FTIR spectroscopic analysis of sputum: preliminary findings on a potential novel diagnostic marker for COPD.

COPD is a common, progressively disabling disease and a major health burden worldwide. Fourier transform infrared (FTIR) spectroscopy provides for sensitive analysis of complex biological samples. COPD pathogenesis involves quantitative and qualitative changes in sputum biosynthesis. This first study explores whether FTIR can produce distinct spectral profiles of human sputum, and capture differences between COPD and health. Sputum obtained from 15 COPD patients and 15 healthy volunteers was analysed using FTIR spectroscopy; differences in peak positions, height and configuration were identified and measured. All samples gave reproducible characteristic IR absorption spectra. The most relevant regions identified were the amide and glycogen rich regions, showing crucial spectral differences between health and COPD relating to peak position shifts or intensity alteration. These novel preliminary findings support further exploration of FTIR sputum profiling in a clinical study to determine its potential as a practical method for monitoring COPD.

RhoA signaling modulates cyclin D1 expression in human lung fibroblasts; implications for idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic Pulmonary Fibrosis (IPF) is a debilitating disease characterized by exaggerated extracellular matrix deposition and aggressive lung structural remodeling. Disease pathogenesis is driven by fibroblastic foci formation, consequent on growth factor overexpression and myofibroblast proliferation. We have previously shown that both CTGF overexpression and myofibroblast formation in IPF cell lines are dependent on RhoA signaling. As RhoA-mediated regulation is also involved in cell cycle progression, we hypothesise that this pathway is key to lung fibroblast turnover through modulation of cyclin D1 kinetic expression. METHODS: Cyclin D1 expression was compared in primary IPF patient-derived fibroblasts and equivalent normal control cells. Quantitative real time PCR was employed to examine relative expression levels of cyclin D1 mRNA; protein expression was confirmed by western blotting. Effects of Rho signaling were investigated using transient transfection of constitutively active and dominant negative RhoA constructs as well as pharmacological inhibitors. Cellular proliferation of lung fibroblasts was determined by BrdU incorporation ELISA. To further explore RhoA regulation of cyclin D1 in lung fibroblasts and associated cell cycle progression, an established Rho inhibitor, Simvastatin, was incorporated in our studies. RESULTS: Cyclin D1 expression was upregulated in IPF compared to normal lung fibroblasts under exponential growth conditions (p < 0.05). Serum deprivation inhibited cyclin D1 expression, which was restored following treatment with fibrogenic growth factors (TGF-beta1 and CTGF). RhoA inhibition, using a dominant negative mutant and a pharmacological inhibitor (C3 exotoxin), suppressed levels of cyclin D1 mRNA and protein in IPF fibroblasts, with significant abrogation of cell turnover (p < 0.05). Furthermore, Simvastatin dose-dependently inhibited fibroblast cyclin D1 gene and protein expression, inducing G1 cell cycle arrest. Similar trends were observed in control experiments using normal lung fibroblasts, though exhibited responses were lower in magnitude. CONCLUSION: These findings report for the first time that cyclin D1 expression is deregulated in IPF through a RhoA dependent mechanism that influences lung fibroblast proliferation. This potentially unravels new molecular targets for future anti-IPF strategies; accordingly, Simvastatin inhibition of Rho-mediated cyclin D1 expression in IPF fibroblasts merits further exploitation.

An association study between the Clara cell secretory protein CC16 A38G polymorphism and asthma phenotypes.

BACKGROUND: Previously, an association has been reported between an increased risk of asthma and a polymorphism in the Clara cell secretory protein (CC16) gene [namely, an adenine to guanine substitution in the CC16 gene at position 38 (A38G) downstream from the transcription initiation site within the noncoding region of exon 1]. Homozygous individuals for the polymorphic sequence (AA genotype) were reported to have a significant (6.9 fold) increased risk of developing asthma. This finding has not been confirmed independently. OBJECTIVE: To validate the association of CC16 A38G polymorphism to asthma in a separate well-characterized population through a case-control study. METHODS: We conducted an association study using a sample of 217 unrelated Northern European Caucasians. Individuals were clinically characterized by a validated respiratory questionnaire, spirometry and bronchial reactivity measurement, and genotyped for the A38G polymorphism using PCR and restriction digestion. Association analysis was performed using the nonparametric Chi-squared tests. RESULTS: In the unselected population, 43.3% participants were homozygous for the CC16*G allele and 45.4% were heterozygous (AG). We observed no significant difference in the distribution of positive bronchial reactivity to methacholine (at FEV1 PC20 of

Virus-induced asthma.

Clinical and experimental investigations indicate that respiratory viral infections are important triggers for asthma attacks. Viral upper respiratory infections have been associated with 80% of asthma exacerbations in children and 50% of all asthma episodes in adults. Human Rhinovirus (HRV) has been implicated as the most common virus associated with asthma episodes. The observation that the great majority of wheezing lower respiratory tract illnesses in early life are associated with acute viral infections suggests that viruses may also alter the development of the lungs or of the immune system, acting as co-factors for the inception of asthma. Whilst there is no doubt that viruses are important asthma exacerbation factors, the role of viral infections in the development of asthma still remains controversial.

Simultaneous identification of GSTP1 Ile105-->Val105 and Ala114-->Val114 substitutions using an amplification refractory mutation system polymerase chain reaction assay: studies in patients with asthma.

BACKGROUND: The glutathione S-transferase (GST) enzyme GSTP1 utilizes byproducts of oxidative stress. We previously showed that alleles of GSTP1 that encode the Ile105-->Val105 substitution are associated with the asthma phenotypes of atopy and bronchial hyperresponsiveness (BHR). However, a further polymorphic site (Ala114-->Val114) has been identified that results in the following alleles: GSTP1*A (wild-type Ile105-->Ala114), GSTP1*B (Val105-->Ala114), GSTP1*C (Val105-->Val114) and GSTP1*D (Ile105-->Val114). METHODS: Because full identification of GSTP1 alleles may identify stronger links with asthma phenotypes, we describe an amplification refractory mutation system (ARMS) assay that allows identification of all genotypes. We explored whether the GSTP1 substitutions influence susceptibility to asthma, atopy and BHR. RESULTS: Among 191 atopic nonasthmatic, atopic asthmatic and nonatopic nonasthmatic individuals, none had the BD, CD, or DD genotypes. GSTP1 BC was significantly associated with reduced risk for atopy (P = 0.031). Compared with AA, trend test analysis identified a significant decrease in the frequency of GSTP1 BC with increasing severity of BHR (P = 0.031). Similarly, the frequency of GSTP1 AA increased with increasing BHR. CONCLUSION: These data suggest that GSTP1*B and possibly GSTP1*C are protective against asthma and related phenotypes.

Glutathione-S-transferase family of enzymes.

The loci encoding the glutathione-S-transferase (GST) enzymes comprise a large supergene family located on at least seven chromosomes. The function of the GST enzymes has traditionally been considered to be the detoxication of electrophiles by glutathione conjugation. A wide variety of endogenous (e.g. by-products of reactive oxygen species activity) and exogenous (e.g. polycyclic aromatic hydrocarbons) electrophilic substrates have been identified. Interestingly, recent data has suggested a role, at least for the pi class gene product, in jun kinase inhibition. Since many GST genes are polymorphic, there has been considerable interest in determining whether particular allelic variants are associated with altered risk (or outcome) of a variety of diseases. We describe recent studies in patients with asthma and cutaneous basal cell carcinoma that demonstrate associations between GSTP1 and GSTT1 genotypes and disease phenotypes. Thus, GSTP1val(105)/val(105) was protective against asthma symptoms and GSTT1 null was associated with a subgroup of basal cell carcinoma patients who develop large numbers of primary tumours in clusters. Importantly, these associations were characterised by relatively large odds ratios (0.11 and 7.4, respectively) implying that the allelic variants exert a substantial biological effect. These and other data indicate the importance of GST polymorphism in determining disease phenotype.

Differential mRNA expression of insulin-like growth factor-1 splice variants in patients with idiopathic pulmonary fibrosis and pulmonary sarcoidosis.

Insulin-like growth factor-1 (IGF-1) is a highly mitogenic polypeptide detectable in human lung. Using competitive reverse transcriptase/polymerase chain reaction (RT-PCR), expression of four IGF-1 transcripts was examined in bronchoalveolar lavage cells (BALC) from normal subjects, idiopathic pulmonary fibrosis (IPF), stage I/II (no fibrosis), and stage III/IV (confirmed fibrosis) pulmonary sarcoidosis patients, and fibroblast strains isolated from normal and IPF lungs. Transcripts studied were Class 1 and Class 2 (exons 1 or 2, respectively) with IGF-1Eb or IGF-1Ea (exons 5 or 6, respectively). Total IGF-1 expression was downregulated in BALC of both patients with IPF (p < 0.01) and patients with sarcoidosis (p < 0.04) compared with healthy subjects. In contrast, both constitutive (p < 0.003) and transforming growth factor-beta (TGF-beta)- induced (p < 0.02) IGF-1 expression was higher in fibrotic, compared with normal, fibroblasts. These changes were associated with differential expression of IGF-1 splice variants. Healthy subjects and sarcoidosis patients without fibrosis showed similar expression of Class 1/Class 2 and IGF-1Ea/IGF-1Eb. However, patients with fibrosis demonstrated discordant, increased relative abundance of Class 1 transcripts (p < 0.01). In parallel, all fibrosis patients failed to express Class 2, IGF-1Eb forms and sarcoidosis patients with fibrosis did not express the Class 1, IGF-1Eb variant either. Fibrotic fibroblasts expressed higher constitutive levels of Class 1, IGF-1Ea transcripts compared with normal fibroblasts. Class 2, IGF-1Eb forms were moderately expressed by fibroblasts only after stimulation with TGF-beta, which also further increased levels of Class 1, IGF-1Ea transcripts. Our findings suggest that transition from a healthy to a fibrotic phenotype occurs in association with a changing pattern of IGF-1 mRNA heterogeneity and leads us to hypothesize a potential role for specific IGF-1 variants in fibrogenesis.

Expression of growth hormone-releasing factor, growth hormone, insulin-like growth factor-1 and its binding proteins in human lung.

Reverse transcription PCR showed that mRNA encoding the neurohormones growth hormone-releasing factor (GRF) and GH, and its receptor GH-R, together with IGF-1 splice variants and IGFBPs are expressed by inflammatory cells found in the normal human airway. Unfractionated BALC moderately express GRF, GH and GH-R, IGFBP-2 to IGFBP-6, and IGFBP-rPl. In addition, BALC preferentially express the class 1 IGF-1Ea splice variant of the IGF-1 gene. A similar pattern of expression occurs in purified AM, except they do not appear to express GH-R. In marked contrast, AM precursor peripheral blood monocytes, do not express neuropeptides or IGF-1 and only express IGFBP-1, -4 and -6 and IGFBP-rP1. These data suggest that normal human inflammatory airway cells possess a powerful array of neurohormones and IGFBPs that are available for modulating local IGF-1 bioavailability in the lung.

Polymorphisms at the glutathione S-transferase, GSTP1 locus: a novel mechanism for susceptibility and development of atopic airway inflammation.

A common feature of environmental irritants is their ability to cause local inflammation which could alter airway function. The principal targets of such injury are the epithelial cells lining the airway passages and the lower respiratory gas-exchange areas. While host atopy is a recognized risk factor for airway inflammation, atopy alone cannot cause asthma. We hypothesize that susceptibility to persistent airway inflammation in atopic individuals is characterized by an inherited deficiency in the effectiveness of detoxification of inhaled irritants and products of oxidative stress such as reactive oxygen species (ROS). Our case-control studies show that polymorphisms at the glutathione S-transferase, GSTP1, locus on chromosome 11q13 may account for variation in host response to oxidative stress, a key component of airway inflammation. Frequency of the GSTP1 Val/Val genotype is reduced in atopic subjects compared with nonatopic subjects. Trend analysis also shows a significant decrease of GSTP1 Val/Val (with parallel increase of GSTP1 Ile/Ile) genotype frequency with increasing severity of airflow obstruction/bronchial hyperresponsiveness. The implication of specific polymorphisms at the GSTP1 locus in airway inflammation is entirely novel: however, GST are recognized as a supergene family of enzymes critical in 1) cell protection from the toxic products of ROS-mediated reactions, 2) modulation of eicosanoid synthesis.

Expression of intercellular adhesion molecule-1 (ICAM-1) in nasal epithelial cells of atopic subjects: a mechanism for increased rhinovirus infection?

Since clinical experimental studies indicate that upper respiratory tract viral infections may exacerbate acute asthma symptoms in atopic/asthmatic individuals, we have investigated the expression and modulation of ICAM-1 on human nasal epithelial cells (HNEC) from normal and atopic subjects. ICAM-1 is the attachment molecule for the majority of serotypes of human rhinovirus (HRV), including HRV-14, and is also critical for the migration and activation of immune effector cells. Basal ICAM-1 expression was significantly higher in HNEC obtained by brushings from atopic compared with non-atopic subjects (P = 0.031), and was also significantly increased on atopic HNEC harvested in season compared with out of season (P < 0.05). Atopic HNEC showed further up-regulation in ICAM-1 expression when cultured with clinically relevant allergen (P = 0.032). ICAM-1 levels on normal HNEC were also increased by infection with HRV-14 (P < 0.05). Basal expression of ICAM-1 on atopic nasal polyp epithelial cells (EC) was significantly higher than on both normal and atopic nasal HNEC. This elevated nasal polyp ICAM-1 level was not increased further by allergen, although HRV infection resulted in a small significant increase. Recovered viral titres from HRV-infected nasal polyp EC were 1.5-fold higher than from infected normal nasal HNEC. The data are consistent with the hypothesis that allergen, by enhancing expression of the HRV attachment target on host cells, facilitates viral infection in atopic subjects; simultaneously HRV-induced increases in ICAM-1 levels would favour migration and activation of immune effector cells to the airway, resulting in enhanced atopic inflammation.

Polymorphism at the glutathione S-transferase GSTP1 locus. A new marker for bronchial hyperresponsiveness and asthma.

Most genetic studies of asthma have concentrated on genes on chromosomes 11q and 5q and their association with the key asthma-related phenotypes of bronchial hyperresponsiveness (BHR) and atopy. Although asthma is characterized by airway inflammation, a critical component of which is oxidative stress, few data exist on genes involved in protecting against this insult. We describe an association study designed to examine whether allelic variation at the glutathione-S-transferase GSTP1 locus influences expression of the BHR and atopy phenotypes in asthma. The enzyme encoded by GSTP1 utilizes a variety of lipid and DNA products of oxidative stress, and polymorphic variants of this gene are associated with altered catalytic function of this enzyme. We found that the frequency of GSTP1 Val(105)/Val(105) was significantly lower in asthmatic than in control subjects. Indeed, the presence of this genotype conferred a sixfold lower risk of asthma than did GSTP1 Ile(105)/Ile(105). Remarkably, asthma risk in Val(105) homozygotes was further reduced (by ninefold) after correction for atopic indices, age, and gender. Trend analysis after stratification according to the degree of bronchial reactivity/obstruction showed that the frequency of GSTP1 Val(105)/Val(105) correlates with decreasing severity of airway dysfunction. Furthermore, subjects with GSTP1 Val(105)/Val(105) have four- and 10-fold lower risks, respectively, of exhibiting atopy defined by skin test positivity and IgE level. These data show that GSTP1 polymorphism is strongly associated with asthma and related phenotypes, and provide an alternative explanation for the linkage of chromosome 11q13 with BHR and atopy.

The -403 G-->A promoter polymorphism in the RANTES gene is associated with atopy and asthma.

Asthma is a complex inflammatory condition often associated with bronchial hyperreactivity and atopy. Genetic and environmental factors are implicated and several candidate genes have been implicated. Of these, the chemokine RANTES is responsible for the recruitment of inflammatory cells such as eosinophils and T-lymphocytes. We have recently identified a polymorphism within the RANTES promoter (-403 G-->A) and have examined its role, using a PCR-RFLP assay, in the development of atopy and asthma in 201 Caucasian subjects. Atopic status was determined using skin prick testing and serum IgE levels. Severity of airway dysfunction was assessed using spirometric measurement (FEV1) and methacholine challenge (PC20). The -403 A allele was associated with an increased susceptibility to both atopy and asthma. Thus, the proportion of subjects carrying this allele was higher in each of atopic non-asthmatics, non-atopic asthmatics and atopic asthmatics compared with non-atopic, non-asthmatic controls. In particular, this allele was associated with skin test positivity but not IgE level. Homozygosity for the -403 A allele conferred a 6.5-fold increased risk of moderate/severe airway obstruction (FEV1 < or = 80% predicted), a marker for established asthma. Our data, whilst preliminary, indicate that the association of RANTES genotype with both atopy and asthma reflect independent effects, suggesting different mechanisms for the role of this chemokine in atopy and development of airway obstruction.

Early detection of ozone-induced hydroperoxides in epithelial cells by a novel infrared spectroscopic method.

In the lower atmosphere ozone is a toxic and an unwanted oxidising pollutant causing injury to the airway epithelial cells by lipid peroxidation to yield products such as phospholipid hydroperoxides (PLHP). Measurements of PLHP, which are primary oxidation products, may reflect an early susceptibility of the target cell to oxidative stress. Biphasic cultures of bronchial epithelial cells (BEAS-2B) were exposed to ozone at environmentally relevant concentrations (0.1-1.0 ppm) for 4 and 12 h. Detection of PLHP was made using a novel technique based on fourier transform infrared spectroscopy (FTIR) in combination with high performance thin-layer chromatography (HPTLC). Six phospholipids were identified on the HPTLC plate; lysophosphatidylcholine (LPC), sphingomyelin (SM), phosphatidylcholine (PC), lysophosphatidylethanolamine (LPE), phosphatidylinositol (PI), and phosphatidylethanolamine (PE). From the FTIR spectra, O-O stretching of hydroperoxides was identified in the range 890-820cm(-1). Multivariate data analysis revealed a positive correlation (r = 0.99 for 4 h exposure and r = 0.98 for 12h exposure) between ozone exposure levels and the region of the FTIR-spectrum comprising the main wavelengths for hydroperoxides. These data support this alternative, versatile and novel spectroscopic approach for the early detection of ozone-mediated damage in human airway epithelial cells.

Enhanced insulin-like growth factor binding protein-related protein 2 (Connective tissue growth factor) expression in patients with idiopathic pulmonary fibrosis and pulmonary sarcoidosis.

Connective tissue growth factor is a recently described chemoattractant and fibroblast mitogen which, because of sequence homology and weak binding to insulin-like growth factor (IGF)-1, has been proposed as the eighth member of the IGF binding protein (IGFBP) superfamily, named IGFBP-related protein 2 (IGFBP-rP2). Previous studies have implicated IGFBP-rP2 in a number of heterogeneous fibrotic pathologies, including renal fibrosis, dermal scleroderma, and bleomycin-induced pulmonary fibrosis in mice. Because profibrogenic cytokines may be produced by inflammatory cells, we developed a multiplex competitive reverse transcription/polymerase chain reaction to quantify IGFBP-rP2 transcripts in bronchoalveolar lavage cells from healthy subjects and patients with idiopathic pulmonary fibrosis (IPF) and pulmonary sarcoidosis. IGFBP-rP2 messenger RNA expression was enhanced > 10-fold (P < 0.003) in patients with IPF; > 40-fold (P < 0.006) in stage I/II sarcoidosis patients, and > 90-fold (P < 0.005) in stage III/IV sarcoidosis patients by comparison with healthy nonsmoking control subjects. We suggest these increases are predominantly associated with lymphocyte- and neutrophil-driven IGFBP-rP2 production. These findings, together with previous reports implicating other IGFBPs in the pathogenesis of pulmonary fibrosis, suggest that the complex network of IGFBPs within the human lung is an important determinant of the outcome of the fibroproliferative response to injury.

Th2 cytokines exert a dominant influence on epithelial cell expression of the major group human rhinovirus receptor, ICAM-1.

Intercellular adhesion molecule (ICAM)-1 is a cell receptor important in both human rhinovirus (HRV) attachment and immune effector cell mobilization. The level of expression of ICAM-1 by epithelial cells (EC) therefore plays a crucial role in the intricate biological phenomena underlying viral binding, host infection and consequent inflammatory events. As T-helper (Th)2 lymphocytes predominate within the asthmatic airway, the influence was evaluated of Th2-associated mediators in the modulation of ICAM-1 expression on uninfected and HRV-infected EC. H292 EC were cultured in vitro, with varying concentrations of interleukin (IL)-4, IL-5, IL-10 and IL-13 for 24 h and then infected with live HRV-14. Surface ICAM-1 expression was assessed by immunocytochemistry. Infection with HRV-14 resulted in a twofold increase in ICAM-1 expression. IL-4, IL-5, IL-10 and IL-13 produced a 2.7-5.1-fold enhancement of ICAM-1 expression of uninfected cells and caused approximately a further twofold increase in infected cells over the expression induced by HRV infection itself. Interferon-gamma in combination with each Th2-associated cytokine only slightly reduced, but did not override, the Th2-induced level of ICAM-1 expression on both uninfected and virus-infected EC. These data suggest that the effects of Th2-associated cytokines on intercellular adhesion molecule-1 expression and recovery of infectious virus are dominant over the effects of the Th1-associated cytokines such as interferon-gamma. Since the airway mucosa in atopic asthma is predominantly infiltrated by Th2 lymphocytes, these results could explain both the increased susceptibility to human rhinovirus infection in asthmatic patients and the associated exacerbation of asthma symptoms.

Expression of insulin-like growth factor binding proteins in bronchoalveolar lavage fluid of patients with pulmonary sarcoidosis.

Pulmonary sarcoidosis involves development of parenchymal granulomata that usually resolve spontaneously; however, it remains unclear what pathogenic mechanisms are responsible for the progression to local or diffuse fibrosis with irreversible lung remodeling that occurs in 20% of patients. Alveolar macrophages have a pivotal role in sarcoidosis, releasing mediators including insulin-like growth factor (IGF)-1, a potent profibrogenic molecule. IGF-1 bioavailability in the lung is dependent on at least six high-affinity IGF-binding proteins (IGFBP), which mainly inhibit IGF-1 action. We have investigated their presence in patients with established stage III sarcoidosis to determine whether IGF-1 and IGFBP contribute to the fibrogenic process in these patients and as such contribute to the (clinical) progression of the disease. The fibroblast mitogenic potential of bronchoalveolar lavage fluid (BALF) was more than 3-fold higher (P < 0.005) in sarcoid patients. Sarcoid BALF-induced activity could be inhibited (P < 0.0005) by neutralizing antibodies to IGF-1. We established the IGFBP profile of BALF with Western ligand analysis and quantified expression of IGFBP-3 by immunoblotting. IGFBP-2 and IGFBP-4 predominate in normal and sarcoid BALF, but IGFBP-3 occurs only as a modified, smaller, 29-kD form, expression of which was raised (P < 0.003) in sarcoid patients. Gene expression of IGF-1 and IGFBP-3 was demonstrated by reverse transcription-polymerase chain reaction in BAL cells. Thus, local production of pro-fibrogenic IGF-1 may be subject to substantial post-translational regulation by associated IGFBP and IGFBP proteases that may contribute to enhanced fibrogenesis in sarcoidosis patients with evidence of progression or (development) of fibrosis.

Effect of oral antibiotics on lung mucociliary clearance during exacerbation of chronic obstructive pulmonary disease.

It has been well established that lung mucociliary clearance is depressed in patients with chronic obstructive pulmonary disease. This study examines whether oral antibiotics have a detectable effect on this clearance mechanism during exacerbation in patients with such disease. Twelve patients with a mean +/- SE age of 63 +/- 2 years participated in a randomized, double-blind, parallel group study to assess the effect of 1 week of treatment with amoxycillin (500 mg t.d.s.) or ciprofloxacin (500 mg b.d.) on lung mucociliary clearance during exacerbation. Lung mucociliary clearance rates were measured by a non-invasive radioaerosol technique. Both drugs on average resulted in small, non-significant, enhancement of mucociliary clearance. Following treatment, the numbers of coughs were reduced in both groups and significantly (P < 0.05) after treatment with ciprofloxacin. Sputum production was also significantly reduced (P < 0.01) in both groups. The magnitude of improvement in lung mucociliary clearance was relatively modest following 1 week of treatment with either antibiotic. Since the number of coughs was significantly less after ciprofloxacin treatment the measured enhancement of lung mucociliary transport is probably, however, an underestimate.

A biological model to explain the association between human rhinovirus respiratory infections and bronchial asthma.

Human rhinoviruses (HRVs) are a frequent cause or upper respiratory tract infections in children and adults, and can exacerbate existing pulmonary disease. The major group of HRV attach to the receptor intercellular adhesion molecule (ICAM)-1, which is expressed on many cell types including epithelial cells. To study the influence of biological mediators on ICAM-1 expression, and consequently HRV attachment and infection, we have established an in vitro model system to evaluate the effects or pre-exposure to different cytokines on surface expression of ICAM-1 of uninfected and HRV-14-infected epithelial cells. The results of our studies show that the cytokines interleukin (IL)-1 beta, IL-8 and tumour necrosing factor (TNF)alpha increased ICAM-1 expression on epithelial cells. Epithelial cells infected with live HRV-14 displayed a significant upregulation of ICAM-1 compared to baseline. In contrast, interferon (IFN)gamma, whilst increasing the level of ICAM-1 expression on uninfected cells, induced a marked persistent downregulation of ICAM-1 expression on HRV-infected epithelial cells. In addition, IFN gamma appeared to completely override the ICAM-1 upregulation induced by IL-1 beta, IL-8 and TNF alpha, during HRV infection. We have further demonstrated that type 2 T-helper cell (Th2)-associated cytokines, predominantly IL-13, induce a marked upregulation or epithelial cell surface ICAM-1, thus increasing cellular binding sites for HRV attachment. As the airway mucosa or asthmatic subjects is predominantly infiltrated by activated type 2 T-helper cells with a simultaneous decrease of type 1 T-helper cells, our observations could explain the increased susceptibility to human rhinovirus infection observed in asthma.