Tracheal Cells Research Articles

Brush cells fine-tune neurogenic inflammation in the airways.

Airway epithelial cells, once considered a simple barrier layer, are now recognized as providing an active site for antigen sensing and immune response initiation. Most mucosal sites contain chemosensory epithelial cells, rare and specialized cells gaining recognition for their unique functions in sensing and directing the immune response symphony. In this issue of the JCI, Hollenhorst, Nandigama, et al. demonstrated that tracheal chemosensory brush cells detected bitter-tasting substances, including quorum-sensing molecules (QSMs) generated by pathogenic Pseudomonas aeruginosa. The authors used various techniques, including genetic deletion of brush cells, genetic manipulation of brush cell signaling, deletion of sensory neurons, in vivo imaging, and infection models with P. aeruginosa, to show that QSMs increased vascular permeability and innate immune cell influx into the trachea. These findings link the recognition of bacterial QSMs to the innate immune response in the airways, with translational implications for airway inflammation and infectious pathology.

Metabolic control of progenitor cell propagation during Drosophila tracheal remodeling

Adult progenitor cells in the trachea of Drosophila larvae are activated and migrate out of niches when metamorphosis induces tracheal remodeling. Here we show that in response to metabolic deficiency in decaying tracheal branches, signaling by the insulin pathway controls the progenitor cells by regulating Yorkie (Yki)-dependent proliferation and migration. Yki, a transcription coactivator that is regulated by Hippo signaling, promotes transcriptional activation of cell cycle regulators and components of the extracellular matrix in tracheal progenitor cells. These findings reveal that regulation of Yki signaling by the insulin pathway governs proliferation and migration of tracheal progenitor cells, thereby identifying the regulatory mechanism by which metabolic depression drives progenitor cell activation and cell division that underlies tracheal remodeling.

Cellular mechanism underlying the facilitation of contractile response induced by IL-25 in mouse tracheal smooth muscle.

Asthma is a common heterogeneous respiratory disease characterized by airway inflammation and airway hyperresponsiveness (AHR) which is associated with abnormality in smooth muscle contractility. The epithelial cell-derived cytokine IL-25 is implicated in type 2 immune pathology including asthma, whereas the underlying mechanisms have not been fully elucidated. This study aims to investigate the effects of IL-25 on mouse tracheal smooth muscle contractility and elucidate the cellular mechanisms. Incubation with IL-25 augmented the contraction of mouse tracheal smooth muscles, which could be suppressed by the L-type voltage-dependent Ca2+ channel (L-VDCC) blocker nifedipine. Furthermore, IL-25 enhanced the cytosolic Ca2+ signals and triggered the upregulation of α1C L-VDCC (CaV1.2) in primary cultured mouse tracheal smooth muscle cells. Knocking down IL-17RA/IL-17RB receptors or inhibiting the transforming growth factor-β-activated kinase 1 (TAK1)-tumor progression locus 2 (TPL2)-MAPK kinase 1/2 (MEK1/2)-ERK1/2-activating protein-1 (AP-1) signaling pathways suppressed the IL-25-elicited upregulation of CaV1.2 and hyperreactivity in tracheal smooth muscles. Moreover, inhibition of TPL2, ERK1/2 or L-VDCC alleviated the AHR symptom induced by IL-25 in a murine model. This study revealed that IL-25 potentiated the contraction of tracheal smooth muscle and evoked AHR via activation of TPL2-ERK1/2-CaV1.2 signaling, providing novel targets for the treatment of asthma with a high-IL-25 phenotype.

NKX2‐1 directly regulates Efnb2 to generate a boundary that allocates tracheal cells and defines the position of tracheoesophageal separation

Tracheoesophageal (TE) fistula is a human congenital condition characterized by an improper connection between the esophagus and trachea. These two tubular organs originate from a single foregut tube and separate from one another beginning at the site of lung bud evagination and progressing rostrally. Genetic studies in mice have identified signaling pathways that initiate dorsoventral patterning of the foregut; NKX2‐1 marks ventral, presumptive tracheal cells whereas high levels of SOX2 expression predominantly mark the dorsal, esophageal‐fated cells. Perturbation of either factor results in loss of that identity, increased expression of the opposing transcription factor, and failure of TE separation. Currently, the downstream mechanisms required to effect morphogenetic separation at the cellular and tissue level remain largely unknown. Here, we show that EPHRIN‐B2, a member of the EPH/EPHRIN signaling family, is restricted to the dorsal, esophageal‐fated cells, and that this domain of expression is required for TE separation and for maintaining the correct allocation of tracheal cells to the nascent trachea. Further, we demonstrate that NKX2‐1 directly binds at the Efnb2 locus and negatively regulates its expression, establishing an NKX2‐1/EPHRIN‐B2 TE boundary. Nkx2‐1 mosaic mutants exhibit ectopic NKX2‐1/EPHRIN‐B2 boundaries which drive ectopic tracheal separation morphogenesis in an EPHRIN‐B2 dependent manner. These results establish NKX2‐1 as a regulator of TE cell sorting and tissue separation, through modulation of EPH/EPHRIN signaling, which then serves to translate tissue identity information into morphogenetic outcomes.

Investigating Cell Fate Specification upon Nkx2‐1Loss

Nkx2‐1 has long been known to be essential for proper respiratory development, as its loss in mice results in an unseparated trachea with underdeveloped cystic lungs. However, how NKX2‐1 regulates distinct cell fates in the proximal airways is unknown. While Nkx2‐1 has been extensively studied for its role in lung adenocarcinomas, few studies have described the effects of Nkx2‐1 loss on the trachea beyond its role in early tracheal separation morphogenesis. In previously published work we have shown that the majority of genes differentially expressed in the trachea and esophagus, during early foregut development are not regulated by NKX2‐1 and maintain their expected dorsoventral expression patterns in the unseparated foregut upon NKX2‐1 loss. These results suggested that Nkx2‐1 may not be a master regulator of early respiratory fate, but may instead regulate specific cell fate programs during tracheal development. To address this question, we examined E16.5 and E18.5 Nkx2‐1Δ/Δand control mouse tracheas by immunofluorescent staining for tracheal cell types. Secretory cells (MUC5AC), multiciliated cells (acetylated α‐TUBULIN and FOXJ1), and Club cells (SCGB1A1) were all found in Nkx2‐1Δ/Δventral foreguts by immunofluorescence. While all expected proximal airway cell types were present in Nkx2‐1Δ/Δ tracheas, a disproportionate number of multiciliated cells were present in the Nkx2‐1Δ/Δforegut compared to control. This was not a consequence of effects on early respiratory induction, because tamoxifen‐induced loss of NKX2‐1 in Nkx2‐1CreER/loxembryos at E12.5 also resulted in overabundance of FOXJ1 positive cells at E16.5. Based on these results, while we conclude that NKX2‐1 is not required to define the early proximal respiratory epithelial lineage, it may instead be required for specification of proper proportions and distribution of respiratory cell types.

Protective Effects of N-Acetylcysteine on Lipopolysaccharide-Induced Respiratory Inflammation and Oxidative Stress.

As the leading cause of bovine respiratory disease (BRD), bacterial pneumonia can result in tremendous losses in the herd farming industry worldwide. N-acetylcysteine (NAC), an acetylated precursor of the amino acid L-cysteine, has been reported to have anti-inflammatory and antioxidant properties. To explore the protective effect and underlying mechanisms of NAC in ALI, we investigated its role in lipopolysaccharide (LPS)-induced bovine embryo tracheal cells (EBTr) and mouse lung injury models. We found that NAC pretreatment attenuated LPS-induced inflammation in EBTr and mouse models. Moreover, LPS suppressed the expression of oxidative-related factors in EBTr and promoted gene expression and the secretion of inflammatory cytokines. Conversely, the pretreatment of NAC alleviated the secretion of inflammatory cytokines and decreased their mRNA levels, maintaining stable levels of antioxidative gene expression. In vivo, NAC helped LPS-induced inflammatory responses and lung injury in ALI mice. The relative protein concentration, total cells, and percentage of neutrophils in BALF; the level of secretion of IL-6, IL-8, TNF-α, and IL-1β; MPO activity; lung injury score; and the expression level of inflammatory-related genes were decreased significantly in the NAC group compared with the LPS group. NAC also ameliorated LPS-induced mRNA level changes in antioxidative genes. In conclusion, our findings suggest that NAC affects the inflammatory and oxidative response, alleviating LPS-induced EBTr inflammation and mouse lung injury, which offers a natural therapeutic strategy for BRD.

Thrombin Induces COX-2 and PGE2 Expression via PAR1/PKCalpha/MAPK-Dependent NF-kappaB Activation in Human Tracheal Smooth Muscle Cells.

The inflammation of the airway and lung could be triggered by upregulation cyclooxygenase (COX)-2 and prostaglandin E2 (PGE2) induced by various proinflammatory factors. COX-2 induction by thrombin has been shown to play a vital role in various inflammatory diseases. However, in human tracheal smooth muscle cells (HTSMCs), how thrombin enhanced the levels of COX-2/PGE2 is not completely characterized. Thus, in this study, the levels of COX-2 expression and PGE2 synthesis induced by thrombin were determined by Western blot, promoter-reporter assay, real-time PCR, and ELISA kit. The various signaling components involved in the thrombin-mediated responses were differentiated by transfection with siRNAs and selective pharmacological inhibitors. The role of NF-κB was assessed by a chromatin immunoprecipitation (ChIP) assay, immunofluorescent staining, as well as Western blot. Our results verified that thrombin markedly triggered PGE2 secretion via COX-2 upregulation which were diminished by the inhibitor of thrombin (PPACK), PAR1 (SCH79797), Gi/o protein (GPA2), Gq protein (GPA2A), PKCα (Gö6976), p38 MAPK (SB202190), JNK1/2 (SP600125), MEK1/2 (U0126), or NF-κB (helenalin) and transfection with siRNA of PAR1, Gqα, Giα, PKCα, JNK2, p38, p42, or p65. Moreover, thrombin induced PAR1-dependent PKCα phosphorylation in HTSMCs. We also observed that thrombin induced p38 MAPK, JNK1/2, and p42/p44 MAPK activation through a PAR1/PKCα pathway. Thrombin promoted phosphorylation of NF-κB p65, leading to nuclear translocation and binding to the COX-2 promoter element to enhance promoter activity, which was reduced by Gö6976, SP600125, SB202190, or U0126. These findings supported that COX-2/PGE2 expression triggered by thrombin was engaged in PAR1/Gq or Gi/o/PKCα/MAPK-dependent NF-κB activation in HTSMCs.

The wing imaginal disc.

The Drosophila wing imaginal disc is a tissue of undifferentiated cells that are precursors of the wing and most of the notum of the adult fly. The wing disc first forms during embryogenesis from a cluster of ∼30 cells located in the second thoracic segment, which invaginate to form a sac-like structure. They undergo extensive proliferation during larval stages to form a mature larval wing disc of ∼35,000 cells. During this time, distinct cell fates are assigned to different regions, and the wing disc develops a complex morphology. Finally, during pupal stages the wing disc undergoes morphogenetic processes and then differentiates to form the adult wing and notum. While the bulk of the wing disc comprises epithelial cells, it also includes neurons and glia, and is associated with tracheal cells and muscle precursor cells. The relative simplicity and accessibility of the wing disc, combined with the wealth of genetic tools available in Drosophila, have combined to make it a premier system for identifying genes and deciphering systems that play crucial roles in animal development. Studies in wing imaginal discs have made key contributions to many areas of biology, including tissue patterning, signal transduction, growth control, regeneration, planar cell polarity, morphogenesis, and tissue mechanics.

Tracheal separation is driven by NKX2-1-mediated repression of Efnb2 and regulation of endodermal cell sorting.

SUMMARYThe mechanisms coupling fate specification of distinct tissues to their physical separation remain to be understood. The trachea and esophagus differentiate from a single tube of definitive endoderm, requiring the transcription factors SOX2 and NKX2-1, but how the dorsoventral site of tissue separation is defined to allocate tracheal and esophageal cell types is unknown. Here, we show that the EPH/EPHRIN signaling gene Efnb2 regulates tracheoesophageal separation by controlling the dorsoventral allocation of tracheal-fated cells. Ventral loss of NKX2-1 results in disruption of separation and expansion of Efnb2 expression in the trachea independent of SOX2. Through chromatin immunoprecipitation and reporter assays, we find that NKX2-1 likely represses Efnb2 directly. Lineage tracing shows that loss of NKX2-1 results in misallocation of ventral foregut cells into the esophagus, while mosaicism for NKX2-1 generates ectopic NKX2-1/EPHRIN-B2 boundaries that organize ectopic tracheal separation. Together, these data demonstrate that NKX2-1 coordinates tracheal specification with tissue separation through the regulation of EPHRIN-B2 and tracheoesophageal cell sorting.

First person – Carolina Camelo

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Carolina Camelo is first author on ‘ Tracheal tube fusion in Drosophila involves release of extracellular vesicles from multivesicular bodies’, published in JCS. Carolina is a PhD student in the lab of Stefan Luschnig at Institute of Integrative Cell Biology and Physiology, University of Münster, Germany, where she is interested in understanding how cells organize themselves to form functional tissues and organisms, particularly during development. In this work, I was interested in understanding how tracheal tube fusion is regulated by specialized tracheal fusion cells.

A medium composition containing normal resting glucose that supports differentiation of primary human airway cells

Primary cells isolated from the human respiratory tract are the state-of-the-art for in vitro airway epithelial cell research. Airway cell isolates require media that support expansion of cells in a basal state to maintain the capacity for differentiation as well as proper cellular function. By contrast, airway cell differentiation at an air–liquid interface (ALI) requires a distinct medium formulation that typically contains high levels of glucose. Here, we expanded and differentiated human basal cells isolated from the nasal and conducting airway to a mature mucociliary epithelial cell layer at ALI using a medium formulation containing normal resting glucose levels. Of note, bronchial epithelial cells expanded and differentiated in normal resting glucose medium showed insulin-stimulated glucose uptake which was inhibited by high glucose concentrations. Normal glucose containing ALI also enabled differentiation of nasal and tracheal cells that showed comparable electrophysiological profiles when assessed for cystic fibrosis transmembrane conductance regulator (CFTR) function and that remained responsive for up to 7 weeks in culture. These data demonstrate that normal glucose containing medium supports differentiation of primary nasal and lung epithelial cells at ALI, is well suited for metabolic studies, and avoids pitfalls associated with exposure to high glucose.

An endosome-associated actin network involved in directed apical plasma membrane growth.

Membrane trafficking plays many roles in morphogenesis, from bulk membrane provision to targeted delivery of proteins and other cargos. In tracheal terminal cells of the Drosophila respiratory system, transport through late endosomes balances membrane delivery between the basal plasma membrane and the apical membrane, which forms a subcellular tube, but it has been unclear how the direction of growth of the subcellular tube with the overall cell growth is coordinated. We show here that endosomes also organize F-actin. Actin assembles around late endocytic vesicles in the growth cone of the cell, reaching from the tip of the subcellular tube to the leading filopodia of the basal membrane. Preventing nucleation of endosomal actin disturbs the directionality of tube growth, uncoupling it from the direction of cell elongation. Severing actin in this area affects tube integrity. Our findings show a new role for late endosomes in directing morphogenesis by organizing actin, in addition to their known role in membrane and protein trafficking.

The apoptotic and proliferative effects of tulathromycin and gamithromycin on bovinetracheal epithelial cell culture

Gamithromycin and tulathromycin are commonly used in the treatment of bovine respiratory bacterial diseases. The current work was undertaken to establish the apoptotic, necrotic, and cytotoxic effects of these antibiotics in the target animal. Cells with apoptosis and necrosis were determined by dual staining method, cytotoxic effects were determined by MTT assay, cell proliferative effects were examined by XCelligence real-time cell analysis system (RTCA-SP). The comparison between gamithromycin and tulathromycin concentrations on tracheal cells in terms of % cell viability was found to be significantly different. While the cell viability percentage of gamithromycin was higher at 150 μg/mL, 180 μg/mL, and 240 μg/mL than tulathromycin, and at 2 μg/mL, 4 μg/mL, 10 μg/mL, 20 μg/mL, and 50 μg/mL concentrations tulathromycin cell viability was higher than gamithromycin (p < 0.05). When the staining method data were evaluated, the difference between the results of % apoptotic index at 20 μg/mL concentration was significant and it was found that gamithromycin had more apoptotic effect than tulathromycin (p < 0.05). It was seen that tulathromycin and gamithromycin applied on tracheal epithelial cells at concentrations of 2 and 10 ?g/mL increased the viability depending on time. The increase in epithelial cell proliferation of gamithromycin and tulathromycin due to time shows that these antibiotics can maintain longterm prophylactic treatment against diseases.

Radiotherapy for Tracheal Squamous Cell Carcinoma: A Review

Tracheal cancer is a rare localization without official recommendation nether European nor French. This review summarizes the data published about tracheal squamous cell carcinoma treated with radiotherapy in terms of dose and fractionation, techniques, volume, toxicity, overall survival and local control.

Isolation, Ex Vivo Culture, and Stimulation of Tracheal and Nasal Chemosensory Cells.

Brush cells are chemosensory epithelial cells present at most mucosal surfaces.Brush cells are a dominant source of cysteinyl leukotrienes and IL-25 in the airway epithelium and are equipped with the machinery to generate prostaglandins and acetylcholine. Activation of innate type 2 lymphoid cells and dendritic cells triggered by brush cell-derived mediators skew the immune response in the airway to type 2 inflammation that underlies atopic disease such as asthma. This chapter describes an effective method of brush cell isolation from the mouse trachea for transcriptional analysis and from the nasal cavity for transcriptional analysis and ex vivo stimulation.The nasal or tracheal mucosa is first incubated in a dispase solution for easy mechanical separation of the epithelial layer from the underlying submucosa. The detached epithelium is then digested with a papain solution. This method provides high yields of viable brush cells in a single-cell suspension, which can be used for flow cytometric analysis, single-cell sorting, cell culture, and functional assays.In the nose, where brush cells are more abundant, we present two methods of isolation of brush cells: (1) using fluorescent reporter mice that mark brush cells or (2) using a combination of high expression of EpCAM and low expression of CD45 to obtain a population of cells that is enriched for nasal chemosensory brush cells.

Characterization of the Role of Extracellular Vesicles Released from Chicken Tracheal Cells in the Antiviral Responses against Avian Influenza Virus.

During viral respiratory infections, the innate antiviral response engages a complex network of cells and coordinates the secretion of key antiviral factors, such as cytokines, which requires high levels of regulation and communication. Extracellular vesicles (EVs) are particles released from cells that contain an array of biomolecules, including lipids, proteins, and RNAs. The contents of EVs can be influenced by viral infections and may play a role in the regulation of antiviral responses. We hypothesized that the contents of EVs released from chicken tracheal cells are influenced by viral infection and that these EVs regulate the function of other immune cells, such as macrophages. To this end, we characterized the protein profile of EVs during avian influenza virus (AIV) infection and evaluated the impact of EV stimulation on chicken macrophage functions. A total of 140 differentially expressed proteins were identified upon stimulation with various stimuli. These proteins were shown to be involved in immune responses and cell signaling pathways. In addition, we demonstrated that EVs can activate macrophages. These results suggest that EVs play a role in the induction and modulation of antiviral responses during viral respiratory infections in chickens.

BMP4 and Wnt signaling interact to promote mouse tracheal mesenchyme morphogenesis.

Tracheobronchomalacia and complete tracheal rings are congenital malformations of the trachea associated with morbidity and mortality for which the etiology remains poorly understood. Epithelial expression of Wls (a cargo receptor mediating Wnt ligand secretion) by tracheal cells is essential for patterning the embryonic mouse trachea's cartilage and muscle. RNA sequencing indicated that Wls differentially modulated the expression of BMP signaling molecules. We tested whether BMP signaling, induced by epithelial Wnt ligands, mediates cartilage formation. Deletion of Bmp4 from respiratory tract mesenchyme impaired tracheal cartilage formation that was replaced by ectopic smooth muscle, recapitulating the phenotype observed after epithelial deletion of Wls in the embryonic trachea. Ectopic muscle was caused in part by anomalous differentiation and proliferation of smooth muscle progenitors rather than tracheal cartilage progenitors. Mesenchymal deletion of Bmp4 impaired expression of Wnt/β-catenin target genes, including targets of WNT signaling: Notum and Axin2. In vitro, recombinant (r)BMP4 rescued the expression of Notum in Bmp4-deficient tracheal mesenchymal cells and induced Notum promoter activity via SMAD1/5. RNA sequencing of Bmp4-deficient tracheas identified genes essential for chondrogenesis and muscle development coregulated by BMP and WNT signaling. During tracheal morphogenesis, WNT signaling induces Bmp4 in mesenchymal progenitors to promote cartilage differentiation and restrict trachealis muscle. In turn, Bmp4 differentially regulates the expression of Wnt/β-catenin targets to attenuate mesenchymal WNT signaling and to further support chondrogenesis.

Crosstalk between basal extracellular matrix adhesion and building of apical architecture during morphogenesis.

ABSTRACTTissues build complex structures like lumens and microvilli to carry out their functions. Most of the mechanisms used to build these structures rely on cells remodelling their apical plasma membranes, which ultimately constitute the specialised compartments. In addition to apical remodelling, these shape changes also depend on the proper attachment of the basal plasma membrane to the extracellular matrix (ECM). The ECM provides cues to establish apicobasal polarity, and it also transduces forces that allow apical remodelling. However, physical crosstalk mechanisms between basal ECM attachment and the apical plasma membrane remain understudied, and the ones described so far are very diverse, which highlights the importance of identifying the general principles. Here, we review apicobasal crosstalk of two well-established models of membrane remodelling taking place during Drosophila melanogaster embryogenesis: amnioserosa cell shape oscillations during dorsal closure and subcellular tube formation in tracheal cells. We discuss how anchoring to the basal ECM affects apical architecture and the mechanisms that mediate these interactions. We analyse this knowledge under the scope of other morphogenetic processes and discuss what aspects of apicobasal crosstalk may represent widespread phenomena and which ones are used to build subsets of specialised compartments.

Ultraviolet-A light increases mitochondrial anti-viral signaling protein in confluent human tracheal cells via cell-cell signaling.

Mitochondrial antiviral signaling (MAVS) protein mediates innate antiviral responses, including responses to certain coronaviruses such as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). We have previously shown that ultraviolet-A (UVA) therapy can prevent virus-induced cell death in human ciliated tracheal epithelial cells (HTEpC) infected with coronavirus-229E (CoV-229E), and results in increased intracellular levels of MAVS. In this study, we explored the mechanisms by which UVA light can activate MAVS, and whether local UVA light application can activate MAVS at locations distant from the light source (e.g. via cell-to-cell communication). MAVS levels were compared in HTEpC exposed to 2 mW/cm2 narrow band (NB)-UVA for 20 min and in unexposed controls at 30–40% and at 100% confluency, and in unexposed HTEpC treated with supernatants or lysates from UVA-exposed cells or from unexposed controls. MAVS was also assessed in different sections of confluent monolayer plates where only one section was exposed to NB-UVA. Our results showed that UVA increases the expression of MAVS protein. Further, cells in a confluent monolayer exposed to UVA conferred an elevation in MAVS in cells adjacent to the exposed section, and also in cells in the most distant sections which were not exposed to UVA. In this study, human ciliated tracheal epithelial cells exposed to UVA demonstrate increased MAVS protein, and also appear to transmit this influence to confluent cells not exposed to UVA, likely via cell-cell signaling.

Cellular Mechanism Underlying the Facilitation of Contractile Response Induced by Tumor Necrosis Factor-α in Mouse Tracheal Smooth Muscle

The proinflammatory cytokine tumor necrosis factor-α (TNF-α) augments intracellular Ca2+ signaling and contractile responses of airway smooth muscles, leading to airway hyperresponsiveness. However, the underlying mechanism has not been fully elucidated. This study aimed to investigate the cellular mechanism of the potentiated contraction of mouse tracheal smooth muscle induced by TNF-α. The results showed that TNF-α triggered facilitation of mouse tracheal smooth muscle contraction in an epithelium-independent manner. The TNF-α-induced hypercontractility could be suppressed by the protein kinase C inhibitor GF109203X, the tyrosine kinase inhibitor genistein, the Src inhibitor PP2, or the L-type voltage-dependent Ca2+ channel blocker nifedipine. Following TNF-α incubation, the α1C L-type Ca2+ channel (CaV1.2) was up-regulated in cultured primary mouse tracheal smooth muscle cells. Pronounced phosphotyrosine levels were observed in mouse tracheas. In conclusion, this study shows that TNF-α enhanced airway smooth muscle contraction via protein kinase C-Src-CaV1.2 pathways, which provides novel insights into the pathologic role of proinflammatory cytokines in mediating airway hyperresponsiveness.