Samia Yazid

Egle Solito

Helen Christian

Simon Mcarthur

Nicolas Goulding

R J Flower
email: r.j.flower@qmul.ac.uk

Cromoglycate Drugs Suppress Eicosanoid Generation in U937 Cells by Promoting the Release of Anx-A1

Keywords: Sodium nedocromil, glucocorticoids, okadaic acid, PKC, PP2A phosphatase

A c c e p t e d M a n u s c r i p t 2 Using biochemical, epifluorescence and electron microscopic techniques in a U937 model system, we investigated the effect of the anti-allergic drugs di-sodium cromoglycate and sodium nedocromil on the trafficking and release of the antiinflammatory protein Annexin-A1 (Anx-A1) when this was triggered by glucocorticoid (GC) treatment. GCs alone produced a rapid (within 5 min) concentration-dependent activation of PKCα/β (Protein Kinase C; EC 2.7.11.13) and phosphorylation of Anx-A1 on Ser 27 . Both phospho-proteins accumulated at the plasma membrane and Anx-A1 was subsequently externalised thereby inhibiting thromboxane (Tx) B 2 generation. When administered alone, cromoglycate or nedocromil had little effect on this pathway however, in the presence of a fixed sub-maximal concentration of GCs, increasing amounts of the cromoglycate-like drugs caused a striking concentration-dependent enhancement of Anx-A1 and PKCα/β phosphorylation, membrane recruitment and Anx-A1 release from cells resulting in greatly enhanced inhibition of TxB 2 generation. GCs also stimulated phosphatase accumulation at the plasma membrane of U937 cells. Both cromoglycate and nedocromil inhibited this enzymatic activity as well as that of a highly purified PP2A phosphatase preparation. We conclude that stimulation by the cromoglycate-like drugs of intracellular Anx-A1 trafficking and release (hence inhibition of eicosanoid release) is secondary to inhibition of a phosphatase PP2A (phosphoprotein phosphatase; EC 3.1.3.16), which probably forms part of a control loop to limit Anx-A1 release. These experiments provide a basis for a novel mechanism of action for the cromolyns, a group of drugs that have long puzzled investigators. Anx-A1, a 37kDa member of the annexin super-family (13 proteins in mammals), and its N-terminal peptide N-acetyl 2-26 [START_REF] Cirino | Antiinflammatory actions of an N-terminal peptide from human lipocortin 1[END_REF][START_REF] Perretti | Lipocortin-1 fragments inhibit neutrophil accumulation and neutrophil-dependent edema in the mouse. A qualitative comparison with an anti-CD11b monoclonal antibody[END_REF], have been shown by us, and by other laboratories to possess powerful anti-inflammatory actions in a wide variety of animal models of acute [START_REF] Perretti | Pharmacology of interleukin-1induced neutrophil migration[END_REF][START_REF] Perretti | Modulation of IL-1-induced neutrophil migration by dexamethasone and lipocortin 1[END_REF][START_REF] Cirino | Human recombinant lipocortin 1 has acute local anti-inflammatory properties in the rat paw edema test[END_REF][START_REF] Perretti | Anti-inflammatory lipocortin-derived peptides[END_REF][START_REF] Wu | Lipocortin 1 mediates the inhibition by dexamethasone of the induction by endotoxin of nitric oxide synthase in the rat[END_REF][START_REF] Harris | Alteration of neutrophil trafficking by a lipocortin 1 N-terminus peptide[END_REF][START_REF] Mancuso | Leukocyte transmigration, but not rolling or adhesion, is selectively inhibited by dexamethasone in the hamster post-capillary venule. Involvement of endogenous lipocortin 1[END_REF][START_REF] Perretti | Measurement of lipocortin 1 levels in murine peripheral blood leukocytes by flow cytometry: modulation by glucocorticoids and inflammation[END_REF][START_REF] Ferreira | Role of lipocortin-1 in the anti-hyperalgesic actions of dexamethasone[END_REF][START_REF] Cuzzocrea | Lipocortin 1 protects against splanchnic artery occlusion and reperfusion injury by affecting neutrophil migration[END_REF][START_REF] Lim | Promoting detachment of neutrophils adherent to murine postcapillary venules to control inflammation: effect of lipocortin 1[END_REF][START_REF] D'amico | Lipocortin 1 reduces myocardial ischemia-reperfusion injury by affecting local leukocyte recruitment[END_REF][START_REF] La | Annexin 1 peptides protect against experimental myocardial ischemiareperfusion: analysis of their mechanism of action[END_REF][START_REF] La | Analysis of the protection afforded by annexin 1 in ischaemia-reperfusion injury: focus on neutrophil recruitment[END_REF][START_REF] Bandeira-Melo | A novel effect for annexin 1-derived peptide ac2-26: reduction of allergic inflammation in the rat[END_REF] or chronic [START_REF] Yang | Antiinflammatory effect of lipocortin 1 in experimental arthritis[END_REF][START_REF] Yang | Inhibitory effect of annexin I on synovial inflammation in rat adjuvant arthritis[END_REF][START_REF] Yang | Modulation of inflammation and response to dexamethasone by Annexin 1 in antigen-induced arthritis[END_REF] inflammation.

The biologically active pool, in this context, is the extracellular protein. Anx-A1 is both induced and secreted from cells under the influence of GCs [START_REF] Goulding | Anti-inflammatory lipocortin 1 production by peripheral blood leucocytes in response to hydrocortisone[END_REF][START_REF] Peers | Glucocorticoid-and nonglucocorticoid induction of lipocortins (annexins) 1 and 2 in rat peritoneal leucocytes in vivo[END_REF][START_REF] Ahluwalia | Induction of lipocortin 1 by topical steroid in rat skin[END_REF][START_REF] Perretti | Mobilizing lipocortin 1 in adherent human leukocytes downregulates their transmigration[END_REF]. The release, as opposed to the induction, of cytosolic Anx-A1 is increased by GCs acting in a receptordependent, non-genomic manner. This GC-induced secretory event is preceded by Ser 27 phosphorylation apparently as a result of PKC (Protein Kinase C; EC 2.7.11.13) activation [START_REF] Croxtall | Glucocorticoids act within minutes to inhibit recruitment of signalling factors to activated EGF receptors through a receptor-dependent, transcription-independent mechanism[END_REF][START_REF] John | Kinasedependent regulation of the secretion of thyrotrophin and luteinizing hormone by glucocorticoids and annexin 1 peptides[END_REF][START_REF] Solito | Dexamethasone induces rapid serine-phosphorylation and membrane translocation of annexin 1 in a human folliculostellate cell line via a novel nongenomic mechanism involving the glucocorticoid receptor, protein kinase C, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase[END_REF]. Indeed, the Anx-A1 Ser 27 -Ala 27 mutant is not secreted from cells and has a different intracellular distribution [START_REF] Solito | Posttranslational modification plays an essential role in the translocation of annexin-A1 from the cytoplasm to the cell surface[END_REF]. Once on the cell surface, Anx-A1 can act in an autocrine (or paracrine) fashion to inhibit cell activation probably by interaction with receptors of the FPR family, specifically FPR-L1 (ALXR; [START_REF] Gavins | Leukocyte antiadhesive actions of annexin 1: ALXR-and FPR-related anti-inflammatory mechanisms[END_REF][START_REF] Perretti | Annexin 1 and the biology of the neutrophil[END_REF][START_REF] Hayhoe | Annexin 1 and its bioactive peptide inhibit neutrophil-endothelium interactions under flow: indication of distinct receptor involvement[END_REF]).

The 'cromoglycate-like' anti-allergic drugs (cromolyns) are a group of compounds of which sodium cromoglycate and sodium nedocromil are the exemplars. The family also embraces lodoxamide, traxanol and amlexanox as well as some H 1 antagonists such as ketotifen, azelastine, pemirolast and olopatidine, many of which appear to share a similar pharmacology (or exhibit cross-tachyphylaxis) with cromoglycate [START_REF] Cook | Mechanisms of antihistamines and mast cell stabilizers in ocular allergic inflammation[END_REF].

Contemporary reviewers are unanimous in attributing the anti-asthmatic activity of the cromoglycate-like drugs to their anti-inflammatory properties (see refs. [START_REF] Barnes | Anti-inflammatory therapy for asthma[END_REF][START_REF] Viscardi | Cromolyn sodium prophylaxis inhibits pulmonary proinflammatory cytokines in infants at high risk for bronchopulmonary dysplasia[END_REF][START_REF] Bleecker | Clinical effects of nedocromil sodium on allergen-related mechanisms[END_REF]), although the exact mechanism of action of this group of drugs has proved elusive. Early experiments [START_REF] Cox | Disodium cromoglycate (FPL 670) ('Intal'): a specific inhibitor of reaginic antibody-antigen mechanisms[END_REF][START_REF] Cox | Recent developments concerned with the mode of action of disodium cromoglycate (Intal)[END_REF][START_REF] Cox | Nature and modes of action of disodium cromoglycate (Lomudal)[END_REF][START_REF] Cox | Mode of action of disodium cromoglycate, studies on immediate type hypersensitivity reactions using 'double sensitization' with two antigenically distinct rat reagins[END_REF][START_REF] Orr | Development of preclinical models for testing antiasthma drugs[END_REF] led to the concept that these drugs acted mainly on mast cells to suppress mediator release, but the balance of evidence now suggests that this is unlikely to be their only clinically significant action and that mast cells are not their sole target.

Of particular relevance to this study is the observation that they can also suppress eicosanoid generation [START_REF] Mattoli | Nedocromil sodium prevents the release of 15-hydroxyeicosatetraenoic acid from human bronchial epithelial cells exposed to toluene diisocyanate in vitro[END_REF][START_REF] Radeau | Effect of nedocromil sodium on sulfidopeptide leukotrienes-stimulated human alveolar macrophages in asthma[END_REF].

A c c e p t e d M a n u s c r i p t 4 Here we report that the mechanism whereby the latter effect is accomplished in differentiated U937 cells depends upon the enhanced intracellular trafficking and release of Anx-A1, and that these drugs synergise strongly with agents which activate PKC, such as GCs, to bring about this effect. Our findings highlight a novel mechanism of action for the cromolyns as well as providing a compelling rationale for a combination antiallergy therapy.

Materials and methods

U937 cell culture.

U937 cells were obtained from the American Type Culture Collection and cultured in RPMI 1640 supplemented with 10% FCS, 1% L-glutamine, 1% non essential amino acids and 0.1% gentamicin at 37ºC (Sigma-Aldrich, Poole, UK) in 5% CO 2 atmosphere.

Proliferating monocytic cells were removed from the flask when 70 to 80% confluence was achieved, transferred to 12-well plates at a density of 10 6 cells/well and preincubated with 10ng/ml phorbol 12-myristate 13-acetate (PMA) for 24 h to promote monocytic differentiation after which period they acquire sensitivity to GCs [START_REF] Solito | Dexamethasone induces the expression of the mRNA of lipocortin 1 and 2 and the release of 1 and 5 in differentiated, but not undifferentiated U-937 cells[END_REF].

For assessment of drug effects, GCs were tested alone or in combination with the antiallergic drugs for different times. Three different procedures were used to prepare samples for analysis. To analyse proteins of interest, U937 cells were normally lysed at the end of the treatment period and the total protein content used for further analysis. To do this U937 cells in suspension following drug treatment, were decanted into 1.5ml

Eppendorf tubes and gently centrifuged (300 xg) for 5 min. The supernatant was removed and the resultant pellet resuspended in 500 µl of lysis buffer containing 1mM EDTA (which removes Anx-A1 attached to cell membranes), 200mM NaCl, 20mM Tris-HCl (pH 8.0), 1mM protease and 1mM phosphatase inhibitors (equimolar mixture of Na 3 VO 4 , β-glycerophosphate, NaF) and 0.1% Triton-X.

In some experiments, where we studied the differential abundance of proteins in the cell cytosol and pellet, the cells were first ruptured by freeze-thawing in liquid nitrogen 3-4 times and the crude fractions separated by centrifugation at 13,000 xg for 5 min. The A c c e p t e d M a n u s c r i p t 5 supernatant and pellet fractions were then prepared for analysis in lysis buffer as above prior to western blotting.

To prepare U937 plasma membranes, cell organelles were separated by ultracentrifugation. Cells ruptured by sonication were centrifuged at 300 xg for 5 min to remove coarse debris and intact cells and the supernatant was removed and resuspended in 1ml lysis buffer. An initial centrifugation at 13,000 xg separated nuclei, mitochondria and other dense material. The supernatant from this step was then resuspended in 0.5ml lysis buffer and centrifuged for 1h at 100,000 xg. The resulting pellet was resuspended in lysis buffer containing 0.1% Triton-X.

The cellular protein content of different fractions was analysed to determine the total protein concentration using the BioRad Protein Assay Method (Bio-Rad Laboratories, Hemel Hempstead, UK) according to the manufacturer's instructions.

Transfection of U937 cells with GFP-Anx-A1 construct and fluorescence microscopy.

U937 cells were stably transfected with a mouse Anx-A1 cDNA open reading frame-GFP as previously reported [START_REF] Solito | Posttranslational modification plays an essential role in the translocation of annexin-A1 from the cytoplasm to the cell surface[END_REF]. Cells transfected with EGFP 'empty' plasmid were used as controls. Stably transfected clones were kept in culture for no longer than 20 passages.

For transfection, the cells were plated in 6 well plates at a concentration of 10 6 /well in DMEM. Transient transfection was performed the following day, using a Dharmafect reagent 2 (Dharmacon-Perbio Science, Cramlington, UK), a liposoluble agent that fuses with the membrane , according to the manufacturer's protocol. These cells grow in suspension and after a period of 2 weeks in G418 selection, cells were sorted by FACS to enrich the GFP positive clones followed by a serial dilution in order to facilitate a clonal expansion.

To visualize export of Anx-A1-GFP cells were stimulated with GCs and/or cromoglycate-like drugs for 5-10 min, fixed in 4% paraformaldehyde (PFA), and stained The slides were mounted in Mowiol (Sigma-Aldrich, Poole, Dorset, UK). Fluorescence micrographs were obtained with a Cool-Snap-Pro of colour camera (Media Cybernetics, Finchampstead, Berkshire, UK) and image processing software (Image Pro Plus 4.5) linked to a Nikon Eclipse E800 microscope. The filter used to detect GFP-fluorescence was an excitation band-pass filter (450 -490 nM), a dichroic mirror (510 nM), and an emission band filter (515-560 nM). Z-stack images at 0.5µm separation were collected on an inverted epifluorescence TE2000 U Nikon microscope, and were subjected to nearest neighbour deconvolution using Openlab 5.5 software (Improvision, Coventry, UK)

Western blotting.

The total cellular protein was determined and the supernatant analysed by conventional western blotting techniques. Immunodetection was accomplished using different antibodies recognizing either the full-length Anx-A1 protein, (polyclonal anti-anx-A1 antibody; Invitrogen, Paisley, UK), Anx-A1 phosphorylated on Ser 27 , (polyclonal anti-Ser 27 -Anx-A1 antibody; Neosystem, Strasbourg, France) and α-tubulin (monoclonal antiα-tubulin; Sigma-Aldrich, Poole, UK). A horseradish peroxidase-conjugated secondary antibody (Sigma-Aldrich, Poole, UK) detected bands related to the proteins of interest and these were revealed using ECL reagents and quantitated using the Image J densitometry program. All data were normalised to α-tubulin and expressed as percentage of control (differentiated cells stimulated with 10ng/mL PMA for 24 h).

ELISA for Anx-A1

Anx-A1 protein levels in conditioned medium were determined by ELISA as reported by Goulding et al. [START_REF] Goulding | Anti-inflammatory lipocortin 1 production by peripheral blood leucocytes in response to hydrocortisone[END_REF]. Briefly, 96-well flat-bottomed ELISA plates (Greiner, Gloucestershire, UK) were coated with 1µg anti-Anx-A1 mAb 1B in bicarbonate buffer (pH 9.6) and incubated overnight at 4°C. After washing in the bicarbonate buffer, A c c e p t e d M a n u s c r i p t 7 potentially uncoated sites were blocked with 100µL of PBS containing 1% BSA for 1h at room temperature. Sample aliquots (100µL) or Anx-A1 standard solutions (prepared in 0.1% Tween-20 in PBS; concentration ranging between 10 and 0.001 µg/mL) were added for 1h at 37°C. After extensive washing in PBS/Tween-20, 100µL of a polyclonal rabbit anti-human Anx-A1 serum (Zymed cat no 71-3400, Invitrogen, Paisley, UK; diluted 1:1000 in PBS/Tween-20) were added (1h at 37°C) before incubation with donkey antirabbit IgG conjugated to alkaline phosphatase (1:1000; Sigma-Aldrich, Poole, UK). The colour was developed by addition of 100µL p-nitrophenyl phosphate (Sigma-Aldrich, Poole, UK; 1mg/mL in bicarbonate buffer, pH 9.6). Absorbance was read at 405nm (with a 620-nm reference filter) in a microplate reader (Titertek™, Vienna, Austria). Anx-A1 levels in the study samples were read against the standard curve and expressed as ng/ml.

Electron microscopy

After drug treatment as described above, U937 cells were fixed with a mixture of freshly prepared 3% (weight/volume) paraformaldehyde and 0.5% (volume/volume) glutaraldehyde in PBS (pH 7.2) for 4 h at 4°C, washed briefly in PBS, and transferred to a solution of 2.3M sucrose (in PBS) at 4°C overnight. The cyroprotected cells were slamfrozen (Reichert MM80E; Leica, Milton Keynes, UK), freeze-substituted at -80°C in methanol for 48 h, and embedded at -20°C in LRGold acrylic resin (Agar Scientific, Stansted, UK) in a Reichert freeze-substitution system. Ultrathin sections (50-80 nm) were prepared using a Reichert Ultracut-S ultratome and incubated at room temperature for 2 h with a well-characterised in-house polyclonal sheep anti-Anx-A1 antibody (dilution 1:200) followed by a second antibody labelled with immunogold (British Biocell, Cardiff, UK). The serum and antiserum were diluted in 0.1M phosphate buffer containing 0.1% egg albumin. After immunolabelling, sections were lightly counterstained with uranyl acetate and lead citrate (British Biocell, Cardiff, UK) and examined with a JEOL 1010 transmission electron microscope (JEOL, Peabody, MA).

The number of cytoplasmic and membrane 15 nm gold particles were counted in 30 cells and calculated as particles/µm 2 by dividing the total number of gold particles counted by the cell area. For measurement of cell area micrographs of each cell were taken at a A c c e p t e d M a n u s c r i p t 8 magnification of x 4,000. The cell areas were analysed from scanned micrographs using Axiovision software, version 3.4 (Zeiss, Hemel Hempstead, UK). In all cases the analyst was blind to the sample code.

Measurement of Tx B 2 activity

Supernatants (2ml) from individual samples were concentrated (x10) using Centricon centrifugal filter devices (Millipore, Watford, UK) with Ultracel YM-10 membrane centrifuged at 5000 xg for 1 h. An enzyme immunoassay was established to detect and quantify Tx B 2-released in the supernatant (Biotrak Assay, Amersham, UK). The method was conducted following the manufacturer protocols. A standard curve ranging from 0.5-64 pg/well was prepared using the reagent provided and the optical density was then read at 450nm in a microplate reader, within 30 min.

PKC activity assay

The assay was performed using the PKC Kinase Activity Assay Kit (Stressgen, Cambridge Bioscience, Cambridge, UK) as described in the manufacturer's protocol: each sample was loaded on to a pre-coated plate with a substrate peptide for PKC and the reaction initiated by adding ATP. The phosphospecific substrate antibody (rabbit polyclonal) was added and detected by an HRP-conjugated anti-rabbit IgG and the colour developed with a TMB substrate in proportion to PKC phosphotransferase activity. The reaction was stopped with 100 µl of 1M H 2 SO 4 and the colour was measured on a microplate reader at 450nm. The kinase activity in the cell lysate was calculated as a ratio between the average of absorbance in each sample (subtracted by the absorbance in the blank) and the amount of protein loaded per assay. A recombinant active protein kinase C was used as a positive control.

Phosphatase activity assay

To detect protein phosphatase (phosphoprotein phosphatase; EC 3.1.3.16) activity in the samples, we used the colorimetric Sensolyte pNPP Protein Phosphatase kit (ANASPEC, San Jose, CA, USA). Membrane or recombinant PP2A samples were prepared according to the protocols suggested by the manufacturer. The colorimetric substrate p-nitrophenyl A c c e p t e d M a n u s c r i p t 9 phosphate (pNPP) was used to assess the activity of generic phosphatase activity in our samples, yielding a yellow colour that can be quantified at 405 nm from which the substrate hydrolysis calculated from the molar extinction coefficient supplied. For a kinetic reading, the absorbance was measured every 5 min for 30 min. Samples containing drug alone without enzyme were monitored to check that they had no influence on the colour reaction.

Drugs and materials

The following chemicals (EDTA, glutaraldehyde, β-glycerophosphate, H 2 SO 4, methanol, NaCl, NaF, Na 3 VO 4 , paraformaldehyde, PMA, sucrose, Tris-HCl and 0.1% Triton-X) and drugs (betamethasone, dexamethasone, hydrocortisone, 5-methylprednisolone and prednisolone, PI3 kinase inhibitor (LY 294002), MAP-Kinase inhibitor (PD98059), mifepristone (RU-486), okadaic acid and disodium cromoglycate) were purchased from Sigma-Aldrich, Poole, Dorset, UK. Highly purified (>90%) bovine PP2A 1800.0 U/mg was obtained from Calbiochem (Merck Chemicals, Nottingham, UK). Sodium nedocromil was a generous gift from Sanofi-Aventis.

All drugs were diluted in incubation medium immediately before use to a final concentration that did not exceed 0.04% (w/v).

Data Analysis

For electron microscopy, all values for immunogold particles counted represent the mean ± S.E.M: n=30 cells per group. Preliminary analysis confirmed that the data were normally distributed. Subsequent analysis was undertaken by one-way ANOVA with post hoc analysis performed using Scheffe's test.

Elsewhere, all data are presented as mean ± S.E.M. and were tested for normality prior to analysis. Statistical differences between groups were established using one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test.

In all cases differences were considered significant if P<0.05. GCs increase both the synthesis (genomic) as well as the release (non-genomic) of Anx-A1 from cells. As most previous studies have focussed upon the former we determined, in a series of pilot studies, the time course of GC action on these separate processes in U937 cells (data not shown). Treatment with 1µM dexamethasone strongly stimulated the production of the Ser 27 phosphorylated species within 5 min and this was sustained for up to 30 min. There were no changes in the total mass of Anx-A1 protein in cells prior to the 60 min time point after which steadily increasing amounts accumulated during the following 24 h. We therefore chose 5 min as the optimal time point for most of our subsequent assays as strong non-genomic effects of the GC could be easily observed without detectable genomic actions occurring. We tested dexamethasone, hydrocortisone, prednisolone, methylprednisolone and betamethasone in this system finding that all stimulated Anx-A1 Ser 27 phosphorylation in a qualitatively comparable fashion, however prednisolone, methylprednisolone and betamethasone were not examined further in our assay systems.

We then established the concentration dependency of GC-induced Anx-A1 phosphorylation in U937 cell lysates at 5 min. Figure 1A (upper panel) shows that increasing concentrations of dexamethasone (0.02-5.0 nM) produced a corresponding augmentation (~4 fold, by densitometry) of Anx-A1 Ser 27 phosphorylation. We examined the concentration-response curves of dexamethasone, prednisolone and hydrocortisone in detail and determined the relative EC 50 concentrations to be 0.2, 0.4 and 1nM respectively. In this paper we report data obtained mainly using dexamethasone but we observed that hydrocortisone exhibited qualitatively identical behaviour in terms of its interaction with the cromolyns. To determine if PKC was the sole kinase responsible for Anx-A1 phosphorylation in this system, we examined the effects of a panel of inhibitors. PKCα/β (80-82 kDa). When we probed our blots with a panel of isoform specific antiphospho PKC antisera, no PKC δ (Ser 643 ) or PKC θ Thr 538 was detectable. Some PKC δ

Thr 505 was detected but with a lower MW (78kDa) than the GC stimulated species seen in our blots. However, the specific anti phospho-PKCα/β Thr 638/641 antibody showed good reactivity with a band that increased with GC treatment over a period of 5-30 min treatment (Figure 1D). We therefore concluded that the main kinase responsible for Anx-A1 phosphorylation in this system was almost certainly PKCα/β. We next assessed the effect of sodium cromoglycate and nedocromil on Anx-A1 and PKCα/β phosphorylation in our U937 cell system. Figure 2A shows that the administration of nedocromil (0.02-5.0 nM) alone had a negligible or only a weak action on the phosphorylation of Anx-A1 (<1.5 fold) or PKCα/β (1-2 fold) when compared with dexamethasone (see Figure 1A &B). Cromoglycate alone (0.02-5.0 nM) was similarly only very weakly active in this system (data not shown). Surprisingly however, in the presence of a fixed concentration (2 nM) of a GC, these drugs exhibited a further strong concentration-dependent increase in both Anx-A1 and PKCα/β phosphorylation (> 3-4 fold; Figure 2B) that exceeded the maximum stimulation caused by the GCs alone. In this paper we mainly report data obtained using nedocromil but we observed that cromoglycate exhibited qualitatively the same effect in all our assay systems.

Translocation of phosphorylated species.

PKCα/β is enriched at the plasma membrane when activated and Anx-A1 is likewise recuited to the membrane upon phosphorylation at Ser 27 . Figure 2C shows that in the presence of a fixed concentration of dexamethasone, escalating concentrations of nedocromil cause the translocation of Ser 27 phospho Anx-A1 from the (13,000 xg) supernatant to the particulate fraction of the U937 cells. We refined this analysis by using differential centrifugation to prepare some 100,000 xg membranes from cells treated with drug combinations. Figure 2D shows that, relative to either drug given alone, the combination of nedocromil and dexamethasone increased (2 fold) the amount of activated phospho PKCα/β in the membrane fraction as determined by western blotting and also increased (P<0.05) the catalytic activity of the enzyme in this compartment.

Cromoglycate-like drugs potentiate the action GCs on eicosanoid release.

The ability of GCs to arrest eicosanoid synthesis depends on the release of Anx-A1 from target cells in many systems. As the cromoglycate-like drugs are also reported to inhibit eicosanoid formation, we next determined whether these drugs alone, or in combination Figure 3A shows that whilst nedocromil (0.5 nM) alone had only a small (<10%; NS) effect on TxB 2 release it greatly potentiated (P<0.001) the inhibitory effect of 2nM dexamethasone (P<0.01) alone. This correlated with a potentiation of Anx-A1 release into the medium as assessed by the ELISA assay (P<0.001). Figure 3C shows that this is also true of Anx-A1 release. In the presence of 2 nM dexamethasone, nedocromil (0.2-10.0 nM) caused a concentration dependent inhibition of TxB 2 generation that was correlated exactly with the increasing amounts (7-8 fold) of Anx-A1 released onto the outside of the cell (see blot insert).

To confirm that the drug-induced inhibition of TxB 2 was caused entirely by increased Anx-A1 in this system we tested the effect of a neutralising anti-Anx-A1 monoclonal (mab 1A) and isotype-matched irrelevant monoclonal antibody. Figure 3D shows that addition of the mabs alone had no effect but that the neutralising, but not the irrelevant monoclonal antibodies completely reversed (P<0.001) the inhibitory effect of the dexamethasone-nedocromil combination on TxB 2 release.

GCs and cromoglycate-like drugs promote intracellular trafficking and membrane localisation of Anx-A1 in U937 cells.

A c c e p t e d M a n u s c r i p t 14

To investigate the effect of the drugs on the intracellular movement of Anx-A1 at a fine level of detail, we utilised U937 cells that had been transfected with an Anx-A1-GFP construct and analysed the effects of drug treatment by confocal microscopy.

The western blots in Figure 4A shows that only GFP protein (27kDa) was seen in U937 cells transfected with the mock plasmids E1, E2 and E3 whereas in U937 cells transfected with Anx-A1-GFP constructs G1 and G2 a 64 kDa band corresponding to the GFP-Anx-A1 conjugate was observed. Clones G1, G2, E1 and E3 were selected for this study (upper panel probed with anti-GFP and lower with anti-Anx-A1 antibodies).

Figure 4B shows an experiment where we used Anx-A1 -GFP transfected U937 cells in which the plasma membrane has been stained with the red fluorescent Alexa Fluor ® 594 wheat germ agglutinin (WGA) labelling reagent that specifically stains membrane phospholipids. The Anx-A1-GFP in vehicle treated cells is localised in the cytoplasm (row a). Not much change is seen with nedocromil (5nM) treatment alone (row b), however, treatment with dexamethasone (2 nM) alone induces some degree of Anx-A1 redistribution, as shown by a partial co-localisation of Anx-A1 with the membrane (row c, merged image). When dexamethasone is combined with 5 nM nedocromil, however a striking degree of co-localisation of Anx-A1with the membrane is observed (row d) and evidence of membrane 'bubbling' (possible related to secretory activity) may be inferred from the presence of GFP-Anx-A1 outside the cell membrane.

Although we observed that the non-genomic actions of these drugs were evident at 5 min, we were curious about events that might take place over a shorter time-span. To investigate this we used a slightly different experimental design in which time-lapse video-microscopy was used. GFP tagged Anx-A1 transfected U937 cells were filmed for 3 min under various experimental conditions, and the frames examined for changes that might reflect the release of Anx-A1 in response to drugs. effect. Treatment with dexamethasone (2 nM) alone produced some evidence of secretory activity at 60 s congruent with our other data. However, when a combination of dexamethasone and 5 nM nedocromil was administered was administered, the GFPtagged protein was released from focal sites on the membrane within 30 s and release from multiple sites was very pronounced within 3 min.

Electron microscopy studies.

The experiments with the transfected U937 cells could be compromised by the fact that Anx-A1 was conjugated to GFP and therefore that its intracellular trafficking might be atypical. To address this we used conventional electron microscopic and immunochemistry techniques to follow the fate of Anx-A1 in response to various drug treatments. promotes association of the protein with the membrane (P<0.01). In the presence of both GCs and nedocromil however (panel 6D), there was a significant (P<0.01) increase of membrane-associated Anx-A1 and a corresponding decrease in cytosolic and vesicular associated material.

Cromoglycate-like drugs inhibit membrane phosphatase activity.

All the data generated so far pointed to a mechanism whereby Anx-A1, translocated to the membrane after phosphorylation on Ser 27 under the influence of activated PKCα/β, was released from the cell to exert an extracellular inhibitory effect on eicosanoid synthesis and that this process was somehow potentiated by the cromoglycate-like drugs.

The catalytic activity of PKCα/β is limited by membrane phosphatases, in particular PP2A, and we hypothesised that the cromoglycate like drugs inhibited the activity of the generation and release of Ser 27 phospho Anx-A1. Since okadaic acid is a wellcharacterised inhibitor of PP2A (and PP1 in higher concentrations), we investigated its action in the U937 system. Figure 7A shows that this inhibitor (1 µM) does indeed (P<0.001) share the ability of cromoglycate to potentiate Anx-A1 release in this system.

We next measured the net phosphatase activity in U937 100,000 xg membranes. In the experiment depicted in Figure 7B, we prepared 100,000 xg membranes from U937 dexamethasone-treated cells (as described above) and then pre-incubated them for 5 min with either 5 nM nedocromil or 1 µM okadaic acid before assessing their phosphatase activity at 10 min (the time point that gave maximal readings in pilot studies; data not shown). Strikingly, phosphatase activity was almost completely inhibited in the presence of either nedocromil or okadaic acid.

Finally, we tested the effect of cromoglycate and nedocromil on a highly purified PP2A preparation from bovine kidney. Fig 7C shows that both nedocromil and cromoglycate inhibited the catalytic activity of this enzyme in a concentration-dependent manner after pre-incubation with the phosphatase for 5 min with IC 50 s of approximately 0.65 nM and ~1.7 nM respectively. As expected, okadaic acid was also strongly inhibitory (IC 50 <<1 µM).

Discussion

The Anx-A1 system in undifferentiated myelomonocytic U937 cells does not respond to glucocorticoids and it is necessary to pre-treat the cells with low concentrations of PMA to render them responsive [START_REF] Solito | Dexamethasone induces the expression of the mRNA of lipocortin 1 and 2 and the release of 1 and 5 in differentiated, but not undifferentiated U-937 cells[END_REF]. Using this model system, we have studied the rapid nongenomic effects of GCs on Anx-A1 intracellular trafficking and export and investigated the eicosanoid inhibitory mechanism of action of the cromoglycate-like drugs.

Concerning the former, we have confirmed and extended earlier reports (e.g. [START_REF] Croxtall | Glucocorticoids act within minutes to inhibit recruitment of signalling factors to activated EGF receptors through a receptor-dependent, transcription-independent mechanism[END_REF][START_REF] Solito | Dexamethasone induces rapid serine-phosphorylation and membrane translocation of annexin 1 in a human folliculostellate cell line via a novel nongenomic mechanism involving the glucocorticoid receptor, protein kinase C, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase[END_REF]) that GCs exhibit two distinct actions on the synthesis and intracellular distribution of Anx-A1 with a rapid non-genomic, receptor-dependent, action seen within 5 min of treatment GC effects in U937 cells were evident within minutes demonstrating how rapidly these agents can act through the Anx-A1 system. Whilst the 'classical' genomic actions of GCs have been considered to take hours to develop, there is increasing evidence in a number of systems (e.g. [START_REF] Long | Rapid nongenomic inhibitory effects of glucocorticoids on phagocytosis and superoxide anion production by macrophages[END_REF][START_REF] Lovell | Acute modulation of adrenal chromaffin cell BK channel gating and cell excitability by glucocorticoids[END_REF][START_REF] Buttgereit | The molecular basis for the effectiveness, toxicity, and resistance to glucocorticoids: focus on the treatment of rheumatoid arthritis[END_REF][START_REF] Urbach | Rapid anti-secretory effects of glucocorticoids in human airway epithelium[END_REF]) that these rapid non-genomic effects, including many which require GR ligation (e.g. [START_REF] Lowenberg | Rapid immunosuppressive effects of glucocorticoids mediated through Lck and Fyn[END_REF][START_REF] Lowenberg | Glucocorticoids cause rapid dissociation of a T-cell-receptor-associated protein complex containing LCK and FYN[END_REF]) are common, and may be crucial to the homeostatic or therapeutic action of these drugs . Interestingly, we observed some 'background' Anx-A1 phosphorylation in our cultured U937 cells in the absence of GC stimulation. At least part of this effect may have caused by endogenous GCs in the culture media since it was reduced in the presence of RU 486 -and may have been reduced even further if charcoal-stripped serum had been used. However, stimuli other than GCs can also cause Anx-A1 phosphorylation [START_REF] Solito | Posttranslational modification plays an essential role in the translocation of annexin-A1 from the cytoplasm to the cell surface[END_REF], which often appears in response to cellular 'stress'. In vivo, where there is a constant GC 'tone', one would anticipate some action of the cromolyns without further stimulation.

Our novel finding that the cromoglycate-like drugs greatly potentiated the effect of GCs on Anx-A1 release and eicosanoid inhibition throws interesting light upon the way in which this anti-inflammatory protein is externalised as well as providing the basis for a novel mechanistic explanation for the eicosanoid inhibitory activity of these anti-allergic drugs. According to this new paradigm for cromolyn action, we propose that these drugs act by potentiating the release of the anti-inflammatory protein Anx-A1 from cells. They accomplish this by promoting phosphorylation of Anx-A1 but only when this has been triggered by GCs or some other stimulus such as cell activation. This is amply demonstrated by the imaging techniques and the EM studies which are in excellent agreement with the western blotting and ELISA data concerning the fate of Anx-A1 following treatment with these drugs.

A c c e p t e d M a n u s c r i p t

18 Anx-A1 is present in several subcellular compartments of cells including the nucleus, the cytoplasm, the plasma and other membranes as well as attached to the cytoskeleton [START_REF] Peers | Glucocorticoid-and nonglucocorticoid induction of lipocortins (annexins) 1 and 2 in rat peritoneal leucocytes in vivo[END_REF][START_REF] Seemann | Annexin I targets S100C to early endosomes[END_REF][START_REF] Bianchi | Subcellular localization of S100A11 (S100C) in LLC-PK1 renal cells: Calcium-and protein kinase cdependent association of S100A11 with S100B and vimentin intermediate filaments[END_REF]. It has been repeatedly observed that 'activation' of macrophages and other cells can lead to an accumulation of Anx-A1 at the plasma membrane where it can (for example) block EGF [START_REF] Croxtall | Glucocorticoids act within minutes to inhibit recruitment of signalling factors to activated EGF receptors through a receptor-dependent, transcription-independent mechanism[END_REF] and LPS induced ERK activation [START_REF] Alldridge | The annexin protein lipocortin 1 regulates the MAPK/ERK pathway[END_REF] and undoubtedly fulfil other intracellular roles (e.g. [START_REF] Seemann | Annexin I targets S100C to early endosomes[END_REF][START_REF] Futter | Annexin I is phosphorylated in the multivesicular body during the processing of the epidermal growth factor receptor[END_REF][START_REF] White | EGF stimulates annexin 1-dependent inward vesiculation in a multivesicular endosome subpopulation[END_REF]).

The actual mechanism of Anx-A1 secretion itself has not been addressed in this paper and remains unclear. The protein does not contain a canonical signal sequence and it is generally accepted that secretion occurs through a non-classical pathway. There is some evidence that the ABC transporter system could be involved [START_REF] Morris | Steroid effects on secretion from subsets of lactotrophs: role of folliculostellate cells and annexin 1[END_REF][START_REF] Chapman | Evidence for a role of the adenosine 5'-triphosphate-binding cassette transporter A1 in the externalization of annexin I from pituitary folliculo-stellate cells[END_REF][START_REF] Wein | Mediation of annexin 1 secretion by a probenecid-sensitive ABC-transporter in rat inflamed mucosa[END_REF] but other potential mechanisms could include a 'diffusion controlled' process facilitated by binding of the exported species to extracellular proteoglycans as suggested for fibroblast growth factor 2 [START_REF] Nickel | Unconventional secretion: an extracellular trap for export of fibroblast growth factor 2[END_REF]. A recent paper suggests that ceramide platforms in cells may preferentially bind Anx-A1 and could be important for externalisation [START_REF] Babiychuk | Fluorescent annexin A1 reveals dynamics of ceramide platforms in living cells[END_REF].

Whichever mechanism operates, phosphorylation of the protein at Ser 27 is clearly a crucial prelude to membrane recruitment and subsequent export and so it is probable that any stimulus that activates PKCα/β will lead to the marginalisation and release of Anx-A1 from cells in which this signalling system is supported. However, it is also evident that not all marginalised Anx-A1 is immediately exported suggesting a further control over this process acting at the level of the membrane itself.

Several authors have reported that GC receptor ligation activates PKC thus promoting its translocation to the plasma membrane (see [START_REF] Qiu | Nongenomic mechanism of glucocorticoid inhibition of bradykinin-induced calcium influx in PC12 cells: possible involvement of protein kinase C[END_REF][START_REF] Ishizuka | Effect of dexamethasone and prednisolone on insulin-induced activation of protein kinase C in rat adipocytes and soleus muscles[END_REF][62][START_REF] Plotkin | Glucocorticoids induce osteocyte apoptosis by blocking focal adhesion kinase-mediated survival. Evidence for inside-out signaling leading to anoikis[END_REF]). Previous work from our group suggests that in some systems, phosphatidylinositol 3-kinase may play a key role [START_REF] Solito | Dexamethasone induces rapid serine-phosphorylation and membrane translocation of annexin 1 in a human folliculostellate cell line via a novel nongenomic mechanism involving the glucocorticoid receptor, protein kinase C, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase[END_REF] in activating PKC. PKC activity is ultimately terminated by dephosphorylation at the membrane probably by the Ser/Thr PP2A phosphatase [START_REF] Hansra | 12-O-Tetradecanoylphorbol-13-acetate-induced dephosphorylation of protein kinase Calpha correlates with the presence of a membrane-associated protein phosphatase 2A heterotrimer[END_REF] and ubiquitination. In many cell types, including the macrophage, PKCα (and some other isoforms) activate (and may associate with) PP2A (see review [START_REF] Sim | Targeting of PKA, PKC and protein phosphatases to cellular microdomains[END_REF]). PKCα is reciprocally controlled by the activity PP2A itself is a heterotrimeric enzyme comprising one each of two variant catalytic and structural sub-units together with one (of a family of about twenty) modulatory/targeting sub-units. The nature of the latter determines the targeting and substrate specificity of the assembled enzyme complex. C-terminal carboxymethylation of the catalytic subunit at Leu 309 activates PP2A probably by facilitating the formation of trimeric complexes [START_REF] Lee | Identification of proteins interacting with the catalytic subunit of PP2A by proteomics[END_REF][START_REF] Yoo | Subunit composition and developmental regulation of hepatic protein phosphatase 2A (PP2A)[END_REF][START_REF] Ortega-Gutierrez | Targeted disruption of the PME-1 gene causes loss of demethylated PP2A and perinatal lethality in mice[END_REF]. At the membrane, the enzyme may be phosphorylated on Tyr 307 by receptor or other tyrosine kinase action [START_REF] Chen | Polymerase chain reactions using Saccharomyces, Drosophila and human DNA predict a large family of protein serine/threonine phosphatases[END_REF] that may inhibit the phosphatase. However, the significance of these post-translational modifications to enzyme activity in vitro and in vivo is not yet entirely clear [START_REF] Sim | Targeting of PKA, PKC and protein phosphatases to cellular microdomains[END_REF].

The degree of Anx-A1 phosphorylation, and hence the amount exported, will therefore depend upon the net catalytic activity of the PKCα-PP2A complex which, in turn will result from the reciprocal interactions between the two enzymes. The cumulative evidence from biochemical, immunocytochemical and imaging studies presented here all support the notion that whilst the cromoglycate-like drugs alone have only weak activity they are able greatly to accelerate the phosphorylation and release of Anx-A1 when this is primed (in this case) by GCs, which promote PKC activation and some degree of Anx-A1 phosphorylation and membrane localisation. Our analysis points to a direct inhibitory action of these drugs on PP2A enzyme activity as the most likely explanation for this synergism although we cannot entirely rule out the participation of PP1, another phosphatase, or the possibility that which ever phosphatase is involved, it acts directly to dephosphorylate Anx-A1 itself rather than PKC. Supporting the notion that PP2A is the actual target however is our observation that the PP2A inhibitor okadaic acid [START_REF] Takai | Inhibition of specific binding of okadaic acid to protein phosphatase 2A by microcystin-LR, calyculin-A and tautomycin: method of analysis of interactions of tight-binding ligands with target protein[END_REF], which has been shown in many systems to potentiate the effects of PKC mediated events, mimics the effect of the cromoglycate-like drugs and promoted GC-stimulated Anx-A1 export in our system.

A c c e p t e d M a n u s c r i p t 20 Experiments using anti-Anx-A1 neutralising antibodies [START_REF] Perretti | Modulation of IL-1-induced neutrophil migration by dexamethasone and lipocortin 1[END_REF][START_REF] Wu | Lipocortin 1 mediates the inhibition by dexamethasone of the induction by endotoxin of nitric oxide synthase in the rat[END_REF][START_REF] Mancuso | Leukocyte transmigration, but not rolling or adhesion, is selectively inhibited by dexamethasone in the hamster post-capillary venule. Involvement of endogenous lipocortin 1[END_REF][START_REF] Perretti | Acute inflammatory response in the mouse: exacerbation by immunoneutralization of lipocortin 1[END_REF][START_REF] Duncan | The local antiinflammatory action of dexamethasone in the rat carrageenin oedema model is reversed by an antiserum to lipocortin 1[END_REF][START_REF] Croxtall | Lipocortin 1 mediates dexamethasone-induced growth arrest of the A549 lung adenocarcinoma cell line[END_REF], anti-sense RNA [START_REF] Taylor | An antisense oligodeoxynucleotide to lipocortin 1 reverses the inhibitory actions of dexamethasone on the release of adrenocorticotropin from rat pituitary tissue in vitro[END_REF][START_REF] Croxtall | Antisense oligonucleotides to human lipocortin-1 inhibit glucocorticoid-induced inhibition of A549 cell growth and eicosanoid release[END_REF] or transgenic animals [START_REF] Yang | Modulation of inflammation and response to dexamethasone by Annexin 1 in antigen-induced arthritis[END_REF][START_REF] Roviezzo | The annexin-1 knockout mouse: what it tells us about the inflammatory response[END_REF][START_REF] Hannon | Aberrant inflammation and resistance to glucocorticoids in annexin 1-/-mouse[END_REF][START_REF] Chatterjee | Annexin 1-deficient neutrophils exhibit enhanced transmigration in vivo and increased responsiveness in vitro[END_REF][START_REF] Damazo | Critical protective role for annexin 1 gene expression in the endotoxemic murine microcirculation[END_REF] which over-or under-express the Anx-A1 gene, have conclusively demonstrated that this protein is a key mediator of GC action in the innate as well as the adaptive [START_REF] D'acquisto | Annexin 1 mediates the immuno-modulatory effects of glucocorticoids on T cell activation and differentiation[END_REF][START_REF] D'acquisto | Impaired T cell activation and increased Th2 lineage commitment in Annexin-1deficient T cells[END_REF] immune system as well as in other aspects of physiology [START_REF] Taylor | Evidence from immunoneutralization and antisense studies that the inhibitory actions of glucocorticoids on growth hormone release in vitro require annexin 1 (lipocortin 1)[END_REF][START_REF] John | Annexin 1 and the regulation of endocrine function[END_REF] and cell biology [START_REF] Croxtall | Attenuation of glucocorticoid functions in an Anx-A1-/-cell line[END_REF][START_REF] Yona | Stimulus-specific defect in the phagocytic pathways of annexin 1 null macrophages[END_REF][START_REF] Yona | Macrophage biology in the Anx-A1-/-mouse[END_REF].

Although there are inflammatory models in which Anx-A1 is not efficacious [START_REF] Cirino | Human recombinant lipocortin 1 has acute local anti-inflammatory properties in the rat paw edema test[END_REF], our observation that cromoglycate drugs promote Anx-A1 externalisation may explain several of their disparate actions including their ability to inhibit leukocyte activation [START_REF] Kay | Disodium cromoglycate inhibits activation of human inflammatory cells in vitro[END_REF], 'priming' or migration [START_REF] Bruijnzeel | Effects of nedocromil sodium on in vitro induced migration, activation, and mediator release from human granulocytes[END_REF][START_REF] Szkudlinska | Chemotactic activity of neutrophils from atopic and non-atopic subjects--effect of sodium cromoglycate (DSCG)][END_REF], mediator action [START_REF] Dahlen | Dual inhibitory action of nedocromil sodium on antigen-induced inflammation[END_REF], macrophage activation [START_REF] Joseph | Basic research on nedocromil sodium][END_REF],

tachykinin action [START_REF] Yamawaki | Inhaled cromoglycate reduces airway neurogenic inflammation via tachykinin antagonism[END_REF], eicosanoid [START_REF] Mattoli | Nedocromil sodium prevents the release of 15-hydroxyeicosatetraenoic acid from human bronchial epithelial cells exposed to toluene diisocyanate in vitro[END_REF][START_REF] Radeau | Effect of nedocromil sodium on sulfidopeptide leukotrienes-stimulated human alveolar macrophages in asthma[END_REF] and cytokine release [START_REF] Rusznak | Ozone-induced mediator release from human bronchial epithelial cells in vitro and the influence of nedocromil sodium[END_REF] as well as adhesion molecule expression [START_REF] Sacco | Modulation of HLA-DR antigen and ICAM-1 molecule expression on airway epithelial cells by sodium nedocromil[END_REF]. We can only conjecture as to whether our putative mechanism of action accounts for the ability of the cromoglycate-like drugs to inhibit the release of mast cell histamine but it is certainly true that Anx-A1 has potent inhibitory effects on mast cells [START_REF] Bandeira-Melo | A novel effect for annexin 1-derived peptide ac2-26: reduction of allergic inflammation in the rat[END_REF] and that these cells are a prime site for synthesis of this protein [START_REF] Oliani | An immunocytochemical and in situ hybridization analysis of annexin 1 expression in rat mast cells: modulation by inflammation and dexamethasone[END_REF].

Whilst our hypothesis is novel, there have been several previous attempts to link cromolyn action to activation of signalling pathways and modification of potential downstream molecular targets. Treatment of mast cells with cromoglycate results in the phosphorylation of (at least) four intracellular protein substrates including the erythrocyte band 4.1 group protein moesin [START_REF] Theoharides | Cloning and cellular localization of the rat mast cell 78-kDa protein phosphorylated in response to the mast cell "stabilizer" cromolyn[END_REF][START_REF] Theoharides | Antiallergic drug cromolyn may inhibit histamine secretion by regulating phosphorylation of a mast cell protein[END_REF] and there have been scattered reports of an interaction between cromoglycate and PKC stretching back over some years (e.g. [START_REF] Bansal | Increased levels of protein kinase C in lymphocytes in asthma: possible mechanism of regulation[END_REF][START_REF] Lucas | Cromolyn inhibition of protein kinase C activity[END_REF][START_REF] Sagi-Eisenberg | Possible role for a calcium-activated phospholipid-dependent protein kinase in mode of action of DSCG[END_REF]). However, most researchers seem to have considered that these drugs inhibit, rather than stimulate, this enzyme, although differing experimental protocols and crucially, timing, obscure clear interpretation of this issue. Congruent with our findings there is a report that some biological effects of methylprednisolone can be augmented using okadaic acid, an inhibitor of PP2A [START_REF] Uzunoglu | Augmentation of methylprednisolone-induced differentiation of myeloid leukemia cells by serine/threonine protein phosphatase inhibitors[END_REF].

There have also been previous reports [START_REF] Oyama | Two distinct anti-allergic drugs, amlexanox and cromolyn, bind to the same kinds of calcium binding proteins, except calmodulin, in bovine lung extract[END_REF][START_REF] Shishibori | Three distinct anti-allergic drugs, amlexanox, cromolyn and tranilast, bind to S100A12 and S100A13 of the S100 protein family[END_REF] of a link between the cromoglycate drugs and the annexin system in that these drugs have an affinity for some S100 proteins One practical implication of this work arises from the observation that GCs and cromoglycate-like drugs exhibit a strong synergistic action that might be applicable to anti-inflammatory therapy. The notion that these drugs can inhibit PP2A may also suggest other uses for these agents in other conditions where PKC activation plays a significant role.

  Alexa Fluor ® 594 wheat germ agglutin (WGA: Image-IT™ live plasma labelling kit, Molecular Probes, Leiden, The Netherlands) to visualise the plasma membrane of live Anx-A1 -GFP transfected cells.

1

 1 GCs alone stimulate Anx-A1 phosphorylation through a PKCα α α α/β β β β dependentmechanism.

A c c e p t e d M a n u s c r i p t 11 Previous

 11 studies have implicated the enzyme PKC in this GC-induced phosphorylation of Anx-A1. We tested this in the present system using a pan-specific anti-phospho PKC antibody, finding that the abundance of phosphorylated PKC was increased in a similar concentration-dependent fashion (>3 fold; Fig 1A lower panel) by these drugs to that of Ser27 phospho Anx-A1.To confirm that the GC effect was dependent upon GR occupation we tested the effect of the GR antagonist RU486 on the ability of dexamethasone to stimulate phosphorylation of Anx-A1. Fig1Bshows that 2 nM dexamethasone was unable to stimulate Anx-A1 Ser27 production in the presence of 1 µM of this drug (P<0.01). It is noteworthy that the background Anx-A1 phosphorylation sometimes observed in untreated cells was itself reduced by RU 486 (P<0.05) perhaps implying that residual GCs in the medium may drive a low level phosphorylation of the protein.

  Figure 1C shows the effect of the PI3 kinase inhibitor LY294002 (10nM), a MAP kinase inhibitor PD98059 (5µM) and an inhibitor of PKC, PKC19-31 (5µM) on the phosphorylation response. Only PKC 19-31 was able significantly (P<0.001) to reverse the dexamethasone-induced increase in Anx-A1 phosphorylation. The MW of the phosphorylated PKC enzyme detected by western blotting using the pan-specific PKC antibody was in the range 76-82 kDa suggesting the relevant isoform was either PKC δ (78 kDa), PKC θ (76 kDa) or

A c c e p t e d M a n u s c r i p t 12 3. 2

 122 Cromoglycate-like drugs synergise with GCs to stimulate Anx-A1 phosphorylation and release.

A c c e p t e d M a n u s c r i p t 13 with

 13 GCs, enhanced Anx-A1 release thereby producing a concomitant decrease in the generation of TxB 2 , a prominent eicosanoid spontaneously released from U937 cells.

Figures

  Figures 3B & C show the concentration dependency of this effect. In Fig 3B, the maximum inhibition achieved by dexamethasone at 5 min (<20%: 0.2-20 nM) alone was evident at a concentration of 1 nM. When nedocromil (0.2-20 nM) was added to 2 nM dexamethasone however, a concentration-dependent increase in the inhibitory activity was observed which maximally inhibited TxB 2 release at concentrations of 20 nM nedocromil.

Figure 5

 5 Figure 5 shows a panel of individual 6 frames taken at 30 s intervals throughout these experiments during which drug (or vehicle) treatments were administered. Treatment of

Figure

  Figure 6A-D shows the results of this investigation together with a graphical analysis of the quantitation of immunogold particles in Figure 6E. In untreated U937 cells immunogold labelled Anx-A1 was found predominantly in the peri-nuclear region and the cytosol, often in combination with vesicular structures (panel 6A) with little discernable at the membrane. Nedocromil (5 nM) had little effect on this pattern of distribution (panel 6B) but dexamethasone (2 nM; panel 6C) or hydrocortisone (2 nM)

A c c e p t e d M a n u s c r i p t 16 these

 16 enzymes thus prolonging the catalytic action of PKCα/β and indirectly potentiating

A c c e p t e d M a n u s c r i p t 17 followed

 17 by a 'slow' genomic action of GCs with a much longer latency, as judged by the increasing intracellular mass of Anx-A1 seen at time points longer than 1h. We have also confirmed a mandatory role for Ser 27 phosphorylation of Anx-A1 prior to membrane translocation and export and established that the kinase most likely to drive this process is the PKCα/β isoform of PKC.

A c c e p t e d M a n u s c r i p t 19 of

 19 the phosphatase, which limits the catalytic action of the kinase by dephosphorylation[START_REF] Boudreau | Protein phosphatase 2A and protein kinase Calpha are physically associated and are involved in Pseudomonas aeruginosainduced interleukin 6 production by mast cells[END_REF][START_REF] Ricciarelli | Regulation of recombinant PKC alpha activity by protein phosphatase 1 and protein phosphatase 2A[END_REF][START_REF] Lee | Ahnak protein activates protein kinase C (PKC) through dissociation of the PKC-protein phosphatase 2A complex[END_REF][START_REF] Zhang | Protein kinase C delta negatively regulates tyrosine hydroxylase activity and dopamine synthesis by enhancing protein phosphatase-2A activity in dopaminergic neurons[END_REF][START_REF] Faulkner | Protein phosphatase 2A enhances activation of human immunodeficiency virus type 1 by phorbol myristate acetate[END_REF].

A c c e p t e d M a n u s c r i p t 21 that

 21 are intracellular binding partners for members of the annexin family of proteins. It is not clear if, or how these data relate to the current findings.

u s c r i p t 33 Figure 1 .

 331 Figure 1. Dexamethasone increases the phosphorylation of Anx-A1 and PKC in U937 cells within 5 min in a concentration dependent fashion.

A c c e p t e d M a n u s c r i p t 35 Figure 2 .

 352 Figure 2. Nedocromil potentiates the effect of dexamethasone on Anx-A1 and PKCα/β phosphorylation and protein translocation to the plasma membrane fraction.

A c c e p t e d M a n u s c r i p t Page 37 of 53 A c c e p t e d M a n u s c r i p t 37 Figure 3 .

 53373 Figure 3. Nedocromil potentiates dexamethasone inhibition of TxB 2 release from U937 cells.

A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t 43 *

 43 * P <0.01 membrane Anx-A1 increased relative to control values. § P <0.05, § § P <0.02 Anx-A1 changed relative to control values.

A c c e p t e d M a n u s c r i p t 44 Figure 7 .

 447 Figure 7. The cromoglycate drugs inhibit PP2A phosphatase activity.

Figure A c c e p t e d M a n u s c r i p

  Figure

Figure

  Figure

Figure A c c e p t e d M a n u s c r i p

  Figure

Figure

  Figure

Acknowledgements

We acknowledge the support of the Wellcome Trust, the Research Advisory Board of St Bartholomew's and the Royal London School of Medicine and Dentistry. The time-lapse microscope was funded by a grant from the Wellcome Trust (N072634/Z/08/Z).

Nedocromil was a generous gift from Sanofi-Aventis. We thank Dr Phuong Vo for helpful advice and input into this project.

Data are expressed as mean ± S.E.M. Statistical differences between groups were established using one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test.

Conflict of interest.

The authors have no conflicting financial interests. Upper figure. The western blot, probed with an anti-GFP antibody, shows that only GFP protein (27kDa) was seen in U937 cells transfected with the mock plasmids E-1, E-2 and E-3 whereas in U937 cells transfected with Anx-A1-GFP constructs GFP-1 and GFP-2 a 64kDa band corresponding to the GFP-Anx-A1 conjugate was observed. Clones GFP-1, GFP-2, EGFP-1 and EGFP-3 were used for this study.

Lower figure. This blot shows the complementary experiment to that in panel A, where the samples were probed with the anti-Anx-A1 antibody.

Panel B.

In these experiments, the plasma membrane of U937 cells transfected with the Anx-A1-GFP construct, was stained red with Alexa Fluor © 594 WGA dye and the cells subjected to various drug treatments protocols. The four columns represent the information from the two colour channels, the merged channel and a magnified (x 90) section of the membrane.

Rows.

Row a. Shows the situation when the transfected U937 cells are treated with vehicle alone. Anx-A1 is mainly contained within the cytoplasmic compartment and the plasma membrane stains red.

Row b.

There appears to be little evidence of co-localisation after treatment with 5nM nedocromil alone.

Row c. Treatment with dexamethasone alone 2nM clearly promotes co-localisation of

Anx-A1 and membrane phospholipids. Panel A. Vehicle treatment. U937 cells treated with vehicle alone for 5 min were fixed and processed as described in materials and methods. In this image (note a slightly lower power than the others to provide an overview of distribution) immunogold labelled Anx-A1 (arrowed) was mainly to be found in the perinuclear region or in the cytoplasm apparently associated with vesicles (arrowed). Little was seen on the plasma membrane.


Katrin Schuh

Dr Andreas Pahl
email: andreas.pahl@pharmakologie.med.uni-erlangen.de

From

Inhibition of the MAP kinase ERK protects from lipopolysaccharideinduced lung injury By

Keywords: Lung, Inflammation, Lipopolysaccharide, ERK, MEK-inhibitor, COPD

The pathogenesis of chronic obstructive pulmonary disease (COPD) is characterized by pulmonary inflammation associated with lung neutrophilia and elevated levels of proinflammatory mediators in the bronchoalveolar lavage fluid or sputum of patients. Recent findings revealed that mitogen-activated protein kinase (MAPK) signaling cascade is involved in the inflammatory response of lung injury. In the present study we could elucidate the role of extracellular signal-related MAPK in the murine model of LPS-induced acute lung injury by using U0126, a specific inhibitor of MEK1/2, upstream kinases of ERK. Phosphorylation of ERK was inhibited by U0126 in vivo as well as in vitro. In freshly isolated human peripheral blood mononuclear cells U0126 dose-dependently blocked the release of IL-2 and TNF-α. For in vivo studies mice were exposed to aerosolized LPS to induce an acute lung injury mimicking some aspects of COPD. This led a recruitment of neutrophils to the lung and to the release of pro-inflammatory cytokines into bronchoalveolar lavage. Pretreatment of mice with U0126 significantly reduced lung neutrophilia and diminished levels of TNF-α and chemotactic MIP-2 and KC in bronchoalveolar fluid. U0126 also decreased albumin levels in BAL fluid, a marker of vascular leakage. Histological examination of lung tissues revealed that ERK MAPK inhibition using U0126 efficiently attenuated LPS-induced pulmonary inflammatory responses. These data suggest that ERK signaling plays an important role in acute lung injury and pharmacologic inhibition of ERK provides a promising new therapeutic strategy for lung inflammatory diseases and in particular COPD.

A c c e p t e d M a n u s c r i p t

Introduction

Chronic obstructive pulmonary disease (COPD) is characterized by airflow obstruction that is not fully reversible. It is a major global health problem with growing incidence, which places an increasing burden on health services in industrialized and developing countries [START_REF] Mannino | Global burden of COPD: risk factors, prevalence, and future trends[END_REF]. The term COPD encompasses emphysema, chronic obstructive bronchitis and a chronic inflammation in the smaller airways and lung parenchyma [START_REF] Barnes | Chronic obstructive pulmonary disease[END_REF][START_REF] Puljic | Lipopolysaccharide-induced lung inflammation is inhibited by neutralization of GM-CSF[END_REF]. The local inflammation observed in COPD patients is predominated by neutrophilic granulocytes [START_REF] Barnes | Immunology of asthma and chronic obstructive pulmonary disease[END_REF]. Furthermore increased numbers of macrophages and T-cells, predominantly CD8+ T-cells, are found in the alveolar walls of COPD patients [START_REF] O'donnell | Inflammatory cells in the airways in COPD[END_REF].

Cigarette smoke is the major risk factor for the development of COPD, but also exposure to air pollution and genetic factors play an important role [START_REF] Barnes | Chronic obstructive pulmonary disease[END_REF]. Tobacco and cigarette smoke contain high levels of lipopolysaccharide (LPS), a cell wall component of gramnegative bacteria, which is a potent inflammatory stimulus [START_REF] Hasday | Bacterial endotoxin is an active component of cigarette smoke[END_REF]. Inhalation of LPS causes acute and chronic inflammation in murine airways [START_REF] Brass | Subchronic endotoxin inhalation causes persistent airway disease[END_REF], which is accompanied by recruitment of neutrophils to the lung and increased levels of pro-inflammatory cytokines and chemokines in the lung. Acute or chronic inhalation of LPS mimics some relevant features of COPD in mice and serves as a model for preclinical analysis of possible drug candidates [START_REF] Shapiro | Animal models for COPD[END_REF].

LPS induced lung injury is mediated through Toll-like-receptor 4 (TLR4) and CD14 ligation, that leads to the activation of mitogen-activated protein kinase (MAPK) signaling cascades, which ultimately results in the secretion of tumor necrosis factor α (TNF-α) and other pro-inflammatory cytokines [START_REF] Togbe | Toll-like receptor and tumour necrosis factor dependent endotoxin-induced acute lung injury[END_REF][START_REF] Zhao | The role of mitogen-activated protein kinase phosphatase-1 in the response of alveolar macrophages to lipopolysaccharide: attenuation of proinflammatory cytokine biosynthesis via feedback control of p38[END_REF]. The MAPK families are ubiquitous and highly conserved serine-threonine kinases, including p38MAPK, extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK). The signaling cascade is initiated by three-tired sequential phosphorylation steps (MAPKKK, MAPKK, MAPK). MEK1 and MEK2 are Compound 37, a specific p38 α,β MAPK inhibitor, attenuated LPS-induced bronchoconstriction and neutrophil recruitment into the lungs in a dose-dependent manner [START_REF] Schnyder-Candrian | Dual effects of p38 MAPK on TNF-dependent bronchoconstriction and TNFindependent neutrophil recruitment in lipopolysaccharide-induced acute respiratory distress syndrome[END_REF]. It was concluded, that p38 MAPK mediates lung neutrophilia, cytokine and chemokine production and bronchoconstriction [START_REF] Schnyder-Candrian | Dual effects of p38 MAPK on TNF-dependent bronchoconstriction and TNFindependent neutrophil recruitment in lipopolysaccharide-induced acute respiratory distress syndrome[END_REF][START_REF] Kim | p38 Mitogen-activated protein kinase upregulates LPS-induced NF-kappaB activation in the development of lung injury and RAW 264.7 macrophages[END_REF].

It has been shown that ERK1/2 pathway plays a pivotal role in IL-13-induced asthmalike lung inflammation [START_REF] Lee | ERK1/2 mitogen-activated protein kinase selectively mediates IL-13-induced lung inflammation and remodeling in vivo[END_REF]. We hypothesized that the ERK1/2 pathway also plays an important role in LPS-mediated pulmonary inflammation and that inhibition of ERK signaling pathway may have anti-inflammatory effects. To this end we investigated the impact of U0126, a selective and potent MEK1/2 inhibitor, in the mouse model of acute LPS-induced lung injury and examined weather pharmacologic inhibition of ERK attenuates disease severity.

A c c e p t e d M a n u s c r i p t 5

Materials and Methods

Animals

All animal protocols were performed in accordance with the national animal protection rules and permitted by the local governmental authority (Regierung von Mittelfranken, Germany). BALB/c mice 6-8 weeks of age weighing 22-25 g (purchased from Harlan, Borchen, Germany) were used. The animals were fed on a normal standard diet (food pellets purchased from Altromin, Lage, Germany) with tap water ad libitum and were housed in a 12-hour light/dark cycle, at 20 to 21°C and with 40-60% humidity levels.

Mouse model of LPS-induced lung inflammation

The animals were treated intraperitoneally with U0126 (30mg/kg; purchased from Tocris Biosciences, Ellisville, USA), PD98059 (30mg/kg; Tocris Biosciences, Ellisville, USA) or vehicle (200 µl PBS with 5% DMSO, Invitrogen, Karlsruhe, Germany) 2 h prior to LPS challenge. U0126, PD98059 or vehicle-treated mice were exposed to aerosolized LPS (10 mg; 1.5 mg/ml saline; Lipopolysaccharides from Escherichia coli Serotype 026:B6, Sigma, St.Louis, USA) using the Buxco-Nebulizer (Buxco, Wilmington, USA). One inhalation cycle encompassed 9.9 minutes of LPS (25% duty) and followed by 5 minutes of drying. This was repeated four times. Animals were sacrificed 24 hours or at the indicated time point after LPS challenge by putting them into a CO 2 rich atmosphere. Tracheotomy was performed and a 24G cannula (InSyte 24G, BD Biosciences, Heidelberg, Germany) was inserted in the trachea.

In each animal bronchoalveolar lavage (BAL) was performed by flushing the airways and lungs (6*0,5ml) with cold Hank´s balanced solution (Gibco, Karlsruhe, Germany) supplemented with Na-EDTA and HEPES (Sigma, Steinheim, Germany). The supernatants of A c c e p t e d M a n u s c r i p t 6 the first lavage were collected and stored at -20°C for further analysis. The cells of pooled BAL of each animal were sedimented by centrifugation at 300*g for 5 min. The cell pellets were re-suspended in 2 ml RPMI-1640 (Invitrogen, Karlsruhe, Germany) and total BAL cell counts of each animal were determined using a haemocytometer (Sysmex microcellcounter F300, Norderstedt, Germany). For cytological examination, cytospin slides were prepared using a cytospin centrifuge (Thermo Shandon, Frankfurt, Germany) and stained with Diff-Quick (Dade-Behring, Marburg, Germany). Differential cell count was performed with at least 400 cells/slide.

Cytokine and chemokine measurements

Levels of tumor-necrosis factor α (TNF-α), macrophage inflammatory protein 2 (MIP-2) and keratinocyte-derived chemokine (KC) in BAL fluid were measured using a murine TNF-α OptEIA ELISA kit (BD Biosciences, San Diego, USA) and murine MIP-2 and murine KC DuoSet ELISA Development kits (R&D Systems, Minneapolis, USA). Levels of TNF-α and Interleukin 2 (IL-2) in supernatants of stimulated PBMC were determined using a human TNF-α OptEIA ELISA Set and a human IL-2 OptEIA ELISA kit (BD Biosciences, San Diego, USA). Cytokine and chemokine levels were assayed according to the manufacturer´s recommendations.

Phospho-p44/42 MAPK and Phospho-p38 MAPK Sandwich ELISA

Mice were treated intraperitoneally with U0126 (30mg/kg) or vehicle two hours prior to LPS challenge. Two hours later mice were sacrificed the lungs were removed and stored in liquid

Lung histological examination

Lungs were instilled intratracheally with 15 ml 4% buffered formaldehyde (Roth, Karlsruhe, Germany) using a syringe pump (sp200i, WPI, Berlin, Germany) and fixed overnight in formaldehyde. The tissues were embedded in paraffin and 4 micrometer slices were cut. To examine bronchial inflammation sections were stained with conventional hematoxylin and eosin (Roth, Karlsruhe, Germany). Bronchial inflammation was determined using a semiquantitative score as described by Myou [START_REF] Myou | Blockade of inflammation and airway hyperresponsiveness in immune-sensitized mice by dominant-negative phosphoinositide 3-kinase-TAT[END_REF]. The severity of inflammation was graded in five categories; 0, normal lung structure; 1, few inflammatory cells; 2, a ring of inflammatory cells 1 cell layer deep; 3, a ring of inflammatory cells 2-4 cells deep; 4, a ring of inflammatory cells of >4 cells deep. The score was determined in a blinded manner.

Myeloperoxidase assay

Determination of myeloperoxidase activity was adapted from Matos et.al [START_REF] Matos | Effects of tachykinin NK1 or PAF receptor blockade on the lung injury induced by scorpion venom in rats[END_REF]. The lungs were flash frozen in liquid nitrogen immediately after removing and stored at -80°C until examination. The frozen lungs were homogenized with an ultra turrax in 1 ml Na 2 PO 4 buffer (0.05 M, pH5.4) and centrifuged for 10 min at 20000*g at 4°C. The supernatants were discarded. The pellet was resuspended in 1 ml Na 2 PO 4 buffer containing 0.5% hexadecyltrimethylammonium bromide (Sigma-Aldrich, St.Louis, USA) and homogenized. This was followed by three freeze-thaw cycles using liquid nitrogen. After centrifugation at

Vascular leakage

The vascular leakage was determined by measurement of total albumin levels in supernatants of bronchoalveolar lavage using a Bradford-Assay (Bio-Rad, München, Germany).

Isolation and treatment of peripheral blood mononuclear cells (PBMC)

PBMC were isolated from EDTA-anticoagulated venous blood from healthy human volunteers by using density-gradient centrifugation over Ficoll-Histopaque (Sigma, St.Louis, USA). In brief, blood samples were diluted 1:1 with Hank´s balanced salt solution and carefully layered over Histopaque. After centrifugation at 742*g for 20 min the PBMC rich film above the Histopaque was collected and washed twice with Hanks buffer. The number of PBMC was determined using a haemocytometer and afterwards re-suspended at a density of 1*10 6 cells/ml in RPMI 1640. The cells were plated in 24-well-plates and cultured for 24 h at 37°C in a 5% CO 2 atmosphere.

For flow cytometry analysis cells were preincubated with U0126 (1µM) for 30 min and then stimulated with TPA (Phorbol-12-myristate-13-acetate) (0.1µg/ml) (Sigma, St. Louis USA).

15 min later cells were fixed with 1.5% formaldehyde and then stained intracellular with specific antibodies.

For cytokine measurements cells were preincubated with various concentrations of U0126 (1µM-30mM) for 30 min. Cells were stimulated with LPS (1mg/ml) for TNF-α analysis or A c c e p t e d M a n u s c r i p t 9 anti-CD3/CD28-beads (2µl/well) (Invitrogen Dynal, Oslo, Norway) for measurement of IL-2 levels. 24 h later supernatants were collected and stored at -80°C until analysis.

Flow cytometric analysis (FACS)

PBMC were fixed with 1.5% formaldehyde and permeabilized with ice-cold 100% methanol.

Intracellular staining was performed after Fc-block with Phosflow antibodies: ERK 1/2 AF488, p38 MAPK AF488 and isotype control antibody (BD Biosciences, San Diego, USA).

Cells were analyzed using a FACScan-flow cytometer and CellQuest Software (BD Biosciences). Geometrical mean and mean were used for analysis.

Statistical analysis

GraphPad Prism (Version 5) software was used for analysis of data. Data are presented as means ± SEM. Statistical significance was determined by the Student´s unpaired t-test for comparison of two groups. P-values of p < 0.05 were considered statistically significant.

U0126 inhibits ERK activation in human primary cells

The specificity of U0126 was determined by analyzing phosphorylated MAPK in stimulated human PBMC using flow cytometry. Stimulation of cells with LPS yielded only a weak activation under these conditions. Due to the low signal to noise ratio, we decided to use the robust and strong stimulus TPA for pharmacological characterization of U0126 in vitro. The phosphorylated forms of ERK and p38 were stained intracellularly with phosphospecific antibodies. Stimulation with TPA increased the levels of phosphorylated ERK as well as of phosphorylated p38 (Fig. 1). Pretreatment of human PBMC with U0126 (1µM) significantly reduced levels of phosphorylated ERK (p = 0.049) (Fig. 1A). In contrast, activation of p38 was not affected by cell treatment with U0126 (1µM) (Fig. 1B). Cell viability was analysed by the WST assay. No cytotoxicity was detected up to the highest concentration of U0126 (30µM) (data not shown).

Dose dependent inhibition of cytokine induction in stimulated PBMC by U0126

Next, we analyzed whether the specific inhibition of pERK by U0126 affects cytokine release.

Previously it has been shown that LPS activates ERK in macrophage cell lines and human monocytes [START_REF] Scherle | Inhibition of MAP kinase kinase prevents cytokine and prostaglandin E2 production in lipopolysaccharide-stimulated monocytes[END_REF] and that CD3/CD28 stimulation activates MAPK in T-cells [START_REF] Chialda | Inhibitors of mitogen-activated protein kinases differentially regulate costimulated T cell cytokine production and mouse airway eosinophilia[END_REF]. We stimulated human monocytes with LPS and lymphocytes with anti-CD3/CD28 beads in absence or presence of various concentrations of U0126. This compound dose dependently inhibited LPS induced TNF-α with an IC 50 of 0.21µM (Fig. 2A). Likewise IL-2 release in stimulated lymphocytes was dose dependently inhibited by U0126 with an IC 50 of 0.9µM (Fig. 2B).

A c c e p t e d M a n u s c r i p t 11

Effects of U0126 on LPS-induced lung injury in vivo

To evaluate the effects of MEK1/2 inhibition in vivo the model of acute LPS induced lung injury was used. BAL fluid was collected 24 h hours after LPS aerosol challenge and total and differential cell counts were determined. LPS challenge led to massive influx (20-fold increase compared to negative control) of inflammatory cells into the airways. Pretreatment of mice with U0126 reduced total cell numbers recovered in BAL fluid significantly (p = 0.009; Fig. 3A). The increased cell infiltration consisted primarily of neutrophils. U0126 inhibited neutrophil numbers recovered in BAL fluid significantly as compared to vehicle treated mice (p < 0.001) (Fig. 3B). In addition decreases in macrophage (p = 0.046) and lymphocyte (p = 0.026) populations were detected as well (Fig. 3B). The reduced neutrophil numbers among U0126-treated mice were consistent with decreased MPO activity in lung tissue (p = 0.044) (Fig. 3C). Furthermore U0126 administration caused significant reduced albumin levels (p = 0.047) in BAL fluid supernatants as a marker for decreased lung permeability and inflammation (Fig. 3D).

Effect of U0126 on ERK activation in vivo

In order to analyze the pharmacodynamic action of U0126 we assessed levels of phosphorylated ERK in lung tissue. LPS challenge caused substantially increased phosphorylation of MAPK (Fig. 4A). Lungs obtained from U0126-treated mice showed significantly reduced phosphorylated ERK level (p = 0.04) indicating the inhibition of phosphorylated ERK by U0126 in the lungs of LPS treated mice (Fig. 4A). In contrast, phosphorylation of p38 in lung tissue was not affected by U0126. Levels of phosphorylated A c c e p t e d M a n u s c r i p t 12 p38 of lungs obtained from U0126-treated mice were comparable with positive controls (Fig. 4B).

Effect of U0126 on cytokine and chemokine levels in BAL fluid

It has been shown that MIP-2, KC and TNF-α play a pivotal role in LPS-induced lung injury [START_REF] Jeyaseelan | Transcriptional profiling of lipopolysaccharide-induced acute lung injury[END_REF]. To evaluate this in vivo BAL fluid samples were collected 2h after LPS challenge and levels of these cytokines and chemokines were determined. LPS inhalation induced elevated cytokine release into BAL fluid as compared to untreated mice. Treatment of mice with the MEK 1/2 inhibitor U0126 prior to LPS challenge led to significantly reduced levels of KC (p = 0.015), MIP-2 (p = 0.049) and TNF-α (p = 0.008) as compared to vehicle treated animals (Fig. 5A-C).

Effect of U0126 on lung inflammation in vivo

The effect of U0126 on LPS induced lung inflammation was analyzed morphologically by histological analysis. Lung tissue was collected 24 h after LPS challenge. Light microscopy of haematoxylin-eosin stained lung slices revealed marked infiltration of inflammatory cells into peribronchial and perivascular connective tissues after LPS aerosol challenge as compared to naive mice (Fig. 6A,B compared to E,F). Edematous changes of the alveolar walls, swelling of alveolar epithelial cells and massive influx of polymorphonuclear cells were observed. The majority of the infiltrated cells were neutrophils. Administration of U0126 (30mg/kg) markedly attenuated the neutrophil-rich leukocyte infiltration as compared to vehicle control (p = 0.007) (Fig. 6C,D,G).

A c c e p t e d

M a n u s c r i p t 13

Effects of PD98059 on LPS-induced lung injury in vivo

To rule out that the effects of MEK blockade by U0126 are non-specific another structurally unrelated MEK inhibitor was tested. Administration of PD98059 significantly reduced total cell numbers in BAL fluid (p = 0.029), lung neutrophilia (p = 0.025) and MPO activity in lung tissue (p = 0,024) as compared to vehicle controls (Fig. 7).

A c c e p t e d M a n u s c r i p t

Discussion

The lack of effective pharmacological intervention is still a major problem in chronic inflammatory diseases of the lung, especially COPD. LPS-induced acute lung injury features some relevant characteristics of the disease and therefore could serve as a preliminary model for COPD [START_REF] Shapiro | Animal models for COPD[END_REF]. Inhibition of MAPK pathways may provide novel anti-inflammatory therapies.

There are several reports that the MAPK p38 plays a pivotal role in acute lung injury [START_REF] Schnyder-Candrian | Dual effects of p38 MAPK on TNF-dependent bronchoconstriction and TNFindependent neutrophil recruitment in lipopolysaccharide-induced acute respiratory distress syndrome[END_REF][START_REF] Kim | p38 Mitogen-activated protein kinase upregulates LPS-induced NF-kappaB activation in the development of lung injury and RAW 264.7 macrophages[END_REF].

Moreover evidence has accumulated over the past years that the MAPK ERK is activated by LPS in vitro [START_REF] Scherle | Inhibition of MAP kinase kinase prevents cytokine and prostaglandin E2 production in lipopolysaccharide-stimulated monocytes[END_REF][START_REF] Guha | Lipopolysaccharide activation of the MEK-ERK1/2 pathway in human monocytic cells mediates tissue factor and tumor necrosis factor alpha expression by inducing Elk-1 phosphorylation and Egr-1 expression[END_REF]. These observations led us to hypothesize that the ERK1/2 signaling pathway also plays a decisive role in LPS mediated pulmonary inflammation and that inhibition of ERK may have anti-inflammatory effects. To our knowledge, in the present study we have demonstrated for the first time that pharmacological inhibition of the ERK pathway using U0126, a selective and potent MEK1/2 inhibitor, attenuated inhaled LPSinduced lung injury in an animal model of acute lung injury. These results were corroborated by showing that an unrelated inhibitor of the ERK cascade inhibited acute lung injury as well.. U0126 is a small molecule compound, that was initially identified as an inhibitor of AP-1 transactivation in a cell-based reporter assay [START_REF] Favata | Identification of a novel inhibitor of mitogen-activated protein kinase kinase[END_REF]. U0126 directly inhibits MEK1/2 through inhibition of the catalytic activity of the active enzyme. We have demonstrated that U0126 is specific inhibitor of ERK phosphorylation in vitro as well as in vivo. TNF-α production in LPS-treated monocytes has been shown to be MAPK dependent, which can be blocked by ERK and p38 inhibitors [START_REF] Foey | Regulation of monocyte IL-10 synthesis by endogenous IL-1 and TNF-alpha: role of the p38 and p42/44 mitogen-activated protein kinases[END_REF]. We observed similar results in freshly isolated human PBMC. The production of TNF-α was dose-dependently inhibited by U0126 via ERK blockade. The observed IC 50 value of 0.21µM corresponds to findings in a previous study [START_REF] Scherle | Inhibition of MAP kinase kinase prevents cytokine and prostaglandin E2 production in lipopolysaccharide-stimulated monocytes[END_REF]. Recent studies demonstrated that CD3/CD28 ligation in T cells activates ERK and upregulates IL-2 production [START_REF] Kogkopoulou | Conditional up-regulation of IL-2 production by p38 MAPK inactivation is mediated by increased Erk1/2 activity[END_REF]. In agreement with a previous report U0126 [START_REF] Chialda | Inhibitors of mitogen-activated protein kinases differentially regulate costimulated T cell cytokine production and mouse airway eosinophilia[END_REF] was capable of reducing IL-2 production dose-dependently (IC 50 = 0.9µM) in stimulated human PBMC.

A c c e p t e d M a n u s c r i p t 15 Duan and coworkers previously showed that U0126 (using a concentration of 30mg/kg) effectively reduced lung inflammation, mucus plugging and pulmonary eosinophilia in an asthma mouse model [START_REF] Duan | Anti-inflammatory effects of mitogen-activated protein kinase kinase inhibitor U0126 in an asthma mouse model[END_REF]. To investigate the inhibitory effects of U0126 in vivo we used a murine model of a COPD-like acute disease, wherein aerosolized LPS induces an acute lung injury. In lung tissue U0126 specifically blocked phosphorylation of ERK whereas activation of p38 was not affected demonstrating the specificity of the inhibitor used.

Consistent with previous studies exposure to LPS led to massive influx of inflammatory cells, primarily neutrophils peaking 24 h after LPS exposure [START_REF] Puljic | Lipopolysaccharide-induced lung inflammation is inhibited by neutralization of GM-CSF[END_REF]. Neutrophils undergo directed migration to an inflamed area along a gradient of chemokines. LPS is known to induce the production of several inflammatory and chemotactic cytokines [START_REF] Strieter | Chemokines in lung injury: Thomas A. Neff Lecture[END_REF]. We observed that concurrently with the massive influx of neutrophils and mononuclear cells, LPS exposure led to a massive increase of the levels of the pro-inflammatory cytokine TNF-α and the neutrophil chemotactic chemokines KC and MIP-2 in BAL fluid. This mimics the clinical situation, whereas neutrophilia and elevated cytokine levels in sputum and BAL fluid are typical clinical findings among patients with acute lung injury. Administration of U0126 inhibited effectively inflammatory cell influx in the BAL fluid of mice exposed to LPS. Since administration of another structurally unrelated MEK inhibitor PD98059 was efficiently in reducing lung neutrophilia as well, non-specific effects of U0126 can be ruled out. An increase of alveolar epithelial permeability is a characteristic feature of several chronic lung diseases like asthma or COPD. It is well known that LPS injures lung endothelial and epithelial cells and enhances lung permeability [START_REF] Li | Lipopolysaccharide-induced alveolar epithelial permeability: the role of nitric oxide[END_REF]. Recently it has been shown that acute lung injury can be attenuated by reduction of lung hyperpermeability [START_REF] Speyer | Regulatory effects of iNOS on acute lung inflammatory responses in mice[END_REF]. In this study we observed that endotoxin inhalation led to elevated albumin levels in bronchoalveolar lavage, which is marker of increased lung epithelial permeability. U0126 was highly efficient in reducing LPS induced albumin levels in BAL fluid. Thus the reduction of lung hyperpermeability may contribute to therapeutic effects of U0126 on acute lung injury.

Emphysematous alterations of alveolar walls and destruction of lung parenchyma accompanied by massive infiltration of inflammatory cells are hallmarks of COPD. Long term LPS exposure leads to a prominent infiltration of macrophages and CD8+ lymphocytes to peripheral airways [START_REF] Vernooy | Long-term intratracheal lipopolysaccharide exposure in mice results in chronic lung inflammation and persistent pathology[END_REF]. In contrast short term exposure to LPS entails rapid influx of neutrophils that peaks 24 hours after challenge and diminishes near baseline 72 hours later [START_REF] Vernooy | Intratracheal instillation of lipopolysaccharide in mice induces apoptosis in bronchial epithelial cells: no role for tumor necrosis factor-alpha and infiltrating neutrophils[END_REF]. Similar results were obtained in our study, where histological analysis of the lung revealed a massive infiltration of neutrophils. Blocking ERK by U0126 successfully abated lung inflammation and reduced tissue neutrophilia. This corroborates our findings in BAL and indicates that pulmonary neutrophil infiltration is ERK dependent.

In conclusion, the ERK signaling pathway is crucial for LPS-induced lung injury. Furthermore, we could demonstrate that inhibition of ERK by using a specific inhibitor effectively attenuated acute lung injury. We could show, that the anti-inflammatory action of U0126 may be mediated by reduced lung neutrophilia and hyperpermeability, as well as decreased levels of pro-inflammatory cytokines and chemokines. Since effective antiinflammatory pharmacotherapy for COPD is still not available and MAPK inhibitors are in Mice were treated intraperitoneally with U0126 (30mg/kg) or vehicle 2 h prior to LPS challenge. 2 h after LPS challenge mice were sacrificed and the lungs were removed. Lung lysates were analyzed for phosphorylated ERK (A) and p38 (B) levels using sandwich ELISA. The absorbance at 450nm was measured and is shown as optical density ± SEM (n=6). # p < 0.05 as compared to vehicle without LPS stimulation; *p < 0.05 as compared to LPS stimulkated vehicle control.

Fig. 5. Effect of U0126 on cytokine and chemokine levels in bronchoalveolar fluid.

Animals were treated 2 h prior to LPS challenge intraperitoneally with U0126 (30mg/kg) or vehicle control. 2h later bronchoalveolar lavage was performed. The lavage was centrifuged and the supernatants were used for ELISA. Levels of murine KC (A), MIP-2 (B) and TNF-α (C) were assessed by standard sandwich ELISA with a detection limit of 15.6pg/ml for each cytokine. Data are expressed as means ± SEM (n=4). Statistically significant differences at *p < 0.05, **p < 0.01, as compared to vehicle control.

  MAPKK) of ERK1 and ERK2. Recently a number of studies revealed that p38 MAPK is an important enzyme for the pathogenesis of LPS-induced lung injury.

7 (

 7 nitrogen. After homogenization by ultra turrax the lysate was microcentrifuged and the supernatant was used for detection of pERK and pp38 (PathScan Phospho-p44/42 MAPK A c c e p t e d M a n u s c r i p t Thr202/Tyr204) Sandwich ELISA Kit and PathScan Phospho p38α MAPK (Thr180/Tyr182) Sandwich ELISA Kit (Cell Signaling, Danvers, USA)) according to the manufacturers recommendations. Absorbance at 450nm is proportional to the quantity of p44/42 MAPK phosphorylated at Thr202/Tyr204 and p38 MAPK phosphorylated at Thr180/Tyr182.

  for 10 min at 4°C the supernatants were collected. Tetramethylbenzidine (TMB) and hydrogen peroxide (TMB Substrate Reagent Set, BD Biosciences, San Diego, USA) were added to the supernatants and the reaction was stopped after 5 min with 1M H 3 PO 4 . The activity of MPO was quantified by measuring absorbance at 450nm.

Furthermore, inhibition

  of ERK MAPK resulted in decreased production of the proinflammatory cytokine TNF and the neutrophil chemoattractants KC and MIP-2. It is tempting to speculate that inhibition of these chemokines by the MEK1/2 inhibitor may contribute to the decreased lung neutrophilia. Concomitant with diminished lung neutrophilia U0126 led to reduced MPO activity in LPS challenged mice. A number of reports revealed an important role for MPO in the microbicidal activity of neutrophils and that it is released during neutrophil activation [25, A c c e p t e d M a n u s c r i p t 16 26]. Our finding that inhibition of ERK resulted in reduced both lung neutrophilia and MPO activity support the efficiency of this inhibitor.

A 21 Figure legends Fig. 1 .

 21legends1 Figure legends

Fig. 2 .

 2 Fig. 2. U0126 dose dependently inhibits cytokine release from stimulated human PBMC.

Fig. 3 .

 3 Fig. 3. U0126 reduces LPS-induced cellular infiltration into bronchoalveolar lavage

A

  c c e p t e d M a n u s c r i p t 22 BAL fluid (D) Data are expressed as means ± SEM (A, B, C n=10 mice/group, D n=7). * p < 0.05, ** p < 0.01 as compared to vehicle control.

Fig. 4 .

 4 Fig. 4. Inhibition of phosphorylated ERK by U0126 in lung tissue.

Fig. 6 .

 6 Fig. 6. Effects of U0126 on lung inflammation.

A

  c c e p t e d M a n u s c r i p t 23 negative controls (E, F). Scale bars represent 100µm. Data are presented as means ± SEM. *p < 0.05, ** p < 0.01 as compared with positive control.

Fig. 7 .

 7 Fig. 7. PD98059 reduces LPS-induced cellular infiltration into bronchoalveolar lavage

A

  c c e p t e d M a n u s c r i p t Graphical Abstract Inhibition of the MAP kinase ERK reduces inflammation in a murine model of acute lung injury. Graphical Abstract A c c e p t e d M a n u s c r i p t

Figure 2 A

 2 Figure 2

Figure 3 A

 3 Figure 3

Figure 4 A

 4 Figure 4

Figure 5 A

 5 Figure 5

Figure 6 A

 6 Figure 6

Figure 7

 7 Figure 7

Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (SFB 643).

The authors thank Alexandra Wandersee for her expert technical assistance and Claudia Urbanger for animal care.

Abbreviations: COPD, chronic obstructive pulmonary disease; MAPK(-K; -KK), mitogenactivated protein kinase (kinase; kinase kinase); LPS, lipopolysaccharide; ERK, extracellular signal-related kinase; MEK, extracellular signal-regulated kinase kinase; TNF, tumor necrosis factor, PBMC, peripheral blood mononuclear cells; MIP, macrophage inflammatory protein; KC, keratinocyte-derived chemokine, BAL, bronchoalveolar lavage; MPO, myeloperoxidase;