Allergenic proteases cleave the chemokine CX3CL1 directly from the surface of airway epithelium and augment the effect of rhinovirus.

CX3CL1 has been implicated in allergen-induced airway CD4+ T-lymphocyte recruitment in asthma. As epidemiological evidence supports a viral infection-allergen synergy in asthma exacerbations, we postulated that rhinovirus (RV) infection in the presence of allergen augments epithelial CX3CL1 release. Fully differentiated primary bronchial epithelial cultures were pretreated apically with house dust mite (HDM) extract and infected with rhinovirus-16 (RV16). CX3CL1 was measured by enzyme-linked immunosorbent assay and western blotting, and shedding mechanisms assessed using inhibitors, protease-activated receptor-2 (PAR-2) agonist, and recombinant CX3CL1-expressing HEK293T cells. Basolateral CX3CL1 release was unaffected by HDM but stimulated by RV16; inhibition by fluticasone or GM6001 implicated nuclear factor-κB and ADAM (A Disintegrin and Metalloproteinase) sheddases. Conversely, apical CX3CL1 shedding was stimulated by HDM and augmented by RV16. Although fluticasone or GM6001 reduced RV16+HDM-induced apical CX3CL1 release, heat inactivation or cysteine protease inhibition completely blocked CX3CL1 shedding. The HDM effect was via enzymatic cleavage of CX3CL1, not PAR-2 activation, yielding a product mitogenic for smooth muscle cells. Extracts of Alternaria fungus caused similar CX3CL1 shedding. We have identified a novel mechanism whereby allergenic proteases cleave CX3CL1 from the apical epithelial surface to yield a biologically active product. RV16 infection augmented HDM-induced CX3CL1 shedding-this may contribute to synergy between allergen exposure and RV infection in triggering asthma exacerbations and airway remodeling.

Efficacy and safety of etanercept in moderate-to-severe asthma: a randomised, controlled trial.

Increased tumour necrosis factor-α levels have been observed in bronchial biopsies and induced sputum from subjects with severe asthma. We investigated etanercept (ETN) as a therapeutic option for treating moderate-to-severe persistent asthma. In this 12-week, randomised, double-blind, placebo-controlled, phase 2 trial, subjects (n=132) with moderate-to-severe persistent asthma received subcutaneous injections of 25 mg ETN or placebo twice weekly, and were evaluated at baseline, and at weeks 2, 4, 8 and 12. The primary end-point was the change from baseline to week 12 in pre-bronchodilator forced expiratory volume in 1 s (FEV1)% predicted. Secondary end-points included morning peak expiratory flow, FEV1% pred, Asthma Control Questionnaire (5-item version), asthma exacerbations, provocative concentration of methacholine causing a 20% decrease in FEV1, and the Asthma Quality of Life Questionnaire. No significant differences were observed between ETN and placebo for any of the efficacy end-points. ETN treatment was well tolerated, with no unexpected safety findings observed during the study. Clinical efficacy of ETN was not shown in subjects with moderate-to-severe persistent asthma over 12 weeks. However, ETN treatment was a well-tolerated therapy. Studies in specific subsets of patients with asthma with longer-term follow-up may be needed to fully evaluate the clinical efficacy of ETN in this population.

Frmd7 expression in developing mouse brain.

AIMS: Mutations in the FERM domain containing 7 (FRMD7) genes are known to cause a significant number of cases of congenital idiopathic nystagmus (CIN). Only limited expression data exist suggesting low levels of expression in all tissues. In this study, we assess the expression profile of the murine homologue of FRMD7 (Frmd7) in tissue from three murine organs during development. METHODS: cDNA was extracted from heart, lung, and brain tissues of MF-1 mice at 12 developmental time points, embryonic days 11-19, postnatal days 1 and 8, and from adult mice. Relative expression of Frmd7 mRNA was calculated using quantitative real-time PCR techniques with two normalising genes (Gapdh and Actb). RESULTS: Expression of Frmd7 was low in all tissues consistent with earlier reports. In heart and lung tissues, expression remained very low with an increase only in adult samples. In brain tissue, expression levels were higher at all time points with a significant increase at embryonic day 18, with no gender-specific influence on Frmd7 expression. CONCLUSIONS: Frmd7 is expressed at low levels in all tissues studied suggesting a role in many tissue types. However, higher overall expression and a sharp increase at ED18 in the murine brain suggest a different role in this tissue.Earlier studies have shown that genes expressed in the murine brain during development exhibit temporal functional clustering. The temporal pattern of Frmd7 expression found in this study mirrors that of genes involved in synapse formation/function, and genes related to axon growth/guidance. This suggests a role for Frmd7 in these processes and should direct further expression studies.

Chronological expression of Ciliated Bronchial Epithelium 1 during pulmonary development.

Ciliated Bronchial Epithelium (CBE) 1 is a novel gene, which is expressed in ciliated cells. As cilia are important during embryogenesis, the present authors characterised the murine homologue of CBE1 (Cbe1) and compared its temporal expression during murine and human lung development. Cbe1 cDNA was cloned and characterised using sequencing, standard PCR and Western blotting. Mouse and human embryonic/fetal lungs (HELs) were harvested for mRNA analysis and protein localisation in vivo and in vitro using RT-PCR and immunohistochemistry. The Cbe1 amino acid sequence was >75% identical with CBE1 and its alternative splicing and tissue distribution were highly conserved. Pulmonary expression of Cbe1 mRNA was increased at embryonic day (E)16, 1 day later than Foxj1, which is consistent with a role in ciliogenesis. In HELs, CBE1 mRNA was detectable at 8-9 weeks post-conception and increased in explant culture. CBE1 protein expression was weak at 10 weeks post-conception but strong at 12.3 weeks post-conception, in parallel with cilia formation. Additionally, Cbe1 mRNA was expressed at E11 (4-5 weeks post-conception in HELs) in the absence of Foxj1, implying a distinct role in early development. Chronological regulation of CBE1/Cbe1 expression during pulmonary differentiation suggests involvement in ciliogenesis, with an additional role during early lung development.

The role of LTA4H and ALOX5AP polymorphism in asthma and allergy susceptibility.

BACKGROUND: Leukotrienes (LTs) have been identified as central mediators in asthma and allergy. Pharmacological inhibition of cysteinyl-LT activity improves asthma symptoms and control. Accumulating evidence suggests a role for the dihydroxy leukotriene LTB(4) in airway disease. LTA(4) hydrolase and 5-lipoxygenase activating protein have key roles in LTB(4) production. Single nucleotide polymorphism (SNPs) and haplotypes spanning the LTA4H and ALOX5AP genes have been associated with LTB(4) production and myocardial infarction (MI). OBJECTIVE: To assess the contribution of LTA4H and ALOX5AP polymorphism to asthma and allergy susceptibility. METHODS: Three hundred and forty-one Caucasian families (two asthmatic siblings) were genotyped for eight SNPs spanning ALOX5AP and five SNPs spanning LTA4H. Association analyses of asthma and related phenotypes (total IgE, atopy, bronchial hyper-responsiveness, FEV(1)) were undertaken using the Family Based Association Test. RESULTS: Single point analyses identified association (P < 0.05) between SNPs SG13S114, SG13S89, SG13S41 (ALOX5AP), rs1978331 (LTA4H) and asthma and/or related phenotypes. Haplotype analyses using all LTA4H SNPs identified a single key risk haplotype for the development of asthma (P = 0.006) and related phenotypes (P = 0.042-0.005). Haplotype analyses using all ALOX5AP SNPs identified several asthma and atopy risk and protective haplotypes. There was limited correlation with previously identified MI risk haplotypes in both genes. Carriers of both ALOX5AP SG13S41 and LTA4H rs1978331 alleles had an increased risk of developing asthma (OR 2.17, CI 1.41-3.32). CONCLUSIONS: These data provide evidence for the role of SNPs spanning the ALOX5AP and LTA4H genes in asthma and atopy susceptibility in the Caucasian population and support a role for LTB(4) in disease pathogenesis.

Cellular localization of interleukin 13 receptor alpha2 in human primary bronchial epithelial cells and fibroblasts.

BACKGROUND: Interleukin (IL) 13 is a key cytokine in asthma, regulating fibrosis, airway remodeling, induction of immunoglobulin E synthesis by B cells, bronchial hyperresponsiveness, and mucus production. IL-13 signals through the type II IL-4 receptor (IL-4R), which is composed of the IL-4Ralpha and the IL-13Ralpha1 chains. Another IL-13 binding chain, IL-13Ralpha2, binds IL-13 with high affinity but has no known signaling capability and is thought to serve as a decoy receptor providing tight regulation of IL-13 responses. METHODS: In this study, we investigated the cellular localization of IL-13Ralpha2 in human primary bronchial epithelial cells and fibroblasts using flow cytometry and confocal microscopy, as well as the in vivo expression of IL-13Ralpha2 in the human bronchial mucosa by means of immunohistochemistry. RESULTS: IL-13Ralpha2 is predominantly an intracellular rather than a membrane-bound molecule in both human primary bronchial epithelial cells and fibroblasts and displays a diffuse granular cytoplasmic distribution in both cell types. IL-13Ralpha2 protein is expressed in vivo in the human bronchial mucosa with its expression being higher in bronchial epithelial cells than bronchial fibroblasts both in vivo and in vitro. CONCLUSIONS: IL-13Ralpha2 is expressed by both human primary bronchial epithelial cells and fibroblasts as an intracellular protein with a diffuse cytoplasmic distribution. In vivo, IL-13Ralpha2 is expressed in the human airway mucosa mainly by bronchial epithelial cells.

The role of a soluble TNFalpha receptor fusion protein (etanercept) in corticosteroid refractory asthma: a double blind, randomised, placebo controlled trial.

AIM: Tumour necrosis factor alpha (TNFalpha) is a cytokine recognised as a therapeutic target in chronic inflammatory diseases. METHODS: A randomised, double blind, placebo controlled parallel group trial is reported of etanercept (an IgG1-TNF p75 receptor fusion protein), administered once weekly for 12 weeks in 39 patients with severe corticosteroid refractory asthma. Efficacy was measured by change from the pretreatment baseline in Asthma Related Quality of Life (AQLQ) and Asthma Control (ACQ) Questionnaire scores (the primary endpoints), lung function, peak expiratory flow (PEF) and bronchial hyperresponsiveness (BHR). Sputum and serum inflammatory cells and cytokines, serum albumin and C reactive protein (CRP) as biomarkers of inflammation were also assessed. RESULTS: There was a small but significant difference in reduction of ACQ scores between treatment and placebo (-1.11 (95% CI -1.56 to -0.75) and -0.52 (95% CI -0.97 to -0.07), respectively, p = 0.037). There was no significant difference in improvements in AQLQ scores, lung function, PEF, BHR or exacerbation rates between the groups. Minor adverse events, including injection site pain and skin rashes, were more frequent with etanercept. There was a significant reduction in sputum macrophages and CRP, and increases in serum TNFalpha and albumin following treatment, but not in other laboratory parameters. CONCLUSION: Etanercept therapy over 12 weeks demonstrated only a small but significant improvement in asthma control and systemic inflammation, as measured by serum albumin and CRP. Larger randomised, placebo controlled trials are required to clarify the role of TNFalpha antagonism in subjects with severe refractory asthma.

Human bronchial epithelial cells express an active and inducible biosynthetic pathway for leukotrienes B4 and C4.

BACKGROUND: Human bronchial epithelial cells synthesize cyclooxygenase and 15-lipoxygenase products, but the 5-lipoxygenase (5-LO) pathway that generates the leukotriene (LT) family of bronchoconstrictor and pro-inflammatory mediators is thought to be restricted to leucocytes. OBJECTIVE: We hypothesized that human bronchial epithelial cells (HBECs) express a complete and active 5-LO pathway for the synthesis of LTB4 and LTC4, either constitutively or after stimulation. METHODS: Flow cytometry, RT-PCR, Western blotting, enzyme immunoassays and reverse-phase high-performance liquid chromatography were used to investigate constitutive and stimulated expression of 5-LO pathway enzymes and the synthesis of LTs B4 and C4 in primary HBECs and in the 16-HBE 14o- cell line. RESULTS: Constitutive mRNA and protein expression for 5-LO, 5-LO-activating protein (FLAP), LTA4 hydrolase and LTC4 synthase were demonstrated in primary HBECs and in the 16-HBE 14o- cell line. In 16-HBE 14o- cells, treatment with calcium ionophore A23187, bradykinin or LPS up-regulated the expression of these enzymes. The up-regulation of 5-LO was blocked by the anti-inflammatory glucocorticoid dexamethasone. Human bronchial epithelial cells were shown to generate bioactive LTs, with primary HBECs generating 11-fold more LTC4 and five-fold more LTB4 than 16-HBE 14o- cells. LT production was enhanced by ionophore treatment and blocked by the FLAP inhibitor MK-886. CONCLUSIONS: Expression of an active and inducible 5-LO pathway in HBEC suggests that damaged or inflamed bronchial epithelium may synthesize LTs that contribute directly to bronchoconstriction and leucocytosis in airway inflammation.

Local genetic and environmental factors in asthma disease pathogenesis: chronicity and persistence mechanisms.

While asthma is an inflammatory disorder of the airways usually associated with atopy, an important additional component is involvement of the epithelium and underlying mesenchyme acting as a trophic unit (EMTU). In addition to allergens, a wide range of environmental factors interact with the EMTU, such as virus infections, environmental tobacco smoke and pollutants, to initiate tissue damage and aberrant repair responses that are translated into remodelling of the airways. While candidate gene association studies have revealed polymorphic variants that influence asthmatic inflammation, positional cloning of previously unknown genes is identifying a high proportion of novel genes in the EMTU. Dipeptidyl peptidase (DPP) 10 and disintegrin and metalloproteinase (ADAM)33 are newly identified genes strongly associated with asthma that are preferentially expressed in the airway epithelium and underlying mesenchyme, respectively. Also of increasing importance is the recognition that genes associated with asthma and atopy have important interactions with the environment through epigenetic mechanisms that influence their expression. This type of research will not only identify biomarkers of different types of asthma across the full range of phenotypic expression, but will also identify novel therapeutic targets that could influence the natural history of the heterogenes lung disease.

Current research priorities in chronic fatigue syndrome/myalgic encephalomyelitis: disease mechanisms, a diagnostic test and specific treatments.

Chronic fatigue syndrome (CFS) is an illness characterised by disabling fatigue of at least 6 months duration, which is accompanied by various rheumatological, infectious and neuropsychiatric symptoms. A collaborative study group has been formed to deal with the current areas for development in CFS research--namely, to develop an understanding of the molecular pathogenesis of CFS, to develop a diagnostic test and to develop specific and curative treatments. Various groups have studied the gene expression in peripheral blood of patients with CFS, and from those studies that have been confirmed using polymerase chain reaction (PCR), clearly, the most predominant functional theme is that of immunity and defence. However, we do not yet know the precise gene signature and metabolic pathways involved. Currently, this is being dealt with using a microarray representing 47,000 human genes and variants, massive parallel signature sequencing and real-time PCR. It will be important to ensure that once a gene signature has been identified, it is specific to CFS and does not occur in other diseases and infections. A diagnostic test is being developed using surface-enhanced, laser-desorption and ionisation-time-of-flight mass spectrometry based on a pilot study in which putative biomarkers were identified. Finally, clinical trials are being planned; novel treatments that we believe are important to trial in patients with CFS are interferon-beta and one of the anti-tumour necrosis factor-alpha drugs.

The cysteinyl-leukotriene type 1 receptor polymorphism 927T/C is associated with atopy severity but not with asthma.

BACKGROUND: The cysteinyl-leukotriene receptor type 1 (CysLT1) mediates the bronchoconstrictor and pro-inflammatory actions of cysteinyl-leukotrienes (LTC4, LTD4, LTE4) in asthma and is the molecular target of the lukast class of oral anti-leukotriene drugs. We screened the CYSLTR1 gene on chromosome Xq13-21 for coding region polymorphisms, and investigated their associations with allergy and asthma. METHODS: Solid-phase chemical cleavage was used to screen polymorphisms in the coding region of CYSLTR1. A TaqMan allelic discrimination assay was used to genotype a 927T/C SNP and oligonucleotide ligation assays were used to genotype the previously reported 617T/C and 898G/A SNPs of CYSLTR1 in 341 asthmatic families from the UK. Associations with asthma diagnosis, atopic status, serum-specific IgE and severity of allergy and asthma were examined. RESULTS: Family-based association tests showed that the 927 T allele was associated with atopy severity, especially in female subjects, but not with asthma diagnosis or severity, atopic status, bronchial hyper-responsiveness to methacholine or forced expiratory volume in 1 s. CONCLUSION: Mutation screening identified only one polymorphism, 927T/C, in the coding region of the CysLT1 receptor. This polymorphsim is predictive of atopy severity, but not associated with asthma.

Enhanced upregulation of smooth muscle related transcripts by TGF beta2 in asthmatic (myo) fibroblasts.

BACKGROUND: Transforming growth factor beta (TGF beta) upregulates a number of smooth muscle specific genes in (myo)fibroblasts. As asthma is characterised by an increase in airway smooth muscle, we postulated that TGFbeta(2) favours differentiation of asthmatic (myo)fibroblasts towards a smooth muscle phenotype. METHODS: Primary fibroblasts were grown from bronchial biopsy specimens from normal (n = 6) and asthmatic (n = 7) donors and treated with TGF beta2 to induce myofibroblast differentiation. The most stable genes for normalisation were identified using RT-qPCR and the geNorm software applied to a panel of 12 "housekeeping" genes. Expression of alpha-smooth muscle actin (alpha SMA), heavy chain myosin (HCM), calponin 1 (CPN 1), desmin, and gamma-actin were measured by RT-qPCR. Protein expression was assessed by immunocytochemistry and western blotting. RESULTS: Phospholipase A2 and ubiquitin C were identified as the most stably expressed and practically useful genes for normalisation of gene expression during myofibroblast differentiation. TGF beta2 induced mRNA expression for all five smooth muscle related transcripts; alpha SMA, HCM and CPN 1 protein were also increased but desmin protein was not detectable. Although there was no difference in basal expression, HCM, CPN 1 and desmin were induced to a significantly greater extent in asthmatic fibroblasts than in those from normal controls (p = 0.041 and 0.011, respectively). CONCLUSIONS: Although TGF beta2 induced the transcription of several smooth muscle related genes, not all were translated into protein. Thus, while TGF beta2 is unable to induce a bona fide smooth muscle cell phenotype, it may "prime" (myo)fibroblasts for further differentiation, especially if the cells are derived from asthmatic airways.

The contribution of transforming growth factor-beta and epidermal growth factor signalling to airway remodelling in chronic asthma.

Asthma is increasing in prevalence in the developing world, affecting approximately 10% of the world's population. It is characterised by chronic lung inflammation and airway remodelling associated with wheezing, shortness of breath, acute bronchial hyperresponsiveness to a variety of innocuous stimuli and a more rapid decline in lung function over time. Airway remodelling, involving proliferation and differentiation of mesenchymal cells, particularly myofibroblasts and smooth muscle cells, is generally refractory to corticosteroids and makes a major contribution to disease chronicity. Transforming growth factor-beta is a potent profibrogenic factor whose expression is increased in the asthmatic airways and is a prime candidate for the initiation and persistence of airway remodelling in asthma. This review highlights the role of transforming growth factor-beta in the asthmatic lung, incorporating biosynthesis, signalling pathways and functional outcome. In vivo, however, it is the balance between transforming growth factor-beta and other growth factors, such as epidermal growth factor, which will determine the extent of fibrosis in the airways. A fuller comprehension of the actions of transforming growth factor-beta, and its interaction with other signalling pathways, such as the epidermal growth factor receptor signalling cascade, may enable development of therapies that control airway remodelling where there is an unmet clinical need.

Tumour necrosis factor (TNFalpha) as a novel therapeutic target in symptomatic corticosteroid dependent asthma.

BACKGROUND: Tumour necrosis factor alpha (TNFalpha) is a major therapeutic target in a range of chronic inflammatory disorders characterised by a Th1 type immune response in which TNFalpha is generated in excess. By contrast, asthma is regarded as a Th2 type disorder, especially when associated with atopy. However, as asthma becomes more severe and chronic, it adopts additional characteristics including corticosteroid refractoriness and involvement of neutrophils suggestive of an altered inflammatory profile towards a Th1 type response, incriminating cytokines such as TNFalpha. METHODS: TNFalpha levels in bronchoalveolar lavage (BAL) fluid of 26 healthy controls, 42 subjects with mild asthma and 20 with severe asthma were measured by immunoassay, and TNFalpha gene expression was determined in endobronchial biopsy specimens from 14 patients with mild asthma and 14 with severe asthma. The cellular localisation of TNFalpha was assessed by immunohistochemistry. An open label uncontrolled clinical study was then undertaken in 17 subjects with severe asthma to evaluate the effect of 12 weeks of treatment with the soluble TNFalpha receptor-IgG1Fc fusion protein, etanercept. RESULTS: TNFalpha levels in BAL fluid, TNFalpha gene expression and TNFalpha immunoreative cells were increased in subjects with severe corticosteroid dependent asthma. Etanercept treatment was associated with improvement in asthma symptoms, lung function, and bronchial hyperresponsiveness. CONCLUSIONS: These findings may be of clinical significance in identifying TNFalpha as a new therapeutic target in subjects with severe asthma. The effects of anti-TNF treatment now require confirmation in placebo controlled studies.

Gene expression in peripheral blood mononuclear cells from patients with chronic fatigue syndrome.

BACKGROUND: Chronic fatigue syndrome (CFS) is a multisystem disease, the pathogenesis of which remains undetermined. AIMS: To test the hypothesis that there are reproducible abnormalities of gene expression in patients with CFS compared with normal healthy persons. METHODS: To gain further insight into the pathogenesis of this disease, gene expression was analysed in peripheral blood mononuclear cells from 25 patients with CFS diagnosed according to the Centers for Disease Control criteria and 25 normal blood donors matched for age, sex, and geographical location, using a single colour microarray representing 9522 human genes. After normalisation, average difference values for each gene were compared between test and control groups using a cutoff fold difference of expression > or = 1.5 and a p value of 0.001. Genes showing differential expression were further analysed using Taqman real time polymerase chain reaction (PCR) in fresh samples. RESULTS: Analysis of microarray data revealed differential expression of 35 genes. Real time PCR confirmed differential expression in the same direction as array results for 16 of these genes, 15 of which were upregulated (ABCD4, PRKCL1, MRPL23, CD2BP2, GSN, NTE, POLR2G, PEX16, EIF2B4, EIF4G1, ANAPC11, PDCD2, KHSRP, BRMS1, and GABARAPL1) and one of which was downregulated (IL-10RA). This profile suggests T cell activation and perturbation of neuronal and mitochondrial function. Upregulation of neuropathy target esterase and eukaryotic translation initiation factor 4G1 may suggest links with organophosphate exposure and virus infection, respectively. CONCLUSION: These results suggest that patients with CFS have reproducible alterations in gene regulation.

Risk factors and characteristics associated with severe and difficult to treat asthma phenotype: an analysis of the ENFUMOSA group of patients based on the ECRHS questionnaire.

BACKGROUND: Severe and difficult to treat asthma impairs health status and accounts for about half of asthma expenditure. In 1994, a European Network For Understanding Mechanisms of Severe Asthma (ENFUMOSA) was formed. A large group of patients from nine European countries has been selected. OBJECTIVE: To examine the risk factors and symptoms associated with a phenotype of severe/difficult to treat asthma. METHODS: The present report presents data assessed through the use of the European Community Respiratory Health Survey (ECRHS) Questionnaire in 148 mild-moderate controlled and 155 severe asthmatics from the ENFUMOSA group. RESULTS: There is a negative association of severe asthma with reported allergy and with a family history of allergy (Odds ratio (OR)=0.45). Sharing a bedroom before the age of five is associated with a higher risk of severe asthma (OR=1.5) while childhood infections, play school attendance and exposure to allergens or animals are not. A larger proportion of severe asthma patients report symptoms at work (OR=2.7) or have to change jobs (OR=4.3) and fewer severe than mild patients are currently employed (OR=0.39). Smoking and exposure to smoke is similar in mild and severe asthma. Dietary habits do not differ between the groups, but severe asthmatics report eating less savoury snacks and there is a trend for lower intake of sweets. CONCLUSIONS: Analysis of the ECRHS questionnaire in the ENFUMOSA study shows that severe asthma patients experience more symptoms and their health status is impaired by their inability to work and perhaps eat freely. Personal and maternal history of allergy is associated with mild but not severe asthma. Other than sharing a bedroom before the age of 5 years, no childhood exposure risk factors associated with severe asthma could be identified from this analysis.

Altered protein tyrosine phosphorylation in asthmatic bronchial epithelium.

A disease-related, corticosteroid-insensitive increase in the expression of epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase in asthmatic bronchial epithelium has been shown previously by the current authors. To determine whether this is associated with enhanced intracellular signalling, the aim of this study was to evaluate epithelial tyrosine phosphorylation. Bronchial biopsies were analysed for the presence of phosphotyrosine by immunohistochemistry. Bronchial epithelial cells were exposed to EGF, hydrogen peroxide or tumour necrosis factor-alpha in vitro for measurement of tyrosine phosphorylated signalling intermediates and interleukin (IL)-8 release. Phosphotyrosine was increased significantly in the epithelium of severe asthmatics when compared with controls or mild asthmatics; however, in mild asthma, phosphotyrosine levels were significantly decreased when compared with controls. There was no significant difference between phosphotyrosine levels before or after 8 weeks of treatment with budesonide. Stimulation of bronchial epithelial cells resulted in tyrosine phosphorylation of several proteins, including EGFR, Shc and p42/p44 mitogen-activated protein kinase. In the presence of salbutamol, a transient partial suppression of EGFR phosphorylation occurred, whereas dexamethasone was without effect. Neither salbutamol nor dexamethasone inhibited EGF-stimulated IL-8 release. These data indicate that regulation of protein tyrosine kinase activity is abnormal in severe asthma. The epidermal growth factor receptor and/or other tyrosine kinase pathways may contribute to persistent, corticosteroid-unresponsive inflammation in severe asthma.

Epithelial stress and structural remodelling in childhood asthma.

BACKGROUND: In adult asthma the bronchial epithelium shows high expression of the epidermal growth factor receptor (EGFR) and the cyclin dependent kinase inhibitor, p21waf, linked to ongoing stress and injury. METHODS: To determine if these are early markers of disease, sections of bronchial specimens obtained post mortem or by bronchoscopy from non-asthmatic (n = 7), moderate (n = 7), or severe (n = 9) asthmatic children aged 5-15 years were examined immunohistochemically. All severe and one moderately asthmatic children were receiving inhaled corticosteroids. RESULTS: The lamina reticularis of the asthmatic biopsy sections was found to be thicker (p = 0.01) than normal with increased deposition of collagen III (p = 0.007); submucosal eosinophil numbers did not differ between groups. As in adults, there was an asthma-related increase in epithelial EGFR (p<0.002) but there was no evidence of proliferation, with Ki67 being reduced (p = 0.001) and p21waf increased (p<0.004). The thickness of the lamina reticularis was significantly correlated with epithelial EGFR (rho = 0.77, p<0.001). CONCLUSIONS: These data provide evidence that, in asthmatic children, the epithelium is stressed or injured without significant eosinophilic inflammation. This change in the epithelial phenotype is associated with collagen deposition in the lamina reticularis, suggesting that the epithelial mesenchymal trophic unit is active early in, and may contribute to, the pathogenesis of asthma.