Search

Severe or refractory asthma affects 5 to 15% of all patients with asthma, but is responsible for more than half of the health burden associated with the disease. Severe asthma is characterized by a dramatic increase in smooth muscle and airway inflammation. Although glucocorticoids are the mainstay of treatment in asthma, they are unable to fully control the disease in individuals with severe asthma. We found that airway smooth muscle cells (ASMCs) from individuals with severe asthma showed elevated activities of the ERK1/ERK2 and p38 MAPK pathways despite treatment with oral and inhaled glucocorticoids, which increased the expression of DUSP1, a phosphatase shown to limit p38 MAPK activity. In ex vivo ASMCs, TNF-α but not IL-17A induced expression of the neutrophil chemoattractant CXCL8. Moreover, TNF-α led to up-regulation of the ERK1/ERK2 and p38 MAPKs pathways, with only the latter being sensitive to pretreatment with the glucocorticoid dexamethasone. In contrast to epithelial and endothelial cells, TNF-α-stimulated CXCL8 synthesis was dependent on ERK1/ERK2 but not on p38 MAPK. Moreover, suppressing ERK1/ERK2 activation prevented neutrophil recruitment by ASMCs, whereas suppressing p38 MAPK activity had no impact. Taken together, these results highlight the ERK1/ERK2 MAPK cascade as a novel and attractive target in severe asthma because the activation of this pathway is insensitive to the action of glucocorticoids and is involved in neutrophil recruitment, contributing the to inflammation seen in the disease.

TGF-β is one of the main mediators involved in tissue remodeling in the asthmatic lung. This profibrotic cytokine is produced by a number of cells, including macrophages, epithelial cells, fibroblasts, and eosinophils. High expression of TGF-β in patients with asthma was reported by many investigators. However, controversy remains whether the concentration of TGF-β correlates with disease severity. TGF-β is believed to play an important role in most of the cellular biological processes leading to airway remodeling. It was shown to be involved in epithelial changes, subepithelial fibrosis, airway smooth muscle remodeling, and microvascular changes. Here, sources of TGF-β, as well as its role in the development of airway remodeling, will be reviewed. Therapeutic strategies that modulate TGF-β will also be discussed.

The leukotriene modifiers are a novel generation of therapeutic agents in the treatment of allergic asthma. However, the mechanisms by which the cysteinyl (cys) leukotrienes (LTs) participate in allergen-induced airway eosinophilia and airway hyperresponsiveness (AHR) are still unclear. In the present study, we have investigated the role of cys-LTs in ovalbumin (OVA)-induced airway responses in a murine model of asthma. Montelukast (3 or 10 mg/kg), a selective cys-LT1 receptor antagonist, reduced airway eosinophilia and AHR after OVA challenge. The levels of interleukin (IL)-5 and eotaxin in the bronchoalveolar lavage fluid (BALF) from montelukast-treated (3 mg/kg) mice were unaffected, although a decrease in IL-5 was observed with a dose of 10 mg/kg. LTD4 (50 ng) instilled intranasally to immunized mice augmented macrophages in the BALF, but in conjunction with OVA challenge it caused BALF eosinophilia and neutrophilia when given before challenge and BALF neutrophilia but not eosinophilia when given 2 h after challenge. However, there were no increases of IL-5 or eotaxin in BALF following LTD4 treatment. Repeated instillations of LTD4 to immunized mice, mimicking allergen challenge, did not induce AHR but in conjunction with OVA challenge LTD4 enhanced AHR. These results indicate that allergen-induced eosinophilia and AHR are in part mediated by the cys-LT1 receptor, and that, although LTD4 alone has no effect on airway eosinophilia, in conjunction with antigenic stimulation it potentiates the degree of airway inflammation and AHR.

We studied the role of interleukin (IL)-4, IL-5, and allergen-specific immunoglobulin (Ig) E in the development of allergen-induced sensitization, airway inflammation, and airway hy-perresponsiveness (AHR). Normal, IL-4-, and IL-5-deficient C57BL/6 mice were sensitized intraperitoneally to ovalbumin (OVA) and repeatedly challenged with OVA via the airways. After allergen sensitization and airway challenge, normal and IL-5-deficient, but not IL-4-deficient, mice developed increased serum levels of total and antigen-specific IgE levels and increased IL-4 production in the lung tissue compared with nonsensitized control mice. Only normal mice showed significantly increased IL-5 production in the lung tissue and an eosinophilic infiltration of the peribronchial regions of the airways, whereas both IL-4- and IL-5-deficient mice had little or no IL-5 production and no significant eosinophilic airway inflammation. Associated with the inflammatory responses in the lung, only normal mice developed increased airway responsiveness to methacholine after sensitization and airway challenge; in both IL-4- and IL-5-deficient mice, airway responsiveness was similar to that in nonsensitized control mice. Reconstitution of sensitized, IL-4-deficient mice before allergen airway challenge with IL-5, but not with allergen-specific IgE, restored eosinophilic airway inflammation and the development of AHR. These data demonstrate the importance of IL-4 for allergen-driven airway sensitization and that IL-5, but not allergen-specific IgE, is required for development of eosinophilic airway inflammation and AHR after this mode of sensitization and challenge.

Interleukin (IL)-9 has recently been shown to play an important role in allergic disease because its expression is strongly associated with the degree of airway responsiveness and the asthmatic-like phenotype. IL-9 is a pleiotropic cytokine that is active on many cell types involved in the allergic immune response. Mucus hypersecretion is a clinical feature of chronic airway diseases; however, the mechanisms underlying the induction of mucin are poorly understood. In this report, we show that IL-9 regulates the expression of a subset of mucin genes in lung cells both in vivo and in vitro. In vivo, the constitutive expression of IL-9 in transgenic mice results in elevated MUC2 and MUC5AC gene expression in airway epithelial cells and periodic acid-Schiff-positive staining (reflecting mucous glycogenates). Similar results were observed in C57BL/6J mice after IL-9 intratracheal instillation. In contrast, instillation of the T helper 1-associated cytokine interferon gamma failed to induce mucin production. In vitro, our studies showed that IL-9 also induces expression of MUC2 and MUC5AC in human primary lung cultures and in the human muccoepidermoid NCI-H292 cell line, indicating a direct effect of IL-9 on inducing mucin expression in these cells. Altogether, these results suggest that upregulation of mucin by IL-9 might contribute to the pathogenesis of human inflammatory airway disorders, such as asthma. These data extend the role of the biologic processes that IL-9 has on regulating the many clinical features of asthma and further supports the IL-9 pathway as a key mediator of the asthmatic response.

Eosinophil differentiation is thought to occur by the action of interleukin (IL)-5 on CD34(+) progenitor cells. The allergen-induced increase in eosinophil numbers in isolated airway preparations in vitro, and detection of increased numbers of circulating CD34(+) cells in atopic subjects, led us to the hypothesis that the eosinophil infiltration of the airway in asthma may result from local mucosal differentiation, in addition to recruitment from the bone marrow. We examined CD34(+) cell numbers by immunohistochemistry and IL-5 receptor alpha (IL-5Ralpha) messenger RNA (mRNA) expression by in situ hybridization in bronchial biopsies from atopic asthmatic patients, and from atopic and nonatopic control subjects. CD34(+) cell numbers were increased in the airway in atopic asthmatic and atopic nonasthmatic subjects. In contrast, CD34(+)/ IL-5Ralpha mRNA+ cells were increased in asthmatic subjects when compared with both atopic and nonatopic control subjects. Airway numbers of CD34(+)/IL-5Ralpha mRNA+ cells were correlated to airway caliber in asthmatic subjects and to eosinophil numbers. These findings support the concept that eosinophils may differentiate locally in the airway in asthma.

We have recently shown the increased mRNA expression of interleukin (IL)-4 and IL-13 in sinus biopsies from allergic subjects with chronic sinusitis (ACS), whereas only IL-13 mRNA was elevated in biopsies obtained from nonallergic subjects with chronic sinusitis (NCS). In the lymph nodes and spleen, these cytokines may promote IgE production through transcriptional activation of the germline IgE heavy chain promoter, an event which precedes immunoglobulin isotype switching to IgE in B cells. We hypothesized that local expression of IL-4 and/or IL-13 might act by inducing germline IgE heavy chain transcript expression locally in the sinus mucosa of chronic sinusitis patients. Mucosal sinus biopsies were obtained from 13 patients with ACS, 12 subjects with NCS, and 11 normal control individuals. The numbers of B cells in the sinus mucosa were studied by immunocytochemistry with anti-CD20 monoclonal antibodies. In situ hybridization was performed using antisense radiolabeled riboprobes complementary to the IgE epsilon -heavy chain germline (Iepsilon) and heavy chain constant region (Cepsilon) gene transcripts. Riboprobes specific for the IgG gamma-heavy chain constant region (Cgamma) were used as an isotype control. Immunocytochemical analysis indicated augmented numbers of CD20-positive B cells in the biopsies obtained from ACS patients compared with NCS subjects (P < 0.05) and normal control subjects (P < 0.01). Statistically significant increases were observed in the numbers of cells expressing Iepsilon and Cepsilon transcripts in the sinus mucosa of ACS patients compared with those with NCS (P < 0. 001) and normal controls (P < 0.001), while Cgamma RNA expression did not differ significantly between the groups. In three randomly selected ACS biopsies, 92-100% of cells expressing Cepsilon transcripts and 100% of Iepsilon RNA-positive cells coexpressed CD20 immunoreactivity. Cells expressing Cepsilon transcripts were also significantly increased in NCS compared with normal controls (P < 0. 05). The results of this study suggest that local IgE class switching occurs in the pathogenesis of ACS and that ACS and NCS are both associated with increased expression of Cepsilon transcripts.

The histopathology of bronchial asthma is associated with structural changes within the airways, including subepithelial fibrosis, as well as chronic eosinophilic inflammation. The mechanisms responsible for this tissue remodeling, and in particular the role of inflammatory cells, remain to be established. Transforming growth factor-beta (TGF-beta) is a potent profibrotic cytokine which may contribute to the thickening of the reticular lamina by the deposition of collagen fibers. To investigate the molecular mechanisms underlying these structural changes, we have investigated the expression of TGF-beta1 mRNA and immunoreactivity within the bronchial mucosa of mild to severe asthmatic individuals and normal control subjects using the techniques of in situ hybridization and immunocytochemistry. As eosinophils are prominent within the asthmatic airway and are known to synthesize pro-inflammatory cytokines, the presence of TGF-beta1 mRNA and immunoreactive protein in eosinophils was also examined. Asthmatic individuals exhibited a greater expression of TGF-beta1 mRNA and immunoreactivity in the airways submucosa than normal control subjects (P < 0.05), and these increases were directly related to the severity of the disorder. The extent of airways fibrosis, as detected histochemically, was also increased in asthmatics compared with normal control subjects (P < 0.005). In asthmatic subjects, the presence of subepithelial fibrosis was associated with the severity of the disease and correlated with the decline in forced expiratory volume in 1 s (r2 = 0.78; P < 0.05). Within the asthmatic airways, EG2-positive eosinophils represented the major source of TGF-beta1 mRNA and immunoreactivity. These results provide evidence that TGF-beta1 may play a role in the fibrotic changes occurring within asthmatic airways and that activated eosinophils are a major source of this cytokine.

Asthma is characterized by the presence of activated CD4+ cells in the airways. We hypothesized that the newly characterized cytokine interleukin (IL)-16 is involved in the pathogenesis of asthma through its ability to selectively induce CD4+ cell recruitment within the inflamed bronchial wall. We investigated the expression of IL-16 in bronchial biopsies obtained from subjects with mild asthma (n = 10), atopic nonasthmatic individuals (n = 6), and normal control subjects (n = 10). Cryostat sections from 4% paraformaldehyde-fixed fiberoptic bronchial biopsies were immunostained using a specific antibody that recognizes human IL-16. IL-16 mRNA expression was determined by in situ hybridization. IL-16 immunoreactivity and mRNA were demonstrated mainly in bronchial epithelial cells in all subjects. IL-16 immunoreactivity and IL-16 mRNA expression within the epithelium were significantly higher in bronchial biopsies obtained from asthmatic subjects as compared to both atopic nonasthmatic and normal controls (P < 0.001). The numbers of subepithelial IL-16 immunoreactive cells and IL-16 mRNA-positive cells were also greater in the bronchial biopsies obtained from asthmatic subjects as compared to both atopic nonasthmatic and normal controls (P < 0.001). Epithelial expression of IL-16 immunoreactivity and mRNA correlated with the CD4+ cell infiltration (r2 = 0.70, P < 0.001). There were significant associations between epithelial and subepithelial IL-16 immunoreactivity and airway responsiveness to methacholine. This study demonstates that IL-16 is expressed in airway tissues, particularly in the epithelial cells, and that up-regulation of its expression is a feature of allergic asthma. These results suggest an in vivo role for IL-16 in the pathogenesis of asthma, possibly through the recruitment of CD4+ cells, and support the increasing evidence for the participation of epithelial cells in regulating inflammatory responses.

The allergen-induced late nasal response (LNR) is associated with high expression of interleukin-4 (IL-4) and IL-5 messenger RNA (mRNA) in the nasal mucosa, suggesting a role for Th2-type cytokines in the development of the LNR. Moreover, topical corticosteroid-mediated inhibition of the LNR is accompanied by inhibition of IL-4, but not IL-5, mRNA expression, IL-13 shares a number of functions with IL-4, including IgE switching and vascular cell adhesion molecule-1 (VCAM-1) upregulation. We investigated the expression of IL-13 mRNA and immunoreactivity in nasal biopsies from 10 normal subjects and 20 subjects with allergic rhinitis. IL-4 mRNA expression was examined in the same subjects. The allergic rhinitis patients were randomized to receive a 6-wk treatment with either topical fluticasone propionate (n = 10) or placebo (n = 10) nasal spray twice daily. A nasal biopsy was taken before treatment and 24 h after local nasal allergen provocation with a grass-pollen extract. Before treatment, there was no significant difference between the allergic rhinitis patients and controls in the expression of IL-13 mRNA and immunoreactivity. After allergen provocation, we observed a significant increase in IL-13 mRNA-positive and immunoreactive cells at 24 h only in subjects given placebo (P < 0.001). Inhibition of the LNR after corticosteroid treatment was associated with a marked decrease in allergen-induced IL-13 mRNA-positive (P < 0.001) and immunoreactive cells (P < 0.001). In subjects given placebo, 76.9 +/- 5.5% of IL-13 mRNA-positive cells observed after allergen were CD3+, whereas 11.2 +/- 2.7% coexpressed immunoreactivity for mast-cell tryptase. In these subjects, increases in cells expressing IL-13 mRNA were greater than for IL-4 mRNA (P = 0.001), and double in situ hybridization studies revealed that 100% of the IL-4 mRNA-positive cells coexpressed IL-13 mRNA, whereas 66.6 +/- 10.5% of IL-13 mRNA-positive cells coexpressed IL-4 transcripts after allergen challenge. The results of this study suggest that IL-13 expression is a prominent feature of the LNR, and that inhibition of the LNR following steroid therapy may be partly attributable to inhibition of IL-13 expression.

We have previously shown that inducible nitric oxide (iNO)-synthase immunoreactivity is expressed in bronchial epithelium and increased in asthma which suggests a possible role for NO in airway hyperresponsiveness. We tested the hypothesis that exposure of a sensitized animal to antigen could account for the increased expression of iNO-synthase in the airways. We examined the expression of iNO-synthase mRNA and immunoreactivity in the lungs of ovalbumin (OA) sensitized Brown Norway (BN) rats 8 h after antigen challenge by in situ hybridization and immunocytochemistry. Sensitized and unchallenged or bovine serum albumin (BSA) challenged rats, or unsensitized and OA challenged rats served as controls. With the use of an iNO-synthase probe we found a higher expression of iNO-synthase mRNA in BN rat airways after antigen challenge with OA but not after antigen challenge with BSA or in other controls. Most of the expression was in the epithelium of the airways with few cells positive in the subepithelial inflammatory infiltrate or in lung lavage. Very strong iNO-synthase immunoreactivity was observed in the airway epithelium of sensitized and OA challenged rats. No significant immunoreactivity was observed in the inflammatory infiltrate of the airways or in lung parenchyma. In conclusion, iNO-synthase increases in the airways of sensitized rats after exposure to antigen, the major source being from airway epithelial cells. NO may have a role in the development of the late airway response and bronchial hyperresponsiveness.

Localization and characterization of endothelin-producing cells in the developing (fetal and postnatal) and adult human lung was investigated using the technics of immunocytochemistry and in situ hybridization. Immunoreactivity for endothelin was seen mainly in pulmonary endocrine cells of developing human lung. Immunoreactivity was also seen in the airway epithelium in fewer cases (about 50%) of human adults. In situ hybridization with 35S- or 32P-labeled RNA probes complementary to endothelin-1, -2, and -3, showed that endothelin mRNAs were expressed in a number of cells that were in similar sites to endocrine cells. Immunocytochemistry and in situ hybridization employed on pairs of reverse-face serial sections showed the presence of endothelin immunoreactivity and mRNAs in the same endocrine cell. Correlative studies revealed that endothelin is co-localized with general endocrine markers (synaptophysin, chromogranin, protein gene product 9.5) and regulatory peptides (e.g., gastrin-releasing peptide). The density (cells/mm2) of endocrine cells containing immunoreactivity or mRNAs was highest during fetal life and started to decline before birth, and was minimal in adults. Endothelin-like immunoreactivity and mRNAs were also expressed in endothelial cells. From these results, it is concluded that endothelin is synthesized in endocrine cells of human lung and the change of developmental expression of this peptide suggests it may play a part in growth regulation in addition to its putative vasoconstrictor role in human lung.