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BTS/BLF Young Investigators Prize Symposium

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M. Patel, D. Xu, P. Kewin, B. Choo-Kang, C. McSharry, N. C. Thomson, F. Y. Liew.Division of Immunology, Infection, & Inflammation, University of Glasgow and Western Infirmary, Glasgow G11 6NT, UK

Toll-like receptors (TLRs) are primary sensors of both innate and adaptive immune systems where they play a pivotal role in the response directed against structurally conserved components of pathogens. Synthetic bacterial lipopeptide Pam3CSK4 (BLP) is a TLR2 agonist capable of modulating Th1 and Th2 responses. Asthma is a chronic inflammatory condition of the airways characterised by airway hyperresponsiveness, inflammatory infiltrates in the bronchial wall containing eosinophils, and elevated serum IgE levels. Th2 lymphocytes are thought to play a key role in the initiation and perpetuation of this airway inflammation.

This study examines the therapeutic effect of Pam3CSK4 in established airways inflammation in a murine model of asthma. In mice previously sensitised and challenged with OVA, Pam3CSK4 given intraperitoneally markedly reduced the total inflammatory cell infiltrate and eosinophilia in bronchoalveolar lavage fluid (see fig). Pam3CSK4 therapy was associated with a reduction in OVA induced IL-4 and IL-5 secretion from thoracic lymph node culture, airway inflammation, bronchial hyperresponsiveness, and serum levels of IgE. Pam3CSK4 therapy was also associated with an increase in OVA induced IFNγ, IL-12, and IL-10 production. However, the anti-inflammatory effect of Pam3CSK4 was independent of IL-10 or TGFβ, and by extension, unlikely to involve regulatory T cells. The therapeutic effect of Pam3CSK4 was critically dependent on IL-12 and IFNγ. IL-12 production by dendritic cells was enhanced by Pam3CSK4, which induced increased IFNγ production by CD4+ T in vitro.

Abstract T1

Pam3CSK4 has a therapeutic effect on established allergic airways disease. (A) Experimental protocol. BALB/c mice were injected i.p. with 100 μg OVA and 100 μl 2% Alum on day 0 and day 14. The mice were boosted i.n. with 100 μg OVA on day 14. All the mice were then challenged i.n. on three consecutive days beginning on day 25. Pam3CSK4 (100 g per mouse) was administered i.p. only once on either day 25, 26, or 27. Pam3CSK4 was given two hours after challenge. Penh was determined on day 28 and mice were sacrificed on day 29. Serum, BAL, and lymphoid cells were collected and lung histology studied. (B) BAL total cell concentration and eosinophil proportion fell markedly after Pam3CSK4 therapy. Even mice given 3× i.n. challenges (on days 25–27) and then given one dose of Pam3CSK4 two hours after the last i.n. challenge demonstrated a highly significant fall in their BAL indices (**p<0.01 compared with mice given OVA alone). Enhanced Pause (Penh), after a two minute nebulisation with 50 mg/ml methacholine, shows decreased measure of airways hyperreactivity in Pam3CSK4 treated mice (*p<0.05). Lung cytokines were not detected. Data are mean (SEM), n = 10, and are representative of four independent experiments. (C) H&E sections of lung (×20 magnification) demonstrate that airways inflammation was completely ameliorated by Pam3CSK4 therapy. Pictures (day 26) are representative of 10 mice per group.

TLR2 activation skews the immune response to a Th1 profile which decreased Th2 cell differentiation and eosinophilic airways inflammation. Our results provide direct evidence that Pam3CSK4 could represent a novel therapeutic agent in allergic airways disease.


C. M. O’Kane, P. T. G. Elkington, J. S. Friedland.Department of Infectious Diseases, Imperial College, Hammersmith Campus, London, UK

Background: Oncostatin M (OSM), a monocyte or T cell derived cytokine member of the IL-6 superfamily, drives matrix degradation from stromal cells in extrapulmonary sites by inducing matrix metalloproteinases (MMP) -1/-3. There are no data on OSM induced matrix degradation in the lung, nor on OSM as an important cytokine in the pathogenesis of tuberculosis (TB). We investigated monocyte secretion of OSM on infection with Mycobacterium tuberculosis (Mtb), and the functional effects of such OSM on pulmonary fibroblasts.

Methods: Monocytes isolated by density centrifugation and adherence were infected with Mtb. Conditioned media from Tb infected monocytes (CoMTb) was harvested at 24 hours and filtered to remove bacilli. OSM was measured by ELISA and MAP kinase phosphorylation by western blot. Human lung fibroblasts were grown in collagen gels and stimulated with OSM. MMP-1 was measured by casein zymography, MMP-3 by western blot and Tissue Inhibitors of Metalloproteinases (TIMPs, which inhibit MMP activity in vivo) by ELISA.

Results: Live MTb stimulated OSM secretion by monocytes and monocyte derived macrophages (MDMs) in a dose dependent manner over 72 hours. CoMTb also induced OSM secretion from uninfected monocytes (285 (SD 17) pg/ml v 50 (SD 5) pg/ml), implicating a paracrine loop. G-protein coupled receptor (GPCR) inhibition by pertussis reduced TB and CoMTb induced OSM (from 314 (SD 30) pg/ml v 136 (SD 15) pg/ml, p = 0.03) Stimulation of monocytes with cholera induced OSM (653 (SD 48) v 165 (SD 14) pg/ml, p<0.001). TB infection of monocytes resulted in ERK phosphorylation within 15 minutes, and inhibition of the ERK MAP kinase pathway reduced Tb induced OSM (from 314 (SD 30) to 66 (SD 5) pg/ml). Prostaglandins, which act via GPCRs and ERK, are produced by monocytes in response to MTb. PGE2 and its downstream mediator B2cAMP both stimulated OSM secretion by monocytes (224 (SD 27) v 50 (SD 5) pg/ml). Indomethacin reduced TB induced OSM (from 275 (SD 71) to 163 (SD 8) pg/ml, p = 0.05). The functional effects of monocyte OSM secretion were investigated. OSM alone had little effect on fibroblast MMP-1 or -3 mRNA or secretion. TNFalpha alone induced moderate MMP-1and -3 gene expression and secretion. TNFalpha and OSM had a synergistic effect on MMP-1 and -3 (see fig). Secretion of the major inhibitors of MMP-1 and -3 (TIMPs-1/-2) was reduced in response to the combination of OSM and TNFα. TNFα was sufficient to markedly reduce TIMP-1, while neither TNF nor OSM alone had any effect on TIMP-2 but in combination downregulated its secretion (see fig).

Conclusion: MTb induces OSM secretion from monocytes via an ERK and prostaglandin dependent paracrine loop. OSM synergises with monocyte derived TNFα to induce unopposed MMP-1 and -3 secretion by fibroblasts. Such monocyte-fibroblast networks involving OSM may drive tissue destruction in TB.


F. Hollins, D. Kaur, R. Saunders, L. Woodman, P. Berger, A. Sutcliffe, C. Brightling, P. Bradding.Department of Infection, Immunity and Inflammation, Institute for Lung Health, University of Leicester, Leicester, UK

Mast cells are located within the airway smooth muscle (ASM) bundle of asthmatic patients, which may be a key factor in the development of variable airflow obstruction and airway hyperresponsiveness. Our aim was to investigate whether human ASM could support the survival and proliferation of human lung mast cells (HLMCs) and to examine the role of cytokines implicated in mast cell survival. HLMCs were purified from normal lung obtained at surgery for carcinoma using immunomagnetic affinity selection. Human ASM was microdissected from large airway specimens also obtained at lung resection surgery. HLMC (n = 4, 3.9±0.26′104 cells/well) and human ASM cells (n = 6) were co-cultured at 1:4 ratios in normal ASM media without serum or cytokines for up to 10 days. HLMC controls were set up in parallel including mast cells alone, mast cells with stem cell factor (SCF, 100 ng/ml) or interleukin (IL)-6 (50 ng/ml), and mast cells with SCF (100 ng/ml), IL-6 (50 ng/ml) and 10% fetal calf serum (FCS). Additional experiments with HLMC (n = 1) in co-culture with ASM (n = 3) were also performed as above with neutralising antibodies to IL-6 (0.6 mg/ml) and SCF (1 mg/ml). After 10 days no HLMCs survived in serum-free medium in the absence of SCF and IL-6. In the presence of cytokines and FCS the number of HLMCs at day 10 was 5.3±0.67′104 cells/well (p = 0.09). The number of HLMCs increased markedly in co-culture with ASM (14.3±0.11′104 cells/well; p = 0.02). The addition of IL-6 and SCF neutralising antibodies partially reduced the survival/proliferation in the co-culture at day 10 (4.3±0.27′104 cells/well; p = 0.02). In conclusion, ASM supports HLMC survival and proliferation, which is in-part mediated by IL-6 and SCF. This may have important implications for the pathogenesis and treatment of asthma.


A. Gunnell1, J. D. Moffatt2, N. A. Wilson1, R. H. Johns1, P. Sasikumar1, W. C. Yu3, G. J. Laurent1, R. C. Chambers1, Y. C. G. Lee1.1Centre for Respiratory Research, University College London; 2Department of Pharmacology, King’s College London; 3Princess Margaret Hospital, Hong Kong; 4Oxford Centre for Respiratory Medicine, UK

Introduction: Pleural infection (empyema) affects over 65 000 patients in UK and USA each year, with a mortality of 20%. Pleural adhesions and collagen deposition contribute to development of loculations, which prohibit adequate drainage of pleural pus. The mechanism of pleural adhesion formation is unclear. TGFβ is a potent profibrotic cytokine, and our previous work showed that intrapleural administration of TGFβ potently induced pleural fibrosis. Recent evidence suggests that thrombin and its receptor, PAR-1 (proteinase-activated receptor-1), hold an important role in tissue inflammation and fibrosis. We hypothesised that pleural infection results in intrapleural thrombin accumulation, which stimulates mesothelial cell release of TGFβ, in part via activation of PAR-1.

Aims: To show (1) that thrombin is present in human pleural fluid of infective aetiology, (2) that PAR-1 is present on mesothelial cells; and (3) that thrombin induces TGFβ release from mesothelial cells via PAR-1.

Methods and Results: (1) Biologically active levels of thrombin were found in infective human pleural fluids (n = 25) and these levels were higher than in transudative controls (n = 19), (230 v 160 mU/l, p<0.05 ANOVA). Likewise, active TGFβ levels were increased in infective human pleural fluids. To confirm that serosal infection induces thrombin accumulation, C57BL/6 mice were injected with LPS (10 μg/g) intraperitoneally. This induced a time dependent increase in thrombin levels in the lavage fluid at 3 and 24 hours (both p<0.05 v saline controls, ANOVA).

(2) PAR-1 was found to be expressed by human mesothelial cells (Met5A) and by mesothelial cells in resected human pleural tissues by immunohistochemistry. RT-PCR confirmed PAR-1 expression in benign mesothelial (Met5A) and malignant mesothelioma cell lines (JU-77, ONE-58, CRL-2081, and CRL-5951).

(3) Thrombin (1–40 nM) stimulated a significant dose dependent release of active and total TGFβ (up to sixfold) from mesothelial cells, as measured by a luciferase based bioassay. This response is at least in part mediated via PAR-1, as the TGFβ release was also induced in a dose dependent fashion by the PAR-1 agonist peptide, TFLLR-NH2 (1–50 μM) but not the reverse peptide RLLFT-NH2 (10 μM).

Conclusion: Taken together these data support the hypothesis that during pleural infection, thrombin contributes significantly, via PAR-1 activation, to the mesothelial release and intrapleural accumulation of active TGFβ, a cytokine known to induce pleural adhesions and fibrosis.


G. Cruse, S. M. Duffy, C. E. Brightling, P. Bradding.Institute for Lung Health, Department of Infection, Immunity and Inflammation, University of Leicester

Mast cells play a critical role in the immunopathology of asthma through the sustained release of a plethora of autacoid mediators, proteases, and cytokines. Importantly, mast cells infiltrate the airway smooth muscle bundles of asthmatic bronchi, but not those of eosinophilic bronchitics or normal subjects. Thus infiltration of mast cells into the airway smooth muscle is likely to be critical in the development of the disordered airway physiology in asthma. Human lung mast cells (HLMC) express the K+ channel IKCa1 which modulates mediator release in these cells, and is proposed to facilitate the retraction of the cell body during migration of several cell types. This action seems to require both opening and closing of the channel which is possibly influenced by intracellular calcium oscillations. We have tested the hypothesis that blockade of IKCa1 could attenuate HLMC migration. HLMC were isolated and purified from lung resected for bronchial carcinoma. HLMC chemotaxis was assayed using a modified Boyden chamber technique with Transwell chambers (8 μm pore size). HLMC migration towards the chemokine CXCL10 (100 ng/ml) was inhibited significantly with the non-selective IKCa1 blocker charybdotoxin (100 nmol/l) and the highly specific IKCa1 blocker TRAM-34 (200 nmol/l) in a dose dependent manner when added to the cells in the upper chamber. Thus with the addition of 100 nmol/l charybdotoxin, CXCL10-induced HLMC migration was inhibited by 78 (SD 14) % (n = 6, p = 0.002), while 200 nmol/l TRAM-34 inhibited CXCL10-induced migration by 80 (SD 7) % (n = 6, p = 0.0001). Similar results were seen when SCF was used as the chemoattractant indicating that the regulation of migration by IKCa1 is not restricted to an interaction with G protein coupled chemoattractants. Since modulation of IKCa1 can inhibit both chemotaxis and HLMC mediator release, the use of IKCa1 blockers such as TRAM-34 (a relatively non-toxic derivative of the hepatotoxic drug clotrimazole) could provide novel therapeutic strategies for mast cell mediated diseases such as asthma.

Supported by the Wellcome Trust.


K. G. Blyth1,2, B. A. Groenning2, J. J. Morton3, P. B. Mark2, J. E. Foster2, H. J. Dargie2, A. J. Peacock1.1Scottish Pulmonary Vascular Unit, 2Glasgow Cardiac Magnetic Resonance Unit, Western Infirmary, Glasgow, UK; 3Clinical Biochemistry, University of Glasgow, UK

Objectives: Serum brain natriuretic peptide levels (as [NTproBNP]) are elevated in patients with pulmonary hypertension (PHT). [NTproBNP] correlates with invasive pulmonary haemodynamics, falls in response to appropriate therapy and has proven to be of prognostic significance in patients presenting with PHT (Yap et al, Chest 2004). We sought to determine the relation between [NTproBNP] and high fidelity measurements of right ventricular (RV) performance in patients presenting with PHT.

Methods: Twenty five consecutive patients attending for baseline assessment of PHT and without any history of left sided or coronary heart disease underwent cardiovascular magnetic resonance (CMR) imaging and venous blood sampling for [NTproBNP], in addition to routine right heart catheterisation. Right and left ventricular (RV and LV) dimensions (indexed to BSA), RV and LV mass, RV mass index (RVMI  =  RV mass/LV mass) and RV and LV ejection fraction were determined at CMR. Venous blood for NTproBNP measurement was sampled after 15 minutes of recumbent rest and analysed using a chemiluminescent assay with a CV<5% (Roche, UK). CMR was performed on a 1.5T Siemens Sonata using a standard PHT protocol described in detail recently (Blyth et al. EHJ 2005).

Results: [NTproBNP] was logarithmically distributed in the population studied. Mean log 10 [NTproBNP] was 2.7 (SD 0.8) pg/ml. RV end-diastolic volume (RVEDV/BSA), RV end-systolic volume (RVESV/BSA) and RVMI were increased and RV ejection was depressed in PHT patients relative to normal values. All LV measurements were within normal limits. log 10 [NTproBNP] correlated linearly with RVEDV (r2 = 0.36, p = 0.003), RVESV (r2 = 0.424, p = 0.0003) and RVMI (r2 = 0.49, p<0.0001) and inversely with RVEF (r2 = 0.43, p = 0.0003). No significant relations between log 10 [NTproBNP] and LV measurements were identified. log 10 [NTproBNP] correlated with invasive pulmonary haemodynamics as previously reported.

Conclusion: [NTproBNP] in patients presenting with PHT appears closely related to the extent of RV remodeling (that is, RV dilatation and RV hypertrophy) and RV systolic dysfunction present in these patients. Since RV performance is closely linked to outcome in PHT, the relations we describe may explain the prognostic significance of [NTproBNP] in PHT recently reported (Nagaya et al. JACC 2001).

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