Abstract: Acidic mammalian chitinase (AMCase) is expressed in an exaggerated fashion in epithelial cells at sites of pulmonary T helper cell type 2 inflammation and plays important roles in the pathogenesis of anti-parasite and asthma-like responses. However, the mechanisms that control epithelial cell AMCase secretion and its effector responses have not been adequately defined. To address these issues, we used in vivo and in vitro experimental systems to define the pathways of epithelial AMCase secretion and its epithelial regulatory effects. Here we demonstrate that, in murine T helper cell type 2 modeling systems, AMCase colocalizes with the epidermal growth factor receptor (EGFR) and ADAM17 (a membrane disintegrin and metallopeptidase 17) in lung epithelial cells. In vitro cotransfection experiments in A549 cells demonstrated that AMCase and EGFR physically interact with each other. Cotransfection of AMCase and EGFR also increased, whereas EGFR inhibition decreased AMCase secretion. Interestingly, AMCase secretion was not significantly altered by treatment with EGF but was significantly decreased when the upstream EGFR transactivator ADAM17 was inhibited. AMCase secretion was also decreased when the EGFR-downstream Ras was blocked. Transfected and recombinant AMCase induced epithelial cell production of CCL2, CCL17, and CXCL8. These studies demonstrate that lung epithelial cells secrete AMCase via an EGFR-dependent pathway that is activated by ADAM17 and mediates its effects via Ras. They also demonstrate that the AMCase that is secreted feeds back in an autocrine and/or paracrine fashion to stimulate pulmonary epithelial cell chemokine production.

Abstract: Cystic fibrosis (CF) lung disease is characterized by infection with Pseudomonas aeruginosa and a sustained accumulation of neutrophils. In this study, we analyzed 1) the expression of MyD88-dependent TLRs on circulating and airway neutrophils in P. aeruginosa-infected CF patients, P. aeruginosa-infected non-CF bronchiectasis patients, and noninfected healthy control subjects and 2) studied the regulation of TLR expression and functionality on neutrophils in vitro. TLR2, TLR4, TLR5, and TLR9 expression was increased on airway neutrophils compared with circulating neutrophils in CF and bronchiectasis patients. On airway neutrophils, TLR5 was the only TLR that was significantly higher expressed in CF patients compared with bronchiectasis patients and healthy controls. Studies using confocal microscopy and flow cytometry revealed that TLR5 was stored intracellularly in neutrophils and was mobilized to the cell surface in a protein synthesis-independent manner through protein kinase C activation or after stimulation with TLR ligands and cytokines characteristic of the CF airway microenvironment. The most potent stimulator of TLR5 expression was the bacterial lipoprotein Pam(3)CSK(4). Ab-blocking experiments revealed that the effect of Pam(3)CSK(4) was mediated through cooperation of TLR1 and TLR2 signaling. TLR5 activation enhanced the phagocytic capacity and the respiratory burst activity of neutrophils, which was mediated, at least partially, via a stimulation of IL-8 production and CXCR1 signaling. This study demonstrates a novel mechanism of TLR regulation in neutrophils and suggests a critical role for TLR5 in neutrophil-P. aeruginosa interactions in CF lung disease.

Abstract: Various inflammatory diseases are characterized by tissue infiltration of neutrophils. Chemokines recruit and activate leukocytes, but neutrophils are traditionally known to be restricted in their chemokine receptor (CR) expression repertoire. Neutrophils undergo phenotypic and functional changes under inflammatory conditions, but the mechanisms regulating CR expression of infiltrated neutrophils at sites of chronic inflammation are poorly defined. Here we show that infiltrated neutrophils from patients with chronic inflammatory lung diseases and rheumatoid arthritis highly express CR on their surface that are absent or only marginally expressed on circulating neutrophils, i.e., CCR1, CCR2, CCR3, CCR5, CXCR3, and CXCR4, as measured by flow cytometry, immunohistochemistry, and confocal microscopy. The induction of CR surface expression on infiltrated neutrophils was functionally relevant, because receptor activation by chemokine ligands ex vivo modulated neutrophil effector functions such as respiratory burst activity and bacterial killing. In vitro studies with isolated neutrophils demonstrated that the surface expression of CR was differentially induced in a cytokine-mediated, protein synthesis-dependent manner (CCR1, CCR3), through Toll-like (CXCR3) or NOD2 (CCR5) receptor engagement, through neutrophil apoptosis (CCR5, CXCR4), and/or via mobilization of intracellular CD63(+) granules (CXCR3). CR activation on infiltrated neutrophils may represent a key mechanism by which the local inflammatory microenvironment fine-tunes neutrophil effector functions in situ. Since the up-regulation of CR was exclusively found on infiltrated neutrophils at inflammatory sites in situ, the targeting of these G protein-coupled receptors may have the potential to site-specifically target neutrophilic inflammation.

Abstract: BACKGROUND: Asthma is characterized by a T(H)2 immune response. CD4(+)CD25(hi) regulatory T cells (Tregs) have been proposed to prevent allergic diseases through suppression of T(H)2 responses. OBJECTIVE: We sought to investigate the role of CD4(+)CD25(hi) T cells in children with asthma. METHODS: CD4(+)CD25(hi) Tregs and forkhead/winged-helix transcription factor FOXP3 mRNA levels were quantified in peripheral blood and bronchoalveolar lavage fluid (BALF) of 18 children with asthma, 10 children with chronic cough, and 13 control subjects without lung diseases. CD4(+)CD25(hi) T cells were isolated from peripheral blood and BALF of asthmatic patients and control subjects, and their capacity to suppress proliferation and cytokine/chemokine production of autologous responder T cells was analyzed. RESULTS: CD4(+)CD25(hi) T cells were decreased in BALF of asthmatic children compared with values in children with cough or control subjects. In children with asthma, inhaled corticosteroid treatment was associated with increased percentages of CD4(+)CD25(hi) T cells in peripheral blood and BALF. Isolated BALF and peripheral blood CD4(+)CD25(hi) T cells from nonasthmatic subjects suppressed proliferation and cytokine/chemokine production by CD4(+)CD25(-) responder T cells. BALF CD4(+)CD25(hi) T cells from asthmatic subjects failed to suppress proliferation and production of T(H)2-associated cytokines and chemokines by CD4(+)CD25(-) responder T cells, which was restored after use of inhaled corticosteroids. CONCLUSION: These findings provide the first evidence that pulmonary CD4(+)CD25(hi) Tregs are impaired in pediatric asthma. CLINICAL IMPLICATIONS: Pulmonary Tregs might represent a therapeutic target in pediatric asthma.

Abstract: Interleukin-8 (IL-8) activates neutrophils via the chemokine receptors CXCR1 and CXCR2. However, the airways of individuals with cystic fibrosis are frequently colonized by bacterial pathogens, despite the presence of large numbers of neutrophils and IL-8. Here we show that IL-8 promotes bacterial killing by neutrophils through CXCR1 but not CXCR2. Unopposed proteolytic activity in the airways of individuals with cystic fibrosis cleaved CXCR1 on neutrophils and disabled their bacterial-killing capacity. These effects were protease concentration-dependent and also occurred to a lesser extent in individuals with chronic obstructive pulmonary disease. Receptor cleavage induced the release of glycosylated CXCR1 fragments that were capable of stimulating IL-8 production in bronchial epithelial cells via Toll-like receptor 2. In vivo inhibition of proteases by inhalation of alpha1-antitrypsin restored CXCR1 expression and improved bacterial killing in individuals with cystic fibrosis. The cleavage of CXCR1, the functional consequences of its cleavage, and the identification of soluble CXCR1 fragments that behave as bioactive components represent a new pathophysiologic mechanism in cystic fibrosis and other chronic lung diseases.

Abstract: BACKGROUND: Asthma is characterized by a T(H)2 immune response. CD4(+)CD25(hi) regulatory T cells (Tregs) have been proposed to prevent allergic diseases through suppression of T(H)2 responses. OBJECTIVE: We sought to investigate the role of CD4(+)CD25(hi) T cells in children with asthma. METHODS: CD4(+)CD25(hi) Tregs and forkhead/winged-helix transcription factor FOXP3 mRNA levels were quantified in peripheral blood and bronchoalveolar lavage fluid (BALF) of 18 children with asthma, 10 children with chronic cough, and 13 control subjects without lung diseases. CD4(+)CD25(hi) T cells were isolated from peripheral blood and BALF of asthmatic patients and control subjects, and their capacity to suppress proliferation and cytokine/chemokine production of autologous responder T cells was analyzed. RESULTS: CD4(+)CD25(hi) T cells were decreased in BALF of asthmatic children compared with values in children with cough or control subjects. In children with asthma, inhaled corticosteroid treatment was associated with increased percentages of CD4(+)CD25(hi) T cells in peripheral blood and BALF. Isolated BALF and peripheral blood CD4(+)CD25(hi) T cells from nonasthmatic subjects suppressed proliferation and cytokine/chemokine production by CD4(+)CD25(-) responder T cells. BALF CD4(+)CD25(hi) T cells from asthmatic subjects failed to suppress proliferation and production of T(H)2-associated cytokines and chemokines by CD4(+)CD25(-) responder T cells, which was restored after use of inhaled corticosteroids. CONCLUSION: These findings provide the first evidence that pulmonary CD4(+)CD25(hi) Tregs are impaired in pediatric asthma. CLINICAL IMPLICATIONS: Pulmonary Tregs might represent a therapeutic target in pediatric asthma.