Altered allergen-induced eosinophil trafficking and physiological dysfunction in airways with preexisting virus-induced injury

Although both asthmatics and allergic rhinitics develop an acute inflammatory response to lower airway allergen challenge, only asthmatics experience airway obstruction resulting from chronic environmental allergen exposure. Hypothesizing that asthmatic airways have an altered response to chronic allergic inflammation, we compared the effects of repeated low-level exposures to inhaled Alternaria extract in sensitized rats with preexisting chronic postbronchiolitis airway dysfunction versus sensitized controls with normal airways. Measurements of air space (bronchoalveolar lavage) inflammatory cells, airway goblet cells, airway wall collagen, airway wall eosinophils, airway alveolar attachments, and pulmonary physiology were conducted after six weekly exposures to aerosolized saline or Alternaria extract. Postbronchiolitis rats, but not those starting with normal airways, had persistent increases in airway wall eosinophils, goblet cell hyperplasia in small airways, and loss of lung elastic recoil after repeated exposure to aerosolized Alternaria extract. Despite having elevated airway wall eosinophils, the postbronchiolitis rats had no eosinophils in bronchoalveolar lavage at 5 days after the last allergen exposure, suggesting altered egression of tissue eosinophils into the air space. In conclusion, rats with preexisting airway pathology had altered eosinophil trafficking and allergen-induced changes in airway epithelium and lung mechanics that were absent in sensitized control rats that had normal airways before the allergen exposures.     

Increased bronchovascular permeability after allergen exposure in sensitive asthmatics

To investigate the possibility that an increase in bronchovascular permeability is associated with allergen exposure in sensitive asthmatics we evaluated the amounts of serum proteins in bronchoalveolar lavage (BAL) effluents before and after local challenge with allergen. After exposure of sensitive asthmatic airways (n = 15) to allergen significant increases in total protein compared with controls were observed: 0.08 +/- 0.01 mg/ml in control airways and 0.13 +/- 0.02 mg/ml in challenged airways; P less than 0.05. The greatest changes induced by allergen exposure involved small-molecular-weight proteins (less than 345,000) and an inverse correlation was observed between log molecular weight and percent increase in the concentrations of the specific proteins; r = -0.61. BAL-serum distribution coefficients of serum proteins in airway fluids reflected a greater diffusability of low-molecular-weight proteins immediately after allergen exposure. We also evaluated the movement of serum proteins into lung after local allergen exposure using intravenously administered 99mTc-albumin (n = 10) and found an immediate 3.8-fold increase in amounts of radioactive albumin in allergen exposed airways compared with airways exposed to diluent. Most of the radioactivity was recovered in the first 5 ml of aliquot withdrawn, suggesting a marked increase in the permeability of the bronchial (large airway) vascular-epithelial membrane. An increase in serum proteins was also observed in BAL fluid of asthmatics 2-4 h after aerosol challenge (n = 4), including all proteins in the molecular weight range 45,000-900,000. These studies suggest that allergen exposure in sensitive asthmatics causes an acute increase in bronchovascular permeability to serum proteins.     

IL-4 and IL-13 form a negative feedback circuit with surfactant protein-D in the allergic airway response

The innate immune molecule surfactant protein-D (SP-D) plays an important regulatory role in the allergic airway response. In this study, we demonstrate that mice sensitized and challenged with either Aspergillus fumigatus (Af) or OVA have increased SP-D levels in their lung. SP-D mRNA and protein levels in the lung also increased in response to either rIL-4 or rIL-13 treatment. Type II alveolar epithelial cell expression of IL-4Rs in mice sensitized and challenged with Af, and in vitro induction of SP-D mRNA and protein by IL-4 and IL-13, but not IFN-gamma, suggested a direct role of IL-4R-mediated events. The regulatory function of IL-4 and IL-13 was further supported in STAT-6-deficient mice as well as in IL-4/IL-13 double knockout mice that failed to increase SP-D production upon allergen challenge. Interestingly, addition of rSP-D significantly inhibited Af-driven Th2 cell activation in vitro whereas mice lacking SP-D had increased numbers of CD4(+) cells with elevated IL-13 and thymus- and activation-regulated chemokine levels in the lung and showed exaggerated production of IgE and IgG1 following allergic sensitization. We propose that allergen exposure induces elevation in SP-D protein levels in an IL-4/IL-13-dependent manner, which in turn, prevents further activation of sensitized T cells. This negative feedback regulatory circuit could be essential in protecting the airways from inflammatory damage after allergen inhalation.     

Inhibition of neutrophil elastase attenuates airway hyperresponsiveness and inflammation in a mouse model of secondary allergen challenge: neutrophil elastase inhibition attenuates allergic airway responses

Background

Chronic asthma is often associated with neutrophilic infiltration in the airways. Neutrophils contain elastase, a potent secretagogue in the airways, nonetheless the role for neutrophil elastase as well as neutrophilic inflammation in allergen-induced airway responses is not well defined. In this study, we have investigated the impact of neutrophil elastase inhibition on the development of allergic airway inflammation and airway hyperresponsiveness (AHR) in previously sensitized and challenged mice.

Methods

BALB/c mice were sensitized and challenged (primary) with ovalbumin (OVA). Six weeks later, a single OVA aerosol (secondary challenge) was delivered and airway inflammation and airway responses were monitored 6 and 48 hrs later. An inhibitor of neutrophil elastase was administered prior to secondary challenge.

Results

Mice developed a two-phase airway inflammatory response after secondary allergen challenge, one neutrophilic at 6 hr and the other eosinophilic, at 48 hr. PAR-2 expression in the lung tissues was enhanced following secondary challenge, and that PAR-2 intracellular expression on peribronchial lymph node (PBLN) T cells was also increased following allergen challenge of sensitized mice. Inhibition of neutrophil elastase significantly attenuated AHR, goblet cell metaplasia, and inflammatory cell accumulation in the airways following secondary OVA challenge. Levels of IL-4, IL-5 and IL-13, and eotaxin in BAL fluid 6 hr after secondary allergen challenge were significantly suppressed by the treatment. At 48 hr, treatment with the neutrophil elastase inhibitor significantly reduced the levels of IL-13 and TGF-β1 in the BAL fluid. In parallel, in vitro IL-13 production was significantly inhibited in spleen cells from sensitized mice.

Conclusion

These data indicate that neutrophil elastase plays an important role in the development of allergic airway inflammation and hyperresponsiveness, and would suggest that the neutrophil elastase inhibitor reduced AHR to inhaled methacholine indicating the potential for its use as a modulator of the immune/inflammatory response in both the neutrophil- and eosinophil-dominant phases of the response to secondary allergen challenge.     

Distribution of human PLUNC/BPI fold-containing (BPIF) proteins

Although gene expression studies have shown that human PLUNC (palate, lung and nasal epithelium clone) proteins are predominantly expressed in the upper airways, nose and mouth, and proteomic studies have indicated they are secreted into airway and nasal lining fluids and saliva, there is currently little information concerning the localization of human PLUNC proteins. Our studies have focused on the localization of three members of this protein family, namely SPLUNC1 (short PLUNC1), SPLUNC2 and LPLUNC1 (long PLUNC1). Western blotting has indicated that PLUNC proteins are highly glycosylated, whereas immunohistochemical analysis demonstrated distinct patterns of expression. For example, SPLUNC2 is expressed in serous cells of the major salivary glands and in minor mucosal glands, whereas SPLUNC1 is expressed in the mucous cells of these glands. LPLUNC1 is a product of a population of goblet cells in the airway epithelium and nasal passages and expressed in airway submucosal glands and minor glands of the oral and nasal cavities. SPLUNC1 is also found in the epithelium of the upper airways and nasal passages and in airway submucosal glands, but is not co-expressed with LPLUNC1. We suggest that this differential expression may be reflected in the function of individual PLUNC proteins.     

Two-dimensional electrophoresis protein profiling and identification in rat bronchoalveolar lavage fluid following allergen and endotoxin challenge

The protein content of bronchoalveolar lavage fluid (BALF) from actively sensitised Brown Norway (BN) rats challenged with allergen (ovalbumin, OA) and from na?ve Brown Norway rats challenged with endotoxin (lipopolysaccharide, LPS) was analyzed and compared to healthy controls treated with vehicle only. BALF proteins were analyzed by one-dimensional (1-D) and two-dimensional (2-D) gel electrophoresis and identified by peptide mass fingerprinting matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) or nanoliquid chromatography-tandem MS (nanoLC-MS/MS) after in-gel trypsin digestion of selected 2-D gel spots. Our study shows that the BALF protein profile is significantly different in animals after allergen (OA) or endotoxin (LPS) challenge as compared to controls, concerning the content of proteins derived from plasma or produced locally in the lung. In both challenges the following proteins presented patterns which differed qualitatively compared to control: T-kininogen I and II, alpha-1-antitrypsin, calgranulin A, fetuin A and B, and haptoglobin. Other proteins were diminished in both challenges, such as Clara cell 10 kDa secretory protein (CC10) and pulmonary surfactant associated protein B (SP-B); c-reactive protein increased in the OA-challenge and decreased in the LPS-challenge, and pulmonary surfactant associated protein A (SP-A) was decreased in the OA-challenge and was not significantly changed in the LPS-challenge. The identified proteins could be important not only for the diagnosis but have also interesting implications for medical treatment of lung inflammatory conditions. Furthermore, even if based on a limited number of animals, our results are of interest for the identification of lung protein markers and a better understanding of the mechanisms involved in the pathogenesis of lung diseases.     

A broad-spectrum caspase inhibitor attenuates allergic airway inflammation in murine asthma model

Asthma is characterized by acute and chronic airway inflammation, and the severity of the airway hyperreactivity correlates with the degree of inflammation. Many of the features of lung inflammation observed in human asthma are reproduced in OVA-sensitized/challenged mice. T lymphocytes, particularly Th2 cells, are critically involved in the genesis of the allergic response to inhaled Ag. In addition to antiapoptotic effects, broad-spectrum caspase inhibitors inhibit T cell activation in vitro. We investigated the effect of the broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), on airway inflammation in OVA-sensitized/challenged mice. OVA-sensitized mice treated with z-VAD-fmk immediately before allergen challenge showed marked reduction in inflammatory cell infiltration in the airways and pulmonary blood vessels, mucus production, and Th2 cytokine production. We hypothesized that the caspase inhibitor prevented T cell activation, resulting in the reduction of cytokine production and eosinophil infiltration. Treatment with z-VAD-fmk in vivo prevented subsequent T cell activation ex vivo. We propose that caspase inhibitors may offer a novel therapeutic approach to T cell-dependent inflammatory airway diseases.     

Reduced endothelial nitric oxide synthase in lungs of chronically ventilated preterm lambs

Nitric oxide (NO), produced in lung vascular endothelium and airway epithelium, has an important role in regulating smooth muscle cell growth and tone. Chronic lung disease, a frequent complication of premature birth, is characterized by excess abundance, tone, and reactivity of smooth muscle in the pulmonary circulation and conducting airways, leading to increased lung vascular and airway resistance. Whether these structural and functional changes are associated with diminished pulmonary expression of endothelial nitric oxide synthase (eNOS) protein is unknown. Both quantitative immunoblot analysis and semiquantitative immunohistochemistry showed that there was less eNOS protein in the endothelium of small intrapulmonary arteries and epithelium of small airways of preterm lambs that were mechanically ventilated for 3 wk compared with control lambs born at term. No significant differences were detected for other proteins (inducible NOS, alpha-smooth muscle actin, and pancytokeratin). Lung vascular and respiratory tract resistances were greater in the chronically ventilated preterm lambs compared with control term lambs. These results support the notion that decreased eNOS in the pulmonary circulation and respiratory tract of preterm lambs may contribute to the pathophysiology of chronic lung disease.     

gammadelta T cells provide a breath of fresh air for asthma research.

gammadelta T cells downregulate airways responsiveness to allergen challenge, possibly by controlling the 'repair' response of the airway epithelium to alphabeta T cell-mediated damage (pages 1150-1156).     

Sex differences in the development of airway epithelial tolerance to naphthalene

Exposure to air pollution has been linked to pulmonary diseases. Naphthalene (NA), an abundant polycyclic aromatic hydrocarbon in tobacco smoke and urban air, is a model toxicant for air pollution effects in the lung. Repeated exposures to NA in male mice result in tolerance, defined as the emergence of a resistant cell phenotype after prior exposure. Tolerance has not been studied in females. Females have sex differences in airway epithelial responses and in the prevalence of certain airway diseases. Male and female mice were exposed to a tolerance-inducing regimen of NA, and lungs were examined by airway level to characterize the cellular changes associated with repeated NA exposure and to assess the expression of genes and proteins involved in NA bioactivation and detoxification. The airway epithelium in treated males resembled that of controls. Females in the tolerant state were characterized by dense populations of ciliated cells in midlevel, distal, and bifurcating airways and a lower abundance of Clara cells at all airway levels. Cytotoxicity following a secondary challenge dose was also greater in females than males. Furthermore, females had decreased gene/protein expression of CYP2F2, a P-450 that metabolizes NA to a toxic epoxide, and glutamate-cysteine ligase, the rate-limiting enzyme in glutathione synthesis, than NA-tolerant males at all airway levels examined. We conclude that, while females develop tolerance, sex differences exist in the tolerant state by airway level, and females remain more susceptible than males to repeated exposures to NA.     

CC chemokine ligand 1 promotes recruitment of eosinophils but not Th2 cells during the development of allergic airways disease

One of the characteristic features of allergic asthma is recruitment of large numbers of inflammatory cells including eosinophils and Th2 lymphocytes to the lung. This influx of inflammatory cells is thought to be a controlled and coordinated process mediated by chemokines and their receptors. It is thought that distinct, differential expression of chemokine receptors allows selective migration of T cell subtypes in response to the chemokines that bind these receptors. Th2 cells preferentially express CCR8 and migrate selectively to its ligand, CC chemokine ligand (CCL)1. We studied the role of the CCR8 ligand, CCL1, in the specific recruitment of Th2 cells and eosinophils to the lung in a murine model of allergic airway disease. We have demonstrated for the first time that CCL1 is up-regulated in the lung following allergen challenge. Moreover, a neutralizing Ab to CCL1 reduced eosinophil migration to the lung, but had no effect on recruitment of Th2 cells following allergen challenge. In addition, there was no change in airway hyperresponsiveness or levels of Th2 cytokines. In a Th2 cell transfer system of pulmonary inflammation, anti-CCL1 also failed to affect recruitment of Th2 cells to the lung following allergen challenge. Significantly, intratracheal instillation of rCCL1 increased recruitment of eosinophils but not Th2 cells to the lung in allergen-sensitized and -challenged mice. In summary, our results indicate that CCL1 is important for the pulmonary recruitment of eosinophils, rather than allergen-specific Th2 cells, following allergen challenge.     

Human bronchoalveolar lavage: biofluid analysis with special emphasis on sample preparation

Respiratory diseases are an important health problem throughout the world. Whether caused by industrial pollutants, infections, smoking, cancer or metabolic diseases, damage to the lungs and airways often lead to morbidity or death. Bronchoalveolar lavage (BAL) obtained by fiber-optic bronchoscopy is a biofluid mirroring the expression of normally secreted pulmonary proteins and the products of activated cells and destructive processes. The characterization of the proteome within this compartment provides an opportunity to establish temporal and prognostic indicators of airway disease. The objective of this study was to develop methods of analysis of BAL samples, which achieved the highest level of annotation of the expression map of this proteome. We have optimized the process of sample preparation after investigating a variety of techniques including dialysis, ultramembrane filtration, precipitation and gel filtration. We have further studied methods to remove albumin from BAL in order to unmask proteins hidden on two-dimensional gels. In a pilot application of the method, BAL protein profiles obtained from healthy nonsmokers and smokers at risk for developing chronic obstructive pulmonary disease showed distinct differences.     

Downregulation of leukotriene biosynthesis by thymoquinone attenuates airway inflammation in a mouse model of allergic asthma

Chronic airway inflammation is a key feature of bronchial asthma. Leukotrienes are potent inflammatory mediators that play a role in the pathophysiology of asthma, and their levels are elevated in the airways in response to allergen challenge. We examined the anti-inflammatory effect of thymoquinone (TQ), the active principle in the volatile oil of Nigella sativa seeds, on leukotriene (LT) biosynthesis in a mouse model of allergic asthma. Mice sensitized and challenged with ovalbumin (OVA) antigen had an increased amounts of leukotriene B4 and C4, Th2 cytokines, and eosinophils in bronchoalveolar lavage (BAL) fluid. In addition, there was also a marked increase in lung tissue eosinophilia and goblet cell numbers. Administration of TQ before OVA challenge inhibited 5-lipoxygenase, the main enzyme in leukotriene biosynthesis, expression by lung cells and significantly reduced the levels of LTB4 and LTC4. This was accompanied by a marked decrease in Th2 cytokines and BAL fluid and lung tissue eosinophilia, all of which are characteristics of airway inflammation. These results demonstrate the anti-inflammatory effect of TQ in experimental asthma.     

Interleukin-5-mediated allergic airway inflammation inhibits the human surfactant protein C promoter in transgenic mice

Allergen challenge in the lung of humans and animals is associated with surfactant dysfunction, but the mechanism of this effect has not been established. By using a murine model of asthma we now report the effect of allergen-induced airway inflammation on the expression of transgenes regulated by the human surfactant protein (hSP)-C promoter. The hSP-C 3.7-kilobase pair promoter was used to direct the expression of eotaxin, an eosinophil-selective chemokine, into the lungs of several transgenic lines. As expected, the transgenic mice expressed increased amounts of eotaxin mRNA and protein compared with wild-type mice. Surprisingly, following allergen challenge, there was a marked down-regulation of transgene mRNA in three independent transgenic lines. The down-regulation was in contrast to other related proteins such as endogenous eotaxin and surfactant protein D levels, which were both increased following allergen challenge. Consistent with specific down-regulation of the eotaxin transgene, there was no increase in pulmonary eosinophil levels in the transgenic mice above that found in wild-type mice. Analysis of hSP-C transgenic mice with distinct reporter genes and 3'-untranslated regions revealed that allergen challenge was directly affecting the hSP-C promoter. We hypothesized that allergen-induced down-regulation of the hSP-C promoter was related to the eosinophilic inflammation. To test this, we blocked eosinophilic inflammation in the lungs by treating mice with neutralizing antiserum against interleukin-5. Interestingly, this treatment also blocked allergen-induced inhibition of the hSP-C promoter. These results establish that allergic airway inflammation is associated with up-regulation of the surfactant proteins primarily involved in immunity, whereas down-regulation of the surfactant protein primarily involved in maintaining airway patency. Furthermore, the marked down-regulation of the hSP-C promoter is interleukin-5-dependent, implying a critical role for eosinophilic inflammation. These results suggest that alterations in surfactant protein levels may contribute to immune and airway dysfunction in asthma.     

Overexpression of lunatic fringe does not affect epithelial cell differentiation in the developing mouse lung

The Notch/Notch-ligand pathway regulates cell fate decisions and patterning in various tissues. Several of its components are expressed in the developing lung, suggesting that this pathway is important for airway cellular patterning. Fringe proteins, which modulate Notch signaling, are crucial for defining morphogenic borders in several organs. Their role in controlling cellular differentiation along anterior-posterior axis of the airways is unknown. Herein, we report the temporal-spatial expression patterns of Lunatic fringe (Lfng) and Notch-regulated basic helix-loop-helix factors, Hes1 and Mash-1, during murine lung development. Lfng was only expressed during early development in epithelial cells lining the larger airways. Those epithelial cells also expressed Hes1, but at later gestation Hes1 expression was confined to epithelium lining the terminal bronchioles. Mash-1 displayed a very characteristic expression pattern. It followed neural crest migration in the early lung, whereas at later stages Mash-1 was expressed in lung neuroendocrine cells. To clarify whether Lfng influences airway cell differentiation, Lfng was overexpressed in distal epithelial cells of the developing mouse lung. Overexpression of Lfng did not affect spatial or temporal expression of Hes1 and Mash-1. Neuroendocrine CGRP and protein gene product 9.5 expression was not altered by Lfng overexpression. Expression of proximal ciliated (beta-tubulin IV), nonciliated (CCSP), and distal epithelial cell (SP-C, T1alpha) markers also was not influenced by Lfng excess. Overexpression of Lfng had no effect on mesenchymal cell marker (alpha-sma, vWF, PECAM-1) expression. Collectively, the data suggest that Lunatic fringe does not play a significant role in determining cell fate in fetal airway epithelium.     

Alternaria induces STAT6-dependent acute airway eosinophilia and epithelial FIZZ1 expression that promotes airway fibrosis and epithelial thickness

The fungal allergen, Alternaria, is specifically associated with severe asthma, including life-threatening exacerbations. To better understand the acute innate airway response to Alternaria, naive wild-type (WT) mice were challenged once intranasally with Alternaria. Naive WT mice developed significant bronchoalveolar lavage eosinophilia following Alternaria challenge when analyzed 24 h later. In contrast to Alternaria, neither Aspergillus nor Candida induced bronchoalveolar lavage eosinophilia. Gene microarray analysis of airway epithelial cell brushings demonstrated that Alternaria-challenged naive WT mice had a >20-fold increase in the level of expression of found in inflammatory zone 1 (FIZZ1/Retnla), a resistin-like molecule. Lung immunostaining confirmed strong airway epithelial FIZZ1 expression as early as 3 h after a single Alternaria challenge that persisted for ≥5 d and was significantly reduced in STAT6-deficient, but not protease-activated receptor 2-deficient mice. Bone marrow chimera studies revealed that STAT6 expressed in lung cells was required for epithelial FIZZ1 expression, whereas STAT6 present in bone marrow-derived cells contributed to airway eosinophilia. Studies investigating which cells in the nonchallenged lung bind FIZZ1 demonstrated that CD45(+)CD11c(+) cells (macrophages and dendritic cells), as well as collagen-1-producing CD45(-) cells (fibroblasts), can bind to FIZZ1. Importantly, direct administration of recombinant FIZZ1 to naive WT mice led to airway eosinophilia, peribronchial fibrosis, and increased thickness of the airway epithelium. Thus, Alternaria induces STAT6-dependent acute airway eosinophilia and epithelial FIZZ1 expression that promotes airway fibrosis and epithelial thickness. This may provide some insight into the uniquely pathogenic aspects of Alternaria-associated asthma.     

Recombinant human platelet-activating factor-acetylhydrolase inhibits airway inflammation and hyperreactivity in mouse asthma model

Numerous in vitro and in vivo studies in both animal models and human asthmatics have implicated platelet-activating factor (PAF) as an important inflammatory mediator in asthma. In a murine asthma model, we examined the anti-inflammatory activities of recombinant human PAF-acetylhydrolase (rPAF-AH), which converts PAF to biologically inactive lyso-PAF. In this model, mice sensitized to OVA by i.p. and intranasal (i.n.) routes are challenged with the allergen by i.n. administration. The OVA challenge elicits an eosinophil infiltration into the lungs with widespread mucus occlusion of the airways and results in bronchial hyperreactivity. The administration of rPAF-AH had a marked effect on late-phase pulmonary inflammation, which included a significant reduction in airway eosinophil infiltration, mucus hypersecretion, and airway hyperreactivity in response to methacholine challenge. These studies demonstrate that elevating plasma levels of PAF-AH through the administration of rPAF-AH is effective in blocking the late-phase pulmonary inflammation that occurs in this murine allergen-challenge asthma model. These results suggest that rPAF-AH may have therapeutic effects in patients with allergic airway inflammation.     

V gamma 4+ gamma delta T cells regulate airway hyperreactivity to methacholine in ovalbumin-sensitized and challenged mice

The Vgamma4(+) pulmonary subset of gammadelta T cells regulates innate airway responsiveness in the absence of alphabeta T cells. We now have examined the same subset in a model of allergic airway disease, OVA-sensitized and challenged mice that exhibit Th2 responses, pulmonary inflammation, and airway hyperreactivity (AHR). In sensitized mice, Vgamma4(+) cells preferentially increased in number following airway challenge. Depletion of Vgamma4(+) cells before the challenge substantially increased AHR in these mice, but had no effect on airway responsiveness in normal, nonchallenged mice. Depletion of Vgamma1(+) cells had no effect on AHR, and depletion of all TCR-delta(+) cells was no more effective than depletion of Vgamma4(+) cells alone. Adoptively transferred pulmonary lymphocytes containing Vgamma4(+) cells inhibited AHR, but lost this ability when Vgamma4(+) cells were depleted, indicating that these cells actively suppress AHR. Eosinophilic infiltration of the lung and airways, or goblet cell hyperplasia, was not affected by depletion of Vgamma4(+) cells, although cytokine-producing alphabeta T cells in the lung increased. These findings establish Vgamma4(+) gammadelta T cells as negative regulators of AHR and show that their regulatory effect bypasses much of the allergic inflammatory response coincident with AHR.     

LRP1 loss in airway epithelium exacerbates smoke-induced oxidative damage and airway remodeling

The LDL receptor-related protein 1 (LRP1) partakes in metabolic and signaling events regulated in a tissue-specific manner. The function of LRP1 in airways has not been studied. We aimed to study the function of LRP1 in smoke-induced disease. We found that bronchial epithelium of patients with chronic obstructive pulmonary disease and airway epithelium of mice exposed to smoke had increased LRP1 expression. We then knocked out LRP1 in human bronchial epithelial cells in vitro and in airway epithelial club cells in mice. In vitro, LRP1 knockdown decreased cell migration and increased transforming growth factor β activation. Tamoxifen-inducible airway-specific LRP1 knockout mice (club Lrp1^?/?) induced after complete lung development had increased inflammation in the bronchoalveolar space and lung parenchyma at baseline. After 6 months of smoke exposure, club Lrp1^?/? mice showed a combined restrictive and obstructive phenotype, with lower compliance, inspiratory capacity, and forced expiratory volume_0.05/forced vital capacity than WT smoke-exposed mice. This was associated with increased values of Ashcroft fibrotic index. Proteomic analysis of room air exposed-club Lrp1^?/? mice showed significantly decreased levels of proteins involved in cytoskeleton signaling and xenobiotic detoxification as well as decreased levels of glutathione. The proteome fingerprint created by smoke eclipsed many of the original differences, but club Lrp1^?/? mice continued to have decreased lung glutathione levels and increased protein oxidative damage and airway cell proliferation. Therefore, LRP1 deficiency leads to greater lung inflammation and damage and exacerbates smoke-induced lung disease.     

Distribution of Clara cell secretory protein expression in the tracheobronchial airways of rhesus monkeys

Clara cell secretory protein (CCSP) is a protective lung protein that is believed to have antioxidant, immunomodulatory, and anticarcinogenic properties; to be present in all adult mammals; and to be well conserved in rodents, humans, and nonhuman primates. The rationale for this study is to define the distribution and abundance of CCSP in the airway epithelium and lavage fluid of the adult rhesus monkey and to provide information for evaluating CCSP as a marker of Clara cells and as a biomarker of lung health. Lung tissue and lavage fluid from 3-yr-old rhesus monkeys were examined using histopathology and immunohistochemistry. Proximal bronchi, midlevel bronchi, and terminal/respiratory bronchioles were compared for immunohistochemical localization of CCSP in three-dimensional whole mounts as well as in paraffin and Araldite sections. Immunoreactive CCSP was found in nonciliated cells throughout the airway epithelium. Proximal and midlevel airways had the highest labeling. CCSP decreased in distal airways, and respiratory bronchioles had little to no CCSP. CCSP in the most distal airways was in tall cuboidal cells adjacent to the pulmonary artery. Although a large number of cells were present in the terminal bronchioles that would be classified as Clara cells based on morphology (nonciliated cells with apical protrusions), only a small number stained positively for immunoreactive CCSP. Semiquantitative analysis of Western blots indicated that changes in lavage CCSP are consistent with, and may be predictive of, overall CCSP levels in the airway epithelium in this primate species that is phylogenetically similar to humans.