T1 Lymphocytic bronchoalveolitis in idiopathic chronic cough

S.S. Birring, C.E. Brightling, F.A. Symon, S. Barlow, A.J. Wardlaw, I.D. Pavord. Institute For Lung Health, Glenfield Hospital, Leicester

We have recently reported an excess of cases of organ specific autoimmune diseases amongst patients with idiopathic chronic cough and have suggested that the cough may be due to homing of activated lymphocytes from the primary site of autoimmune inflammation to the lung. We tested this hypothesis in comparative immunopathology study of 19 patients with idiopathic chronic cough recruited over a two year period (mean age 54y, 79% female, mean duration of cough 2y), 11 healthy subjects and 14 patients with explained chronic cough of similar severity. Organ specific autoimmune disease was present in six of idiopathic cough patients (32%) but no normals/explained cough subjects. All subjects had a bronchoscopy, bronchoalveolar lavage (BAL) and bronchial biopsy using standard techniques.

We obtained cytospins from the BAL for a differential cell count and studied BAL T-cell status (CD4/8), activation (CD103, CD25, CD49a, HLA-DR) and chemokine receptors (CCR3,5,6,11, CXCR3) using 3-colour flow cytometry. Bronchial biopsies were embedded in glycolmethacrylate and immunohistochemistry for CD3,4,8 (lymphocytes), CD14 (monocytes), CD45 (leukocytes), CD56 (NK cells), EG2 (eosinophils), Neutrophil Elastase (neutrophils), AA1 (mast cells), Interferon-γ, IL5, 3H4(IL4) was performed. The mean (SEM) BAL differential lymphocyte count was 6.8 (1.3)% in normals, 15.1 (2.6)% in idiopathic cough (mean difference from normals 8.3%; 95% Confidence Interval 1.6 to 15.0; p<0.02) and 7.0(1.7)% in explained cough. The proportion of BAL T-cells expressing CD4 was similar in all three groups and there were no differences in activation status of T-cells or chemokine receptor expression. CD56 and IFNγ expression in biopsies (cells/mm² submucosa) were reduced in subjects with idiopathic cough compared to normals and subjects with explained cough (p=0.03 and 0.047: Kruskal-Wallis). EG2 and 3H4 expression were raised in explained cough (p=0.02 & 0.03) but there were no differences in expression of CD3 and other markers between groups. In conclusion, we have made a novel observation of lymphocytic bronchoalveolar inflammation in patients with idiopathic chronic cough. The association with organ specific autoimmune disease suggests that this might be due to homing of activated lymphocytes from primary sites of autoimmune inflammation or a hitherto unrecognised autoimmune bronchitis. Further studies are required to investigate the interaction between T-cells and the cough reflex.

T2 Tgf-β activation is diminished following bleomycin-induced lung injury in mice lacking neutrophil elastase

F. Chua¹, S.E. Dunsmore¹, A.W. Segal², J. Roes², G.J. Laurent¹. ¹Centre for Respiratory Research; ²Department of Medicine, Royal Free and University College London School of Medicine, London WC1E 6JJ

Transforming growth factor-beta (TGF-β) is widely implicated in the pathogenesis of pulmonary fibrosis. We have previously reported that mice lacking neutrophil elastase (NE^-/-) are resistant to pulmonary fibrosis induced by bleomycin instillation. We hypothesised that decreased TGF-β activation may contribute to the resistance of these null animals against fibrosis. Active TGF-β was quantitated in bronchoalveolar lavage fluid (BALF) and lung tissue from wild type (WT) and NE^-/- mice seven days following instillation of 0.05 unit bleomycin or saline. Levels of active TGF-β in bleomycin-treated WT BALF averaged 0.36 ± 0.02 mg/ml, two-fold greater than in saline controls (p<0.001). In contrast, active TGF-β levels in bleomycin-treated NE^-/- BALF (0.23 ± 0.01 mg/ml, p<0.001 v WT values) were not different from saline controls. Conversely, a greater amount of TGF-β was detected in lung tissue from bleomycin-treated NE^-/- mice (p<0.05 v WT). No differences in inflammatory cellularity, alveolar-capillary leak, TGF-β₁ or TGF-β₃ mRNA expression were apparent between the two genotypes at this time point. Furthermore, in bleomycin-treated WT lungs, immunocytochemical staining for active TGF-β (LC1–30 antibody, Dr. K. Flanders, NIH) was widespread, with prominent localization to areas of inflamed or damaged alveoli. In contrast, staining for active TGF-β in bleomycin-treated NE^-/- lungs was minimal, and limited to peribronchial and perivascular locations. In conclusion, TGF-β activation was diminished in alveolar fluid from bleomycin-treated NE^-/- mice and correlated with decreased staining for active TGF-β in lung tissue. These data provide the first in vivo evidence that neutrophil elastase may play a critical role in modulating TGF-β activation. In particular, this study suggests that neutrophil elastase inhibitors may have a therapeutic potential in abrogating TGF-β-mediated fibrotic lung disease.

Supported by the Wellcome Trust, MRC and the British Lung Foundation.

T3 The cost effectiveness of an asthma management strategy directed at normalising the induced sputum eosinophil count

R.H. Green, C.E. Brightling, S. McKenna, B. Hargadon, D. Parker, P. Bradding, A.I. Wardlaw, I.D. Pavord. Institute for Lung Health, Glenfield Hospital, Leicester, UK

We have recently shown that a management strategy directed at normalising the induced sputum eosinophil count reduces asthma exacerbations and hospital admissions compared to a traditional approach. We now report the results of a concurrent health economic evaluation designed to determine the cost effectiveness of this sputum management strategy. Seventy four subjects with moderate to severe asthma recruited from hospital clinics were randomised into two groups: one managed by standard British Thoracic Society asthma guidelines (BTS management group) and one managed using an algorithm aimed at normalising the induced sputum eosinophil count as well as minimising symptoms (sputum management group). Patients were seen nine times over 12 months and on each occasion sputum was induced and processed, but the results were not disclosed in the BTS guidelines group. Throughout the study patients completed daily diary cards recording medication use, days off work, emergency GP or hospital visits and hospital admissions. The overall cost of each management strategy was calculated using our estimates of the cost of sputum induction and processing, the 2001 Unit Costs of Health and Social Care, the Department of Health 2001 reference costs and the British National Formulary. Total costs for each patient were calculated as the sum of the costs of hospital out-patient appointments, primary care visits, hospital admissions and medication use throughout the 12 months, with the addition of the costs of sputum induction and processing for patients in the sputum management group only. Patients in the sputum management group experienced significantly fewer severe asthma exacerbations than patients in the BTS management group (35 v 109, p=0.01) and significantly fewer patients were admitted to hospital with asthma (1 v 6, p=0.047). Amongst the 49 (24 v 25) patients in regular employment, the mean (SEM) days off work was 2.2 (0.8) in the sputum management group and 8.3 (2.1) in the BTS management group (p=0.01). The estimated annual total mean (SEM) cost per patient was (£1755 (119) in the sputum management group and £1954 (164) in the BTS management group (p=0.30). A treatment strategy directed at normalising the induced sputum eosinophil count reduces asthma exacerbations and hospital admissions without incurring additional costs to the health service. In addition, by reducing work days lost due to asthma, this approach may result in significant cost savings to society.

T4 The αvβ5 intgerin induces anoikis in squamous cell carcinoma (scc) cells by activating the intrinsic and extrinsic death pathways and inhibiting an akt/pkb survival signal

S.M. Janes, F.M. Watt. Cancer Research UK, 44 Lincoln's Inn Fields, London, WC2A 3PX, UK

Introduction: Focal or extensive loss of αvβ5 is a feature of the most poorly differentiated SCCs, while an increase in αvβ6 is associated with invasiveness and metastatic spread (

). Epithelial cells normally undergo apoptosis on detachment from their extracellular matrix (anoikis), but transformed cells do not. Hence failure to express a particular integrin may render tumour cells “deaf” to specific signals determining apoptosis. We hypothesised that expression of αv on H357 cells (which completely lack αv) and SCC4 cells (which have low expression) would restore their ability to undergo anoikis.

Methods: αv or α4 integrin subunits were transfected into H357 and SCC4 cells using the pBabepuro retroviral vector giving high expressing polyclonal populations. Anoikis was assessed by FACS analysis for sub-G1 cells and by TUNEL staining. Signalling pathways were investigated using MEK, p38MAPK, and PI3K inhibitors, and a constituitively active Akt construct. The apoptotic pathway was investigated using specific caspase inhibitors, Western blotting for activated pro and anti apoptotic molecules and a dominant negative FADD construct. Anoikis activation was examined using a chimeric molecule with the cytoplasmic domain of β5 attached to the extracellular domain of β6.

Results: The αv subunit formed a functional heterodimer with β5 as measured by FACS and adhesion to vitronectin. Anoikis was dramatically increased in the αv infected cells (both H357s and SCC4s) compared to the parental populations, empty vector controls and α4 infected cells at 48 and 72 hours (p<0.01). Anoikis was not increased in αvβ6 expressing cells. The pathways involved in αvβ5 induced anoikis include a suppression of activation of the survival factor Akt/PKB, possibly via the cytoplasmic domain of β5 and both the intrinsic (mitochondrial) and extrinsic (death receptor mediated) cell death pathways.

Conclusion: Re-introduction of the αv integrin subunit increased SCCs ability to undergo anoikis via the αvβ5 heterodimer. Both the intrinsic and extrinsic apoptotic pathways are required, and a cell survival mechanism of activating Akt/PKB is suppressed.

T5 Nasal mucociliary clearance is normal in children with CF: evidence against a primary cftr-related mechanism in the upper airway

D. McShane, J.C. Davies, T. Wodehouse, A. Bush, D. Geddes, E. Alton. Departments of Paediatrics and Gene Therapy, Royal Brompton Hospital, London, UK

Studies in adult CF patients have reported impaired mucociliary clearance (MCC) in both the nose and lungs, which has been implicated in the pathogenesis of airway plugging and bacterial lung infection. We studied young children to explore a primary versus secondary cause of impaired nMCC. Saccharin clearance times were measured in 18 children with CF (median age 11y (9.5B15y)) and 21 non-CF children (median age 12y (9.5B16y)). Eleven children with Primary Ciliary Dyskinesia (PCD) served as positive controls (median age 12y (9 B 14.7y)). A 2 mm piece of saccharin was placed on the inferior turbinate and the time taken to taste measured up to 30 mins. Nasal lavage (NL) was performed from the other nostril with 4ml sterile PBS. No difference was seen in nMCC times between the CF and non-CF groups (CF median (IQR) 11(8;15) min; non-CF median 11 (9;16) min), whereas those in the PCD group all had nMCC times of >30 minutes (p<0.05 for both CF & non-CF). There was no evidence of excess inflammation in the CF NL samples as assessed by levels of the neutrophil chemoattractant, IL-8 (CF 52 (3;75) pg/ml NLF;non-CF 37 (5; 108) pg/ml NLF). Interestingly,the children with PCD had significantly raised levels of IL-8 (377 (244; 598) pg/ml NLF; p<0.01 compared with both CF & non-CF). We next measured nMCC in adult CF patients with and without chronic sinus (CS) disease (CS+ or CS−). CS+ CF patients had significantly longer clearance times than CS− subjects (CS+ n=12:18 (11; 24) mins;CS− n=16: 14 (6; 16) mins; p<0.05). IL-8 was significantly higher in the CF CS+ group than in the healthy adult group (CF CS+45 (15; 76) pg/ml NLF; healthy adults: 10 (1; 64) pg/ml NLF; p=0.01) and there was a trend with regards to the CF CS− group (15 (1; 39) pg/ml NLF; p=0.06). In conclusion, the delayed MCC reported in the CF nose was not seen in a group of young children,but became apparent in adult patients with chronic sinus disease. This suggests a role for mucosal inflammation in the aetiology of delayed MCC in the upper and proximal airways but not distal airways where the ciliated epithelium is more sparse,and low volume ASL may be more detrimental on the clearance process.

T6 Phosphodiesterase 4 isoforms differentially regulate lps stimulated macrophage function: the role of PDE4b2.

M.C. Shepherd^{1 2}, N.C. Thomson², M.D. Houslay¹. ¹Institute of Biological and Life Sciences, University of Glasgow, Glasgow, UK; ²Department of Asthma research, Medicine and Therapeutics, Gartnavel General Hospital Glasgow UK

Phosphodiesterase4 (PDE4), a family of cAMP hydrolysing enzymes is widely expressed in immune cells. Four genes encode PDE4A, PDE4B, PDE4C, and PDE4D. Differential mRNA splicing produces variability within these families. Structural differences between isoforms suggest specific roles in cell regulation. I have shown PDE4 isoform expression regulation with macrophage differentiation. Such monocytes lose PDE4D expression, but gain PDE4A and PDE4B isoforms. I hypothesised important roles for PDE4 isoforms in macrophage behaviour.

Methods: RAW 264.7 cells were treated with 10ng/ml lps +/− the PDE4 inhibitor rolipram, PDE3 inhibitor cilostamide or signal transduction inhibitors. Cyclo-oxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, PDE4 isoform activity or ERK 1/2 activity was measured. ERK activity was assessed by the phospho-ERK/Total ERK ratio on western blot. Total PDE4, PDE3, and PDE4 isoform activity from immunoprecipitates were assessed, TNFα and prostaglandin E2 (PGE2) production were measured in growth medium by ELISA. RAP-1A G-protein activity mutants were used to investigate rolipram's action.

Results: Rolipram, not cilostamide caused indomethacin sensitive increase in iNOS expression, but indomethacin resistant increase in COX-2 expression in lps stimulated macrophages. PGE2 production was dose dependently increased by rolipram while TNFα production was inhibited in an indomethacin resistant fashion. Rolipram caused an increased and early activation of ERK 2. Lps led to a MEK dependent increase in PDE4 activity by 40% and PDE4B by 42%. Transfected RAP-1A activity mutants did not influence inflammatory mediator production.

Discussion: Rolipram increases the expression of COX-2, PGE2, and iNOS while inhibiting TNFα. TNFα inhibition is not due to increased PGE2 production. Complex crosstalk between the cAMP and MAPKinase signal cascades suggests increased PDE4B activity acts to regulate pro-inflammatory signal propagation. Rolipram alters the response of ERK 2 to lps, promoting a “proliferative” response. The G-protein Rap-1A did not mediate the interaction between cAMP and MAPKinase.