Research Article Intracellular Secretory...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 560141 18 pageshttpdxdoiorg1011552013560141

Research ArticleIntracellular Secretory Leukoprotease Inhibitor ModulatesInositol 145-Triphosphate Generation and Exerts anAnti-Inflammatory Effect on Neutrophils of Individuals withCystic Fibrosis and Chronic Obstructive Pulmonary Disease

Emer P Reeves Nessa Banville Dorothy M Ryan Niamh OrsquoReilly David A BerginKerstin Pohl Kevin Molloy Oliver J McElvaney Khalifah Alsaleh Ahmed Aljorfi OsamaKandalaft Eimear OrsquoFlynn Patrick Geraghty Shane J OrsquoNeill and Noel G McElvaney

Respiratory Research Division Department of Medicine Royal College of Surgeons in Ireland Education amp Research CentreBeaumont Hospital Dublin 9 Ireland

Correspondence should be addressed to Emer P Reeves emerreevesrcsiie

Received 2 April 2013 Revised 1 July 2013 Accepted 15 July 2013

Academic Editor Edineia Lemos de Andrade

Copyright copy 2013 Emer P Reeves et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Secretory leukoprotease inhibitor (SLPI) is an anti-inflammatory protein present in respiratory secretions Whilst epithelial cellSLPI is extensively studied neutrophil associated SLPI is poorly characterised Neutrophil function including chemotaxis anddegranulation of proteolytic enzymes involves changes in cytosolic calcium (Ca2+) levels which is mediated by production ofinositol 145-triphosphate (IP

3) in response to G-protein-coupled receptor (GPCR) stimuliThe aim of this study was to investigate

the intracellular function of SLPI and the mechanism-based modulation of neutrophil function by this antiprotease Neutrophilswere isolated from healthy controls (119899 = 10) individuals with cystic fibrosis (CF) (119899 = 5) or chronic obstructive pulmonary disease(COPD) (119899 = 5) Recombinant human SLPI significantly inhibited fMet-Leu-Phe (fMLP) and interleukin(IL)-8 induced neutrophilchemotaxis (119875 lt 005) and decreased degranulation of matrix metalloprotease-9 (MMP-9) hCAP-18 andmyeloperoxidase (MPO)(119875 lt 005)Themechanismof inhibition involvedmodulation of cytosolic IP

3production and downstreamCa2+ fluxThe described

attenuation of Ca2+ flux was overcome by inclusion of exogenous IP3in electropermeabilized cells Inhibition of IP

3generation

and Ca2+ flux by SLPI may represent a novel anti-inflammatory mechanism thus strengthening the attractiveness of SLPI as apotential therapeutic molecule in inflammatory airway disease associated with excessive neutrophil influx including CF non-CFbronchiectasis and COPD

1 Introduction

The ability of neutrophils to mobilise rapidly to a site ofinfection is paramount to successful elimination of foreignmicrobes however an excessive infiltrative response can elicitextensive tissue damage Indeed neutrophil migration tothe airways is a feature of a number of chronic pulmonarydisorders including cystic fibrosis (CF) and chronic obstruc-tive pulmonary disease (COPD) where neutrophil elastase(NE) is largely responsible for protease-mediated damage inthe lung A gradient of attractant molecules including the

bacterial chemotactic peptide fMLP signalling through FPR1and tissue derived IL-8 signalling via the chemokine receptorsCXCR1 and CXCR2 attract neutrophils to migrate towardsthe site of tissue infection in a directional manner referred toas chemotaxis Upon cell migration the normally sphericalcell undergoes lateral polarization to form a leading edge anda trailing tail [1] To achieve this morphological change manyprocesses are initiated including cytoskeletal reorganisationwith protein polymers of filamentous actin microtubulesand intermediate filaments forming at the leading edge anddissolution of polymers occurring at the rear [2]

2 BioMed Research International

Molecular regulation of cytoskeletal rearrangements dur-ing neutrophil signalling associated with chemotaxis anddegranulation of antimicrobial enzymes requires an increasein cytosolic Ca2+ levels [3] This increase is due to the releaseof Ca2+ from the cellrsquos internal store (the calciosome) or influxof Ca2+ from the extracellular environment Signalling viafMLP or IL-8 GPCRs results in activation of the 120573 isoformof phospholipase C in turn yielding production of inositol145 triphosphate (IP

3) [4] IP

3occupancy of receptors on

calciosomes induces a rapid release of stored Ca2+ and ina variety of cell types this cascade has been proposed as apotential target for the treatment of a number of diseasesincluding heart failure and arrhythmias [5 6]

Serine protease inhibitors such as SLPI act locally tomaintain a proteaseantiprotease balance thereby preventingprotease mediated tissue destruction [7] Disruption of thisbalance is responsible for much of the extracellular matrixand subsequent lung tissue damage evident in neutrophildriven lung diseases [8 9] SLPI is a well-characterisedmember of the trappin gene family of proteins [10] It is ahighly basic cationic protein that is produced by epithelialcells of the respiratory tract and is also produced by phago-cytic neutrophils [11] SLPI inhibits a broad range of serineproteases including NE cathepsin G trypsin and tryptaseMoreover SLPI possesses anti-inflammatory characteristicsand in monocytes has been shown to inhibit lipopolysac-charide (LPS)- and lipoteichoic acid-induced nuclear factor(NF)-120581B activation and can compete with p65 for bindingto NF-120581B binding sites [12 13] Moreover SLPI has beenshown to directly bind to bacterial LPS thereby down-regulating production of proinflammatorymediators [14 15]However an area that has received less attention is therole of neutrophil-derived SLPI in modulating inflammatoryresponses

Studies have localised SLPI within the cytosol [11] andalso in secondary granules of neutrophils in turn coreleasedwith lactoferrin [16] Moreover the release of SLPI from neu-trophils upon phorbol ester (PMA) activation suggests thatneutrophil-derived SLPI may regulate the protease antipro-tease balance at sites of tissue inflammation [11] Howeverthe aim of this study was to investigate the intracellularfunction of SLPI within resting and activated neutrophilsThis study has revealed a novel anti-inflammatory role for thisantiprotease and demonstrated the ability of SLPI to modu-late neutrophilmigration and degranulation by inhibiting IP

3

production and Ca2+ ion mobilisation

2 Methods

21 Chemicals and Reagents All chemicals and reagentsincluding inositol 145-triphosphate were purchased fromSigma-Aldrich St Louis MO USA unless indicated other-wise The concentration of SLPI in neutrophil cytosol wasmeasured using the Quantikine Human SLPI Immunoassay(RampD Systems Abingdon Oxon UK) Recombinant humanSLPI (rhSLPI) was obtained from R amp D Systems andrecombinant calpain was obtained from Calbiochem (LaJolla CA USA) Human NE was purchased from Elastin

Products Company Inc (Owensville MO USA) The IP-One HTRF assay kit was purchased from Cisbio Bioassays(Bedford MA 01730 USA)

22 Study Groups Control volunteers (119899 = 10 mean age351 plusmn 18) had no underlying medical illnesses and were notreceiving any medication Prior to recruitment patients withCF (119899 = 5) were exacerbation-free over the preceding 6-weekperiod and patients with COPD were being treated for anacute exacerbation (119899 = 5) There were 25 and 35 malesrecruited to the CF and COPD study respectively The meanage was 225 plusmn 27 and 565 plusmn 27 years for the CF and COPDgroups respectively Informed patient consent was obtainedfor all procedures and ethical approval for the use of bloodsamples from CF and COPD individuals was obtained fromthe Beaumont Hospital Ethics Review Board

23 Preparation of HumanNeutrophils and Cell FractionationBlood was obtained from all donors in 75mL heparinisedS-monovette tubes (Sarstedt Ltd Ireland) and neutrophilswere purified by dextran sedimentation and Lymphoprep(Axis-Shield PoC AS Norway) centrifugation as previouslydescribed [17] Purified cells were resuspended in phosphate-buffered saline (PBS) (pH 74) containing 5mM glucose(PBSG) and used immediately The purity of the neutrophilpopulation was confirmed by flow cytometry measuring theneutrophil membrane marker CD16b and was found to begt99 [18] All in vitro experiments were performed at 37∘CCells were either left unstimulated (control) or activated withPMA (16 120583M) fMLP (1120583M) andor IL-8 (12 nM R amp DSystems) for the indicated time points

For cell fractionation studies isolated neutrophils weresuspended in Lamberts Break Buffer (LBB) (10mM KCl3mM NaCl 4mM MgCl

2 and 10mM piperazine-NN1015840-

bis(2-ethanesulfonic acid) (PIPES) pH 72) containing 10(ww) sucrose and the following protease inhibitors 13 120583Maprotinin 5mM benzamidine 015mM N120572-Tosyl-L-lysinechloromethyl ketone hydrochloride (TLCK) 05mM N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK) 20mMN-(Methoxysuccinyl)-Ala-Ala-Pro-Val-chloromethyl ketone(MeOSuc-AAPV-CMK) 10 120583M soybean trypsin inhibitor(SBTI) orthophenanthroline and 02M pefabloc Cells weresonicated 3 times for 5 sec on ice and centrifuged at 500timesgat 4∘C for 5min to generate a post nuclear supernatant(PNS) The PNS was overlaid on discontinuous sucrosegradients of 175 and 35 (ww) dissolved in LBB This wascentrifuged at 137000timesg for 45min at 4∘C The cytosolicfraction was removed from above the 17 (ww) sucroselayer and membrane fraction was recovered from the top ofthe 34 (ww) sucrose layer The pellet (containing primaryand secondary granules) was resuspended in 10 (ww)sucrose and overlaid on a discontinuous sucrose gradient of30 43 and 55 (ww) This was centrifuged at 137000timesg for1 h at 4∘C Secondary and primary granules were harvestedfrom above the 43 (ww) and 55 (ww) sucrose layersrespectively as previously described [18] The concentrationof protein present in each purified fraction was quantified by

BioMed Research International 3

use of a BCAProtein Assay Kit (Thermo Scientific) accordingto the manufacturerrsquos instructions

Quantification of F- and G-actin was carried out aspreviously described [19] In brief neutrophils were eitheruntreated or treated with SLPI (480 nM) or wortmannin(100 nM) for 10min and then left unactivated or activatedwith IL-8 (12 nM) and fMLP (1 120583M) for 10min Neutrophilswere lysed within an F-actin stabilisation buffer containingATP (1mM) and protease inhibitors (as indicated above) for10min at 37∘C Clarified cell lysates were centrifuged for 2 hat 100000timesg The supernatant was removed and the pelletwas resuspended in an equal volume of buffer containingcytochalasinD (10mM) and incubated on ice for 1 h An equalvolume of supernatant and pellet was electrophoresed andWestern blotted for actin employing a monoclonal anti-actinantibody (10 120583gmL Millipore Billerica MA USA)

24 Neutrophil Uptake Assays Native neutrophil cytosolicSLPI levels were determined by ELISA (SLPI Human ELISACambridge Bioscience UK) Uptake of exogenous recombi-nant human SLPI (rhSLPI 480 nM) by cells (2 times 105mL inPBSG) was performed at 37∘C for 10min After incubationthe SLPI loaded cells were washed with ice cold PBSG andneutrophil levels of cytosolic SLPI measured by ELISA Todetermine the mechanism of SLPI uptake cell (2 times 105mL inPBSG) incubationswere performed at 4∘C for 10min or in thepresence of the endocytosis inhibitors sodium azide (NaN

3

15mM) and sodium fluoride (NaF 10mM) as previouslydescribed [20]

25 SDS-PAGE and Western Blot Analyses Samples weresubjected to SDS-PAGE under denaturing conditions on 4ndash12 (wv) NuPAGE gels (Invitrogen Carlsbad CA USA)following the manufacturerrsquos instructions Routinely 25120583gof neutrophil cytosolic protein 10 120583g of membrane protein5 120583g secondary granule protein and 10 120583g of primary granuleprotein was loaded on each gel After electrophoresis gelswere stained by Coomassie blue R250 for visualizationof proteins or alternatively proteins were transferred onto02 120583m nitrocellulose or PVDF membrane by Western blot-ting using a semidry blotter for 1 h at 100mA Membraneswere blocked for 1 h in 3 dry milk (wv) and 1 (wv)bovine serum albumin (BSA) in PBS containing 005 (vv)Tween 20 Blots were incubated with 10 120583gmL polyclonalrabbit (Rb) anti-SLPI specific antibody (Abcam CambridgeUK) 10 120583gmL polyclonal goat (Gt) anti-SLPI (SynergenInc Boulder Colorado 80301) [21 22] 10 120583gmL polyclonalgoat (Gt) anti-SLPI (RampD Systems) 02 120583gmL polyclonalrabbit anti-myeloperoxidase (MPO) anti-hCAP-18 or anti-lactoferrin antibody (all purchased fromAbcam CambridgeUK) Additional primary antibodies included 02 120583gmLpolyclonal goat antimatrix metalloprotease (MMP)-9 (R amp DSystems) 10 120583gmL monoclonal anti-talin-1 and 10 120583gmLmonoclonal anti-vinculin or monoclonal anti-actin antibody(Millipore)The secondary antibodies were HRP-linked anti-rabbit -goat or -mouse IgG (Cell Signalling TechnologyDanvers MA USA) Immunoreactive protein bands were

visualized employing SuperSignal West Pico Chemilumines-cent Substrate (Pierce Rockford IL USA) after exposure toKodak X-Omat LS Film

26 Neutrophil Electropermeabilization Cell permeabiliza-tion was performed immediately before use of neutrophilsas previously described [23 24] In brief cells (5 times 106)were washed in hypoosmolar buffer (Eppendorf UK Ltd)and then resuspended in 400 120583L of ice-cold hypoosmolarbuffer in the presence or absence of 1 120583M IP

3 The cells were

then transferred into an electroporation cuvette (2mm gap400 120583L volume purchased from Eppendorf) and subjectedto three discharges of 300V using an Eppendorf Multi-porator with gentle stirring between the three pulses bypipetting [24] Permeabilized cells were then incubated atroom temperature for 5min to allow incorporation of IP

3into

electroporated neutrophils immediately washed in PBSGand then employed in chemotaxis assays or for Ca2+ fluxmeasurements Control experiments indicated that the cellpermeabilization procedure did not affect cell viability asdetermined by trypan blue exclusion assays

27 Neutrophil Chemotaxis Assays Chemotaxis assays wereperformed by measuring the percentage of neutrophils (25times 105cells200120583L) migrating towards IL-8 (12 nM) or fMLP(1 120583M)by employing amultiwell chemotaxis chamber (NeuroProbe Inc USA) and polyvinylprrolidone-free polycarbon-ate filters (10 120583m thick with 5 120583m pores) The stimulant wasplaced in the lower chamber (total volume of 90120583L) andpurified neutrophils were placed in the upper chamber (25times 105cells200120583L) with or without SLPI (0 120 240 or480 nM) Neutrophil chemotaxis was quantified over 30minat 37∘C The Neuroprobe chamber was then disassembledand the polycarbonate filter was fixed with methanol andcells were stained using Speedy-Diff solutions (Clin-tech LtdUK) The number of migrated neutrophils was determinedmicroscopically employing a Nikon Eclipse TS100 micro-scope with 10 standardized times 400-high power fields countedfor each well For comparative analysis neutrophils treatedwith IL-8 or fMLP were set at a chemotactic index of 1as indicated Additional experiments were performed afterelectropermeabilization with 1120583M IP

3 Cells were then left

unstimulated (control) or challengedwith fMLP or IL-8 Datawas represented as chemotactic index as previously described[25]

28 Measurement of Intracellular Ca2+ Flux Measurementof intracellular cytosolic Ca2+ was performed employing theFluo-4 NWCalciumAssay Kit (Invitrogen Bio Sciences LtdIreland) in the absence of extracellular Ca2+ according to themanufactures instructions In brief cells (2 times 105mL) wereincubated for 30min in dye loading solution containing theCa2+ sensitive dye Fluo-4 Subsequently cells were either leftuntreated or were exposed to fMLP (1120583M) or IL-8 (12 nM)with changes in fluorescence recorded immediately uponaddition of the stimulant Fluorescence was recorded every10 sec for 5min employing a Victor X3 (PerkinElmer Ire-land) plate reader with excitation wavelength of 490 nm and


emission at 535 nm A subset of experiments was performedby incubating cells with either SLPI (480 nM) the PLC-120573inhibitor U73122 (5120583M) or the GPCR inhibitor pertussistoxin (500 ngmL) prior to stimulation with fMLP (1mM) orIL-8 (12 nM) Additionally experiments were performed bypretreating cells with either SLPI (480 nM) or oxidised SLPI(480 nM) the later oxidised with 20mM hydrogen peroxide(H2O2) as previously described [13]

29 Analysis of Neutrophil Degranulation and Secretion ofSLPI Neutrophils (5 times 106mL per reaction) suspendedin PBSG remained untreated or preloaded with rhSLPI(480 nM) for 10min followed by fMLPIL-8 (used in com-bination at 1120583M and 12 nM respectively to ensure releaseof primary secondary and tertiary granules) stimulation for10 or 20min Cell free supernatants were harvested followingcentrifugation at 500timesg for 5min at 4∘C and analysed fordegranulated proteins MPO as a marker of primary granulerelease hCAP-18 as a marker of secondary granule releaseand MMP-9 as a marker of tertiary granule release byWestern blotting In a subset of experiments the extracellularrelease of native cytosolic SLPI was analysed by Westernblotting following cellular activation with PMA (16 120583M) for0 05 1 or 10min or fMLP (1 120583M) and IL-8 (12 nM) for 0 1020 30 40 or 50 sec Cell free supernatants were harvested asdescribed above and acetone precipitated prior to SDS-PAGEand Western blot analysis for SLPI

210 Neutrophil Elastase (NE) and Calpain Activity AssaysNE (34 nM) activity assays employing the specific sub-strate N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide aspreviously described [26] Liberation of p-nitroaniline wasmeasured at 405 nm at 37∘C at 1min intervals for 5min Inadditional studies the NE inhibitory activity of neutrophilcytosol was assessed before and after immunoprecipitationof SLPI For immunoprecipitation experiments neutrophilcytosols were obtained from 1 times 107 cells and preclearedby incubation with protein A-Sepharose beads for 1 h aspreviously described [18] Goat polyclonal anti-SLPI (5 120583g)or isotype control IgG was then added to precleared cytosolsfor 1 h with rotation at 4∘C SLPI was then removed from thecytosol by incubationwith reconstituted proteinA-Sepharosebeads for 1 h and the anti-NE capacity of the resulting cytosol(25 120583g) was analysed

Kinetic analysis of calpain activity was determinedusing the Calpain-Glo protease assay (Promega CorporationMadison USA) which detects cleavage of the calpain sub-strate Suc-LLVY-aminoluciferin with luminescence recordedas per the manufacturerrsquos instructions The inhibitory effectof rhSLPI and calpastatin was analysed over a range of 0ndash640 nM and 0ndash80 nM respectively

211 Inositol 145-TriphosphateMeasurements For analysis ofIP3we employed a recognised protocol and measured levels

of IP1which accumulates as a stable product of IP

3[27]

Neutrophils (1 times 105) remained untreated or were preloadedwith SLPI (480 nM) as already described As positive controlsthe PLC-120573 inhibitor U73122 (5120583M) and the GPCR inhibitor

pertussis toxin (500 ngmL) were employed prior to stimula-tion with fMLP (1 120583M) or IL-8 (12 nM) for 10 sec Cells werethen lysed and intracellular IP

1levels were determined by

use of an IP-One HTRF assay kit in a final reaction volume of200120583L as per the manufacturerrsquos instructions

212 Flow Cytometry Analysis Flow cytometry was carriedout to evaluate the membrane expression of CD16b as ameasure of cell purity [18] or CD66b (secondary and tertiarygranules) and CD63 (primary granule) as a measure ofdegranulation [24] Cells remained untreated or were stimu-lated with fMLP (1120583M) and IL-8 (12 nM) for 10min at 37∘CNeutrophils were then fixed (4 (wv) paraformaldehyde)and blocked (2 (wv) BSA) for 30min at room temperatureAfter washing (PBS times 2) neutrophils (1 times 106) were incubatedwith 1 120583g100 120583L of mouse monoclonal anti-CD16b (SantaCruz Germany) FITC labelled anti-CD66b or PE labelledanti-CD63 (both from BD Bioscience UK) Control sampleswere exposed to relevant nonspecific isotype control IgGor secondary labeled antibody alone (FITC-labelled bovineanti-mouse) In additional experiments the ability of fMLPto interact with its cognate receptors on the neutrophilmembrane in the presence of rhSLPI was performed aspreviously described [28] In brief purified neutrophils (1times 107mL) were left untreated or exposed to fMLP (10120583M)or rhSLPI (480 nM) for 1min followed by incubation withFITC-labeled fMLP (1120583M) for 1min Cells were washed inPBS and fluorescence was counted using a BD FACSCaliburflow cytometer (BD Bioscience Germany) with a total of10000 events acquired The data were analysed and themean fluorescence intensity (MFI) for each experiment wasdetermined using BD CellQuest Pro software

213 Confocal Immunofluorescence Adherent neutrophilswere produced by placing neutrophils in PBS on Polysinemicroscope slides (AGB Scientific Ltd Ireland) after whichthey were incubated at 37∘C for 5min and then for a further10min at 37∘C in the presence of fMLP (1 120583M) and IL-8(12 nM) or PMA (16 120583M) Cells were then fixed with 4(wv) paraformaldehyde in PBS for 10min and permeabilisedwith 02 (vv) Triton X-100 in PBS for 10min Subsequentlycells were blocked for 1 h with 1 (wv) BSA in PBS andthen incubated with 1 120583gmL FITC-labeled rabbit polyclonalanti-SLPI for 45min and washed in PBS Cells were mountedemploying VECTASHIELD mounting medium with DAPIfor nuclear staining (Vectashield Lab UK) Control sampleswere exposed to non-specific FITC-labeled isotype controlIgG All immunofluorescence slides were viewed and imageswere acquired using a Zeiss LSM710 confocal immunofluo-rescence microscope

214 Statistical Analysis The data was analysed with theGraphPad Prism version 403 for Windows (GraphPad Soft-ware San Diego CA USA) and results were expressed asmeanplusmn standard error (SE) of themeanTheMann-Whitney119880-test or analysis of variance (ANOVA) sample tests followedby Bonferri correction were employed to identify significant


differences Experiments were performed in triplicate and a119875-value lt 005 was deemed significantly different

3 Results and Discussion

31 The Immunomodulatory Effects of Cytosolic SLPI onNeutrophil Migration and Degranulation SLPI has beenpreviously reported to exert an anti-inflammatory effect onimmune cell function [20 29] and in this respect SLPIhas been shown to inhibit monocytic CD4 lymphocyteproliferation and Th1 cytokine (INF-120574) release [30] IndeedT lymphocytes play a key role in the pathogenesis of CF[31] and COPD [32] lung disease and have been identified asthe predominant cell type in subepithelial bronchial tissue ofCF patients [31] Th1 Th2 and Th17 cells may augment thecytokinechemokine profile in the airways of CF patients thuscontributing to the chronic recruitment of neutrophils [31]Thus the objective of this study was to investigate whetherSLPI exerted an anti-inflammatory effect on neutrophilfunction and for this reason it was first necessary to confirmits cellular localisation

Localisation of SLPI within many cell types known toproduce this protein is still being elucidated For exampleit has been reported that cellular SLPI is localised in thenuclei of bronchial epithelial cells exposed to 17120573-estradiolresulting in NF-120581B inhibition and IL-8 gene expression[33] SLPI has also been detected in the nuclei of alveolarmacrophages in patients with pulmonary sepsis but not inhealthy controls [20] indicating that the localisation of SLPIin disease states may differ from healthy control cells Withinthe present study by subcellular fractionation of restingneutrophils and Western blot analysis employing both rabbitand goat polyclonal anti-SLPI antibodies SLPI was identifiedas a component of the cell cytosol and secondary granulefractions (co-localising with lactoferrin) andwas absent fromunstimulated membranes and primary granule fractionscontaining MPO (Figure 1(a)) Localization of SLPI to thesecompartments is in keeping with previously published data[11 16] As the role of cytosolic SLPI was the focus of ensuingexperiments we also confirmed the presence of SLPI incytosolic fractions of CF and COPD cells (Figure 1(b))

Having identified the position of SLPI in the neutrophilfurther experiments were designed to establish whethercytosolic SLPI possessed antiprotease activity Results ofanti-NE kinetic measurements indicated that cytosolic SLPIcontained anti-NE capacity (Figure 1(c)) as both the rateand total level of NE catalytic activity (34 nM) were negatedby freshly isolated neutrophil cytosol (25120583g10 120583L) an effectsignificantly reversed in samples in which SLPI was extractedby immunoprecipitation (Figure 1(c)) At the 240 sec timepoint (4min) cytosol that had been subjected to immuno-precipitation for SLPI removal was 667 plusmn 21 less active ininhibiting NE as compared with complete cytosol (119875 lt 005)and illustrated no significant difference compared to the NEcontrol reaction (Figure 1(d))

To begin to understand the role of intracellular SLPIin neutrophil cell function our approach was to increase

the level of this anti-protease within the cell cytosol Toachieve this cells were resuspended in PBSG containingexogenous rhSLPI for 10min and then washed to removeprotein that had not been taken up by the cells To confirmthat rhSLPI was internalized the concentration of SLPI inneutrophil cytosolic fractions was determined by ELISAResults revealed that addition of 480 nM rhSLPI significantlyraised the concentration of cytosolic SLPI from 96plusmn 63 ng to151 plusmn 48 ng per 1 times 105 cells (119875 lt 005 119899 = 6) indicatingpermeability of the cell membrane to exogenous rhSLPI(Figure 1(e)) To examine internalization of rhSLPI in furtherdetail experiments were repeated at 4∘C thereby applying atemperature block to inhibit transport processes [20] Underthese reduced temperature conditions results indicated thatthe uptake of SLPI and localization to the cell cytosol weresignificantly inhibited by 302 plusmn 34 (Figure 1(e)) The roleof endocytosis was also assessed by pretreating cells withthe endocytosis inhibitors NaN

3(15mM) and NaF (10mM)

[20] These inhibitors had no significant effect on rhSLPIuptake and cytosolic association (Figure 1(e)) Collectivelythese results indicate that exogenously added rhSLPI is takenup by the cell and is localized in the cytosol and is notcontained within endosomes These results are in line withpreviously published data on the internalization of rhSLPI bymononuclear cells [20] In this later publication exogenouslyadded rhSLPI was localized to the cytosol and nucleus ofU937 monocytes SLPI has been characterised as an argininerich cationic molecule [34] and it has been reported thatthe positively charged nature of the protein facilitates itstransduction across negatively charged membranes [20 34]Moreover investigation of the anti-HIV-1 inhibitory activityof SLPI has prompted the identification of membrane pro-teins that are capable of binding to SLPI In this regard SLPIhas been shown to interact with the phospholipid bindingprotein annexin II on the surface of human macrophage cellmembranes [35] and has also been described as a ligand forphospholipid scramblases 1 and 4 (PLSCR1 and PLSCR4)[36] This later interaction suggested that SLPI disruptsinterplay between PLSCR1 and the CD4 receptor on thesurface of CD4+T lymphocytes thus preventing HIV-1 viralinfection [36]

Within the present study subsequent experiments inves-tigated the effect of cytosolic SLPI on directional chemotaxisa fundamental neutrophil cellular response in terms ofpulmonary inflammation Cells (25 times 105200120583L) were firstpreloaded with exogenous rhSLPI (0 120 240 or 480 nM)washed and then exposed to IL-8 (12 nM) or fMLP (1120583M)two stimuli with known effective chemoattractant proper-ties [37 38] As shown in Figures 2(a) and 2(b) rhSLPIinhibited both IL-8 and fMLP-induced neutrophil directionalchemotaxis in a dose dependent manner with an IC

50of

approximately 240 nM recorded for both stimuli Excessiveneutrophil infiltration to the lung is a key pathogenic featurefor disease progression in individuals with CF and COPDFor this reason we examined the effect of rhSLPI on CF andCOPD neutrophil chemotaxis in response to inflammatorystimuli in vitro Results confirmed the inhibitory action oncell migration and demonstrated that cells preloaded withrhSLPI showed a markedly reduced capacity to migrate in


Anti-lactoferrin

6

1417

28

(kDa)

6298

188

38

1 2 3 4Gel

Blots

Anti-MPOAnti-SLPI(Rb Abcam)Anti-SLPI(Gt Synergen)Anti-SLPI(Gt RampD systems)

(a)

(kDa) 1 2 3Gel

1 2 3

Blots14

17

28

62

98

188

38

CF COPD

Anti-SLPI(Gt Synergen)

(b)

0

01

02

03

04

05

06

NE 34 nmTotal cytosolCytosol post-SLPI IPCytosol post-controlIgG IP

2400 60 120 180Time (seconds)

Abso

rban

ce (4

05 nm

)

(c)

NE

NS

Total post-

Cytosol post-

IgG IP

0

01

02

03

04

05

06

Abso

rban

ce (4

05 nm

)

cytosolCytosol

SLPI IPcontrol

lowastlowast

(d)

0

20

40

60

80

100

120

140

160

180

Con +rhSLPI +rhSLPI

NS

lowastlowast

4∘C+rhSLPl+NaN3+NaF

Cyto

solic

SLP

I (ng

105

cells

)

(e)

Figure 1 Localization and activity of native SLPI in peripheral blood neutrophils (a) Coomassie blue stained gel of isolated neutrophilssubjected to subcellular fractionation yielding a cytosol fraction (lane 1) membrane fraction (lane 2) and secondary and primary granulefractions lane 3 and 4 respectively Western blotting employed polyclonal rabbit (Rb) or goat (Gt) antibody to SLPI which localized SLPIto both the cytosolic and secondary granule fractions As controls MPO and lactoferrin were detected using rabbit polyclonal antibodies asmarkers for primary and secondary granules respectively (b) Coomassie blue stained gel of isolated CF and COPD neutrophils subjectedto subcellular fractionation yielding a cytosol fraction (lane 1) membrane fraction (lane 2) and pooled primary and secondary granulefractions (lane 3) Western blotting employed polyclonal Rb antibody confirming cytosolic localization of SLPI in patient samples (c) TheNE inhibitory activity of neutrophil cytosol was assessed before and after immunoprecipitation (IP) of SLPI using Gt polyclonal anti-SLPIantibody (Synergen) The kinetics of inhibition is illustrated in (c) and data of the final time point (240 sec) is plotted in (d) Controlexperiments included isotype control Gt IgG lowast119875 lt 005 betweenNE control (e) Addition of 480 nM rhSLPI to cells (2times 105mL) increased theconcentration of cytosolic SLPI by approximately 50 above the untreated cells (Con) as determined by ELISA Experiments were repeatedat 4∘C or in the presence of NaN

3(15mM) and NaF (10mM) lowast119875 lt 005 between untreated cells (con) Results illustrated in (a) and (b) are

representative gels and blots of 3 separate experiments Results illustrated in panel (c)ndash(e) were performed in triplicate and each bar is themean plusmn SE (NS no significant difference lowast119875 lt 005 calculated by Studentrsquos 119905-test)

response to fMLP and IL-8 (Figures 2(d) and 2(c) respec-tively 119875 lt 005 119899 = 5) An IC

50of 611 nM and 376 nM for

CF cells and an IC50

of 542 nM and 627 nM for COPD cellswere recorded for fMLP and IL-8 stimulation respectivelyThus a greater level of SLPI was required to inhibit CF and

COPD neutrophil chemotaxis compared to control cellsThislatter result may be a consequence of neutrophil primingwithin the circulation of individuals with CF and COPD dueto persistent inflammation Indeed studies have shown thatCFneutrophils are primed and unresponsive to IL-10 induced


Con 120 240 4800

025

05

075

1

SLPI (nM)

lowast

lowastCh

emot

actic

inde

x (fM

LP 1

120583M)

(a)

0

025

05

075

1

Con 120 240 480SLPI (nM)

lowast

lowastlowastChem

otac

tic in

dex

(IL-

8 1

2 nM

)(b)

CF COPD

0

025

05

075

1

lowast lowast

Chem

otac

tic in

dex

(fMLP

1 120583M

)

(c)

CF COPD

0

025

05

075

1

lowast

lowast

Chem

otac

tic in

dex

(IL-

8 1

2 nM

)

(d)

Figure 2 rhSLPI inhibits neutrophil chemotaxis (a) and (b) An increase in chemotactic inhibition efficiency of increasing concentrationsof SLPI (120 240 and 480 nM) The chemotactic index represents the ratio of neutrophils that migrated towards fMLP (1 120583M) or IL-8(12 nM) over 30min at 37∘C (c) and (d) fMLP and IL-8-induced mean chemotactic index of neutrophils (25 times 105cells200 120583L) isolatedfrom individuals with CF (119899 = 5) or individuals with COPD (119899 = 5) in the presence (◻) or absence (◼) of 480 nM SLPI Results illustratedwere performed in triplicate and each bar is the mean plusmn SE lowast119875 lt 005 or lowastlowast119875 lt 001 versus untreated control cells (Con) with statisticalsignificance calculated by Studentrsquos 119905-test

anti-inflammatory signals [39] Pseudomonas alginate [40]TNF-120572 and IL-8 have been shown to be important primingagents forCFneutrophils causing greater release ofMPO [41]and NE compared to neutrophils from control subjects andindividuals with bronchiectasis [42]

The degranulation of proteolytic enzymes and peptidesfrom the neutrophil upon activation is a tightly regulatedprocess in order to prevent unnecessary damage to tissues[43] For this reason we evaluated the inhibitory effect ofrhSLPI on the kinetics of degranulation In this experiment

we used fMLP (1 120583M) and IL-8 (12 nM) in combinationto ensure exocytosis of all three neutrophil granule types(primary secondary and tertiary) and release of granuleproteins was quantified in the extracellular supernatant byimmunoblotting The use of equal cell numbers (5 times 106mL)in each reaction is demonstrated by identical Coomassieblue stained electrophoretic profiles of whole cell lysatesprepared form cells employed in each reaction (Figure 3(a))Levels of cell released MMP-9 from tertiary granules (Fig-ure 3(b)) hCAP-18 from secondary granules (Figure 3(c))


10 20 10 20 10 20Time (min)

614

17

28

(kDa)

6298

38

ConfMLPIL-8

IL-8fMLP

SLPI

(a)

0

1

2

3

4

MM

P-9

(den

sitom

etry

uni

ts)

10 20 10 20 10 20Time (min)

Con fMLPIL-8

fMLP

SLPIIL-8

10 min20 min

lowastlowast

(b)

0

5

10

15

20

hCA

P-18

(den

sitom

etry

uni

ts)

10 20 10 20 10 20Time (min)

Con fMLPIL-8

fMLP

SLPIIL-8

10 min20 min

(c)

0

2

4

6

8

MPO

(d

ensit

omet

ry u

nits)

10 20 10 20 10 20Time (min)

Con fMLPIL-8

fMLP

SLPIIL-8

10 min20 min

lowast

(d)

0

10

20

30

40

50

60

70

CD66

b (M

FI)

Control fMLPIL-8

fMLP

SLPIIL-8

lowastlowastlowast lowastlowastlowast

(e)

CD63

(MFI

)

0

10

20

30

40

Control fMLPIL-8

fMLP

SLPIIL-8


(f)

Figure 3 rhSLPI inhibits neutrophil degranulationNeutrophils (5times 106mL) isolated fromhealthy individuals were either untreated (Con) orexposed to SLPI (480 nM) followed by stimulationwith fMLP (1 120583M) and IL-8 (12 nM) at 37∘CThe use of equal cell numbers in each reactionis demonstrated by the identical electrophoretic profile of whole cell lysates in the Coomassie blue stained gel (a) Cell free supernatants werecollected at 10 (◻) or 20min (◼) and Western blotted for markers of tertiary ((b) MMP-9) secondary ((c) hCAP-18) or primary granulerelease ((d)MPO) (e) and (f) Healthy control neutrophils were fixed in 4 (wv) paraformaldehyde following 10min IL-8fMLP stimulationin the presence or absence of SLPI (480 nM) and analysed by flow cytometry employing a FITC-labeled CD66b (e) or CD63 antibody (f)Data are represented as mean fluorescent intensity (MFI) All results (expressed as relative densitometry units) were performed in triplicateand each bar is the mean plusmn SE A representative Western blot is illustrated lowast119875 lt 005 between SLPI treated and untreated at the respectivetime points calculated by Studentrsquos 119905-test

and MPO from primary granules (Figure 3(d)) were signif-icantly reduced post 20min stimulation in cells preloadedwith 480 nM rhSLPI (a 60 83 and 80 reductionrespectively 119875 lt 005) As tertiary granules are more readilydischarged the inhibitory action of rhSLPI was apparent atthe 10min time point (70 reduction 119875 lt 005) As analternative approach we investigated the membrane expres-sion of CD66b and CD63 CD66b is a membrane receptorthat is exclusively expressed on the membrane of secondaryand tertiary granules and CD63 is present on primarygranule membranes [24] Upon degranulation both CD66b

andCD63 become expressed on the cell surface As illustratedin Figures 3(e) and 3(f) membrane expression of CD66b andCD63 increased by 40 and 180 respectively after 10minfMLPIL-8 combined stimulation compared to unstimulatedcells (119875 lt 0001) In contrast upregulation of CD66b andCD63 to the plasma membrane was significantly decreasedin stimulated neutrophils preloaded with SLPI compared tocells unexposed to SLPI (119875 lt 0001) Collectively this datasuccessfully demonstrates the inhibitory effect of rhSLPI onneutrophil chemotaxis and degranulation via IL-8 and fMLPGPCR signalling


Distribution ratio 24 06minus + + + + + +minusminus minus minus + + minus minusminus

minus minus minus minus minus + +minus

2 1 15 15 24 06

S P S P S P S P

IL-8fMLPSLPI (480 nM)Wort (100 nM)

(a)

Blots

fragment

(kDa)250

98

64

36

fragment

Gels

minus minus+SLPI +

+ +PMA minus minus

+ +fMLPIL-8 minus minus

190 kDa

sim90 kDa

Anti-talin-1

Anti-vinculin

Anti-120573-actin

(b)

0

05

1

15

fMLPIL-8 IL-8

fMLP

SLPI

PMA PMASLPI

Tal

in cl

eava

ge (d

ensit

omet

ry u

nits)

lowastlowast

(c)

fMLPIL-8 IL-8

fMLP

SLPI

PMA PMASLPI

0

05

1

15

Vin

culin

clea

vage

(den

sitom

etry

uni

ts)

lowast

lowast

(d)

Figure 4 SLPI inhibits neutrophil cytoskeletal rearrangements (a) Immunoblot with anti-actin antibody for the distribution of G-actin (inthe supernatant fraction S) and F-actin (in the pellet fraction P) in untreated cells IL-8fMLP (12 nM1 120583M) SLPI (480 nM) or controlwortmannin (Wort 100 nM) treated cellsThe distribution ratios calculated using constants obtained ofWestern blot densitometry values areillustrated (b)The effect of SLPI (480 nM) on talin-1 (190 kDa fragment) or vinculin cleavage (90 kDa fragment) after PMA (16120583M positivecontrol) or IL-8fMLP activation for 10min was analysed by Western blotting using anti-talin-1 and anti-vinculin monoclonal antibodiesEqual sample loading was confirmed by Coomassie blue stained gels (top panels) and the 120573-actin immunoblots (lower panels) Results ofthree separate experiments were expressed as relative densitometry units ((c) and (d)) and each bar is the mean plusmn SE lowast119875 lt 005 betweenSLPI treated and untreated cells for the respective stimuli calculated by Studentrsquos 119905-test

32 Characterisation of the Mode of Action of SLPI Remod-elling of the actin cytoskeleton is a prerequisite for cellchemotaxis and is a crucial event in the degranulationprocess To study the effect of preloading cells with rhSLPIon the redistribution of F-versus G-actin after IL-8fMLPexposure a differential centrifugation assay was employedto analyse in situ F-actin levels Preloading of cells withrhSLPI (480 nM) or exposure to wortmannin (100 nM) asa control [18] suppressed the IL-8fMLP-induced change inthe ratio of G-actin (supernatant fraction) versus F-actin(pellet fraction) (Figure 4(a)) In addition as the C-terminal

fragment of talin (190 kDa) is an actin nucleating proteinwhich binds to G-actin and also the fact that cleavage oftalin-1 is critical to focal adhesion disassembly [44] weinvestigated the possibility that SLPI regulates neutrophilchemotaxis by affecting talin cleavage Neutrophils whichwere either untreated or preloaded with rhSLPI (480 nM1times 107 cells) were stimulated with PMA (positive control) orfMLPIL-8 for 10min and the level of talin-1 cleavage wasassessed by immunoblotting using a mousemonoclonal anti-talin-1 antibody (Figure 4(b)) Site-specific cleavage of talin-1 yielding a 190 kDa fragment was observed in fMLPIL-8


and PMA activated whole cell lysates an effect consistentlyinhibited by preloading cells with rhSLPI (50 and 64inhibition resp) (Figure 4(c) 119875 lt 005) Moreover we eval-uated the effect of rhSLPI on vinculin cleavage a proteininvolved in neutrophil adhesion and pseudopod formation[45] Results revealed that vinculin cleavage in responseto fMLPIL-8 and PMA was significantly reduced in cellspreloaded with rhSLPI as demonstrated by Western blotanalysis (Figure 4(b)) and densitometric quantification (96and 84 inhibition respectively Figure 4(d))

The observation that rhSLPI could inhibit talin andvinculin cleavage prompted us to initially investigate thehypothesis that SLPI inhibited the Ca2+ dependent neutralcysteine protease activity of calpain-1 and calpain-2 whichplay an important role in cell migration by cleaving talin andvinculin [46] SLPI has been shown to inhibit a broad rangeof serine proteases [47] but unlike the antiprotease alpha-1antitrypsin (AAT) SLPI has not been shown to inhibit otherprotease classes For example AAT has been shown to inhibitcaspase-3 activity [48] and to modulate metalloproteaseADAM-17 activity thereby regulating neutrophil chemotaxisin response to soluble immune complexes [18] Howeverunlike AAT or the natural inhibitor calpastatin which havebeen shown to inhibit neutrophil calpain [49] the resultsof the present study demonstrate that SLPI had no directeffect on calpain-1 or calpain-2 activity (see Supplemen-tary Figure 1 in Supplementary Material available online onhttpdxdoiorg1011552013560141)

Upon activation of neutrophils in the absence of extra-cellular Ca2+ the rise in cytosolic Ca2+ levels occurs byrelease from intracellular sites As a result of the rise in Ca2+the neutrophil initiates cytoskeletal rearrangements requiredfor degranulation and chemotaxis [50] Therefore we nextexplored the possibility that SLPI inhibits activation of neu-trophils by regulating Ca2+ mobilisation from intracellularstores Preloading neutrophils (2 times 105mL) isolated fromhealthy individuals with rhSLPI (480 nM) in a Ca2+ freebuffer significantly inhibited the rise in cytosolic Ca2+ levelsfrom intracellular stores triggered by fMLP (Figure 5(a) 119875 lt001 at 10 sec and 119875 lt 005 at 20 sec) and IL-8 (Figure 5(b)119875 lt 005 at 20 and 30 sec) compared to untreated controlcells Of note the observed spike in Ca2+ flux in healthy con-trol cells not treated with SLPI is consistent with previouslypublished data [18] Furthermore neutrophils isolated fromCF stable individuals preloaded with rhSLPI also failed toinitiate aCa2+ spike upon stimulationwith fMLP (Figure 5(c)119875 lt 005 at 10 and 20 sec) or IL-8 (Figure 5(d) 119875 lt001 and 119875 lt 005 at 10 and 20 sec resp) Additionallywhen Ca2+ levels were analysed in neutrophils isolated fromindividuals during an acute exacerbation of COPD there wasa statistically significant decrease in the cytosolic Ca2+ levelsof fMLP and IL-8 stimulated cells preloaded with rhSLPI(480 nM) compared to untreated cells (119875 lt 005 at 10 and20 sec Figures 5(e) and 5(f)) Taken together these resultsindicate that cytosolic SLPI inhibits Ca2+ flux in cells frompatients or healthy donors when exposed to proinflammatorystimuli

To rule out the possibility that SLPI inhibits Ca2+ fluxin cells by preventing IL-8 and fMLP interacting with theirrespective receptors studies on agonist-receptor interactionswere performed Whilst exposure to FITC-labeled fMLPresulted in specific binding of FITC-fMLP on neutrophilmembranes by flow cytometry (Figures 6(a) and 6(b) pre-exposure of cells (1 times 107mL) to unlabeled fMLP preventedFITC-fMLP binding thereby reducing the mean florescenceintensity reading by approximately 75 (4005 plusmn 47 to 1104plusmn 145 MFI Figures 6(a) and 6(b)) In contrast pre-loadingof neutrophils with rhSLPI (480 nM) had no effect on theFITC-fMLP fluorescence reading (4267 plusmn 4164 comparedto 4005 plusmn 4471 in the presence of SLPI) indicating thatthe immuno-regulator activity of SLPI was not a result ofinhibiting fMLP binding to the cell membrane Additionallyresults indicated that preloading cells with rhSLPI did notimpair IL-8 membrane binding (results not shown) More-over reaction with H

2O2 a major oxidant generated by

activated neutrophils oxidizes all fourmethionine residues inSLPI resulting in substantial diminution of its NE inhibitoryactivity [13] In the present study oxidised rhSLPI possesseddecreased inhibitory capacity over intracellular Ca2+ fluxupon activation with fMLP (1 120583M) or IL-8 (12 nM Figures6(c) 6(b) and 6(d)) suggesting the requirement of the activesite of SLPI for the observed inhibitory effect on Ca2+ flux

In response to either fMLP or IL-8 the initial rise inintracellular cytosolic Ca2+ requires IP

3production and sub-

sequent IP3occupancy of receptors on calciosomes induces

a rapid release of Ca2+ [51 52] Therefore we next exploredthe possibility that cytosolic rhSLPI inhibits Ca2+ cytosolicflux by preventing upstream IP

3production For this analysis

we measured levels of IP1which accumulates as a stable

product of IP3[27] Pre-loading of neutrophils (1 times 105mL)

with rhSLPI (480 nM) significantly reduced cumulative levelsof IP1upon fMLP or IL-8 activation (an approximate 75

reduction for both stimuli 119875 lt 005) with similar levels tounstimulated cells observed (Figure 7(a)) Positive controlsfor this experiment included addition of the PLC-120573 inhibitorU73122 (5 120583M) or the GPCR inhibitor pertussis toxin (PTX500 ngmL) with significant inhibition of IP

1accumulation

observed for both (119875 lt 005) Substantiating this result itwas observed that the inhibitory effect of rhSLPI (480 nM)on Ca2+ flux induced by fMLP and IL-8 was overcomeby augmenting cytosolic levels of IP

3(1 120583M) (Figure 7(b))

For this latter experiment IP3was incorporated into elec-

troporated neutrophils and the cells immediately employedin Ca2+ flux measurements Moreover results revealed thatrhSLPI employed at a concentration of either 240 or 480 nMwas unable to reduce the chemotactic index of cells loadedwith IP

3in response to either fMLP (Figure 7(c)) or IL-8

(Figure 7(d))Collectively these results confirm that cytosolic SLPI

did not impede agonist receptor interaction but success-fully modulated IP

3production and subsequent release of

Ca2+ from IP3-regulated internal stores However as the

production of IP3is a direct outcome of PLC-120573 activation

further experiments are required to fully understand howSLPI modulates PLC-120573 activation Moreover it is possible


0 10 20 30 40 500

1

2

3

4

Time (seconds)

Hea

lthy

cells

Ca2

+(r

elat

ive l

evels

)

lowastlowastlowast

(a)

Time (seconds)0 10 20 30 40 50

0

1

2

3

4

Hea

lthy

cells

Ca2

+(r

elat

ive l

evels

)

lowast

lowast

(b)

Time (seconds)0 10 20 30 40 50

0

1

2

3

4

CF ce

lls C

a2+

(rel

ativ

e lev

els)

lowastlowast

(c)

0 10 20 30 40 500

1

2

3

4

Time (seconds)

CF ce

lls C

a2+

(rel

ativ

e lev

els)

lowast

lowastlowast

(d)

0 10 20 30 40 500

2

1

4

3

6

5

7

8

Time (seconds)

COPD

cells

Ca2+

(rel

ativ

e lev

els)

lowast

lowast lowast

fMLP (+SLPI)Con

fMLP (minusSLPI)

(e)

0

1

2

3

4

0 10 20 30 40 50

Time (seconds)

COPD

cells

Ca2+

(rel

ativ

e lev

els)

Con

IL-8 (minusSLPI)IL-8 (+SLPI)

lowastlowast

(f)

Figure 5 SLPI inhibits intracellular Ca2+ flux Neutrophils (2 times 105mL) were isolated from healthy donors ((a) and (b)) individuals withCF ((c) and (d)) or COPD ((e) and (f)) (119899 = 5 for each cohort) Cells remained untreated (control con) or treated with 480 nM SLPI for5min prior to stimulation with fMLP (1120583M) or IL-8 (12 nM) for 10 20 30 40 or 50 sec Intracellular cytosolic Ca2+ was analysed using theInvitrogen Fluo-4NW Calcium Assay kit Each point is the mean plusmn SE (119899 = 5 lowast119875 lt 005 and lowastlowast119875 lt 0001 between untreated and SLPItreated cells calculated by 2 way ANOVA followed by Bonferroni test)

that SLPI affects Ca2+ flux via modulation of other immunesignalling pathways including the mitogen-activated proteinkinase (MAPK) cascade ERK12 and p38 MAPK [53ndash55]modulation of the increase in intracellular Ca2+ associatedwith activation of the transcription factor NF-120581B [56] orby regulation of cAMP-dependent protein kinase A [3]Nevertheless the maintenance of intracellular Ca2+ levelsvia IP

3modulation represents a novel therapeutic strategy

in inflammatory conditions such as COPD and CF whereneutrophil infiltration to the airways causes excessive tissuedamage In line with this theory U73122 a membrane per-meable aminosteroid PLC inhibitor whichwe have been usedas a positive control in this study has previously been shownin an in vivo animal model to significantly inhibit neutrophil

infiltration to the peritoneal cavity upon LPS injection inaddition to reducing IL-8 and leukotriene B

4induced Ca2+

flux in neutrophils [57] The findings of the present study arehighly relevant in the consideration of rhSLPI as a modulatorof Ca2+ flux and therapeuticmodality in chronic neutrophilicairway inflammatory disorders In support of this conceptapplications of aerosolized anti-proteases such as SLPI havebeen investigated as potential therapeutics for people with CFand COPD [58 59] Aerosolized rhSLPI (100mg twice dailyfor 1 week) has previously been administered to individualswith CF [59] with results revealing increased epithelial liningfluid levels of SLPI and significantly reduced levels of activeNE (pretreatment 111 plusmn 18 120583M NE to posttreatment 61 plusmn12 120583M NE) A further in vivo study carried out in rats


Cou

nts

20

40

60

80

100

0

fMLP

SLPI

100 101 102

FL1-H

fMLP998400998400fMLP998400998400

fMLP998400998400

(a)

100

0

20

40

60

80

MFI

Con fMLP SLPIfMLP998400998400

fMLP998400998400 fMLP998400998400

lowastlowast

(b)

0 10 20 30 40 500

1

2

3

4

fMLP

fMLP (+ox-SLPI)fMLP (+SLPI)

Time (seconds)

Cyto

solic

Ca2

+(r

elat

ive l

evel)

lowast

(c)

0

1

2

3

4

IL-8IL-8 (+SLPI)IL-8 (+ox-SLPI)

0 10 20 30 40 50Time (seconds)

Cyto

solic

Ca2

+(r

elat

ive l

evel)

lowast

lowast

(d)

Figure 6 Intracellular nonoxidised SLPI exerts immunomodulatory activity (a) Neutrophils (1 times 107mL) were exposed to unlabeled fMLP(10120583M) or SLPI (480 nM) for 1min followed by FITC-labeled fMLP (fMLP10158401015840) (1 120583M) and the level of bound fMLP10158401015840 quantified by FACSThe negative control (unlabeled cells) is illustrated in dark grey and a total of 10000 events were collected (b) Results in mean fluorescenceintensity units (MFI) demonstrate that pre-incubationwith unlabeled fMLP but not rhSLPI prevented binding of fMLP10158401015840 to the cellmembrane(lowast119875 lt 005 compared to fMLPfMLP10158401015840 cells) (c) and (d) Neutrophils (1times 107mL) remained untreated or treated with 480 nMSLPI or 480 nMoxidised SLPI (ox-SLPI) for 5min prior to stimulation with fMLP ((c) 1120583M) or IL-8 ((d) 12 nM) Ox-SLPI did not inhibit the fMLP or IL-8induced Ca2+ flux (lowast119875 lt 005 between SLPI and ox-SLPI) Each experiment was performed in triplicate each point is the mean plusmn SE andstatistical significance was calculated by 2-way ANOVA followed by Bonferroni test or Studentrsquos 119905-test


0

2

4

6

8

U73122 minus+minus minus minus minus minus+minus minus

PTX minusminus minus minus minus + minusminus minus +

SLPI + minus+minus minus minus minus+minus minus

IL-8 minus minus minus minus minus minus ++ + +

fMLP ++minus minus + + minus minus minus minus

IP1

(rel

ativ

e lev

el)

lowastlowast

lowastlowast

lowast

lowast

(a)

0

1

2

3

4

NS

fMLP minus + + + minus minus minus minus minus

IL-8 minus minus minus minus + + + minus minus

minus minus minus minus minus minus minus + +

SLPI minus minus + + minus + + minus +

NS

NS

IP3

lowast

lowast

Cyto

solic

Ca2

+(r

elat

ive l

evel)

(b)

0 240 480SLPI (nM)

0

05

1

15

Con

NS

NS

lowast lowastlowast

Chem

otac

tic in

dex

(fMLP

1120583

M)

+1120583M IP3

(c)

NS

NS

0 240 480SLPI (nM)

0

05

1

15lowast

lowastlowast

Con+1120583M IP3

Chem

otac

tic in

dex

(IL-

8 1

2 nM

)

(d)

Figure 7 SLPI inhibits production of IP3 (a) Preloading neutrophils (1 times 105mL) with SLPI (480 nM) significantly reduced the accumulative

levels of IP1induced by fMLP (1 120583M) or IL-8 (12 nM) activation (final reaction volume of 200120583L) Positive controls included U73122 (5 120583M)

and pertussis toxin (PTX 500 ngmL) (b) Cells remained untreated or preloaded with SLPI (480 nM) electropermeabilized in the presenceor absence of 1 120583M IP

3and then stimulated with fMLP (1120583M) or IL-8 (12 nM) for 10 sec (c) and (d) Neutrophils were isolated from healthy

individuals and the effect of SLPI (240 or 480 nM) on the cell chemotactic index induced by fMLP (1 120583M) or IL-8 (12 nM) was determinedin the presence (◼) or absence (con ◻) of 1120583M IP

3 SLPI had no significant inhibitory effect on neutrophil chemotaxis in the presence of

augmented cytosolic IP3 Each point is the mean plusmn SE (119899 = 5 NS no significant difference and lowast119875 lt 005 and lowastlowast119875 lt 0001 calculated by

Studentrsquos 119905-test)


Extracellular

ConfMLP

PMA

6

1417

28

(kDa)

496298

188

38

Cytosolic fMLPIL-8 IL-8PMACon

Gel

Blots

Anti-SLPI

(a)

(kDa)

(kDa)

Anti-SLPI

Anti-SLPI

Membranes

Extracellular

Time (min) 0 05 1

0 05 1 10

10

1417

4938

1417

4938

Anti-p47phox

Anti-p47phox

(b)

Con

IL-8fMLP

PMA

(c)

(kDa)

Anti-SLPI

14

28

49

62

98

38

Gel

Blots

CFUn Stim

Control Stim UnUn Stim

COPD

(d)

Extr

acel

lula

r rele

ased

SLP

I

0 10 20 30 40 500

1

2

3

4

Time (sec)

0 10 20 30 40 50Time

(seconds)

0

1

2

3

4Anti-SLPIBlot

(rel

ativ

e den

sitom

etry

uni

ts◼

)

Cyto

solic

Ca2

+(r

elat

ive l

evel

mdash)

lowast

(e)

Figure 8 SLPI is secreted from the cell upon activation (a) Coomassie Blue stained gel (top panel) and immunoblots (bottom 2 panels)employing anti-SLPI antibody (Gt Synergen) for its respective distribution (intracellular or extracellular) in control unstimulated cells (Con)and after IL-8 (12 nM) and fMLP (1120583M) or PMA (16120583M) activation for 10min (b) Donor neutrophils (5 times 106mL) were stimulated withPMA (16120583M) for 0 05 1 or 10min before sonication and preparation of membrane (top panels) and extracellular released protein fractions(bottompanels) SLPI and p47phox translocationwas analysed byWestern blotting employing polyclonal rabbit antibodies (c)The distributionof SLPI in resting unstimulated cells (Con) and after PMA or fMLP (1 120583M) and IL-8 (12 nM) activation for 10min was detected using an FITClabeled rabbit polyclonal anti-SLPI antibody (green fluorescence)The distribution of SLPI was predominantly cytosolic in unstimulated cellsand after stimulation condensed around the margin of the cell (indicated by white arrow) DAPI stained nuclei are represented in blue (times40magnification times10 zoom) (d) Coomassie blue stained gel (top panel) and immunoblot (bottom panel) employing rabbit anti-SLPI antibodyfor detection of released SLPI in the extracellular supernatants of unstimulated (Un) or fMLPIL-8 stimulated (Stim) healthy control (Con)COPD or CF cells (5 times 106mL) (e) Neutrophils isolated from healthy donors were incubated at 37∘C and were treated with fMLPIL-8Cell free supernatants were collected at 0 10 20 30 40 or 50 sec and Western blotted for cell secreted SLPI (top panel) and intracellularcytosolic Ca2+ was analysed using the Invitrogen Fluo-4 Calcium Assay kit Results of three separate experiments were expressed as relativedensitometry units (bar graph) and each bar is the mean plusmn SE lowast119875 lt 005 is between 0 time point and statistical significance was calculatedby Studentrsquos 119905-test

reported that rhSLPI could remain biologically active in thelungs for at least 8 h [60]When administered intratracheally(86mgkg) the reported half-life of SLPI was 4 to 5 hindicating minimal metabolism in the airways rhSLPI has

also been administered intravenously to sheep [21] In thislatter study following an infusion of rhSLPI (1 g) over 10minthe half-life of rhSLPI was reported as 18 h however if therate of infusion was slowed SLPI excretion was significantly


IP3Cytoplasmic domain

Membranedomain

Extracellular domain

Native SLPI

rhSLPI

Agonist ligand

Cell receptor Neutrophil granule

Cytoskeletal rearrangements

Degranulated proteins and peptides

Native SLPIExogenous recombinant human SLPI (rhSLPI)

(a) (b)

rhSLPI

Calciosome

IP3

IP3

PIP2

PIP2

PLC-120573

PLC-120573

PLC-120573

Ca2+

Ca2+

ion channel

Cell activation Treatment with SLPI

Figure 9 SLPI regulates neutrophil activity by modulating IP3production (a) Upon neutrophil stimulation SLPI is secreted from the

cell to the extracellular environment and IP3is formed during PLC-120573 activation IP

3binds to Ca2+ voltage-gated channels on calciosomes

(or endoplasmic reticulum) and allows Ca2+ ions to flow from the lumen of the calciosome toward the cell interior The presence of Ca2+ions within the cytosol allows cytoskeletal rearrangements such as actin polymerization and talin cleavage facilitating the process of celldegranulation and chemotaxis (b) Exogenously added rhSLPI effectively accumulates within the cell and reduces IP

3production thereby

inhibiting degranulation of antimicrobial peptides and proteins and cell migration

decreased with a 3 h infusion associated with 9 excretion[21] Intact SLPI was detected in lymph and epithelial liningfluid which displayed anti-NE capacity in line with the levelsof SLPI [21]

33 Secretion of SLPI from the Neutrophil Coincides withCell Activation The question next investigated was howcells overcome the inhibitory effect of native cytosolic SLPIthereby leading to Ca2+ flux in neutrophils Indeed neu-trophils have previously been shown to secrete SLPI [11]and for this reason ensuing experiments were designed todetermine whether the kinetics of SLPI release upon cellactivation corresponded to the spike of Ca2+ flux ByWesternblotting it was found that neutrophils (5 times 106mL) secretecytosolic SLPI to the outside of the cell in response to bothIL-8 (12 nM) and fMLP (1 120583M) or PMA (16 120583M) activationfor 10min (Figure 8(a)) A reduction in the level of cytosolicSLPI was observed most prominently after PMA activationwith a concomitant increase in the level of extracellularSLPI detected These results are in keeping with a previous

study that recorded significant amounts of SLPI secretedfrom activated neutrophils (3 120583g106 cells24 h) as comparedwith an epithelial and type II pneumocyte cell line [11] Thisset of experiments were expanded and included subcellularfractionation and isolation of membranes following cell (5times 106mL) activation with PMA (16120583M) at 0 05 1 and10min Results revealed that over the timecourse exploredprogressively higher quantities of SLPI became associatedwith the plasma membrane (Figure 8(b) upper panel) fol-lowed by sequential secretion to the outside of the cell (Fig-ure 8(b) lower panel) Translocation of the NADPH oxidasecomponent p47phox from the cytosol to the membrane uponcell activation was used as a positive control [61] howeverp47phox was not released from the cell To corroborate resultsconfocal microscopy was employed and revealed that SLPIwas localized diffusely and uniformly throughout the cytosolof control resting cells (green) In contrast after fMLPIL-8 orPMA stimulation for 10min themajority of SLPI translocatedto the periphery of the cell (Figure 8(c)) Moreover a greaterlevel of SLPI secretion from CF and COPD neutrophils


(5 times 106mL) was detected in extracellular supernatants byWestern blot analysis (Figure 8(d)) This latter result is inagreement with data indicating a requirement for increasedSLPI for modulation of COPD and CF neutrophil activity(Figures 2(c) and 2(d)) possibly due to the occurrence ofcell priming On the assumption that extracellular releaseof SLPI must follow the same time course of seconds asCa2+ flux we monitored release of SLPI from the cell after10 20 30 40 and 50 sec fMLPIL-8 stimulation Releaseof SLPI was quantified in the extracellular supernatantby immunoblotting and results demonstrate that levels ofextracellular SLPI were significantly increased after just 10 secstimulation (Figure 8(e) 119875 lt 005) Although the timecourse of Ca2+ flux peaked at 10 sec a lag period of 30 sec forthe maximum level of detectable cell free SLPI was evidentThis may in part be explained by the time required forimmunodetection of measurable amounts of the protein onthe outside of the cell

SLPI release from the cell following activation has beencomprehensively studied [11] however the signalling eventsincluding possible phosphorylation of SLPI and necessarymembrane receptor interactions required for its release haveyet to be evaluated Nevertheless collectively these resultsindicate that upon exposure to proinflammatory stimulicytosolic SLPI is actively secreted from the cell and the timeof elimination of SLPI coincides with the time of Ca2+ flux

4 Conclusion

This study presents evidence that SLPI is present in thecytosol and also the secondary granules of resting neutrophilsand is secreted from the cell upon exposure to the phagocyteactivator PMA or the chemokines IL-8 and fMLP We showthat neutrophil cytosolic SLPI is effective as a serine proteaseinhibitor with potent activity against NE Moreover withinthe neutrophil SLPI maintains the ability to modulate cellu-lar processes involving cytoskeletal restructuring includingdirectional chemotaxis and degranulation of antimicrobialpeptides and proteases This investigation elucidates a hith-erto undescribed function of intracellular neutrophil cytoso-lic SLPI and indicates that the described anti-inflammatoryeffects of SLPI may be orchestrated through inhibition ofCa2+ flux by modulating IP

3production (Figure 9) Due

to the correlative relationship between neutrophil activationand cytosolic Ca2+ levels inhibition of Ca2+ flux by SLPImay pose as a potential anti-inflammatory therapy duringacute exacerbation of severe chronic obstructive lung diseasethereby reducing neutrophil influx into the airways

SLPI elafin and AAT have been a focus of interest froma therapeutic viewpoint for a number of years Modifyingexcessive neutrophil activation and an overexuberant inflam-matory response would be a relevant treatment objective ina wide variety of conditions including COPD and CF andalso in the modulation of periodontal disease sepsis syn-dromes asthma bronchiectasis and in transplant rejectionRecent studies have employed aerosolized liposomal [62] andhydrogel [63] formulations for successful delivery of SLPIOur finding demonstrating the inhibitory capacity of SLPI on

IP3production and Ca2+ flux strengthens its attractiveness as

a potential therapeutic and the finding that SLPI has an anti-inflammatory role via inhibition of neutrophil chemotaxisand degranulation is a novel concept that opens up a newfieldof investigation

Abbreviations

SLPI Secretory leukoprotease inhibitorCOPD Chronic obstructive pulmonary diseaseCF Cystic fibrosisIP3 Inositol 145-triphosphate

NE Neutrophil elastase

Authorsrsquo Contributions

Emer P Reeves andNessa Banville share joint first authorship

Acknowledgments

The authors thank Professor Clifford Taggart Queenrsquos Uni-versity Belfast for helpful discussions and comments Theauthors are grateful to Dr Warren Thomas and OliveMcCabe Molecular Medicine Laboratories RCSI for pro-viding microscopy support and to all patients with cysticfibrosis and chronic obstructive pulmonary disease who par-ticipated in this study This work was supported by theMedical Research Charities GroupHealth Research BoardScience Foundation Ireland (Grant no 11RFPBMT3094)theUSAlphaOne Foundation and the Program for ResearchinThird Level Institutes (PRTLI) administered by the HigherEducation Authority and Science Foundation Ireland

References

[1] D I Gabrilovich The Neutrophils New Outlook for Old CellsImperial College Press 2nd edition 2005

[2] T D Pollard and J A Cooper ldquoActin a central player in cellshape andmovementrdquo Science vol 326 no 5957 pp 1208ndash12122009

[3] G Tintinger H C Steel and R Anderson ldquoTaming theneutrophil calcium clearance and influx mechanisms as noveltargets for pharmacological controlrdquo Clinical and ExperimentalImmunology vol 141 no 2 pp 191ndash200 2005

[4] G M Bokoch ldquoChemoattractant signaling and leukocyte acti-vationrdquo Blood vol 86 no 5 pp 1649ndash1660 1995

[5] O Binah M Shilkrut G Yaniv and S Larisch ldquoThe Fasreceptor 145-IP3 cascade a potential target for treating heartfailure and arrhythmiasrdquo Annals of the New York Academy ofSciences vol 1015 pp 338ndash350 2004

[6] M J Wacker L M Kosloski W J R Gilbert et al ldquoInhibitionof thromboxane A2-induced arrhythmias and intracellularcalcium changes in cardiacmyocytes by blockade of the inositoltrisphosphate pathwayrdquo Journal of Pharmacology and Experi-mental Therapeutics vol 331 no 3 pp 917ndash924 2009

[7] C Vogelmeier R Buhl R F Hoyt et al ldquoAerosolization ofrecombinant SLPI to augment antineutrophil elastase protec-tion of pulmonary epitheliumrdquo Journal of Applied Physiologyvol 69 no 5 pp 1843ndash1848 1990


[8] P Birrer N G Mcelvaney A Rudeberg et al ldquoProtease-anti-protease imbalance in the lungs of children with cystic fibrosisrdquoAmerican Journal of Respiratory and Critical Care Medicine vol150 no 1 pp 207ndash213 1994

[9] S Suter ldquoThe imbalance between granulocyte neutral proteasesand antiproteases in bronchial secretions from patients withcystic fibrosisrdquo Antibiotics and Chemotherapy vol 42 pp 158ndash168 1989

[10] J Schalkwijk O Wiedow and S Hirose ldquoThe trappin genefamily proteins defined by an N-terminal transglutaminasesubstrate domain and a C-terminal four-disulphide corerdquo Bio-chemical Journal vol 340 part 3 pp 569ndash577 1999

[11] J M Sallenave M S T Har G Cox M Chignard and JGauldie ldquoSecretory leukocyte proteinase inhibitor is a majorleukocyte elastase inhibitor in human neutrophilsrdquo Journal ofLeukocyte Biology vol 61 no 6 pp 695ndash702 1997

[12] C M Greene N G McElvaney S J OrsquoNeill and C C TaggartldquoSecretory leucoprotease inhibitor impairs toll-like receptor 2-and 4-mediated responses in monocytic cellsrdquo Infection andImmunity vol 72 no 6 pp 3684ndash3687 2004

[13] C C Taggart C M Greene N G McElvaney and S OrsquoNeillldquoSecretory leucoprotease inhibitor prevents lipopolysaccharide-induced I120581B120572degradationwithout affecting phosphorylation orubiquitinationrdquo Journal of Biological Chemistry vol 277 no 37pp 33648ndash33653 2002

[14] A Ding NThieblemont J Zhu F Jin J Zhang and S WrightldquoSecretory leukocyte protease inhibitor interferes with uptakeof lipopolysaccharide bymacrophagesrdquo Infection and Immunityvol 67 no 9 pp 4485ndash4489 1999

[15] J W McMichael A Roghanian L Jiang R Ramage and JM Sallenave ldquoThe antimicrobial antiproteinase elafin bindsto lipopolysaccharide and modulates macrophage responsesrdquoAmerican Journal of Respiratory Cell andMolecular Biology vol32 no 5 pp 443ndash452 2005

[16] L C Jacobsen O E Soslashrensen J B Cowland N Borregaardand K Theilgaard-Monch ldquoThe secretory leukocyte proteaseinhibitor (SLPI) and the secondary granule protein lactoferrinare synthesized inmyelocytes colocalize in subcellular fractionsof neutrophils and are coreleased by activated neutrophilsrdquoJournal of Leukocyte Biology vol 83 no 5 pp 1155ndash1164 2008

[17] A W Segal and O T G Jones ldquoRapid incorporation ofthe human neutrophil plasma membrane cytochrome b intophagocytic vacuolesrdquo Biochemical and Biophysical ResearchCommunications vol 92 no 2 pp 710ndash715 1980

[18] D A Bergin E P Reeves P Meleady et al ldquo120572-1 antitrypsinregulates human neutrophil chemotaxis induced by solubleimmune complexes and IL-8rdquo Journal of Clinical Investigationvol 120 no 12 pp 4236ndash4250 2010

[19] R Jayachandran V Sundaramurthy B Combaluzier et alldquoSurvival of Mycobacteria in Macrophages Is Mediated byCoronin 1-Dependent Activation of Calcineurinrdquo Cell vol 130no 1 pp 37ndash50 2007

[20] C C Taggart S A Cryan S Weldon et al ldquoSecretory leuco-protease inhibitor binds to NF-120581B binding sites in monocytesand inhibits p65 bindingrdquo Journal of ExperimentalMedicine vol202 no 12 pp 1659ndash1668 2005

[21] P Birrer N G McElvaney A Gillissen et al ldquoIntra-venous recombinant secretory leukoprotease inhibitor aug-ments antineutrophil elastase defenserdquo Journal of Applied Phys-iology vol 73 no 1 pp 317ndash323 1992

[22] T BMcNeelyMDealyD JDripps JMOrenstein S P Eisen-berg and S M Wahl ldquoSecretory leukocyte protease inhibitor

a human saliva protein exhibiting anti-human immunodefi-ciency virus I activity in vitrordquo Journal of Clinical Investigationvol 96 no 1 pp 456ndash464 1995

[23] M J Herrero-Turrion J Calafat H Janssen M Fukuda and FMollinedo ldquoRab27a regulates exocytosis of tertiary and specificgranules in humanneutrophilsrdquo Journal of Immunology vol 181no 6 pp 3793ndash3803 2008

[24] H W M Niessen and A J Verhoeven ldquoDifferential up-regulation of specific and azurophilic granule membranemark-ers in electropermeabilized neutrophilsrdquoCellular Signalling vol4 no 5 pp 501ndash509 1992

[25] N M Weathington A H Van Houwelingen B D Noerageret al ldquoA novel peptide CXCR ligand derived from extracel-lular matrix degradation during airway inflammationrdquo NatureMedicine vol 12 no 3 pp 317ndash323 2006

[26] G Bergsson E P Reeves PMcNally et al ldquoLL-37 complexationwith glycosaminoglycans in cystic fibrosis lungs inhibits antimi-crobial activity which can be restored by hypertonic salinerdquoJournal of Immunology vol 183 no 1 pp 543ndash551 2009

[27] C M Peres D M Aronoff C H Serezani N Flamand L HFaccioli and M Peters-Golden ldquoSpecific leukotriene receptorscouple to distinct G proteins to effect stimulation of alveolarmacrophage host defense functionsrdquo Journal of Immunologyvol 179 no 8 pp 5454ndash5461 2007

[28] P Anton J OrsquoConnell D OrsquoConnell et al ldquoMucosal subepithe-lial binding sites for the bacterial chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP)rdquo Gut vol 42 no 3pp 374ndash379 1998

[29] P A Henriksen M Hitt Z Xing et al ldquoAdenoviral gene deliv-ery of elafin and secretory leukocyte protease inhibitor atten-uates NF-120581B-dependent inflammatory responses of humanendothelial cells and macrophages to atherogenic stimulirdquoJournal of Immunology vol 172 no 7 pp 4535ndash4544 2004

[30] DGuerrieri N L Tateosian P CMaffıa et al ldquoSerine leucocyteproteinase inhibitor-treated monocyte inhibits human CD4+lymphocyte proliferationrdquo Immunology vol 133 no 4 pp 434ndash441 2011

[31] K Tiringer A Treis P Fucik et al ldquoA Th17- and Th2-skewedcytokine profile in cystic fibrosis lungs represents a potentialrisk factor for Pseudomonas aeruginosa infectionrdquo AmericanJournal of Respiratory and Critical Care Medicine vol 187 no6 pp 621ndash629 2012

[32] K Hoetzenecker A Mitterbauer E Guenova et al ldquoHigh levelsof lung resident CD4+CD28null cells in COPD implications ofautoimmunityrdquoWienKlinWochenschr vol 125 no 5-6 pp 150ndash155 2013

[33] S H Chotirmall C M Greene I K Oglesby et al ldquo17120573-estradiol inhibits IL-8 in cystic fibrosis by up-regulating secre-tory leucoprotease inhibitorrdquo American Journal of Respiratoryand Critical Care Medicine vol 182 no 1 pp 62ndash72 2010

[34] R C Thompson and K Ohlsson ldquoIsolation properties andcomplete amino acid sequence of human secretory leukocyteprotease inhibitor a potent inhibitor of leukocyte elastaserdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 83 no 18 pp 6692ndash6696 1986

[35] G Ma T Greenwell-Wild K Lei et al ldquoSecretory leuko-cyte protease inhibitor binds to annexin II a cofactorfor macrophage HIV-1 infectionrdquo Journal of ExperimentalMedicine vol 200 no 10 pp 1337ndash1346 2004

[36] B Py S Basmaciogullari J Bouchet et al ldquoThe phospholipidscramblases 1 and 4 are cellular receptors for the secretory


leukocyte protease inhibitor and interact with CD4 at theplasma membranerdquo PLoS ONE vol 4 no 3 Article ID e50062009

[37] N C Kaneider A Rabensteiner C M Reinisch S Dunzen-dorfer and C J Wiedermann ldquoInhibition of human neutrophilchemotaxis toward interleukin 8 with six clinical antithrombinconcentrates in vitrordquo Intensive Care Medicine vol 28 no 10pp 1447ndash1452 2002

[38] D McHugh C Tanner R Mechoulam R G Pertwee andR A Ross ldquoInhibition of human neutrophil chemotaxis byendogenous cannabinoids and phytocannabinoids evidence fora site distinct fromCB1 and CB2rdquoMolecular Pharmacology vol73 no 2 pp 441ndash450 2008

[39] A F Petit-Bertron O Tabary H Corvol et al ldquoCirculatingand airway neutrophils in cystic fibrosis display different TLRexpression and responsiveness to interleukin-10rdquo Cytokine vol41 no 1 pp 54ndash60 2008

[40] S S Pedersen A Kharazmi F Espersen and N HoibyldquoPseudomonas aeruginosa alginate in cystic fibrosis sputumand the inflammatory responserdquo Infection and Immunity vol58 no 10 pp 3363ndash3368 1990

[41] D Y Koller R Urbanek andMGotz ldquoIncreased degranulationof eosinophil and neutrophil granulocytes in cystic fibrosisrdquoAmerican Journal of Respiratory and Critical Care Medicine vol152 no 2 pp 629ndash633 1995

[42] C Taggart R J Coakley P Greally G Canny S J OrsquoNeill andN G McElvaney ldquoIncreased elastase release by CF neutrophilsis mediated by tumor necrosis factor-120572 and interleukin-8rdquoAmerican Journal of Physiology vol 278 no 1 pp L33ndashL412000

[43] N Borregaard O E Soslashrensen and K Theilgaard-MonchldquoNeutrophil granules a library of innate immunity proteinsrdquoTrends in Immunology vol 28 no 8 pp 340ndash345 2007

[44] S J FrancoM A Rodgers B J Perrin et al ldquoCalpain-mediatedproteolysis of talin regulates adhesion dynamicsrdquo Nature CellBiology vol 6 no 10 pp 977ndash983 2004

[45] B Yuruker and V Niggli ldquo120572-Actinin and vinculin in humanneutrophils reorganization during adhesion and relation to theactin networkrdquo Journal of Cell Science vol 101 no 2 pp 403ndash414 1992

[46] A Huttenlocher S P Palecek Q Lu et al ldquoRegulation of cellmigration by the calcium-dependent protease calpainrdquo Journalof Biological Chemistry vol 272 no 52 pp 32719ndash32722 1997

[47] S P Eisenberg K K Hale P Heimdal and R C ThompsonldquoLocation of the protease-inhibitory region of secretory leuko-cyte protease inhibitorrdquo Journal of Biological Chemistry vol 265no 14 pp 7976ndash7981 1990

[48] I Petrache I Fijalkowska T R Medler et al ldquo120572-1 antitrypsininhibits caspase-3 activity preventing lung endothelial cellapoptosisrdquo American Journal of Pathology vol 169 no 4 pp1155ndash1166 2006

[49] M Al-Omari E Korenbaum M Ballmaier et al ldquoAcute-phaseprotein 1205721-antitrypsin inhibits neutrophil calpain I and inducesrandom migrationrdquo Molecular Medicine vol 17 no 9-10 pp865ndash874 2011


[51] K H Krause W Schlegel and C B Wollheim ldquoChemotacticpeptide activation of human neutrophils and HL-60 cells Per-tussis toxin reveals correlation between inositol trisphosphategeneration calcium ion transients and cellular activationrdquo

Journal of Clinical Investigation vol 76 no 4 pp 1348ndash13541985

[52] W Schorr D Swandulla and H U Zeilhofer ldquoMechanismsof IL-8-induced Ca2+ signaling in human neutrophil granulo-cytesrdquo European Journal of Immunology vol 29 no 3 pp 897ndash904 1999

[53] L W Chen M W Lin and C M Hsu ldquoDifferent pathwaysleading to activation of extracellular signal-regulated kinase andp38 MAP kinase by formyl-methionyl-leucyl-phenylalanine orplatelet activating factor in human neutrophilsrdquo Journal ofBiomedical Science vol 12 no 2 pp 311ndash319 2005

[54] K R McLeish C Knall R A Ward et al ldquoActivation ofmitogen-activated protein kinase cascades during priming ofhuman neutrophils by TNF-120572 and GM-CSFrdquo Journal of Leuko-cyte Biology vol 64 no 4 pp 537ndash545 1998

[55] Y H Wang W Y Wang J F Liao et al ldquoPreventionof macrophage adhesion molecule-1 (Mac-1)-dependent neu-trophil firm adhesion by taxifolin through impairment ofprotein kinase-dependent NADPH oxidase activation andantagonism of G protein-mediated calcium influxrdquo BiochemicalPharmacology vol 67 no 12 pp 2251ndash2262 2004

[56] F Al-Mohanna S Saleh R S Parhar and K Collison ldquoIL-12-dependent nuclear factor-120581B activation leads to de novosynthesis and release of IL-8 and TNF-120572 in human neutrophilsrdquoJournal of Leukocyte Biology vol 72 no 5 pp 995ndash1002 2002

[57] C Hou T Kirchner M Singer M Matheis D Argentieri andD Cavender ldquoIn vivo activity of a phospholipase C inhibitor1-(6-((17120573 -3-methoxyestra-135(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-25-dione (U73122) in acute and chronic inflam-matory reactionsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 309 no 2 pp 697ndash704 2004

[58] M W Konstan and P B Davis ldquoPharmacological approachesfor the discovery and development of new anti-inflammatoryagents for the treatment of cystic fibrosisrdquo Advanced DrugDelivery Reviews vol 54 no 11 pp 1409ndash1423 2002

[59] N G McElvaney B Doujaiji M J Moan M R Burnham MC Wu and R G Crystal ldquoPharmacokinetics of recombinantsecretory leukoprotease inhibitor aerosolized to normals andindividuals with cystic fibrosisrdquoAmerican Review of RespiratoryDisease vol 148 no 4 part 1 pp 1056ndash1060 1993

[60] A Gast W Anderson A Probst et al ldquoPharmacokineticsand distribution of recombinant secretory leukocyte proteinaseinhibitor in ratsrdquo American Review of Respiratory Disease vol141 no 4 part 1 pp 889ndash894 1990

[61] P G Heyworth J T Curnutte W M Nauseef et al ldquoNeu-trophil nicotinamide adenine dinucleotide phosphate oxidaseassembly Translocation of p47-phox and p67-phox requiresinteraction between p47-phox and cytochrome b558rdquo Journalof Clinical Investigation vol 87 no 1 pp 352ndash356 1991

[62] A M Gibbons N G McElvaney C C Taggart and S ACryan ldquoDelivery of rSLPI in a liposomal carrier for inhalationprovides protection against cathepsin L degradationrdquo Journal ofMicroencapsulation vol 26 no 6 pp 513ndash522 2009

[63] S C Barros J A Martins J C Marcos and A Cavaco-PauloldquoInfluence of secretory leukocyte protease inhibitor-based pep-tides on elastase activity and their incorporation in hyaluronicacid hydrogels for chronic wound therapyrdquo Biopolymers vol 98no 6 pp 576ndash590 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Molecular regulation of cytoskeletal rearrangements dur-ing neutrophil signalling associated with chemotaxis anddegranulation of antimicrobial enzymes requires an increasein cytosolic Ca2+ levels [3] This increase is due to the releaseof Ca2+ from the cellrsquos internal store (the calciosome) or influxof Ca2+ from the extracellular environment Signalling viafMLP or IL-8 GPCRs results in activation of the 120573 isoformof phospholipase C in turn yielding production of inositol145 triphosphate (IP

3) [4] IP

3occupancy of receptors on

calciosomes induces a rapid release of stored Ca2+ and ina variety of cell types this cascade has been proposed as apotential target for the treatment of a number of diseasesincluding heart failure and arrhythmias [5 6]

Serine protease inhibitors such as SLPI act locally tomaintain a proteaseantiprotease balance thereby preventingprotease mediated tissue destruction [7] Disruption of thisbalance is responsible for much of the extracellular matrixand subsequent lung tissue damage evident in neutrophildriven lung diseases [8 9] SLPI is a well-characterisedmember of the trappin gene family of proteins [10] It is ahighly basic cationic protein that is produced by epithelialcells of the respiratory tract and is also produced by phago-cytic neutrophils [11] SLPI inhibits a broad range of serineproteases including NE cathepsin G trypsin and tryptaseMoreover SLPI possesses anti-inflammatory characteristicsand in monocytes has been shown to inhibit lipopolysac-charide (LPS)- and lipoteichoic acid-induced nuclear factor(NF)-120581B activation and can compete with p65 for bindingto NF-120581B binding sites [12 13] Moreover SLPI has beenshown to directly bind to bacterial LPS thereby down-regulating production of proinflammatorymediators [14 15]However an area that has received less attention is therole of neutrophil-derived SLPI in modulating inflammatoryresponses

Studies have localised SLPI within the cytosol [11] andalso in secondary granules of neutrophils in turn coreleasedwith lactoferrin [16] Moreover the release of SLPI from neu-trophils upon phorbol ester (PMA) activation suggests thatneutrophil-derived SLPI may regulate the protease antipro-tease balance at sites of tissue inflammation [11] Howeverthe aim of this study was to investigate the intracellularfunction of SLPI within resting and activated neutrophilsThis study has revealed a novel anti-inflammatory role for thisantiprotease and demonstrated the ability of SLPI to modu-late neutrophilmigration and degranulation by inhibiting IP

3

production and Ca2+ ion mobilisation

2 Methods

21 Chemicals and Reagents All chemicals and reagentsincluding inositol 145-triphosphate were purchased fromSigma-Aldrich St Louis MO USA unless indicated other-wise The concentration of SLPI in neutrophil cytosol wasmeasured using the Quantikine Human SLPI Immunoassay(RampD Systems Abingdon Oxon UK) Recombinant humanSLPI (rhSLPI) was obtained from R amp D Systems andrecombinant calpain was obtained from Calbiochem (LaJolla CA USA) Human NE was purchased from Elastin

Products Company Inc (Owensville MO USA) The IP-One HTRF assay kit was purchased from Cisbio Bioassays(Bedford MA 01730 USA)

22 Study Groups Control volunteers (119899 = 10 mean age351 plusmn 18) had no underlying medical illnesses and were notreceiving any medication Prior to recruitment patients withCF (119899 = 5) were exacerbation-free over the preceding 6-weekperiod and patients with COPD were being treated for anacute exacerbation (119899 = 5) There were 25 and 35 malesrecruited to the CF and COPD study respectively The meanage was 225 plusmn 27 and 565 plusmn 27 years for the CF and COPDgroups respectively Informed patient consent was obtainedfor all procedures and ethical approval for the use of bloodsamples from CF and COPD individuals was obtained fromthe Beaumont Hospital Ethics Review Board

23 Preparation of HumanNeutrophils and Cell FractionationBlood was obtained from all donors in 75mL heparinisedS-monovette tubes (Sarstedt Ltd Ireland) and neutrophilswere purified by dextran sedimentation and Lymphoprep(Axis-Shield PoC AS Norway) centrifugation as previouslydescribed [17] Purified cells were resuspended in phosphate-buffered saline (PBS) (pH 74) containing 5mM glucose(PBSG) and used immediately The purity of the neutrophilpopulation was confirmed by flow cytometry measuring theneutrophil membrane marker CD16b and was found to begt99 [18] All in vitro experiments were performed at 37∘CCells were either left unstimulated (control) or activated withPMA (16 120583M) fMLP (1120583M) andor IL-8 (12 nM R amp DSystems) for the indicated time points

For cell fractionation studies isolated neutrophils weresuspended in Lamberts Break Buffer (LBB) (10mM KCl3mM NaCl 4mM MgCl

2 and 10mM piperazine-NN1015840-

bis(2-ethanesulfonic acid) (PIPES) pH 72) containing 10(ww) sucrose and the following protease inhibitors 13 120583Maprotinin 5mM benzamidine 015mM N120572-Tosyl-L-lysinechloromethyl ketone hydrochloride (TLCK) 05mM N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK) 20mMN-(Methoxysuccinyl)-Ala-Ala-Pro-Val-chloromethyl ketone(MeOSuc-AAPV-CMK) 10 120583M soybean trypsin inhibitor(SBTI) orthophenanthroline and 02M pefabloc Cells weresonicated 3 times for 5 sec on ice and centrifuged at 500timesgat 4∘C for 5min to generate a post nuclear supernatant(PNS) The PNS was overlaid on discontinuous sucrosegradients of 175 and 35 (ww) dissolved in LBB This wascentrifuged at 137000timesg for 45min at 4∘C The cytosolicfraction was removed from above the 17 (ww) sucroselayer and membrane fraction was recovered from the top ofthe 34 (ww) sucrose layer The pellet (containing primaryand secondary granules) was resuspended in 10 (ww)sucrose and overlaid on a discontinuous sucrose gradient of30 43 and 55 (ww) This was centrifuged at 137000timesg for1 h at 4∘C Secondary and primary granules were harvestedfrom above the 43 (ww) and 55 (ww) sucrose layersrespectively as previously described [18] The concentrationof protein present in each purified fraction was quantified by





3





3 The cells were


3into













3[27]



















50of



Anti-lactoferrin

6

1417

28

(kDa)

6298

188

38

1 2 3 4Gel

Blots


(a)

(kDa) 1 2 3Gel

1 2 3

Blots14

17

28

62

98

188

38

CF COPD


(b)

0

01

02

03

04

05

06


2400 60 120 180Time (seconds)

Abso

rban

ce (4

05 nm

)

(c)

NE

NS

Total post-

Cytosol post-

IgG IP

0

01

02

03

04

05

06

Abso

rban

ce (4

05 nm

)

cytosolCytosol

SLPI IPcontrol

lowastlowast

(d)

0

20

40

60

80

100

120

140

160

180

Con +rhSLPI +rhSLPI

NS

lowastlowast


Cyto

solic

SLP

I (ng

105

cells

)

(e)










Con 120 240 4800

025

05

075

1

SLPI (nM)

lowast

lowastCh

emot

actic

inde

x (fM

LP 1

120583M)

(a)

0

025

05

075

1

Con 120 240 480SLPI (nM)

lowast

lowastlowastChem

otac

tic in

dex

(IL-

8 1

2 nM

)(b)

CF COPD

0

025

05

075

1

lowast lowast

Chem

otac

tic in

dex

(fMLP

1 120583M

)

(c)

CF COPD

0

025

05

075

1

lowast

lowast

Chem

otac

tic in

dex

(IL-

8 1

2 nM

)

(d)






10 20 10 20 10 20Time (min)

614

17

28

(kDa)

6298

38

ConfMLPIL-8

IL-8fMLP

SLPI

(a)

0

1

2

3

4

MM

P-9

(den

sitom

etry

uni

ts)

10 20 10 20 10 20Time (min)

Con fMLPIL-8

fMLP

SLPIIL-8

10 min20 min

lowastlowast

(b)

0

5

10

15

20

hCA

P-18

(den

sitom

etry

uni

ts)

10 20 10 20 10 20Time (min)

Con fMLPIL-8

fMLP

SLPIIL-8

10 min20 min

(c)

0

2

4

6

8

MPO

(d

ensit

omet

ry u

nits)

10 20 10 20 10 20Time (min)

Con fMLPIL-8

fMLP

SLPIIL-8

10 min20 min

lowast

(d)

0

10

20

30

40

50

60

70

CD66

b (M

FI)

Control fMLPIL-8

fMLP

SLPIIL-8


(e)

CD63

(MFI

)

0

10

20

30

40

Control fMLPIL-8

fMLP

SLPIIL-8


(f)







2 1 15 15 24 06

S P S P S P S P


(a)

Blots

fragment

(kDa)250

98

64

36

fragment

Gels

minus minus+SLPI +

+ +PMA minus minus


190 kDa

sim90 kDa

Anti-talin-1

Anti-vinculin

Anti-120573-actin

(b)

0

05

1

15

fMLPIL-8 IL-8

fMLP

SLPI

PMA PMASLPI

Tal

in cl

eava

ge (d

ensit

omet

ry u

nits)

lowastlowast

(c)

fMLPIL-8 IL-8

fMLP

SLPI

PMA PMASLPI

0

05

1

15

Vin

culin

clea

vage

(den

sitom

etry

uni

ts)

lowast

lowast

(d)




















1accumulation


3(1 120583M) (Figure 7(b))











0 10 20 30 40 500

1

2

3

4

Time (seconds)

Hea

lthy

cells

Ca2

+(r

elat

ive l

evels

)

lowastlowastlowast

(a)

Time (seconds)0 10 20 30 40 50

0

1

2

3

4

Hea

lthy

cells

Ca2

+(r

elat

ive l

evels

)

lowast

lowast

(b)

Time (seconds)0 10 20 30 40 50

0

1

2

3

4

CF ce

lls C

a2+

(rel

ativ

e lev

els)

lowastlowast

(c)

0 10 20 30 40 500

1

2

3

4

Time (seconds)

CF ce

lls C

a2+

(rel

ativ

e lev

els)

lowast

lowastlowast

(d)

0 10 20 30 40 500

2

1

4

3

6

5

7

8

Time (seconds)

COPD

cells

Ca2+

(rel

ativ

e lev

els)

lowast

lowast lowast

fMLP (+SLPI)Con

fMLP (minusSLPI)

(e)

0

1

2

3

4

0 10 20 30 40 50

Time (seconds)

COPD

cells

Ca2+

(rel

ativ

e lev

els)

Con


lowastlowast

(f)






4induced Ca2+



Cou

nts

20

40

60

80

100

0

fMLP

SLPI

100 101 102

FL1-H

fMLP998400998400fMLP998400998400

fMLP998400998400

(a)

100

0

20

40

60

80

MFI


fMLP998400998400 fMLP998400998400

lowastlowast

(b)

0 10 20 30 40 500

1

2

3

4

fMLP


Time (seconds)

Cyto

solic

Ca2

+(r

elat

ive l

evel)

lowast

(c)

0

1

2

3

4


0 10 20 30 40 50Time (seconds)

Cyto

solic

Ca2

+(r

elat

ive l

evel)

lowast

lowast

(d)



0

2

4

6

8






IP1

(rel

ativ

e lev

el)

lowastlowast

lowastlowast

lowast

lowast

(a)

0

1

2

3

4

NS





NS

NS

IP3

lowast

lowast

Cyto

solic

Ca2

+(r

elat

ive l

evel)

(b)

0 240 480SLPI (nM)

0

05

1

15

Con

NS

NS

lowast lowastlowast

Chem

otac

tic in

dex

(fMLP

1120583

M)

+1120583M IP3

(c)

NS

NS

0 240 480SLPI (nM)

0

05

1

15lowast

lowastlowast

Con+1120583M IP3

Chem

otac

tic in

dex

(IL-

8 1

2 nM

)

(d)










Extracellular

ConfMLP

PMA

6

1417

28

(kDa)

496298

188

38


Gel

Blots

Anti-SLPI

(a)

(kDa)

(kDa)

Anti-SLPI

Anti-SLPI

Membranes

Extracellular

Time (min) 0 05 1

0 05 1 10

10

1417

4938

1417

4938

Anti-p47phox

Anti-p47phox

(b)

Con

IL-8fMLP

PMA

(c)

(kDa)

Anti-SLPI

14

28

49

62

98

38

Gel

Blots

CFUn Stim


COPD

(d)

Extr

acel

lula

r rele

ased

SLP

I

0 10 20 30 40 500

1

2

3

4

Time (sec)

0 10 20 30 40 50Time

(seconds)

0

1

2

3

4Anti-SLPIBlot

(rel

ativ

e den

sitom

etry

uni

ts◼

)

Cyto

solic

Ca2

+(r

elat

ive l

evel

mdash)

lowast

(e)






Membranedomain


Native SLPI

rhSLPI

Agonist ligand





(a) (b)

rhSLPI

Calciosome

IP3

IP3

PIP2

PIP2

PLC-120573

PLC-120573

PLC-120573

Ca2+

Ca2+

ion channel






3production thereby








4 Conclusion







Abbreviations





Acknowledgments


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















3





3 The cells were


3into













3[27]



















50of



Anti-lactoferrin

6

1417

28

(kDa)

6298

188

38

1 2 3 4Gel

Blots


(a)

(kDa) 1 2 3Gel

1 2 3

Blots14

17

28

62

98

188

38

CF COPD


(b)

0

01

02

03

04

05

06


2400 60 120 180Time (seconds)

Abso

rban

ce (4

05 nm

)

(c)

NE

NS

Total post-

Cytosol post-

IgG IP

0

01

02

03

04

05

06

Abso

rban

ce (4

05 nm

)

cytosolCytosol

SLPI IPcontrol

lowastlowast

(d)

0

20

40

60

80

100

120

140

160

180

Con +rhSLPI +rhSLPI

NS

lowastlowast


Cyto

solic

SLP

I (ng

105

cells

)

(e)










Con 120 240 4800

025

05

075

1

SLPI (nM)

lowast

lowastCh

emot

actic

inde

x (fM

LP 1

120583M)

(a)

0

025

05

075

1

Con 120 240 480SLPI (nM)

lowast

lowastlowastChem

otac

tic in

dex

(IL-

8 1

2 nM

)(b)

CF COPD

0

025

05

075

1

lowast lowast

Chem

otac

tic in

dex

(fMLP

1 120583M

)

(c)

CF COPD

0

025

05

075

1

lowast

lowast

Chem

otac

tic in

dex

(IL-

8 1

2 nM

)

(d)






10 20 10 20 10 20Time (min)

614

17

28

(kDa)

6298

38

ConfMLPIL-8

IL-8fMLP

SLPI

(a)

0

1

2

3

4

MM

P-9

(den

sitom

etry

uni

ts)

10 20 10 20 10 20Time (min)

Con fMLPIL-8

fMLP

SLPIIL-8

10 min20 min

lowastlowast

(b)

0

5

10

15

20

hCA

P-18

(den

sitom

etry

uni

ts)

10 20 10 20 10 20Time (min)

Con fMLPIL-8

fMLP

SLPIIL-8

10 min20 min

(c)

0

2

4

6

8

MPO

(d

ensit

omet

ry u

nits)

10 20 10 20 10 20Time (min)

Con fMLPIL-8

fMLP

SLPIIL-8

10 min20 min

lowast

(d)

0

10

20

30

40

50

60

70

CD66

b (M

FI)

Control fMLPIL-8

fMLP

SLPIIL-8


(e)

CD63

(MFI

)

0

10

20

30

40

Control fMLPIL-8

fMLP

SLPIIL-8


(f)







2 1 15 15 24 06

S P S P S P S P


(a)

Blots

fragment

(kDa)250

98

64

36

fragment

Gels

minus minus+SLPI +

+ +PMA minus minus


190 kDa

sim90 kDa

Anti-talin-1

Anti-vinculin

Anti-120573-actin

(b)

0

05

1

15

fMLPIL-8 IL-8

fMLP

SLPI

PMA PMASLPI

Tal

in cl

eava

ge (d

ensit

omet

ry u

nits)

lowastlowast

(c)

fMLPIL-8 IL-8

fMLP

SLPI

PMA PMASLPI

0

05

1

15

Vin

culin

clea

vage

(den

sitom

etry

uni

ts)

lowast

lowast

(d)




















1accumulation


3(1 120583M) (Figure 7(b))











0 10 20 30 40 500

1

2

3

4

Time (seconds)

Hea

lthy

cells

Ca2

+(r

elat

ive l

evels

)

lowastlowastlowast

(a)

Time (seconds)0 10 20 30 40 50

0

1

2

3

4

Hea

lthy

cells

Ca2

+(r

elat

ive l

evels

)

lowast

lowast

(b)

Time (seconds)0 10 20 30 40 50

0

1

2

3

4

CF ce

lls C

a2+

(rel

ativ

e lev

els)

lowastlowast

(c)

0 10 20 30 40 500

1

2

3

4

Time (seconds)

CF ce

lls C

a2+

(rel

ativ

e lev

els)

lowast

lowastlowast

(d)

0 10 20 30 40 500

2

1

4

3

6

5

7

8

Time (seconds)

COPD

cells

Ca2+

(rel

ativ

e lev

els)

lowast

lowast lowast

fMLP (+SLPI)Con

fMLP (minusSLPI)

(e)

0

1

2

3

4

0 10 20 30 40 50

Time (seconds)

COPD

cells

Ca2+

(rel

ativ

e lev

els)

Con


lowastlowast

(f)






4induced Ca2+



Cou

nts

20

40

60

80

100

0

fMLP

SLPI

100 101 102

FL1-H

fMLP998400998400fMLP998400998400

fMLP998400998400

(a)

100

0

20

40

60

80

MFI


fMLP998400998400 fMLP998400998400

lowastlowast

(b)

0 10 20 30 40 500

1

2

3

4

fMLP


Time (seconds)

Cyto

solic

Ca2

+(r

elat

ive l

evel)

lowast

(c)

0

1

2

3

4


0 10 20 30 40 50Time (seconds)

Cyto

solic

Ca2

+(r

elat

ive l

evel)

lowast

lowast

(d)



0

2

4

6

8






IP1

(rel

ativ

e lev

el)

lowastlowast

lowastlowast

lowast

lowast

(a)

0

1

2

3

4

NS





NS

NS

IP3

lowast

lowast

Cyto

solic

Ca2

+(r

elat

ive l

evel)

(b)

0 240 480SLPI (nM)

0

05

1

15

Con

NS

NS

lowast lowastlowast

Chem

otac

tic in

dex

(fMLP

1120583

M)

+1120583M IP3

(c)

NS

NS

0 240 480SLPI (nM)

0

05

1

15lowast

lowastlowast

Con+1120583M IP3

Chem

otac

tic in

dex

(IL-

8 1

2 nM

)

(d)










Extracellular

ConfMLP

PMA

6

1417

28

(kDa)

496298

188

38


Gel

Blots

Anti-SLPI

(a)

(kDa)

(kDa)

Anti-SLPI

Anti-SLPI

Membranes

Extracellular

Time (min) 0 05 1

0 05 1 10

10

1417

4938

1417

4938

Anti-p47phox

Anti-p47phox

(b)

Con

IL-8fMLP

PMA

(c)

(kDa)

Anti-SLPI

14

28

49

62

98

38

Gel

Blots

CFUn Stim


COPD

(d)

Extr

acel

lula

r rele

ased

SLP

I

0 10 20 30 40 500

1

2

3

4

Time (sec)

0 10 20 30 40 50Time

(seconds)

0

1

2

3

4Anti-SLPIBlot

(rel

ativ

e den

sitom

etry

uni

ts◼

)

Cyto

solic

Ca2

+(r

elat

ive l

evel

mdash)

lowast

(e)






Membranedomain


Native SLPI

rhSLPI

Agonist ligand





(a) (b)

rhSLPI

Calciosome

IP3

IP3

PIP2

PIP2

PLC-120573

PLC-120573

PLC-120573

Ca2+

Ca2+

ion channel






3production thereby








4 Conclusion







Abbreviations





Acknowledgments


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























3[27]



















50of



Anti-lactoferrin

6

1417

28

(kDa)

6298

188

38

1 2 3 4Gel

Blots


(a)

(kDa) 1 2 3Gel

1 2 3

Blots14

17

28

62

98

188

38

CF COPD


(b)

0

01

02

03

04

05

06


2400 60 120 180Time (seconds)

Abso

rban

ce (4

05 nm

)

(c)

NE

NS

Total post-

Cytosol post-

IgG IP

0

01

02

03

04

05

06

Abso

rban

ce (4

05 nm

)

cytosolCytosol

SLPI IPcontrol

lowastlowast

(d)

0

20

40

60

80

100

120

140

160

180

Con +rhSLPI +rhSLPI

NS

lowastlowast


Cyto

solic

SLP

I (ng

105

cells

)

(e)










Con 120 240 4800

025

05

075

1

SLPI (nM)

lowast

lowastCh

emot

actic

inde

x (fM

LP 1

120583M)

(a)

0

025

05

075

1

Con 120 240 480SLPI (nM)

lowast

lowastlowastChem

otac

tic in

dex

(IL-

8 1

2 nM

)(b)

CF COPD

0

025

05

075

1

lowast lowast

Chem

otac

tic in

dex

(fMLP

1 120583M

)

(c)

CF COPD

0

025

05

075

1

lowast

lowast

Chem

otac

tic in

dex

(IL-

8 1

2 nM

)

(d)






10 20 10 20 10 20Time (min)

614

17

28

(kDa)

6298

38

ConfMLPIL-8

IL-8fMLP

SLPI

(a)

0

1

2

3

4

MM

P-9

(den

sitom

etry

uni

ts)

10 20 10 20 10 20Time (min)

Con fMLPIL-8

fMLP

SLPIIL-8

10 min20 min

lowastlowast

(b)

0

5

10

15

20

hCA

P-18

(den

sitom

etry

uni

ts)

10 20 10 20 10 20Time (min)

Con fMLPIL-8

fMLP

SLPIIL-8

10 min20 min

(c)

0

2

4

6

8

MPO

(d

ensit

omet

ry u

nits)

10 20 10 20 10 20Time (min)

Con fMLPIL-8

fMLP

SLPIIL-8

10 min20 min

lowast

(d)

0

10

20

30

40

50

60

70

CD66

b (M

FI)

Control fMLPIL-8

fMLP

SLPIIL-8


(e)

CD63

(MFI

)

0

10

20

30

40

Control fMLPIL-8

fMLP

SLPIIL-8


(f)







2 1 15 15 24 06

S P S P S P S P


(a)

Blots

fragment

(kDa)250

98

64

36

fragment

Gels

minus minus+SLPI +

+ +PMA minus minus


190 kDa

sim90 kDa

Anti-talin-1

Anti-vinculin

Anti-120573-actin

(b)

0

05

1

15

fMLPIL-8 IL-8

fMLP

SLPI

PMA PMASLPI

Tal

in cl

eava

ge (d

ensit

omet

ry u

nits)

lowastlowast

(c)

fMLPIL-8 IL-8

fMLP

SLPI

PMA PMASLPI

0

05

1

15

Vin

culin

clea

vage

(den

sitom

etry

uni

ts)

lowast

lowast

(d)




















1accumulation


3(1 120583M) (Figure 7(b))











0 10 20 30 40 500

1

2

3

4

Time (seconds)

Hea

lthy

cells

Ca2

+(r

elat

ive l

evels

)

lowastlowastlowast

(a)

Time (seconds)0 10 20 30 40 50

0

1

2

3

4

Hea

lthy

cells

Ca2

+(r

elat

ive l

evels

)

lowast

lowast

(b)

Time (seconds)0 10 20 30 40 50

0

1

2

3

4

CF ce

lls C

a2+

(rel

ativ

e lev

els)

lowastlowast

(c)

0 10 20 30 40 500

1

2

3

4

Time (seconds)

CF ce

lls C

a2+

(rel

ativ

e lev

els)

lowast

lowastlowast

(d)

0 10 20 30 40 500

2

1

4

3

6

5

7

8

Time (seconds)

COPD

cells

Ca2+

(rel

ativ

e lev

els)

lowast

lowast lowast

fMLP (+SLPI)Con

fMLP (minusSLPI)

(e)

0

1

2

3

4

0 10 20 30 40 50

Time (seconds)

COPD

cells

Ca2+

(rel

ativ

e lev

els)

Con


lowastlowast

(f)






4induced Ca2+



Cou

nts

20

40

60

80

100

0

fMLP

SLPI

100 101 102

FL1-H

fMLP998400998400fMLP998400998400

fMLP998400998400

(a)

100

0

20

40

60

80

MFI


fMLP998400998400 fMLP998400998400

lowastlowast

(b)

0 10 20 30 40 500

1

2

3

4

fMLP


Time (seconds)

Cyto

solic

Ca2

+(r

elat

ive l

evel)

lowast

(c)

0

1

2

3

4


0 10 20 30 40 50Time (seconds)

Cyto

solic

Ca2

+(r

elat

ive l

evel)

lowast

lowast

(d)



0

2

4

6

8






IP1

(rel

ativ

e lev

el)

lowastlowast

lowastlowast

lowast

lowast

(a)

0

1

2

3

4

NS





NS

NS

IP3

lowast

lowast

Cyto

solic

Ca2

+(r

elat

ive l

evel)

(b)

0 240 480SLPI (nM)

0

05

1

15

Con

NS

NS

lowast lowastlowast

Chem

otac

tic in

dex

(fMLP

1120583

M)

+1120583M IP3

(c)

NS

NS

0 240 480SLPI (nM)

0

05

1

15lowast

lowastlowast

Con+1120583M IP3

Chem

otac

tic in

dex

(IL-

8 1

2 nM

)

(d)










Extracellular

ConfMLP

PMA

6

1417

28

(kDa)

496298

188

38


Gel

Blots

Anti-SLPI

(a)

(kDa)

(kDa)

Anti-SLPI

Anti-SLPI

Membranes

Extracellular

Time (min) 0 05 1

0 05 1 10

10

1417

4938

1417

4938

Anti-p47phox

Anti-p47phox

(b)

Con

IL-8fMLP

PMA

(c)

(kDa)

Anti-SLPI

14

28

49

62

98

38

Gel

Blots

CFUn Stim


COPD

(d)

Extr

acel

lula

r rele

ased

SLP

I

0 10 20 30 40 500

1

2

3

4

Time (sec)

0 10 20 30 40 50Time

(seconds)

0

1

2

3

4Anti-SLPIBlot

(rel

ativ

e den

sitom

etry

uni

ts◼

)

Cyto

solic

Ca2

+(r

elat

ive l

evel

mdash)

lowast

(e)






Membranedomain


Native SLPI

rhSLPI

Agonist ligand





(a) (b)

rhSLPI

Calciosome

IP3

IP3

PIP2

PIP2

PLC-120573

PLC-120573

PLC-120573

Ca2+

Ca2+

ion channel






3production thereby








4 Conclusion







Abbreviations





Acknowledgments


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























50of



Anti-lactoferrin

6

1417

28

(kDa)

6298

188

38

1 2 3 4Gel

Blots


(a)

(kDa) 1 2 3Gel

1 2 3

Blots14

17

28

62

98

188

38

CF COPD


(b)

0

01

02

03

04

05

06


2400 60 120 180Time (seconds)

Abso

rban

ce (4

05 nm

)

(c)

NE

NS

Total post-

Cytosol post-

IgG IP

0

01

02

03

04

05

06

Abso

rban

ce (4

05 nm

)

cytosolCytosol

SLPI IPcontrol

lowastlowast

(d)

0

20

40

60

80

100

120

140

160

180

Con +rhSLPI +rhSLPI

NS

lowastlowast


Cyto

solic

SLP

I (ng

105

cells

)

(e)










Con 120 240 4800

025

05

075

1

SLPI (nM)

lowast

lowastCh

emot

actic

inde

x (fM

LP 1

120583M)

(a)

0

025

05

075

1

Con 120 240 480SLPI (nM)

lowast

lowastlowastChem

otac

tic in

dex

(IL-

8 1

2 nM

)(b)

CF COPD

0

025

05

075

1

lowast lowast

Chem

otac

tic in

dex

(fMLP

1 120583M

)

(c)

CF COPD

0

025

05

075

1

lowast

lowast

Chem

otac

tic in

dex

(IL-

8 1

2 nM

)

(d)






10 20 10 20 10 20Time (min)

614

17

28

(kDa)

6298

38

ConfMLPIL-8

IL-8fMLP

SLPI

(a)

0

1

2

3

4

MM

P-9

(den

sitom

etry

uni

ts)

10 20 10 20 10 20Time (min)

Con fMLPIL-8

fMLP

SLPIIL-8

10 min20 min

lowastlowast

(b)

0

5

10

15

20

hCA

P-18

(den

sitom

etry

uni

ts)

10 20 10 20 10 20Time (min)

Con fMLPIL-8

fMLP

SLPIIL-8

10 min20 min

(c)

0

2

4

6

8

MPO

(d

ensit

omet

ry u

nits)

10 20 10 20 10 20Time (min)

Con fMLPIL-8

fMLP

SLPIIL-8

10 min20 min

lowast

(d)

0

10

20

30

40

50

60

70

CD66

b (M

FI)

Control fMLPIL-8

fMLP

SLPIIL-8


(e)

CD63

(MFI

)

0

10

20

30

40

Control fMLPIL-8

fMLP

SLPIIL-8


(f)







2 1 15 15 24 06

S P S P S P S P


(a)

Blots

fragment

(kDa)250

98

64

36

fragment

Gels

minus minus+SLPI +

+ +PMA minus minus


190 kDa

sim90 kDa

Anti-talin-1

Anti-vinculin

Anti-120573-actin

(b)

0

05

1

15

fMLPIL-8 IL-8

fMLP

SLPI

PMA PMASLPI

Tal

in cl

eava

ge (d

ensit

omet

ry u

nits)

lowastlowast

(c)

fMLPIL-8 IL-8

fMLP

SLPI

PMA PMASLPI

0

05

1

15

Vin

culin

clea

vage

(den

sitom

etry

uni

ts)

lowast

lowast

(d)




















1accumulation


3(1 120583M) (Figure 7(b))











0 10 20 30 40 500

1

2

3

4

Time (seconds)

Hea

lthy

cells

Ca2

+(r

elat

ive l

evels

)

lowastlowastlowast

(a)

Time (seconds)0 10 20 30 40 50

0

1

2

3

4

Hea

lthy

cells

Ca2

+(r

elat

ive l

evels

)

lowast

lowast

(b)

Time (seconds)0 10 20 30 40 50

0

1

2

3

4

CF ce

lls C

a2+

(rel

ativ

e lev

els)

lowastlowast

(c)

0 10 20 30 40 500

1

2

3

4

Time (seconds)

CF ce

lls C

a2+

(rel

ativ

e lev

els)

lowast

lowastlowast

(d)

0 10 20 30 40 500

2

1

4

3

6

5

7

8

Time (seconds)

COPD

cells

Ca2+

(rel

ativ

e lev

els)

lowast

lowast lowast

fMLP (+SLPI)Con

fMLP (minusSLPI)

(e)

0

1

2

3

4

0 10 20 30 40 50

Time (seconds)

COPD

cells

Ca2+

(rel

ativ

e lev

els)

Con


lowastlowast

(f)






4induced Ca2+



Cou

nts

20

40

60

80

100

0

fMLP

SLPI

100 101 102

FL1-H

fMLP998400998400fMLP998400998400

fMLP998400998400

(a)

100

0

20

40

60

80

MFI


fMLP998400998400 fMLP998400998400

lowastlowast

(b)

0 10 20 30 40 500

1

2

3

4

fMLP


Time (seconds)

Cyto

solic

Ca2

+(r

elat

ive l

evel)

lowast

(c)

0

1

2

3

4


0 10 20 30 40 50Time (seconds)

Cyto

solic

Ca2

+(r

elat

ive l

evel)

lowast

lowast

(d)



0

2

4

6

8






IP1

(rel

ativ

e lev

el)

lowastlowast

lowastlowast

lowast

lowast

(a)

0

1

2

3

4

NS





NS

NS

IP3

lowast

lowast

Cyto

solic

Ca2

+(r

elat

ive l

evel)

(b)

0 240 480SLPI (nM)

0

05

1

15

Con

NS

NS

lowast lowastlowast

Chem

otac

tic in

dex

(fMLP

1120583

M)

+1120583M IP3

(c)

NS

NS

0 240 480SLPI (nM)

0

05

1

15lowast

lowastlowast

Con+1120583M IP3

Chem

otac

tic in

dex

(IL-

8 1

2 nM

)

(d)










Extracellular

ConfMLP

PMA

6

1417

28

(kDa)

496298

188

38


Gel

Blots

Anti-SLPI

(a)

(kDa)

(kDa)

Anti-SLPI

Anti-SLPI

Membranes

Extracellular

Time (min) 0 05 1

0 05 1 10

10

1417

4938

1417

4938

Anti-p47phox

Anti-p47phox

(b)

Con

IL-8fMLP

PMA

(c)

(kDa)

Anti-SLPI

14

28

49

62

98

38

Gel

Blots

CFUn Stim


COPD

(d)

Extr

acel

lula

r rele

ased

SLP

I

0 10 20 30 40 500

1

2

3

4

Time (sec)

0 10 20 30 40 50Time

(seconds)

0

1

2

3

4Anti-SLPIBlot

(rel

ativ

e den

sitom

etry

uni

ts◼

)

Cyto

solic

Ca2

+(r

elat

ive l

evel

mdash)

lowast

(e)






Membranedomain


Native SLPI

rhSLPI

Agonist ligand





(a) (b)

rhSLPI

Calciosome

IP3

IP3

PIP2

PIP2

PLC-120573

PLC-120573

PLC-120573

Ca2+

Ca2+

ion channel






3production thereby








4 Conclusion







Abbreviations





Acknowledgments


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Anti-lactoferrin

6

1417

28

(kDa)

6298

188

38

1 2 3 4Gel

Blots


(a)

(kDa) 1 2 3Gel

1 2 3

Blots14

17

28

62

98

188

38

CF COPD


(b)

0

01

02

03

04

05

06


2400 60 120 180Time (seconds)

Abso

rban

ce (4

05 nm

)

(c)

NE

NS

Total post-

Cytosol post-

IgG IP

0

01

02

03

04

05

06

Abso

rban

ce (4

05 nm

)

cytosolCytosol

SLPI IPcontrol

lowastlowast

(d)

0

20

40

60

80

100

120

140

160

180

Con +rhSLPI +rhSLPI

NS

lowastlowast


Cyto

solic

SLP

I (ng

105

cells

)

(e)










Con 120 240 4800

025

05

075

1

SLPI (nM)

lowast

lowastCh

emot

actic

inde

x (fM

LP 1

120583M)

(a)

0

025

05

075

1

Con 120 240 480SLPI (nM)

lowast

lowastlowastChem

otac

tic in

dex

(IL-

8 1

2 nM

)(b)

CF COPD

0

025

05

075

1

lowast lowast

Chem

otac

tic in

dex

(fMLP

1 120583M

)

(c)

CF COPD

0

025

05

075

1

lowast

lowast

Chem

otac

tic in

dex

(IL-

8 1

2 nM

)

(d)






10 20 10 20 10 20Time (min)

614

17

28

(kDa)

6298

38

ConfMLPIL-8

IL-8fMLP

SLPI

(a)

0

1

2

3

4

MM

P-9

(den

sitom

etry

uni

ts)

10 20 10 20 10 20Time (min)

Con fMLPIL-8

fMLP

SLPIIL-8

10 min20 min

lowastlowast

(b)

0

5

10

15

20

hCA

P-18

(den

sitom

etry

uni

ts)

10 20 10 20 10 20Time (min)

Con fMLPIL-8

fMLP

SLPIIL-8

10 min20 min

(c)

0

2

4

6

8

MPO

(d

ensit

omet

ry u

nits)

10 20 10 20 10 20Time (min)

Con fMLPIL-8

fMLP

SLPIIL-8

10 min20 min

lowast

(d)

0

10

20

30

40

50

60

70

CD66

b (M

FI)

Control fMLPIL-8

fMLP

SLPIIL-8


(e)

CD63

(MFI

)

0

10

20

30

40

Control fMLPIL-8

fMLP

SLPIIL-8


(f)







2 1 15 15 24 06

S P S P S P S P


(a)

Blots

fragment

(kDa)250

98

64

36

fragment

Gels

minus minus+SLPI +

+ +PMA minus minus


190 kDa

sim90 kDa

Anti-talin-1

Anti-vinculin

Anti-120573-actin

(b)

0

05

1

15

fMLPIL-8 IL-8

fMLP

SLPI

PMA PMASLPI

Tal

in cl

eava

ge (d

ensit

omet

ry u

nits)

lowastlowast

(c)

fMLPIL-8 IL-8

fMLP

SLPI

PMA PMASLPI

0

05

1

15

Vin

culin

clea

vage

(den

sitom

etry

uni

ts)

lowast

lowast

(d)




















1accumulation


3(1 120583M) (Figure 7(b))











0 10 20 30 40 500

1

2

3

4

Time (seconds)

Hea

lthy

cells

Ca2

+(r

elat

ive l

evels

)

lowastlowastlowast

(a)

Time (seconds)0 10 20 30 40 50

0

1

2

3

4

Hea

lthy

cells

Ca2

+(r

elat

ive l

evels

)

lowast

lowast

(b)

Time (seconds)0 10 20 30 40 50

0

1

2

3

4

CF ce

lls C

a2+

(rel

ativ

e lev

els)

lowastlowast

(c)

0 10 20 30 40 500

1

2

3

4

Time (seconds)

CF ce

lls C

a2+

(rel

ativ

e lev

els)

lowast

lowastlowast

(d)

0 10 20 30 40 500

2

1

4

3

6

5

7

8

Time (seconds)

COPD

cells

Ca2+

(rel

ativ

e lev

els)

lowast

lowast lowast

fMLP (+SLPI)Con

fMLP (minusSLPI)

(e)

0

1

2

3

4

0 10 20 30 40 50

Time (seconds)

COPD

cells

Ca2+

(rel

ativ

e lev

els)

Con


lowastlowast

(f)






4induced Ca2+



Cou

nts

20

40

60

80

100

0

fMLP

SLPI

100 101 102

FL1-H

fMLP998400998400fMLP998400998400

fMLP998400998400

(a)

100

0

20

40

60

80

MFI


fMLP998400998400 fMLP998400998400

lowastlowast

(b)

0 10 20 30 40 500

1

2

3

4

fMLP


Time (seconds)

Cyto

solic

Ca2

+(r

elat

ive l

evel)

lowast

(c)

0

1

2

3

4


0 10 20 30 40 50Time (seconds)

Cyto

solic

Ca2

+(r

elat

ive l

evel)

lowast

lowast

(d)



0

2

4

6

8






IP1

(rel

ativ

e lev

el)

lowastlowast

lowastlowast

lowast

lowast

(a)

0

1

2

3

4

NS





NS

NS

IP3

lowast

lowast

Cyto

solic

Ca2

+(r

elat

ive l

evel)

(b)

0 240 480SLPI (nM)

0

05

1

15

Con

NS

NS

lowast lowastlowast

Chem

otac

tic in

dex

(fMLP

1120583

M)

+1120583M IP3

(c)

NS

NS

0 240 480SLPI (nM)

0

05

1

15lowast

lowastlowast

Con+1120583M IP3

Chem

otac

tic in

dex

(IL-

8 1

2 nM

)

(d)










Extracellular

ConfMLP

PMA

6

1417

28

(kDa)

496298

188

38


Gel

Blots

Anti-SLPI

(a)

(kDa)

(kDa)

Anti-SLPI

Anti-SLPI

Membranes

Extracellular

Time (min) 0 05 1

0 05 1 10

10

1417

4938

1417

4938

Anti-p47phox

Anti-p47phox

(b)

Con

IL-8fMLP

PMA

(c)

(kDa)

Anti-SLPI

14

28

49

62

98

38

Gel

Blots

CFUn Stim


COPD

(d)

Extr

acel

lula

r rele

ased

SLP

I

0 10 20 30 40 500

1

2

3

4

Time (sec)

0 10 20 30 40 50Time

(seconds)

0

1

2

3

4Anti-SLPIBlot

(rel

ativ

e den

sitom

etry

uni

ts◼

)

Cyto

solic

Ca2

+(r

elat

ive l

evel

mdash)

lowast

(e)






Membranedomain


Native SLPI

rhSLPI

Agonist ligand





(a) (b)

rhSLPI

Calciosome

IP3

IP3

PIP2

PIP2

PLC-120573

PLC-120573

PLC-120573

Ca2+

Ca2+

ion channel






3production thereby








4 Conclusion







Abbreviations





Acknowledgments


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Con 120 240 4800

025

05

075

1

SLPI (nM)

lowast

lowastCh

emot

actic

inde

x (fM

LP 1

120583M)

(a)

0

025

05

075

1

Con 120 240 480SLPI (nM)

lowast

lowastlowastChem

otac

tic in

dex

(IL-

8 1

2 nM

)(b)

CF COPD

0

025

05

075

1

lowast lowast

Chem

otac

tic in

dex

(fMLP

1 120583M

)

(c)

CF COPD

0

025

05

075

1

lowast

lowast

Chem

otac

tic in

dex

(IL-

8 1

2 nM

)

(d)






10 20 10 20 10 20Time (min)

614

17

28

(kDa)

6298

38

ConfMLPIL-8

IL-8fMLP

SLPI

(a)

0

1

2

3

4

MM

P-9

(den

sitom

etry

uni

ts)

10 20 10 20 10 20Time (min)

Con fMLPIL-8

fMLP

SLPIIL-8

10 min20 min

lowastlowast

(b)

0

5

10

15

20

hCA

P-18

(den

sitom

etry

uni

ts)

10 20 10 20 10 20Time (min)

Con fMLPIL-8

fMLP

SLPIIL-8

10 min20 min

(c)

0

2

4

6

8

MPO

(d

ensit

omet

ry u

nits)

10 20 10 20 10 20Time (min)

Con fMLPIL-8

fMLP

SLPIIL-8

10 min20 min

lowast

(d)

0

10

20

30

40

50

60

70

CD66

b (M

FI)

Control fMLPIL-8

fMLP

SLPIIL-8


(e)

CD63

(MFI

)

0

10

20

30

40

Control fMLPIL-8

fMLP

SLPIIL-8


(f)







2 1 15 15 24 06

S P S P S P S P


(a)

Blots

fragment

(kDa)250

98

64

36

fragment

Gels

minus minus+SLPI +

+ +PMA minus minus


190 kDa

sim90 kDa

Anti-talin-1

Anti-vinculin

Anti-120573-actin

(b)

0

05

1

15

fMLPIL-8 IL-8

fMLP

SLPI

PMA PMASLPI

Tal

in cl

eava

ge (d

ensit

omet

ry u

nits)

lowastlowast

(c)

fMLPIL-8 IL-8

fMLP

SLPI

PMA PMASLPI

0

05

1

15

Vin

culin

clea

vage

(den

sitom

etry

uni

ts)

lowast

lowast

(d)




















1accumulation


3(1 120583M) (Figure 7(b))











0 10 20 30 40 500

1

2

3

4

Time (seconds)

Hea

lthy

cells

Ca2

+(r

elat

ive l

evels

)

lowastlowastlowast

(a)

Time (seconds)0 10 20 30 40 50

0

1

2

3

4

Hea

lthy

cells

Ca2

+(r

elat

ive l

evels

)

lowast

lowast

(b)

Time (seconds)0 10 20 30 40 50

0

1

2

3

4

CF ce

lls C

a2+

(rel

ativ

e lev

els)

lowastlowast

(c)

0 10 20 30 40 500

1

2

3

4

Time (seconds)

CF ce

lls C

a2+

(rel

ativ

e lev

els)

lowast

lowastlowast

(d)

0 10 20 30 40 500

2

1

4

3

6

5

7

8

Time (seconds)

COPD

cells

Ca2+

(rel

ativ

e lev

els)

lowast

lowast lowast

fMLP (+SLPI)Con

fMLP (minusSLPI)

(e)

0

1

2

3

4

0 10 20 30 40 50

Time (seconds)

COPD

cells

Ca2+

(rel

ativ

e lev

els)

Con


lowastlowast

(f)






4induced Ca2+



Cou

nts

20

40

60

80

100

0

fMLP

SLPI

100 101 102

FL1-H

fMLP998400998400fMLP998400998400

fMLP998400998400

(a)

100

0

20

40

60

80

MFI


fMLP998400998400 fMLP998400998400

lowastlowast

(b)

0 10 20 30 40 500

1

2

3

4

fMLP


Time (seconds)

Cyto

solic

Ca2

+(r

elat

ive l

evel)

lowast

(c)

0

1

2

3

4


0 10 20 30 40 50Time (seconds)

Cyto

solic

Ca2

+(r

elat

ive l

evel)

lowast

lowast

(d)



0

2

4

6

8






IP1

(rel

ativ

e lev

el)

lowastlowast

lowastlowast

lowast

lowast

(a)

0

1

2

3

4

NS





NS

NS

IP3

lowast

lowast

Cyto

solic

Ca2

+(r

elat

ive l

evel)

(b)

0 240 480SLPI (nM)

0

05

1

15

Con

NS

NS

lowast lowastlowast

Chem

otac

tic in

dex

(fMLP

1120583

M)

+1120583M IP3

(c)

NS

NS

0 240 480SLPI (nM)

0

05

1

15lowast

lowastlowast

Con+1120583M IP3

Chem

otac

tic in

dex

(IL-

8 1

2 nM

)

(d)










Extracellular

ConfMLP

PMA

6

1417

28

(kDa)

496298

188

38


Gel

Blots

Anti-SLPI

(a)

(kDa)

(kDa)

Anti-SLPI

Anti-SLPI

Membranes

Extracellular

Time (min) 0 05 1

0 05 1 10

10

1417

4938

1417

4938

Anti-p47phox

Anti-p47phox

(b)

Con

IL-8fMLP

PMA

(c)

(kDa)

Anti-SLPI

14

28

49

62

98

38

Gel

Blots

CFUn Stim


COPD

(d)

Extr

acel

lula

r rele

ased

SLP

I

0 10 20 30 40 500

1

2

3

4

Time (sec)

0 10 20 30 40 50Time

(seconds)

0

1

2

3

4Anti-SLPIBlot

(rel

ativ

e den

sitom

etry

uni

ts◼

)

Cyto

solic

Ca2

+(r

elat

ive l

evel

mdash)

lowast

(e)






Membranedomain


Native SLPI

rhSLPI

Agonist ligand





(a) (b)

rhSLPI

Calciosome

IP3

IP3

PIP2

PIP2

PLC-120573

PLC-120573

PLC-120573

Ca2+

Ca2+

ion channel






3production thereby








4 Conclusion







Abbreviations





Acknowledgments


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















10 20 10 20 10 20Time (min)

614

17

28

(kDa)

6298

38

ConfMLPIL-8

IL-8fMLP

SLPI

(a)

0

1

2

3

4

MM

P-9

(den

sitom

etry

uni

ts)

10 20 10 20 10 20Time (min)

Con fMLPIL-8

fMLP

SLPIIL-8

10 min20 min

lowastlowast

(b)

0

5

10

15

20

hCA

P-18

(den

sitom

etry

uni

ts)

10 20 10 20 10 20Time (min)

Con fMLPIL-8

fMLP

SLPIIL-8

10 min20 min

(c)

0

2

4

6

8

MPO

(d

ensit

omet

ry u

nits)

10 20 10 20 10 20Time (min)

Con fMLPIL-8

fMLP

SLPIIL-8

10 min20 min

lowast

(d)

0

10

20

30

40

50

60

70

CD66

b (M

FI)

Control fMLPIL-8

fMLP

SLPIIL-8


(e)

CD63

(MFI

)

0

10

20

30

40

Control fMLPIL-8

fMLP

SLPIIL-8


(f)







2 1 15 15 24 06

S P S P S P S P


(a)

Blots

fragment

(kDa)250

98

64

36

fragment

Gels

minus minus+SLPI +

+ +PMA minus minus


190 kDa

sim90 kDa

Anti-talin-1

Anti-vinculin

Anti-120573-actin

(b)

0

05

1

15

fMLPIL-8 IL-8

fMLP

SLPI

PMA PMASLPI

Tal

in cl

eava

ge (d

ensit

omet

ry u

nits)

lowastlowast

(c)

fMLPIL-8 IL-8

fMLP

SLPI

PMA PMASLPI

0

05

1

15

Vin

culin

clea

vage

(den

sitom

etry

uni

ts)

lowast

lowast

(d)




















1accumulation


3(1 120583M) (Figure 7(b))











0 10 20 30 40 500

1

2

3

4

Time (seconds)

Hea

lthy

cells

Ca2

+(r

elat

ive l

evels

)

lowastlowastlowast

(a)

Time (seconds)0 10 20 30 40 50

0

1

2

3

4

Hea

lthy

cells

Ca2

+(r

elat

ive l

evels

)

lowast

lowast

(b)

Time (seconds)0 10 20 30 40 50

0

1

2

3

4

CF ce

lls C

a2+

(rel

ativ

e lev

els)

lowastlowast

(c)

0 10 20 30 40 500

1

2

3

4

Time (seconds)

CF ce

lls C

a2+

(rel

ativ

e lev

els)

lowast

lowastlowast

(d)

0 10 20 30 40 500

2

1

4

3

6

5

7

8

Time (seconds)

COPD

cells

Ca2+

(rel

ativ

e lev

els)

lowast

lowast lowast

fMLP (+SLPI)Con

fMLP (minusSLPI)

(e)

0

1

2

3

4

0 10 20 30 40 50

Time (seconds)

COPD

cells

Ca2+

(rel

ativ

e lev

els)

Con


lowastlowast

(f)






4induced Ca2+



Cou

nts

20

40

60

80

100

0

fMLP

SLPI

100 101 102

FL1-H

fMLP998400998400fMLP998400998400

fMLP998400998400

(a)

100

0

20

40

60

80

MFI


fMLP998400998400 fMLP998400998400

lowastlowast

(b)

0 10 20 30 40 500

1

2

3

4

fMLP


Time (seconds)

Cyto

solic

Ca2

+(r

elat

ive l

evel)

lowast

(c)

0

1

2

3

4


0 10 20 30 40 50Time (seconds)

Cyto

solic

Ca2

+(r

elat

ive l

evel)

lowast

lowast

(d)



0

2

4

6

8






IP1

(rel

ativ

e lev

el)

lowastlowast

lowastlowast

lowast

lowast

(a)

0

1

2

3

4

NS





NS

NS

IP3

lowast

lowast

Cyto

solic

Ca2

+(r

elat

ive l

evel)

(b)

0 240 480SLPI (nM)

0

05

1

15

Con

NS

NS

lowast lowastlowast

Chem

otac

tic in

dex

(fMLP

1120583

M)

+1120583M IP3

(c)

NS

NS

0 240 480SLPI (nM)

0

05

1

15lowast

lowastlowast

Con+1120583M IP3

Chem

otac

tic in

dex

(IL-

8 1

2 nM

)

(d)










Extracellular

ConfMLP

PMA

6

1417

28

(kDa)

496298

188

38


Gel

Blots

Anti-SLPI

(a)

(kDa)

(kDa)

Anti-SLPI

Anti-SLPI

Membranes

Extracellular

Time (min) 0 05 1

0 05 1 10

10

1417

4938

1417

4938

Anti-p47phox

Anti-p47phox

(b)

Con

IL-8fMLP

PMA

(c)

(kDa)

Anti-SLPI

14

28

49

62

98

38

Gel

Blots

CFUn Stim


COPD

(d)

Extr

acel

lula

r rele

ased

SLP

I

0 10 20 30 40 500

1

2

3

4

Time (sec)

0 10 20 30 40 50Time

(seconds)

0

1

2

3

4Anti-SLPIBlot

(rel

ativ

e den

sitom

etry

uni

ts◼

)

Cyto

solic

Ca2

+(r

elat

ive l

evel

mdash)

lowast

(e)






Membranedomain


Native SLPI

rhSLPI

Agonist ligand





(a) (b)

rhSLPI

Calciosome

IP3

IP3

PIP2

PIP2

PLC-120573

PLC-120573

PLC-120573

Ca2+

Ca2+

ion channel






3production thereby








4 Conclusion







Abbreviations





Acknowledgments


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















2 1 15 15 24 06

S P S P S P S P


(a)

Blots

fragment

(kDa)250

98

64

36

fragment

Gels

minus minus+SLPI +

+ +PMA minus minus


190 kDa

sim90 kDa

Anti-talin-1

Anti-vinculin

Anti-120573-actin

(b)

0

05

1

15

fMLPIL-8 IL-8

fMLP

SLPI

PMA PMASLPI

Tal

in cl

eava

ge (d

ensit

omet

ry u

nits)

lowastlowast

(c)

fMLPIL-8 IL-8

fMLP

SLPI

PMA PMASLPI

0

05

1

15

Vin

culin

clea

vage

(den

sitom

etry

uni

ts)

lowast

lowast

(d)




















1accumulation


3(1 120583M) (Figure 7(b))











0 10 20 30 40 500

1

2

3

4

Time (seconds)

Hea

lthy

cells

Ca2

+(r

elat

ive l

evels

)

lowastlowastlowast

(a)

Time (seconds)0 10 20 30 40 50

0

1

2

3

4

Hea

lthy

cells

Ca2

+(r

elat

ive l

evels

)

lowast

lowast

(b)

Time (seconds)0 10 20 30 40 50

0

1

2

3

4

CF ce

lls C

a2+

(rel

ativ

e lev

els)

lowastlowast

(c)

0 10 20 30 40 500

1

2

3

4

Time (seconds)

CF ce

lls C

a2+

(rel

ativ

e lev

els)

lowast

lowastlowast

(d)

0 10 20 30 40 500

2

1

4

3

6

5

7

8

Time (seconds)

COPD

cells

Ca2+

(rel

ativ

e lev

els)

lowast

lowast lowast

fMLP (+SLPI)Con

fMLP (minusSLPI)

(e)

0

1

2

3

4

0 10 20 30 40 50

Time (seconds)

COPD

cells

Ca2+

(rel

ativ

e lev

els)

Con


lowastlowast

(f)






4induced Ca2+



Cou

nts

20

40

60

80

100

0

fMLP

SLPI

100 101 102

FL1-H

fMLP998400998400fMLP998400998400

fMLP998400998400

(a)

100

0

20

40

60

80

MFI


fMLP998400998400 fMLP998400998400

lowastlowast

(b)

0 10 20 30 40 500

1

2

3

4

fMLP


Time (seconds)

Cyto

solic

Ca2

+(r

elat

ive l

evel)

lowast

(c)

0

1

2

3

4


0 10 20 30 40 50Time (seconds)

Cyto

solic

Ca2

+(r

elat

ive l

evel)

lowast

lowast

(d)



0

2

4

6

8






IP1

(rel

ativ

e lev

el)

lowastlowast

lowastlowast

lowast

lowast

(a)

0

1

2

3

4

NS





NS

NS

IP3

lowast

lowast

Cyto

solic

Ca2

+(r

elat

ive l

evel)

(b)

0 240 480SLPI (nM)

0

05

1

15

Con

NS

NS

lowast lowastlowast

Chem

otac

tic in

dex

(fMLP

1120583

M)

+1120583M IP3

(c)

NS

NS

0 240 480SLPI (nM)

0

05

1

15lowast

lowastlowast

Con+1120583M IP3

Chem

otac

tic in

dex

(IL-

8 1

2 nM

)

(d)










Extracellular

ConfMLP

PMA

6

1417

28

(kDa)

496298

188

38


Gel

Blots

Anti-SLPI

(a)

(kDa)

(kDa)

Anti-SLPI

Anti-SLPI

Membranes

Extracellular

Time (min) 0 05 1

0 05 1 10

10

1417

4938

1417

4938

Anti-p47phox

Anti-p47phox

(b)

Con

IL-8fMLP

PMA

(c)

(kDa)

Anti-SLPI

14

28

49

62

98

38

Gel

Blots

CFUn Stim


COPD

(d)

Extr

acel

lula

r rele

ased

SLP

I

0 10 20 30 40 500

1

2

3

4

Time (sec)

0 10 20 30 40 50Time

(seconds)

0

1

2

3

4Anti-SLPIBlot

(rel

ativ

e den

sitom

etry

uni

ts◼

)

Cyto

solic

Ca2

+(r

elat

ive l

evel

mdash)

lowast

(e)






Membranedomain


Native SLPI

rhSLPI

Agonist ligand





(a) (b)

rhSLPI

Calciosome

IP3

IP3

PIP2

PIP2

PLC-120573

PLC-120573

PLC-120573

Ca2+

Ca2+

ion channel






3production thereby








4 Conclusion







Abbreviations





Acknowledgments


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































1accumulation


3(1 120583M) (Figure 7(b))











0 10 20 30 40 500

1

2

3

4

Time (seconds)

Hea

lthy

cells

Ca2

+(r

elat

ive l

evels

)

lowastlowastlowast

(a)

Time (seconds)0 10 20 30 40 50

0

1

2

3

4

Hea

lthy

cells

Ca2

+(r

elat

ive l

evels

)

lowast

lowast

(b)

Time (seconds)0 10 20 30 40 50

0

1

2

3

4

CF ce

lls C

a2+

(rel

ativ

e lev

els)

lowastlowast

(c)

0 10 20 30 40 500

1

2

3

4

Time (seconds)

CF ce

lls C

a2+

(rel

ativ

e lev

els)

lowast

lowastlowast

(d)

0 10 20 30 40 500

2

1

4

3

6

5

7

8

Time (seconds)

COPD

cells

Ca2+

(rel

ativ

e lev

els)

lowast

lowast lowast

fMLP (+SLPI)Con

fMLP (minusSLPI)

(e)

0

1

2

3

4

0 10 20 30 40 50

Time (seconds)

COPD

cells

Ca2+

(rel

ativ

e lev

els)

Con


lowastlowast

(f)






4induced Ca2+



Cou

nts

20

40

60

80

100

0

fMLP

SLPI

100 101 102

FL1-H

fMLP998400998400fMLP998400998400

fMLP998400998400

(a)

100

0

20

40

60

80

MFI


fMLP998400998400 fMLP998400998400

lowastlowast

(b)

0 10 20 30 40 500

1

2

3

4

fMLP


Time (seconds)

Cyto

solic

Ca2

+(r

elat

ive l

evel)

lowast

(c)

0

1

2

3

4


0 10 20 30 40 50Time (seconds)

Cyto

solic

Ca2

+(r

elat

ive l

evel)

lowast

lowast

(d)



0

2

4

6

8






IP1

(rel

ativ

e lev

el)

lowastlowast

lowastlowast

lowast

lowast

(a)

0

1

2

3

4

NS





NS

NS

IP3

lowast

lowast

Cyto

solic

Ca2

+(r

elat

ive l

evel)

(b)

0 240 480SLPI (nM)

0

05

1

15

Con

NS

NS

lowast lowastlowast

Chem

otac

tic in

dex

(fMLP

1120583

M)

+1120583M IP3

(c)

NS

NS

0 240 480SLPI (nM)

0

05

1

15lowast

lowastlowast

Con+1120583M IP3

Chem

otac

tic in

dex

(IL-

8 1

2 nM

)

(d)










Extracellular

ConfMLP

PMA

6

1417

28

(kDa)

496298

188

38


Gel

Blots

Anti-SLPI

(a)

(kDa)

(kDa)

Anti-SLPI

Anti-SLPI

Membranes

Extracellular

Time (min) 0 05 1

0 05 1 10

10

1417

4938

1417

4938

Anti-p47phox

Anti-p47phox

(b)

Con

IL-8fMLP

PMA

(c)

(kDa)

Anti-SLPI

14

28

49

62

98

38

Gel

Blots

CFUn Stim


COPD

(d)

Extr

acel

lula

r rele

ased

SLP

I

0 10 20 30 40 500

1

2

3

4

Time (sec)

0 10 20 30 40 50Time

(seconds)

0

1

2

3

4Anti-SLPIBlot

(rel

ativ

e den

sitom

etry

uni

ts◼

)

Cyto

solic

Ca2

+(r

elat

ive l

evel

mdash)

lowast

(e)






Membranedomain


Native SLPI

rhSLPI

Agonist ligand





(a) (b)

rhSLPI

Calciosome

IP3

IP3

PIP2

PIP2

PLC-120573

PLC-120573

PLC-120573

Ca2+

Ca2+

ion channel






3production thereby








4 Conclusion







Abbreviations





Acknowledgments


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 10 20 30 40 500

1

2

3

4

Time (seconds)

Hea

lthy

cells

Ca2

+(r

elat

ive l

evels

)

lowastlowastlowast

(a)

Time (seconds)0 10 20 30 40 50

0

1

2

3

4

Hea

lthy

cells

Ca2

+(r

elat

ive l

evels

)

lowast

lowast

(b)

Time (seconds)0 10 20 30 40 50

0

1

2

3

4

CF ce

lls C

a2+

(rel

ativ

e lev

els)

lowastlowast

(c)

0 10 20 30 40 500

1

2

3

4

Time (seconds)

CF ce

lls C

a2+

(rel

ativ

e lev

els)

lowast

lowastlowast

(d)

0 10 20 30 40 500

2

1

4

3

6

5

7

8

Time (seconds)

COPD

cells

Ca2+

(rel

ativ

e lev

els)

lowast

lowast lowast

fMLP (+SLPI)Con

fMLP (minusSLPI)

(e)

0

1

2

3

4

0 10 20 30 40 50

Time (seconds)

COPD

cells

Ca2+

(rel

ativ

e lev

els)

Con


lowastlowast

(f)






4induced Ca2+



Cou

nts

20

40

60

80

100

0

fMLP

SLPI

100 101 102

FL1-H

fMLP998400998400fMLP998400998400

fMLP998400998400

(a)

100

0

20

40

60

80

MFI


fMLP998400998400 fMLP998400998400

lowastlowast

(b)

0 10 20 30 40 500

1

2

3

4

fMLP


Time (seconds)

Cyto

solic

Ca2

+(r

elat

ive l

evel)

lowast

(c)

0

1

2

3

4


0 10 20 30 40 50Time (seconds)

Cyto

solic

Ca2

+(r

elat

ive l

evel)

lowast

lowast

(d)



0

2

4

6

8






IP1

(rel

ativ

e lev

el)

lowastlowast

lowastlowast

lowast

lowast

(a)

0

1

2

3

4

NS





NS

NS

IP3

lowast

lowast

Cyto

solic

Ca2

+(r

elat

ive l

evel)

(b)

0 240 480SLPI (nM)

0

05

1

15

Con

NS

NS

lowast lowastlowast

Chem

otac

tic in

dex

(fMLP

1120583

M)

+1120583M IP3

(c)

NS

NS

0 240 480SLPI (nM)

0

05

1

15lowast

lowastlowast

Con+1120583M IP3

Chem

otac

tic in

dex

(IL-

8 1

2 nM

)

(d)










Extracellular

ConfMLP

PMA

6

1417

28

(kDa)

496298

188

38


Gel

Blots

Anti-SLPI

(a)

(kDa)

(kDa)

Anti-SLPI

Anti-SLPI

Membranes

Extracellular

Time (min) 0 05 1

0 05 1 10

10

1417

4938

1417

4938

Anti-p47phox

Anti-p47phox

(b)

Con

IL-8fMLP

PMA

(c)

(kDa)

Anti-SLPI

14

28

49

62

98

38

Gel

Blots

CFUn Stim


COPD

(d)

Extr

acel

lula

r rele

ased

SLP

I

0 10 20 30 40 500

1

2

3

4

Time (sec)

0 10 20 30 40 50Time

(seconds)

0

1

2

3

4Anti-SLPIBlot

(rel

ativ

e den

sitom

etry

uni

ts◼

)

Cyto

solic

Ca2

+(r

elat

ive l

evel

mdash)

lowast

(e)






Membranedomain


Native SLPI

rhSLPI

Agonist ligand





(a) (b)

rhSLPI

Calciosome

IP3

IP3

PIP2

PIP2

PLC-120573

PLC-120573

PLC-120573

Ca2+

Ca2+

ion channel






3production thereby








4 Conclusion







Abbreviations





Acknowledgments


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Cou

nts

20

40

60

80

100

0

fMLP

SLPI

100 101 102

FL1-H

fMLP998400998400fMLP998400998400

fMLP998400998400

(a)

100

0

20

40

60

80

MFI


fMLP998400998400 fMLP998400998400

lowastlowast

(b)

0 10 20 30 40 500

1

2

3

4

fMLP


Time (seconds)

Cyto

solic

Ca2

+(r

elat

ive l

evel)

lowast

(c)

0

1

2

3

4


0 10 20 30 40 50Time (seconds)

Cyto

solic

Ca2

+(r

elat

ive l

evel)

lowast

lowast

(d)



0

2

4

6

8






IP1

(rel

ativ

e lev

el)

lowastlowast

lowastlowast

lowast

lowast

(a)

0

1

2

3

4

NS





NS

NS

IP3

lowast

lowast

Cyto

solic

Ca2

+(r

elat

ive l

evel)

(b)

0 240 480SLPI (nM)

0

05

1

15

Con

NS

NS

lowast lowastlowast

Chem

otac

tic in

dex

(fMLP

1120583

M)

+1120583M IP3

(c)

NS

NS

0 240 480SLPI (nM)

0

05

1

15lowast

lowastlowast

Con+1120583M IP3

Chem

otac

tic in

dex

(IL-

8 1

2 nM

)

(d)










Extracellular

ConfMLP

PMA

6

1417

28

(kDa)

496298

188

38


Gel

Blots

Anti-SLPI

(a)

(kDa)

(kDa)

Anti-SLPI

Anti-SLPI

Membranes

Extracellular

Time (min) 0 05 1

0 05 1 10

10

1417

4938

1417

4938

Anti-p47phox

Anti-p47phox

(b)

Con

IL-8fMLP

PMA

(c)

(kDa)

Anti-SLPI

14

28

49

62

98

38

Gel

Blots

CFUn Stim


COPD

(d)

Extr

acel

lula

r rele

ased

SLP

I

0 10 20 30 40 500

1

2

3

4

Time (sec)

0 10 20 30 40 50Time

(seconds)

0

1

2

3

4Anti-SLPIBlot

(rel

ativ

e den

sitom

etry

uni

ts◼

)

Cyto

solic

Ca2

+(r

elat

ive l

evel

mdash)

lowast

(e)






Membranedomain


Native SLPI

rhSLPI

Agonist ligand





(a) (b)

rhSLPI

Calciosome

IP3

IP3

PIP2

PIP2

PLC-120573

PLC-120573

PLC-120573

Ca2+

Ca2+

ion channel






3production thereby








4 Conclusion







Abbreviations





Acknowledgments


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

2

4

6

8






IP1

(rel

ativ

e lev

el)

lowastlowast

lowastlowast

lowast

lowast

(a)

0

1

2

3

4

NS





NS

NS

IP3

lowast

lowast

Cyto

solic

Ca2

+(r

elat

ive l

evel)

(b)

0 240 480SLPI (nM)

0

05

1

15

Con

NS

NS

lowast lowastlowast

Chem

otac

tic in

dex

(fMLP

1120583

M)

+1120583M IP3

(c)

NS

NS

0 240 480SLPI (nM)

0

05

1

15lowast

lowastlowast

Con+1120583M IP3

Chem

otac

tic in

dex

(IL-

8 1

2 nM

)

(d)










Extracellular

ConfMLP

PMA

6

1417

28

(kDa)

496298

188

38


Gel

Blots

Anti-SLPI

(a)

(kDa)

(kDa)

Anti-SLPI

Anti-SLPI

Membranes

Extracellular

Time (min) 0 05 1

0 05 1 10

10

1417

4938

1417

4938

Anti-p47phox

Anti-p47phox

(b)

Con

IL-8fMLP

PMA

(c)

(kDa)

Anti-SLPI

14

28

49

62

98

38

Gel

Blots

CFUn Stim


COPD

(d)

Extr

acel

lula

r rele

ased

SLP

I

0 10 20 30 40 500

1

2

3

4

Time (sec)

0 10 20 30 40 50Time

(seconds)

0

1

2

3

4Anti-SLPIBlot

(rel

ativ

e den

sitom

etry

uni

ts◼

)

Cyto

solic

Ca2

+(r

elat

ive l

evel

mdash)

lowast

(e)






Membranedomain


Native SLPI

rhSLPI

Agonist ligand





(a) (b)

rhSLPI

Calciosome

IP3

IP3

PIP2

PIP2

PLC-120573

PLC-120573

PLC-120573

Ca2+

Ca2+

ion channel






3production thereby








4 Conclusion







Abbreviations





Acknowledgments


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Extracellular

ConfMLP

PMA

6

1417

28

(kDa)

496298

188

38


Gel

Blots

Anti-SLPI

(a)

(kDa)

(kDa)

Anti-SLPI

Anti-SLPI

Membranes

Extracellular

Time (min) 0 05 1

0 05 1 10

10

1417

4938

1417

4938

Anti-p47phox

Anti-p47phox

(b)

Con

IL-8fMLP

PMA

(c)

(kDa)

Anti-SLPI

14

28

49

62

98

38

Gel

Blots

CFUn Stim


COPD

(d)

Extr

acel

lula

r rele

ased

SLP

I

0 10 20 30 40 500

1

2

3

4

Time (sec)

0 10 20 30 40 50Time

(seconds)

0

1

2

3

4Anti-SLPIBlot

(rel

ativ

e den

sitom

etry

uni

ts◼

)

Cyto

solic

Ca2

+(r

elat

ive l

evel

mdash)

lowast

(e)






Membranedomain


Native SLPI

rhSLPI

Agonist ligand





(a) (b)

rhSLPI

Calciosome

IP3

IP3

PIP2

PIP2

PLC-120573

PLC-120573

PLC-120573

Ca2+

Ca2+

ion channel






3production thereby








4 Conclusion







Abbreviations





Acknowledgments


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Membranedomain


Native SLPI

rhSLPI

Agonist ligand





(a) (b)

rhSLPI

Calciosome

IP3

IP3

PIP2

PIP2

PLC-120573

PLC-120573

PLC-120573

Ca2+

Ca2+

ion channel






3production thereby








4 Conclusion







Abbreviations





Acknowledgments


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















4 Conclusion







Abbreviations





Acknowledgments


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Intracellular Secretory...

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