Title: CFTR Inhibition Provokes an Inflammatory Response Associated with an Imbalance of the Annexin A1 Pathway
Abstract: Cystic fibrosis (CF), a disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, is characterized by chronic bacterial infections and inflammation in the lung. Having previously shown that deletion of CFTR is associated with lower expression of the endogenous anti-inflammatory protein Annexin A1 (AnxA1), we investigated further this possible functional connection using a validated CFTR inhibitor. Treatment of mice with the CFTR inhibitor-172 (CFTR172) augmented the acute peritonitis promoted by zymosan, an effect associated with lower AnxA1 levels in peritoneal cells. Similar results were obtained with another, chemically distinct, CFTR inhibitor. The pro-inflammatory effect of CFTR172 was lost in AnxA1−/−, as well as CFTR−/− mice. Importantly, administration of hrAnxA1 and its peptido-mimetic to CFTR−/− animals or to animals treated with CFTR172 corrected the exaggerated leukocyte migration seen in these animals. In vitro assays with human Polymorphonuclear leukocyte (PMN) demonstrated that CFTR172 reduced cell-associated AnxA1 by promoting release of the protein in microparticles. We propose that the reduced impact of the counterregulatory properties of AnxA1 in CF cells contributes to the inflammatory phenotype characteristic of this disease. Thus, these findings provide an important insight into the mechanism underlying the inflammatory disease associated with CFTR inhibition while, at the same time, providing a novel pharmacological target for controlling the inflammatory phenotype of CF. Cystic fibrosis (CF), a disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, is characterized by chronic bacterial infections and inflammation in the lung. Having previously shown that deletion of CFTR is associated with lower expression of the endogenous anti-inflammatory protein Annexin A1 (AnxA1), we investigated further this possible functional connection using a validated CFTR inhibitor. Treatment of mice with the CFTR inhibitor-172 (CFTR172) augmented the acute peritonitis promoted by zymosan, an effect associated with lower AnxA1 levels in peritoneal cells. Similar results were obtained with another, chemically distinct, CFTR inhibitor. The pro-inflammatory effect of CFTR172 was lost in AnxA1−/−, as well as CFTR−/− mice. Importantly, administration of hrAnxA1 and its peptido-mimetic to CFTR−/− animals or to animals treated with CFTR172 corrected the exaggerated leukocyte migration seen in these animals. In vitro assays with human Polymorphonuclear leukocyte (PMN) demonstrated that CFTR172 reduced cell-associated AnxA1 by promoting release of the protein in microparticles. We propose that the reduced impact of the counterregulatory properties of AnxA1 in CF cells contributes to the inflammatory phenotype characteristic of this disease. Thus, these findings provide an important insight into the mechanism underlying the inflammatory disease associated with CFTR inhibition while, at the same time, providing a novel pharmacological target for controlling the inflammatory phenotype of CF. Over the past decades our understanding of the underlying causative elements of cystic fibrosis (CF) has significantly improved. We saw a radical shift from innumerable speculations about its origin to a precise definition of causative mutations, specifically those arising in a transmembrane ion channel termed CF transmembrane regulator (CFTR). Clinically childhood mortality has fallen dramatically and the expectancy of CF patients has increased to an average age of more than 30 years.1Quinton PM Physiological basis of cystic fibrosis: a historical perspective.Physiol Rev. 1999; 79: S3-S22PubMed Google Scholar However, there is still no cure and no effective control of the exacerbated chronic inflammatory process that leads to the relentless destruction of the lungs and pancreas exist. Lung disease is associated with the production of a more viscous mucus, resulting from the defect in ion transport across the epithelial cell membrane, and it represents the most life-threatening feature of CF. Several clinical studies indicate that pulmonary neutrophilic inflammation occurs very early in the course of the CF lung disease and often precedes overt signs of colonization or infection. Analysis of bronchoalveolar lavage fluid2Balough K McCubbin M Weinberger M Smits W Ahrens R Fick R The relationship between infection and inflammation in the early stages of lung disease from cystic fibrosis.Pediatr Pulmonol. 1995; 20: 63-70Crossref PubMed Scopus (285) Google Scholar from CF patients of different ages and with a range of disease severity consistently shows a marked rise in neutrophil numbers and elevated amounts of tumor necrosis factor-α, interleukin (IL)−1β and IL-8.3Bonfield TL Panuska JR Konstan MW Hilliard KA Hilliard JB Ghnaim H Berger M Inflammatory cytokines in cystic fibrosis lungs.Am J Respir Crit Care Med. 1995; 152: 2111-2118Crossref PubMed Scopus (599) Google Scholar, 4Corvol H Fitting C Chadelat K Jacquot J Tabary O Boule M Cavaillon JM Clement A Distinct cytokine production by lung and blood neutrophils from children with cystic fibrosis.Am J Physiol Lung Cell Mol Physiol. 2003; 284: L997-L1003Crossref PubMed Scopus (91) Google Scholar, 5Griese M Kappler M Gaggar A Hartl D Inhibition of airway proteases in cystic fibrosis lung disease.Eur Respir J. 2008; 32: 783-795Crossref PubMed Scopus (87) Google Scholar The bronchial epithelium is capable of generating all of these inflammatory modulators in large quantities, except for tumor necrosis factor-α, where activated alveolar macrophages and other leukocytes might contribute significantly to the production of this cytokine in CF.6Khan TZ Wagener JS Bost T Martinez J Accurso FJ Riches DW Early pulmonary inflammation in infants with cystic fibrosis.Am J Respir Crit Care Med. 1995; 151: 1075-1082PubMed Google Scholar During the past decade new lines of research have focused on mechanisms of endogenous anti-inflammation and resolution of inflammation.7Serhan CN Brain SD Buckley CD Gilroy DW Haslett C O'Neill LA Perretti M Rossi AG Wallace JL Resolution of inflammation: state of the art, definitions and terms.FASEB J. 2007; 21: 325-332Crossref PubMed Scopus (847) Google Scholar, 8Serhan CN Savill J Resolution of inflammation: the beginning programs the end.Nat Immunol. 2005; 6: 1191-1197Crossref PubMed Scopus (1794) Google Scholar On an inflammatory challenge, a tightly concerted reaction occurs, leading to the induction of a strong inflammatory response aiming at the deactivation and removal of the initiating insult. In order for this process to prove beneficial to the host, homeostasis needs to be reestablished. In this context, several anti-inflammatory mediators and pathways operate concurrently to assure a strict and timely return to the basal homeostatic state of the inflammatory response. Among these we find the potent anti-inflammatory protein Annexin A1 (AnxA1).9Gilroy DW Lawrence T Perretti M Rossi AG Inflammatory resolution: new opportunities for drug discovery.Nat Rev Drug Discov. 2004; 3: 401-416Crossref PubMed Scopus (642) Google Scholar In resting cells, AnxA1 is predominantly located intracellularly, with a small proportion found on the plasma membrane.10Perretti M D'Acquisto F Annexin A1 and glucocorticoids as effectors of the resolution of inflammation.Nat Rev Immunol. 2009; 9: 62-70Crossref PubMed Scopus (640) Google Scholar On cell activation the protein translocates on the cell surface where it encounters its receptor, a specific 7-trans membrane G-protein coupled receptor, termed formyl peptide receptor 2 or lipoxin A4 receptor (FPR2/ALX).11Perretti M Chiang N La M Fierro IM Marullo S Getting SJ Solito E Serhan CN Endogenous lipid- and peptide-derived anti-inflammatory pathways generated with glucocorticoid and aspirin treatment activate the lipoxin A4 receptor.Nat Med. 2002; 8: 1296-1302Crossref PubMed Scopus (379) Google Scholar Acting in a paracrine/autocrine fashion, AnxA1 the triggers signaling pathways that modulate and down-regulate Polymorphonuclear leukocyte (PMN) activation.10Perretti M D'Acquisto F Annexin A1 and glucocorticoids as effectors of the resolution of inflammation.Nat Rev Immunol. 2009; 9: 62-70Crossref PubMed Scopus (640) Google Scholar, 12Kamal AM Flower RJ Perretti M An overview of the effects of annexin 1 on cells involved in the inflammatory process.Mem Inst Oswaldo Cruz. 2005; 100 Suppl 1: 39-47Crossref PubMed Scopus (71) Google Scholar, 13Hayhoe RP Kamal AM Solito E Flower RJ Cooper D Perretti M Annexin 1 and its bioactive peptide inhibit neutrophil-endothelium interactions under flow: indication of distinct receptor involvement.Blood. 2006; 107: 2123-2130Crossref PubMed Scopus (189) Google Scholar The actions of AnxA1 are terminated, in a time-dependent manner, by the proteolytic cleavage of the N-terminus, most likely by serine proteases (though, cell specific catabolism is also likely to occur).14Vong L D'Acquisto F Pederzoli-Ribeil M Lavagno L Flower RJ Witko-Sarsat V Perretti M Annexin 1 cleavage in activated neutrophils: a pivotal role for proteinase 3.J Biol Chem. 2007; 282: 29998-30004Crossref PubMed Scopus (102) Google Scholar Dysregulation of the AnxA1 system has been associated with exacerbated and prolonged cell activation along with abnormal responses, caused by the lack of “fine tuning” and homeostatic control. Besides modulation of cell activation, other actions recently ascribed to AnxA1 that indicate its multipotent anti-inflammatory properties—and thus might be relevant in the context of chronic inflammation—include: its ability to induced PMN apoptosis15Solito E Kamal A Russo-Marie F Buckingham JC Marullo S Perretti M A novel calcium-dependent proapoptotic effect of annexin 1 on human neutrophils.FASEB J. 2003; 17: 1544-1546PubMed Google Scholar and their removal by the macrophage.16Maderna P Yona S Perretti M Godson C Modulation of phagocytosis of apoptotic neutrophils by supernatant from dexamethasone-treated macrophages and annexin-derived peptide Ac(2-26).J Immunol. 2005; 174: 3727-3733PubMed Google Scholar In addition, AnxA1 up-regulates the anti-inflammatory cytokine IL-10, which in turn leads to the inhibition of inducible Nitric Oxide Synthase mRNA expression and nitric oxide release.17Ferlazzo V D'Agostino P Milano S Caruso R Feo S Cillari E Parente L Anti-inflammatory effects of annexin-1: stimulation of IL-10 release and inhibition of nitric oxide synthesis.Int Immunopharmacol. 2003; 3: 1363-1369Crossref PubMed Scopus (67) Google Scholar Experimental models of CF have relied on the generation of CFTR−/− mouse colonies. One of these colonies revealed a selective absence of the protein AnxA1, a finding associated empirically with exacerbated inflammation (of the gut) and high degree of lethality.18Bensalem N Ventura AP Vallee B Lipecka J Tondelier D Davezac N Dos Santos A Perretti M Fajac A Sermet-Gaudelus I Renouil M Lesure JF Halgand F Laprevote O Edelman A Down-regulation of the anti-inflammatory protein annexin A1 in cystic fibrosis knock-out mice and patients.Mol Cell Proteomics. 2005; 4: 1591-1601Crossref PubMed Scopus (82) Google Scholar The same study showed reduced AnxA1 protein levels in human nasal epithelial cells obtained from CF patients. A more recent study reported formation of a complex between AnxA1 and CFTR, encompassing also S100A10 and cytosolic phospholipase A2, This regulatory complex keeps epithelial cells in check; thus, disruption of this complex in vitro leads to elevated tumor necrosis factor-α mediated eicosanoid release.19Borot F Vieu DL Faure G Fritsch J Colas J Moriceau S Baudouin-Legros M Brouillard F Ayala-Sanmartin J Touqui L Chanson M Edelman A Ollero M Eicosanoid release is increased by membrane destabilization and CFTR inhibition in Calu-3 cells.PLoS One. 2009; 4: e7116Crossref PubMed Scopus (27) Google Scholar The present study sets out with a dual aim: on one hand to establish a model of experimental inflammation linked to CFTR malfunction; on the other hand to extend our knowledge of the potential association between AnxA1 and CFTR to mouse and human neutrophils. This task was fulfilled using the highly selective CFTR inhibitor-172 (CFTR172). Originally identified following a high throughput screening of 50,000 small molecules, CFTR172 is one of six members of the 2-thioxo-4-thiazolidinone class identified as inhibitors. If produces a reversible inhibition of CFTR short-circuit current with a Ki of ∼300 nmol/L.20Ma T Thiagarajah JR Yang H Sonawane ND Folli C Galietta LJ Verkman AS Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin-induced intestinal fluid secretion.J Clin Invest. 2002; 110: 1651-1658Crossref PubMed Scopus (590) Google Scholar Testing this inhibitor in CFTR−/− mice and assessment of the effects of a structurally unrelated CFTR inhibitor, compound GlyH101, reinforced the functional association between CFTR and AnxA1. AnxA1−/−,21Hannon R Croxtall JD Getting SJ Roviezzo F Yona S Paul-Clark MJ Gavins FN Perretti M Morris JF Buckingham JC Flower RJ Aberrant inflammation and resistance to glucocorticoids in annexin 1−/− mouse.FASEB J. 2003; 17: 253-255PubMed Google Scholar backcrossed for more than 10 generations on the C57/Black6 genetic background, and C57/Black6 mice were obtained from B&K Universal (Hull, UK). CFTR−/− mice bred on a C57/Black6 background or their wild-type littermate controls (bred in house) were also used. In all cases mice were 4 to 6 weeks old. Animal work was performed according to UK Home Office regulations (Guidance on the Operation of Animals, Scientific Procedures Act, 1986). CFTR−/− mice22Snouwaert JN Brigman KK Latour AM Malouf NN Boucher RC Smithies O Koller BH An animal model for cystic fibrosis made by gene targeting.Science. 1992; 257: 1083-1088Crossref PubMed Scopus (768) Google Scholar—and wild-type littermates—were injected with 1 mg of zymosan A (Sigma-Aldrich; Poole, UK) in 0.5 ml PBS as reported,23Chatterjee BE Yona S Rosignoli G Young RE Nourshargh S Flower RJ Perretti M Annexin 1-deficient neutrophils exhibit enhanced transmigration in vivo and increased responsiveness in vitro.J Leukoc Biol. 2005; 78: 639-646Crossref PubMed Scopus (106) Google Scholar following the i.v. injection of 1 μg AnxA1 or vehicle in some of the groups. At the 4-hour time point, animals were euthanized by CO2 exposure, and peritoneal cavities were washed with 3 ml of PBS (Oxoid; Basingstoke, UK) containing 3 mmol/L EDTA (Sigma-Aldrich; Poole, UK) and 25 U/ml heparin. Aliquots of lavage fluid were then stained with Turk’s solution and differential cell counts were performed using a Neubauer hemocytometer and a light microscope (Olympus B061): leukocytes were identified as >95% neutrophils by light microscopy determination. Furthermore, cells from the lavage fluid were stained using an anti-GR1 anti-body (clone: RB6 8C5. eBioscience; Hatfield, UK) to determine total number of GR1 positive (GR1+ve) cells present in the peritoneal cavity. Lavage fluids were then centrifuged at 400 × g for 10 minutes and supernatants stored at −80°C for further analysis. In the acute CFTR inhibition experiments, several combinations of the CFTR172 inhibitor (Sigma-Aldrich; Poole, UK) and zymosan were tested. Initially, doses of 10 to 250 μg/kg CFTR172 were given 24 hours and 1 hour before the injection of either one of the following three zymosan doses: 0.1, 0.2, and 1 mg per mouse. In parallel groups, CFTR172 was injected only once, 1 hour before the injection of the different doses of zymosan. Dose range for CFTR172 was selected on a previous publication.20Ma T Thiagarajah JR Yang H Sonawane ND Folli C Galietta LJ Verkman AS Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin-induced intestinal fluid secretion.J Clin Invest. 2002; 110: 1651-1658Crossref PubMed Scopus (590) Google Scholar In separate experiments, AnxA1−/−,21Hannon R Croxtall JD Getting SJ Roviezzo F Yona S Paul-Clark MJ Gavins FN Perretti M Morris JF Buckingham JC Flower RJ Aberrant inflammation and resistance to glucocorticoids in annexin 1−/− mouse.FASEB J. 2003; 17: 253-255PubMed Google Scholar CFTR−/− and C57/Black6 mice were treated with either vehicle or 50 μg/kg CFTR172 24 and 1 hour before treatment with 0.1 mg of zymosan or vehicle. Furthermore, GlyH101 (Calbiochem; Nottingham, UK), another CFTR inhibitor that blocks the channel from its luminal side24Muanprasat C Sonawane ND Salinas D Taddei A Galietta LJ Verkman AS Discovery of glycine hydrazide pore-occluding CFTR inhibitors: mechanism, structure-activity analysis, and in vivo efficacy.J Gen Physiol. 2004; 124: 125-137Crossref PubMed Scopus (229) Google Scholar was also tested, at 60 μg/kg dose (equimolar to 50 μg/kg CFTR172) and a treatment regimen −24 and −1 hour, with 0.1 mg zymosan being injected at time 0. In all cases, cells were collected and the cell counts were determined, with the cell supernatants being extracted and stored as outlined above. To determine the therapeutic benefit of AnxA1 or it peptidomimetic Ac2-26 in controlling the inflammatory processes characterized by CFTR inhibition, mice were treated with 50 μg/kg CFTR172 24 hours and 1 hour before treatment with 0.1 mg of zymosan or vehicle given intraperitoneally; 10 minutes before intraperitoneal treatment mice received a bolus injection of either 1 μg human recombinant (hr)-AnxA1, 50 μg of Ac2-26 or vehicle, all given intravenously (10 ml/kg). The relative abundance of a panel of cytokines was determined using a Mouse Cytokine Array Panel (Panel A: R&D Systems; Abingdon, UK) as outlined in the manufacturers instructions. In brief, membranes were blocked for an hour at room temperature; 200 μl of lavage supernatant were pooled from each group and incubated with the membranes overnight at 4°C following which the membranes were washed and incubated with streptavidin-horseradish peroxidase for 30 minutes at room temperature. Subsequently, each membrane was incubated with an enhanced chemiluminescence detection reagent. The relative abundance of each cytokine kit was ascertained following manufactures instructions, using the internal controls included on each membrane. Changes in IL-6 levels in the lavage fluids were determined using and IL-6 DuoSet (R&D Systems; Abingdon, UK) following manufacturers instructions. In brief 20 μl of lavage supernatant were used from each sample, and incubated in a 96-well high binding plates (Corning, NY), previously coated with an anti-mouse IL-6 capture antibody. Subsequently plates were washed, blocked for 1 hour before addition of 100-μl lavage fluid samples. Following a 2-hour incubation at room temperature, a washing step was included before a 2-hour incubation with the detecting antibody. Streptavidin-horseradish peroxidase was added for 20 minutes and substrate solution for a further 20 minutes; then, reaction was stopped using the Stop Solution provided and the plate read on a microplate reader (NOVOstar; BMG Labtech; Aylesbury, UK). PMN were separated from whole blood as previously described25Perretti M Croxtall JD Wheller SK Goulding NJ Hannon R Flower RJ Mobilizing lipocortin 1 in adherent human leukocytes downregulates their transmigration.Nat Med. 1996; 2: 1259-1262Crossref PubMed Scopus (197) Google Scholar according to a protocol approved by the local Research Ethics Committee (P/00/029 ELCHA) and seeded into 12-well plates at a concentration of 2 × 106 cells per ml. Then vehicle or 20 μmol/L CFTR172 inhibitor19Borot F Vieu DL Faure G Fritsch J Colas J Moriceau S Baudouin-Legros M Brouillard F Ayala-Sanmartin J Touqui L Chanson M Edelman A Ollero M Eicosanoid release is increased by membrane destabilization and CFTR inhibition in Calu-3 cells.PLoS One. 2009; 4: e7116Crossref PubMed Scopus (27) Google Scholar were added for 30 minutes, before stimulation with 10 ng/ml of LPS (E. coli strain 0111:B4; Sigma-Aldrich; Poole, UK) for 4 hours at 37°C in an atmosphere with 5% CO2. Following centrifugation, half of the supernatant was stored at −80°C while the second portion was spun at 100,000 × g for 1 hour to pellet the microparticles26Dalli J Norling LV Renshaw D Cooper D Leung KY Perretti M Annexin 1 mediates the rapid anti-inflammatory effects of neutrophil-derived microparticles.Blood. 2008; 112: 2512-2519Crossref PubMed Scopus (218) Google Scholar following which the microparticle-free supernatant was transferred to fresh tubes and stored at −80°C. For flow cytometric analyses, cell pellets were re-suspended in PBC (a solution containing PBS, 0.15% bovine serum albumin and 1 mmol/L CaCl2; both from Sigma-Aldrich; Poole, UK), whereby the total and cell membrane expression of AnxA1 and FPR2/ALX was determined. Cell membrane immunoreactivity was determined with 5 μg/ml mouse anti-human AnxA1 (clone 1B27Pepinsky RB Sinclair LK Dougas I Liang CM Lawton P Browning JL Monoclonal antibodies to lipocortin-1 as probes for biological function.FEBS Lett. 1990; 261: 247-252Crossref PubMed Scopus (49) Google Scholar), anti-human FPR2/ALX (clone: 6C7-3; kind gift of Dr. Duncan Anderson; Astra Zeneca, UK) or isotype control (IgG1), for 45 minutes on ice. Incubation with a secondary anti-mouse fluorescein isothiocyanate-conjugated antibody (clone:STAR9B. Serotec; Oxford, UK). Total AnxA1 and FPR2/ALX contents were determined in a similar way, though a permeabilization step in 2% paraformaldehyde and 0.03% saponin (30 minutes on ice) was introduced, before incubation with primary and secondary antibodies as above. Flow cytometry was performed with a FACSCalibur (Becton & Dickinson; Oxford, UK), using a photomultiplier tube value of 548 for the FL1 (green) and acquiring ≥10,000 events per sample. In some cases, AnxA1 expression in mouse peritoneal neutrophils (elicited at 4 hours postzymosan and/or CFTR inhibitors) was also determined using the same permeabilization protocol, and a rabbit anti-AnxA1 antibody (Ab; 1:100: Invitrogen; Calne UK) or control rabbit IgG (Dako; Cambridgeshire, UK). Flow cytometry was performed as above, identifying the predominant (>85%) neutrophil population in the lavage fluids by its scatter characteristics. AnxA1 levels in the human PMN supernatants, in the presence or absence of microparticles, was determined by Western blotting following a previously published protocol.26Dalli J Norling LV Renshaw D Cooper D Leung KY Perretti M Annexin 1 mediates the rapid anti-inflammatory effects of neutrophil-derived microparticles.Blood. 2008; 112: 2512-2519Crossref PubMed Scopus (218) Google Scholar In brief, complete or microparticle-free supernatants (50 μl) were boiled with 10 μl of 6× Laemmli buffer for 5 minutes before loading onto a 10% SDS gel. After transfer to polyvinylidene difluoride membrane (Millipore; Watford, UK), and 2 hours blocking using 5% nonfat milk, blots were probed with a mouse anti-human AnxA1 antibody (clone 1B; 0.5 μg/ml) overnight at 4°C before washing and detection with anti-mouse horseradish peroxidase-conjugated antibody (Dako; Cambridgeshire, UK) and enhanced chemiluminescence detection reagent (GE Health care; St. Giles, UK). Subsequently a densitometric analysis was conducted using ImageJ software.28Collins TJ ImageJ for microscopy.Biotechniques. 2007; 43: 25-30Crossref PubMed Google Scholar In some cases, mouse AnxA1 protein in the lavage fluids of the zymosan peritonitis experiments was also determined, whereby 50-μl of cell-free fluids were boiled with 10 μl of 6× Laemlli buffer after which AnxA1 levels were as described above, but using a rabbit anti-AnxA1 Ab (1:5000; InVitrogen; Calne UK) and anti-rabbit horseradish peroxidase-conjugated antibody (Dako; Cambridgeshire, UK) as secondary. Cells obtained from the peritoneal lavage were cytospun onto poly-l-lysine coated slides (VWR International; Leicestershire, UK) before incubation with 10% albumin bovine in PBS (PBSA) to block nonspecific binding. A polyclonal rabbit anti-AnxA1 Ab (InVitrogen; Calne UK) was added (final dilution 1/200 in 1% PBSA) and slides incubated overnight at 4°C. As control for the reaction, some sections were incubated with nonimmune rabbit serum (1/200 working dilution. Sigma-Aldrich; Poole, UK) instead of the primary Ab. After repeated washings in 1% PBSA, a goat anti-rabbit IgG (Fc fragment-specific) Ab conjugated to 5 nm colloidal gold (1/100; British BioCell International, Cardiff, UK) was added. Silver enhancing solution (British BioCell International; Cardiff, UK) was used to augment gold particle staining. At the end of the reaction, slides were washed thoroughly in distilled water, counterstained with hematoxylin, and mounted in BIOMOUNT (British BioCell International; Cardiff, UK). Analysis was conducted with a microscope Nikon equipped with a DXM1200 digital camera, using the software LUCIA (Laboratory Universal Computer Image Analysis; Jencons-PLS, London UK). In vitro experiments with human cells were performed in triplicate using cells from four distinct donors. PMN migration in vivo was conducted using experimental groups of ≥5 mice per group. In all cases data are reported as mean ± SEM. Statistical differences between groups were determined by one-way analysis of variance and, if null hypothesis was rejected, by Student Newman-Keuls test, taking a probability value P < 0.05 as significant. The initial aim of these experiments was to develop a pharmacological model suitable to dissect the potential role of AnxA1 in the propagation of the CF condition. Several treatment combinations of CFTR172 (10, 50, and 250 μg/kg) and zymosan (0.1, 0.2, and 1 mg) were tested in conjunction with different treatment regimens using CFTR172 (−24 hours + −1 hour and −1 hour). As expected, zymosan produced a marked influx of Gr1+ve cells into the peritoneal cavity when given at the top dose of 1 mg per mouse (Figure 1A), a response that was unaffected by CFTR172 co-treatment. Thus, we fine tuned the extent of inflammation, administering 0.2 and 0.1 mg zymosan: in either case a significant cell influx was provoked and CFTR172 augmented the number of Gr1+ve cells recovered at the 4-hour time point (Figure 1, B and C). Out of the combinations tested, 50 μg/kg CFTR172 given as pretreatment (−24 hours and −1 hour) and 0.1 mg zymosan gave robust results (twofold increase over zymosan alone) and was chosen for the subsequent experiments. Of note, mice treated with CFTR172 alone also displayed a modest accumulation of Gr1+ve cells above the virtually null values measured in naïve cavities (Figure 1D). This potentiating effect was not restricted to CFTR172. We tested another CFTR inhibitor, structurally unrelated to CFTR172. Administered at the dose of 60 μg/kg, equimolar to 50 μg/kg CFTR172, compound GlyH101 augmented the peritoneal influx of Gr1+ve cells: 20 ± 3.0 × 105/mouse for zymosan alone and 40 ± 3.2 × 105/mouse for zymosan plus GlyH101 (P < 0.01; five mice per group). Immunohistochemical analysis on cells collected from the peritoneal lavage revealed a significant reduction in cell-associated AnxA1 protein when CFTR172 was administered in conjunction to the low dose zymosan. Figure 2A shows representative images with recruited PMN being highlighted. Using this semiquantitative method cumulative data both for peritoneal neutrophils (Figure 2B, left panel) and macrophages (Figure 2B, right panel) demonstrate that a significant reduction in cell associated AnxA1 immunoreactivity could be detected, in the zymosan + CFTR172 group. Interestingly, in absence of zymosan the CFTR inhibitor appeared to reduced AnxA1 expression in the few PMN present in the peritoneal cavity but not in macrophages (Figure 2B). AnxA1 protein rapidly appeared in cell-free peritoneal lavages following treatment with CFTR172 yielding increased levels (Figure 2B), suggesting a possible process of ‘AnxA1 cell leakage’ into the environment when CFTR is inactive (an observation that was also made with the CFTR−/− animals; data not shown). In contrast, the augmented AnxA1 content in the lavage fluids was significantly reduced on animal treatment with CFTR172 (Figure 2C). This suggests that the strong inflammatory reaction produced by the combination zymosan + CFTR172 subverted the normal mechanisms operative in acute inflammation. Cell-associated AnxA1 levels were reduced also by the other CFTR inhibitor, as quantified by flow cytometry in peritoneal PMN harvested from mice treated with 0.1 mg zymosan alone or together with GlyH101 (60 μg/kg, −24 and −1 hour). Figure 2D reports these data showing an illustrative histogram and the cumulative values out of five mice per group. To underpin a functional role for AnxA1 in the inflammatory response associated with CFTR inhibition, the active combination was tested in AnxA1−/− mice. Figure 3 reports these data, showing that administration of CFTR172 and zymosan again increased total peritoneal (Figure 3A) and Gr1+ve (Figure 3B) cell recruitment in wild-type mice, but were without effect in AnxA1−/− mice. On treatment with zymosan, AnxA1−/− mice exhibited degree of PMN influx which acted as internal control confirming previous publications.23Chatterjee BE Yona S Rosignoli G Young RE Nourshargh S Flower RJ Perretti M Annexin 1-deficient neutrophils exhibit enhanced transmigration in vivo and increased responsiveness in vitro.J Leukoc Biol. 2005; 78: 639-646Crossref PubMed Scopus (106) Google Scholar Interestingly, the mild inflammatory status induced by administration of CFTR172 in the absence of zymosan was again evident in wild-type mice and even more so in AnxA1−/− mice (Figure 3). The analysis of a panel of proteins tested by the array approach revealed that only a few are modulated in these experimental settings. Furthermore, some of these are already increased in AnxA1−/− mice or after CFTR172 treatment (Figure 4). The first differentially modulated marker was solu