S, Sayama K, Shirakata Y, Komatsuzawa H, Ouhara K, Hanakawa Y et al. Induction of keratinocyte migration via transactivation of the epidermal growth factor receptor by the antimicrobial peptide LL-37. J Immunol 2005;175:46624668.
  • 20
    Mookherjee N, Brown KL, Bowdish DM, Doria S, Falsafi R, Hokamp K et al. Modulation of the TLR-mediated inflammatory response by the endogenous human host defense peptide LL-37. J Immunol 2006;176:24552464.
  • 21
    Zhang Z, Cherryholmes G, Chang F, Rose DM, Schraufstatter I, Shively JE. Evidence that cathelicidin peptide LL-37 may act as a functional ligand for CXCR2 on human neutrophils. Eur J Immunol 2009;39:31813194.
  • 22
    Svensson L, Redvall E, Björn C, Karlsson J, Bergin AM, Rabiet MJ et al. House dust mite allergen activates human eosinophils via formyl peptide receptor and formyl peptide receptor-like 1. Eur J Immunol 2007;37:19661977.
  • 23
    Bozza PT, Yu W, Penrose JF, Morgan ES, Dvorak AM, Weller PF. Eosinophil lipid bodies: specific, inducible intracellular sites for enhanced eicosanoid formation. J Exp Med 1997;186:909920.
  • 24
    Bandeira-Melo C, Phoofolo M, Weller PF. Extranuclear lipid bodies, elicited by CCR3-mediated signaling pathways, are the sites of chemokine-enhanced leukotriene C4 production in eosinophils and basophils. J Biol Chem 2001;276:2277922787.
  • 25
    Bozza PT, Melo RC, Bandeira-Melo C. Leukocyte lipid bodies regulation and function: contribution to allergy and host defense. Pharmacol Ther 2007;113:3049.
  • 26
    Melo RC, Sabban A, Weller PF. Leukocyte lipid bodies: inflammation-related organelles are rapidly detected by wet scanning electron microscopy. J Lipid Res 2006;47:25892594.
  • 27
    Cowburn AS, Holgate ST, Sampson AP. IL-5 increases expression of 5-lipoxygenase-activating protein and translocates 5-lipoxygenase to the nucleus in human blood eosinophils. J Immunol 1999;163:456465.
  • 28
    Niyonsaba F, Ushio H, Nagaoka I, Okumura K, Ogawa H. The human beta-defensins (-1, -2, -3, -4) and cathelicidin LL-37 induce IL-18 secretion through p38 and ERK MAPK activation in primary human keratinocytes. J Immunol 2005;175:17761784.
  • 29
    Bowdish DM, Davidson DJ, Speert DP, Hancock RE. The human cationic peptide LL-37 induces activation of the extracellular signal-regulated kinase and p38 kinase pathways in primary human monocytes. J Immunol 2004;172:37583765.
  • 30
    Lin LL, Wartmann M, Lin AY, Knopf JL, Seth A, Davis RJ. cPLA2 is phosphorylated and activated by MAP kinase. Cell 1993;72:269278.
  • 31
    Yu W, Bozza PT, Tzizik DM, Gray JP, Cassara J, Dvorak AM et al. Co-compartmentalization of MAP kinases and cytosolic phospholipase A2 at cytoplasmic arachidonate-rich lipid bodies. Am J Pathol 1998;152:759769.
  • 32
    Sørensen OE, Follin P, Johnsen AH, Calafat J, Tjabringa GS, Hiemstra PS et al. Human cathelicidin, hCAP-18, is processed to the antimicrobial peptide LL-37 by extracellular cleavage with proteinase 3. Blood 2001;97:39513959.
  • 33
    Flamand L, Tremblay MJ, Borgeat P. Leukotriene B4 triggers the in vitro and in vivo release of potent antimicrobial agents. J Immunol 2007;178:80368045.
  • 34
    Neves JS, Radke AL, Weller PF. Cysteinyl leukotrienes acting via granule membrane-expressed receptors elicit secretion from within cell-free human eosinophil granules. J Allergy Clin Immunol 2010;125:477482.
  • 35
    Paul G, Brehm J, Alcorn JF, Holguin F, Aujla S, Celedón JC. Vitamin D and Asthma. Am J Respir Crit Care Med 2012;185:124132.

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflicts of interest
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
all12087-sup-0001-FigureS1-S7.docxWord document1609K

Figure S1. LL-37 mediates LTB4 release from eosinophils (n = 4).

Figure S2. A FPR-2 receptor agonist peptide WKYMVm (Wp), similarly as LL-37, induced cys-LT release from eosinophils (n = 3).

Figure S3. Densitometry analysis of LL-37-induced p-cPLA2 (A) and cPLA2 (B) expression normalized to GAPDH (n = 3).

Figure S4. Densitometry analysis of LL-37-stimulated p-ERK1 and p-ERK2 expression normalized to ERK1 and ERK2 (n = 4).

Figure S5. Densitometry analysis of the effect of PD-98059 on p-cPLA2 expression normalized to cPLA2 (n = 4).

Figure S6. LL-37 induces ECP release from eosinophils (n = 4).

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.