Neutrophil defensins stimulate the release of cytokines by airway epithelial cells: modulation by dexamethasone

OBJECTIVE AND DESIGN: Neutrophils may contribute to recruiting other cells to sites of inflammation by generating chemotactic signals themselves, or by stimulating other cell types to release chemoattractants such as interleukin-8 (IL-8). Recently, we demonstrated that neutrophil-derived alpha-defensins are able to increase IL-8 expression in airway epithelial cells. In addition, it has previously been reported that neutrophil elastase-induced IL-8 synthesis was insensitive to inhibition by the glucocorticoid dexamethasone. The aim of the present study was to investigate the effect of defensins on the expression of various cytokines in cultured airway epithelial cells and to examine the effect of dexamethasone on defensin-induced cytokine synthesis in these cells. METHODS: Cultures of A549 cells and primary bronchial epithelial cells (PBEC) were stimulated with defensins either alone or in the presence of dexamethasone. Supernatants were analyzed for IL-8, ENA-78, IL-6, MCP-1 and GM-CSF by ELISA. In addition, IL-8 and ENA-78 mRNA was detected by Northern blot analysis. RESULTS: Defensins increased IL-8 expression, ENA-78, MCP-1 and GM-CSF release from A549 cells, whereas in PBEC only IL-8 and IL-6 were increased. Pre-treatment with dexamethasone significantly reduced defensin-induced IL-6, IL-8 and ENA-78 synthesis in airway epithelial cells. In addition, dexamethasone also reduced the neutrophil chemotactic activity in supernatants of these cells. CONCLUSIONS: The results from the present study indicate that defensins differentially induce cytokine secretion by A549 cells and PBEC. Glucocorticoids may interfere with the defensin-induced inflammatory process by reducing defensin-induced cytokine secretion in lung epithelial cells.     

Differences between cytokine release from bronchial epithelial cells of asthmatic patients and non-asthmatic subjects: effect of exposure to diesel exhaust particles

BACKGROUND: Recent evidence suggests that the airways of asthmatics are more susceptible to adverse effects of air pollutants than the airways of non-asthmatics, but the underlying mechanisms are not clear. METHODS: We have cultured bronchial epithelial cells (HBEC) from biopsies of atopic mild asthmatic patients and non-atopic non-asthmatic subjects, and investigated constitutive and diesel exhaust particles (DEP)-induced release of several pro-inflammatory mediators. RESULTS: HBEC of asthmatic patients constitutively released significantly greater amounts of IL-8, GM-CSF and sICAM-1 than HBEC of non-asthmatic subjects. RANTES was only released by HBEC of asthmatic patients. Incubation of the asthmatic cultures with 10 micrograms/ml DEP significantly increased the release of IL-8, GM-CSF and sICAM-1 after 24 h. In contrast, only the higher concentrations of 50-100 micrograms/ml DEP significantly increased the release of IL-8 and GM-CSF from HBEC of non-asthmatics. CONCLUSIONS: These results suggest that the increased sensitivity of the airways of asthmatics to air pollutants such as DEP may, at least in part, be a consequence of greater constitutive and pollutant-induced release of specific pro-inflammatory mediators from their bronchial epithelial cells.     

Effect of ozone and nitrogen dioxide on the release of proinflammatory mediators from bronchial epithelial cells of nonatopic nonasthmatic subjects and atopic asthmatic patients in vitro

BACKGROUND: Although studies have suggested that ozone (O3) and nitrogen dioxide (NO2) may play a role in the pathogenesis of asthma, the underlying mechanisms are not clear. OBJECTIVE: We aimed to investigate the effects of O3 and NO2 on the release of IL-8, GM-CSF, RANTES, and soluble intercellular adhesion molecule 1 (sICAM-1) from human bronchial epithelial cells (HBECs) of nonatopic nonasthmatic subjects (nonasthmatic subjects) and atopic subjects with mild asthma (asthmatic subjects) in vitro. METHODS: We cultured HBECs from bronchial biopsy specimens of nonasthmatic and asthmatic subjects; exposed these for 6 hours to air, 10 to 100 ppb O3, or 100 to 400 ppb NO2; and analyzed the release of IL-8, GM-CSF, RANTES, and sICAM-1 after 24 hours' incubation. RESULTS: There was no significant difference between the constitutive release of IL-8, GM-CSF, and sICAM-1 from HBECs of asthmatic and nonasthmatic subjects. RANTES was detected only in HBECs derived from asthmatic subjects. Exposure of HBECs of asthmatic subjects to both 50 to 100 ppb O3 and 200 to 400 ppb NO2 significantly increased the release of IL-8, GM-CSF, RANTES, and sICAM-1 from these cells after 24 hours of incubation. However, 50 to 100 ppb O3 and 200 to 400 ppb NO2 led to a significant increase in release of only IL-8 and sICAM-1 from HBECs of nonasthmatic subjects after 24 hours' incubation. A comparison between the pollutant-induced release of mediators demonstrated that 100 ppb O3-induced release of GM-CSF and sICAM-1 was significantly greater in HBECs of asthmatic subjects (medians, 0.59 and 27.4 pg/microg cellular protein, respectively) than in HBECs of nonasthmatic subjects (medians, 0.27 and 14.4 pg/microg cellular protein, respectively; P < .02). CONCLUSION: These results suggest that O3 and NO2 may modulate airway diseases, such as asthma, by increasing the release of inflammatory mediators from bronchial epithelial cells and that the cells of asthmatic subjects may be more susceptible to the adverse effects of these pollutants.     

Induction of glucocorticoid receptor‐β expression in epithelial cells of asthmatic airways by T‐helper type 17 cytokines

Background Corticosteroid insensitivity in asthmatics is associated with an increased expression of glucocorticoid receptor‐β (GR‐β) in many cell types. T‐helper type 17 (Th17) cytokine (IL‐17A and F) expressions increase in mild and in difficult‐to‐treat asthma. We hypothesize that IL‐17A and F cytokines alone or in combination, induce the expression of GR‐β in bronchial epithelial cells. Objectives To confirm the expression of the GR‐β and IL‐17 cytokines in the airways of normal subjects and mild asthmatics and to examine the effect of cytokines IL‐17A and F on the expression of GR‐β in bronchial epithelial cells obtained from normal subjects and asthmatic patients. Methods The expression of IL‐17A and F, GR‐α and GR‐β was analysed in bronchial biopsies from mild asthmatics and normal subjects by Q‐RT‐PCR. Immunohistochemistry for IL‐17 and GR‐β was performed in bronchial biopsies from normal and asthmatic subjects. The expression of IL‐6 in response to IL‐17A and F and dexamethasone was determined by Q‐RT‐PCR using primary airway epithelial cells from normal and asthmatic subjects. Results We detected significantly higher levels of IL‐17A mRNA expression in the bronchial biopsies from mild asthmatics, compared with normal. GR‐α expression was significantly lower in the biopsies from asthmatics compared with controls. The expression of IL‐17F and GR‐β in biopsies from asthmatics was not significantly different from that of controls. Using primary epithelial cells isolated from normal subjects and asthmatics, we found an increased expression of GR‐β in response to IL‐17A and F in the cells from asthmatics (P0.05). This effect was only partially significant in the normal cells. Dexamethasone significantly decreased the IL‐17‐induced IL‐6 expression in cells from normal individuals but not in those from asthmatics (P0.05). Conclusion Evidence of an increased GR‐β expression in epithelial cells following IL‐17 stimulation suggests a possible role for Th17‐associated cytokines in the mechanism of steroid hypo‐responsiveness in asthmatic subjects. Cite this as: A. Vazquez‐Tello, A. Semlali, J. Chakir, J. G. Martin, D. Y. Leung, D. H. Eidelman and Q. Hamid, Clinical & Experimental Allergy, 2010 (40) 1312–1322.     

Lower airways inflammation in allergic rhinitics: a comparison with asthmatics and normal controls

BACKGROUND: Allergic rhinitis (AR) and asthma represent a continuum of atopic disease. AR is believed to pre-dispose an individual to asthma. Compared with asthmatics and normal controls, the inflammatory response in the lower airways of rhinitics is not fully elucidated. To test the hypothesis that the inflammatory response in the airways of subjects with AR is at a level intermediate between that in normal controls and asthmatics, we have characterized bronchial inflammation and cytokine mRNA levels in non-asthmatic allergic rhinitics and compared it with subjects with allergic asthma and with normal controls. METHODS: Endobronchial mucosal biopsies were obtained at bronchoscopy from 14 allergic rhinitics, 16 asthmatics and 21 normal controls. Biopsies were embedded into glycol methacrylate resin for immunohistochemical analysis of cellular inflammation and snap frozen for semi-quantitative PCR analysis of cytokine mRNA levels. RESULTS: Airway inflammation in rhinitic subjects was characterized by an increase in submucosal eosinophils, mast cells and the mRNA expression of TNF-alpha, at an intermediate level between healthy and asthmatics. In addition, CD3(+) and CD8(+) lymphocytes in the epithelium, the endothelial expression of vascular adhesion molecule-1 and IL-1 beta mRNA were higher in the allergic rhinitics compared with both normal controls and asthmatics, whereas growth-related oncogene alpha-mRNA was decreased in AR compared with both healthy and asthmatics. Airway inflammation in the asthmatic group was characterized by higher numbers of eosinophils and mast cells, together with an increase in TNF-alpha-mRNA compared with both healthy and rhinitics. IFN-gamma mRNA was the highest in normal controls and lowest in the asthmatics. CONCLUSIONS: In individuals with AR the present data suggest an intermediate state of airway inflammation between that observed in normal individuals and subjects with clinical asthma. It is also indicated that IFN-gamma production by CD8(+) T lymphocytes could be protective against the development of airway hyperresponsiveness. Further work is needed to evaluate this hypothesis.     

Exposure to systemic prednisolone for 4 hours reduces ex vivo synthesis of GM-CSF by bronchoalveolar lavage cells and blood mononuclear cells of mild allergic asthmatics

BACKGROUND: In acute severe asthma, the earliest clinical effects of glucocorticosteroids occur from 4 to 5 h after systemic administration, but the mechanisms are unclear. In persistent asthma, corticosteroids are thought to suppress airway inflammation by modulating the expression of adhesion molecules, enzymes, and leucotactic cytokines, including granulocyte-macrophage colony stimulating factor (GM-CSF). GM-CSF is also overexpressed in the airways of symptomatic asthmatics. OBJECTIVES: To examine the early effects of systemic corticosteroids on cytokine expression, we investigated whether ex vivo synthesis of GM-CSF is suppressed in the bronchoalveolar lavage (BAL) cells and peripheral blood mononuclear cells (PBMCs) of normal and mild allergic asthmatic subjects obtained 4 h after a single intravenous dose of prednisolone. METHODS: In a randomized, double-blind, placebo-controlled study, BAL cells and PBMCs were obtained from mild atopic asthmatic patients (n = 9) and normal subjects (n = 9) 4 h after an intravenous bolus dose of 80 mg prednisolone, and cultured for 0-18 h in the presence or absence of lipopolysaccharide (LPS; 10 microg/mL). Enzyme immunoassay was used to assess GM-CSF levels in BAL cell and PBMC culture supernatants, and in BAL fluid. RESULTS: After placebo, GM-CSF synthesis tended to be higher in BAL cells from asthmatics than in normals. LPS stimulation significantly increased median (interquartile range) GM-CSF synthesis by BAL cells ex vivo from 16.4 (23 to 74) to 35.8 (3-148) pg/106 cells in normals (P < 0.05), and from 59 (9 to 204) to 134 (24-288) pg/106 cells in asthmatics (P < 0.01). After intravenous prednisolone, the rise in GM-CSF production induced in BAL cells by LPS was completely abolished in both subject groups. In PBMCs of placebo-treated asthmatics (but not normals), LPS stimulated median GM-CSF synthesis from 164 (110 to 300) to 314 (235-485) pg/106 cells (P = 0.02), and this was blocked by intravenous prednisolone. CONCLUSIONS: LPS-stimulated GM-CSF synthesis ex vivo is abolished in BAL cells of mild asthmatic and normal subjects, and in PBMCs of asthmatics, obtained 4 h after a single intravenous dose of prednisolone. Suppression of GM-CSF synthesis in airway and blood leucocytes may contribute to the early clinical efficacy of systemic glucocorticoids in acute allergic asthma.     

Type III IFN-λ mRNA expression in sputum of adult and school-aged asthmatics

Background The increased susceptibility of asthmatics to rhinovirus infection has recently been related to deficient IFN-λ1 (IL-29) and IFN-λ2/3 (IL-28) production by bronchial epithelial cells and macrophages.
Objectives Here, we studied IFN-λ mRNA expression in the airways of stable asthmatics in comparison with healthy subjects and in relation to asthma symptoms, non-invasive parameters of airway inflammation and lung function parameters.
Methods Airway cells were obtained by sputum induction, in 14 healthy and 35 asthmatic adults and 12 asthmatic school-aged children. IFN-λ was studied at the mRNA level by quantitative RT-PCR.
Results Asthmatic adults have increased sputum IL-28 mRNA but similar IL-29 mRNA expression in comparison with healthy subjects. In asthmatics, both sputum IL-28 and IL-29 mRNA expression correlate with the sputum CD3γ mRNA expression (reflecting infiltrated T cells). IL-28 (but not IL-29) mRNA levels correlate with the relative and absolute number of eosinophils present in the sputum sample. Sputum IL-29 mRNA (but not IL-28) correlates negatively with asthma symptoms in steroid-naïve patients and is significantly higher in steroid-treated than in steroid-naïve patients. Finally, both IL-28 and IL-29 mRNA levels are higher in asthmatic children than in asthmatic adults.
Conclusion Our results show that asthmatic subjects have substantial type III IFN-λ mRNA levels in the airways. Our data furthermore suggest that IL-29 could have an immunoprotective role in the lower airways.     

The role of the epidermal growth factor receptor in sustaining neutrophil inflammation in severe asthma.

BACKGROUND: The extent of epithelial injury in asthma is reflected by expression of the epidermal growth factor receptor (EGFR), which is increased in proportion to disease severity and is corticosteroid refractory. Although the EGFR is involved in epithelial growth and differentiation, it is unknown whether it also contributes to the inflammatory response in asthma. OBJECTIVES: Because severe asthma is characterized by neutrophilic inflammation, we investigated the relationship between EGFR activation and production of IL-8 and macrophage inhibitory protein-1 alpha (MIP-1alpha) using in vitro culture models and examined the association between epithelial expression of IL-8 and EGFR in bronchial biopsies from asthmatic subjects. METHODS: H292 or primary bronchial epithelial cells were exposed to EGF or H2O2 to achieve ligand-dependent and ligand-independent EGFR activation; IL-8 mRNA was measured by real-time PCR and IL-8 and MIP-1alpha protein measured by enzyme-linked immunosorbent assay (ELISA). Epithelial IL-8 and EGFR expression in bronchial biopsies from asthmatic subjects was examined by immunohistochemistry and quantified by image analysis. RESULTS: Using H292 cells, EGF and H2O2 increased IL-8 gene expression and release and this was completely suppressed by the EGFR-selective tyrosine kinase inhibitor, AG1478, but only partially by dexamethasone. MIP-1alpha release was not stimulated by EGF, whereas H2O2 caused a 1.8-fold increase and this was insensitive to AG1478. EGF also significantly stimulated IL-8 release from asthmatic or normal primary epithelial cell cultures established from bronchial brushings. In bronchial biopsies, epithelial IL-8, MIP-1alpha, EGFR and submucosal neutrophils were all significantly increased in severe compared to mild disease and there was a strong correlation between EGFR and IL-8 expression (r = 0.70, P < 0.001). CONCLUSIONS: These results suggest that in severe asthma, epithelial damage has the potential to contribute to neutrophilic inflammation through enhanced production of IL-8 via EGFR- dependent mechanisms.     

House dust mite allergen exerts no direct proinflammatory effects on human keratinocytes

BACKGROUND: Dermatophagoides pteronyssinus is a trigger of atopic dermatitis. Many D pteronyssinus allergens are proteases that can elicit airway inflammation by stimulating the release of cytokines and chemokines by bronchial epithelial cells. OBJECTIVE: We sought to investigate whether D pteronyssinus allergens can exert a similar activity on skin keratinocytes. METHODS: Primary cultures of keratinocytes from healthy subjects or patients with atopic dermatitis and normal human bronchial epithelial cells were compared for cytokine production in response to D pteronyssinus extract. RESULTS: Keratinocytes, but not bronchial epithelial cells, displayed a modest dose-dependent release of IL-1alpha and IL-1 receptor antagonist but no induction of their mRNA after exposure to D pteronyssinus. However, D pteronyssinus also degraded these cytokines. On the other hand, D pteronyssinus extract induced bronchial epithelial cells, but not keratinocytes, to increased expression of IL-8/CXCL8 and GM-CSF mRNA and protein. These effects were efficiently abrogated by a mixture of cysteine and serine protease inhibitors. Both IL-8 and GM-CSF were fully resistant to D pteronyssinus proteolytic attack. No induction of monocyte chemoattractant protein 1/CCL2, RANTES/CCL5, or IFN-gamma-induced protein of 10 kd/CXCL10 was detected in either cell type. Only bronchial epithelial cells expressed protease-activated receptor (PAR) 4 mRNA, whereas PAR-1, PAR-2, and PAR-3 mRNA was found in both cell types. D pteronyssinus did not affect PAR mRNA signals. CONCLUSIONS: Although D pteronyssinus can cause proteolysis-dependent release of cytokines from keratinocytes, it appears incapable of activating de novo expression of cytokines and chemokines, arguing against a direct proinflammatory activity of house dust mite on the skin.     

Cytokine production from sputum cells after allergenic challenge in IgE-mediated asthma

BACKGROUND: Th2 cytokine production from airway cells is thought to govern the eosinophilic airways inflammation in allergic asthma. Induced sputum has become a widely used technique to assess airways inflammation. METHODS: By applying the technique of induced sputum to collect airways cells, we have assessed the spontaneous production of a set of cytokines, including interleukin-4, 6, 10, interferon-gamma and tumour necrosis factor-alpha, 6 h after a bronchial allergenic challenge with Dermatophagoides pteronyssinus (Dpt) in 12 sensitized asthmatics and compared the results obtained after inhalation of saline as control. A group of eight healthy non-allergic subjects was enrolled to control for any non-specific effect of Dpt. Cytokines were measured by a dynamic immunoassay during a 24-h sputum cell culture. RESULTS: Allergen challenge in sensitized asthmatics caused an acute and a late bronchospasm together with a rise in sputum eosinophil counts. Afterwards allergen sputum cells from allergic asthmatics displayed a rise in their production of IL-4 (P < 0.01), IL-6 (P < 0.05) and IL-10 (P < 0.05) when compared to saline. By this time sputum generation of IL-4 in atopic asthmatics was greater than in healthy subjects (P < 0.001). Furthermore, in allergic asthmatics there was a strong correlation between the rise in interleukin-4 production from sputum cells and the rise in sputum eosinophils (r = 0.87, P < 0.001). CONCLUSIONS: Sputum cell culture is a useful model to assess cytokine production in allergic asthmatics who show a marked up-regulation of Th2 cytokines following acute allergen exposure. The rise in sputum eosinophil count following allergen challenge strongly correlates with the rise in IL-4 generation from sputum cells.     

Effect of ozone and nitrogen dioxide on the permeability of bronchial epithelial cell cultures of non-asthmatic and asthmatic subjects

BACKGROUND: Although epidemiological as well as in vivo exposure studies suggest that ozone (O3) and nitrogen dioxide (NO2) may play a role in airway diseases such as asthma, the underlying mechanisms are not clear. OBJECTIVE: Our aim was to investigate the effect of O3 and NO2 on the permeability of human bronchial epithelial cell (HBEC) cultures obtained from non-atopic non-asthmatic (non-asthmatics) and atopic mild asthmatic (asthmatics) individuals. METHODS: We cultured HBECs from bronchial biopsies of non-asthmatics and asthmatics, and exposed these for 6 h to air, 10 to 100 parts per billion (p.p.b.) O3, or to 100 to 400 p.p.b. NO2, and assessed changes in electrical resistance (ER) and movement of 14C-BSA across the cell cultures. RESULTS: Although exposure to either O3 or NO2 did not alter the permeability of HBEC cultures of non-asthmatics, 10 to 100 p.p.b. O3 and 400 p.p.b. NO2 significantly decreased the ER of HBEC cultures of asthmatics, when compared with exposure to air. Additionally, 10, 50 and 100 p.p.b. O3 led to a significant increase in the movement of 14C-BSA across asthmatic HBEC cultures, after 6 h of exposure (medians = 1.73%; P < 0.01, 1.50%; P < 0.05 and 1.53%, P < 0.05, respectively), compared with air exposed cultures (median = 0.89%). Similarly, exposure for 6 h to both 200 and 400 p.p.b. NO2 significantly increased the movement of 14C-BSA across asthmatic HBEC cultures, when compared with air exposure. A comparison of data obtained from the two study groups demonstrated that 10 to 100 p.p.b. O3- and 200 to 400 p.p.b. NO2-induced epithelial permeability was greater in cultures of asthmatics compared with non-asthmatics. CONCLUSION: These results suggest that HBECs of asthmatics may be more susceptible to the deleterious effects of these pollutants. Whether in patients with asthma the greater susceptibility of bronchial epithelial cells to O3 and NO2 contributes to the development of the disease, or is a secondary characteristic of this condition, remains to be determined.     

Transforming growth factor-beta2 induces bronchial epithelial mucin expression in asthma

The transforming growth factor (TGF)-beta family is important for tissue repair in pathological conditions including asthma. However, little is known about the impact of either TGF-beta1 or TGF-beta2 on asthmatic airway epithelial mucin expression. We evaluated bronchial epithelial TGF-beta1 and TGF-beta2 expression and their effects on mucin expression, and the role of TGF-beta1 or TGF-beta2 in interleukin (IL)-13-induced mucin expression. Epithelial TGF-beta1, TGF-beta2, and mucin expression were evaluated in endobronchial biopsies from asthmatics and normal subjects. The effects of TGF-beta1 or TGF-beta2 on mucin MUC5AC protein and mRNA expression, and the impact of IL-13 on epithelial TGF-beta1, TGF-beta2, and MUC5AC were determined in cultured bronchial epithelial cells from endobronchial brushings of both subject groups. In biopsy tissue, epithelial TGF-beta2 expression levels were higher than TGF-beta1 in both asthmatics and normals. TGF-beta2, but not TGF-beta1, was increased in asthmatics compared with normals, and significantly correlated with mucin expression. TGF-beta2, but not TGF-beta1, increased mucin expression in cultured epithelial cells from both subject groups. IL-13 increased the release of TGF-beta2, but not TGF-beta1, from epithelial cells. A neutralizing TGF-beta2 antibody partially inhibited IL-13-induced mucin expression. These data suggest that TGF-beta2 production by asthmatic bronchial epithelial cells may increase airway mucin expression. IL-13-induced mucin expression may occur in part through TGF-beta2 up-regulation.     

Regulation of IL-5 and IL-5 receptor expression in the bone marrow of allergic asthmatics

BACKGROUND: Following consistent demonstrations of the clinical relevance of fluctuations in eosinophil-basophil (Eo-B) progenitors in the blood of patients with a variety of allergic airway disorders, we have turned our attention recently to hemopoietic events occurring in the bone marrow of allergic asthmatic subjects, utilizing a model of airway allergen challenge. METHODS: Flow-cytometric analyses of CD34/45+ progenitors for coexpression of surface alpha-receptor subunits for IL-3, IL-5 and GM-CSF, as well as in situ hybridization and in situ PCR methodologies to detect mRNA for IL-5 and GM-CSF in developing Eo-B in colony and liquid culture assays were employed before and after in vivo allergen challenge. RESULTS: An early, specific upregulation of IL-5R alpha expression on CD34/45 progenitors was observed after allergen challenge, concomitant with the development of the late-phase asthmatic response. Protein and mRNA for both GM-CSF and IL-5 were expressed in a time-dependent manner ex vivo, in developing (beta 7-integrin-positive), colony-derived Eo-B after allergen challenge in vivo. Both retinoic acid and corticosteroids were able to downregulate IL-3- and IL-5-induced expression of IL-5R on cord-blood-derived as well as HL-60 cloned Eo-B progenitors. CONCLUSION: These studies indicate the critical involvement of IL-5 and IL-5R in the induction of Eo-B differentiation and eosinophilic airway inflammation in allergic asthmatics, and point to these events as potential targets for long-term therapy of atopic disease.     

Cytokine pattern in allergic and non-allergic chronic rhinosinusitis in asthmatic children

BACKGROUND: Rhinosinusitis represents one of the most common chronic diseases. The association of rhinosinusitis with asthma has been frequently reported. Eosinophils and Th2 cells play a pathogenic mechanism in asthma. OBJECTIVE: The aims of the study were to evaluate the cytokine pattern in chronic rhinosinusitis in asthmatic children and to compare the findings in allergic vs. non-allergic asthmatics. METHODS: Thirty-five asthmatic children were evaluated, 19 males and 16 females, with an average age of 8.7 years. All children were asthmatic and suffered from chronic rhinosinusitis. Twenty were allergic and 15 were non-allergic. Ten healthy children were studied as normal controls. Evaluated parameters were the levels of the following cytokines: IL-1beta, IL-4, IL-6, IL-8, IL-12, IFN-gamma and TNF-alpha. Cytokines were recovered from rhinosinusal lavage and measured by immunoassays. Nasal cytology was also performed in all subjects and inflammatory cells were counted by conventional staining. RESULTS: Allergic subjects showed a significant increase of IL-4 (P < 0.01) and TNF-alpha (P < 0.05) and a significant decrease of IL-12 (P < 0.05) and of IFN-gamma (P < 0.0001), whereas IL-1beta, IL-6 and IL-8 were not significantly increased. Non-allergic children showed a significant increase of IL-4 (P < 0.05) and a significant decrease of IFN-gamma (P < 0.0001), IL-12 was not significantly decreased, and IL-1beta, IL-6 and IL-8 were not significantly increased. A significant inflammatory infiltrate was present in all asthmatic children. Significant correlations were demonstrated between IL-4 and IL-12 (P < 0.001), IL-12 and IFN-gamma (P < 0.001), IL-8 and neutrophils (P < 0.01), and TNF-alpha and monocytes/macrophages (P < 0.05), in allergic asthmatics. IL-4 and IL-12 were significantly correlated (P < 0.05) as well as IL-8 and neutrophils (P < 0.01) in non-allergic asthmatics. CONCLUSION: This study shows that allergic asthmatic children with chronic rhinosinusitis have a typical Th2 cytokine pattern, but also non-allergic asthmatic children share a similar pattern. These findings would suggest the existence of a common pathophysiological mechanism shared by upper and lower airways and are consistent with the concept of united airways disease.     

Parental tobacco smoking is associated with augmented IL-13 secretion in children with allergic asthma

BACKGROUND: Exposure to environmental tobacco smoke (ETS) has been shown to increase symptoms of allergic bronchial asthma, but direct effects on the expression of inflammatory markers have not been demonstrated thus far. OBJECTIVE: The aim of this study was to assess the correlation of ETS exposure with the expression of proinflammatory mediators in airway secretions, including IFN-gamma and IL-12, as well as IL-5 and IL-13, in allergic asthmatic schoolchildren and healthy control subjects. METHODS: By using the nasopharyngeal aspiration technique, airway secretions were collected from 24 atopic children with asthma (age, 6-16 years) and 26 healthy control subjects, and the concentration of cytokines was measured with immunoenzymatic methods. RESULTS: IL-13 levels were highly increased in patients with asthma (P < .005), and parental tobacco smoke resulted in a significant increase in airway IL-13 secretion in these children compared with that seen in nonexposed children and healthy control subjects (median, 860 pg/mL vs 242 pg/mL and 125 pg/mL, respectively). Furthermore, a positive correlation between IL-13 levels and serum IgE concentrations (r(s) = 0.55) was found in children with allergic asthma. CONCLUSIONS: These results indicate that ETS augments the expression and secretion of IL-13 in allergic asthma and that nasopharyngeal aspiration is a suitable method to assess cytokine measurements in airways in children. Measurements of IL-13 in secretions might be taken into account as a noninvasive marker of airway inflammation and to assess the detrimental effects of ETS.     

Interleukin-1 binds to specific receptors on human bronchial epithelial cells and upregulates granulocyte/macrophage colony-stimulating factor synthesis and release.

Cultured human bronchial epithelial cells constitutively produce granulocyte/macrophage colony-stimulating factor (GM-CSF). An upregulation of the synthesis and release of GM-CSF from those cells might contribute to the persistence of infiltration and local activation of inflammatory cells in some inflammatory diseases of the airways, such as asthma. Increased levels of immunoreactive and biologically active interleukin-1 (IL-1) have been identified in the airway secretions of asthmatic patients, together with an increase in GM-CSF contents. As IL-1 is known to upregulate GM-CSF production in many cell populations, in this study we investigated the ability of IL-1 to bind to specific receptors on bronchial epithelial cells and promote GM-CSF synthesis and release. Bronchial epithelial cells possessed specific single-class surface receptors for recombinant IL-1. The addition of exogenous IL-1 led to a dose-dependent increase in the accumulation of GM-CSF mRNA and release of immunoreactive GM-CSF to the culture medium. Release of IL-1 in the bronchial mucosa during allergic and nonallergic responses may lead to enhanced GM-CSF synthesis and release by epithelial cells, thus promoting airway inflammation.     

CYTOKINE PRODUCTION BY CELL CULTURES FROM BRONCHIAL SUBEPITHELIAL MYOFIBROBLASTS

Myofibroblasts have been previously described beneath the bronchial epithelium and were found to increase in number proportional to the accumulation of extracellular matrix in the bronchial lamina reticularis in asthma. The aim of this study was to assess further the contribution of these structural cells to allergic inflammation in the bronchial mucosa through their cytokine expression. Cell cultures were established from the lamina reticularis of human bronchial biopsies from asthmatic and non-asthmatic subjects. Cytokine secretion was measured by ELISA in supernatants of cultures with or without tumour necrosis factor-α (TNF- α). The mRNA levels for granulocyte–macrophage colony-stimulating factor (GM-CSF) in the cultures were examined by ribonuclease protection assays (RPAs). Bronchial myofibroblasts grown from bronchial biopsies were capable of producing GM-CSF, interleukin-6 (IL-6), interleukin-8 (IL-8), and stem cell factor (SCF) constitutively. The GM-CSF production by myofibroblasts was significantly increased in response to TNF-α simulation with a corresponding increase in GM-CSF mRNA expression. The enhancement of GM-CSF production by TNF-α in myofibroblasts was blocked by the inhibition of RNA synthesis. Prednisolone abolished the GM-CSF production. This study provides evidence for the role of bronchial myofibroblasts in the regulation of inflammatory cell recruitment and activation by interaction in the cytokine network in the bronchial mucosa.