Regulation of Eosinophil Recruitment and Allergic Airway Inflammation by Tropomyosin Receptor Kinase A.

Eosinophilia is a hallmark of allergic airway inflammation (AAI). Identifying key molecules and specific signaling pathways that regulate eosinophilic inflammation is critical for development of novel therapeutics. Tropomycin receptor kinase A (TrkA) is the high-affinity receptor for nerve growth factor. AAI is associated with increased expression of TrkA by eosinophils; however, the functional role of TrkA in regulating eosinophil recruitment and contributing to AAI is poorly understood. This study identifies, to our knowledge, a novel mechanism of eotaxin-mediated activation of TrkA and its role in regulating eosinophil recruitment by using a chemical-genetic approach to specifically inhibit TrkA kinase activity with 1-NM-PP1 in TrkAF592A-knock-in (TrkA-KI) eosinophils. Blockade of TrkA by 1-NM-PP1 enhanced eosinophil spreading on VCAM-1 but inhibited eotaxin-1 (CCL11)-mediated eosinophil migration, calcium flux, cell polarization, and ERK1/2 activation, suggesting that TrkA is an important player in the signaling pathway activated by eotaxin-1 during eosinophil migration. Further, blockade of matrix metalloprotease with BB-94 inhibited eotaxin-1-induced TrkA activation and eosinophil migration, additively with 1-NM-PP1, indicating a role for matrix metalloproteases in TrkA activation. TrkA inhibition in Alternaria alternata-challenged TrkA-KI mice markedly inhibited eosinophilia and attenuated various features of AAI. These findings are indicative of a distinctive eotaxin-mediated TrkA-dependent signaling pathway, which, in addition to other TrkA-activating mediators, contributes to eosinophil recruitment during AAI and suggests that targeting the TrkA signaling pathway to inhibit eosinophil recruitment may serve as a therapeutic strategy for management of eosinophilic inflammation in allergic airway disease, including asthma.

FABP4 regulates eosinophil recruitment and activation in allergic airway inflammation.

Fatty acid binding protein 4 (FABP4), a member of a family of lipid-binding proteins, is known to play a role in inflammation by virtue of its ability to regulate intracellular events such as lipid fluxes and signaling. Studies have indicated a proinflammatory role for FABP4 in allergic asthma although its expression and function in eosinophils, the predominant inflammatory cells recruited to allergic airways, were not investigated. We examined expression of FABP4 in murine eosinophils and its role in regulating cell recruitment in vitro as well as in cockroach antigen (CRA)-induced allergic airway inflammation. CRA exposure led to airway recruitment of FABP4-expressing inflammatory cells, specifically eosinophils, in wild-type (WT) mice. FABP4 expression in eosinophils was induced by TNF-α as well as IL-4 and IL-13. FABP4-deficient eosinophils exhibited markedly decreased cell spreading/formation of leading edges on vascular cell adhesion molecule-1 and significantly decreased adhesion to intercellular adhesion molecule-1 associated with reduced ß2-integrin expression relative to WT cells. Furthermore, FABP4-deficient eosinophils exhibited decreased migration, F-actin polymerization, calcium flux, and ERK(1/2) phosphorylation in response to eotaxin-1. In vivo, CRA-challenged FABP4-deficient mice exhibited attenuated eosinophilia and significantly reduced airway inflammation (improved airway reactivity, lower IL-5, IL-13, TNF-α, and cysteinyl leukotriene C4 levels, decreased airway structural changes) compared with WT mice. In conclusion, expression of FABP4 in eosinophils is induced during conditions of inflammation and plays a proinflammatory role in the development of allergic asthma by promoting eosinophil adhesion and migration and contributing to the development of various aspects of airway inflammation.

Knob protein enhances epithelial barrier integrity and attenuates airway inflammation.

BACKGROUND: Altered epithelial physical and functional barrier properties along with TH1/TH2 immune dysregulation are features of allergic asthma. Regulation of junction proteins to improve barrier function of airway epithelial cells has the potential for alleviation of allergic airway inflammation. OBJECTIVE: We sought to determine the immunomodulatory effect of knob protein of the adenoviral capsid on allergic asthma and to investigate its mechanism of action on airway epithelial junction proteins and barrier function. METHODS: Airway inflammation, including junction protein expression, was evaluated in allergen-challenged mice with and without treatment with knob. Human bronchial epithelial cells were exposed to knob, and its effects on expression of junction proteins and barrier integrity were determined. RESULTS: Administration of knob to allergen-challenged mice suppressed airway inflammation (eosinophilia, airway hyperresponsiveness, and IL-5 levels) and prevented allergen-induced loss of airway epithelial occludin and E-cadherin expression. Additionally, knob decreased expression of TH2-promoting inflammatory mediators, specifically IL-33, by murine lung epithelial cells. At a cellular level, treatment of human bronchial epithelial cells with knob activated c-Jun N-terminal kinase, increased expression of occludin and E-cadherin, and enhanced epithelial barrier integrity. CONCLUSION: Increased expression of junction proteins mediated by knob leading to enhanced epithelial barrier function might mitigate the allergen-induced airway inflammatory response, including asthma.

Assessment of eosinophil peroxidase as a potential diagnostic and prognostic marker in dogs with inflammatory bowel disease.

OBJECTIVE To evaluate a method for identifying intact and degranulated eosinophils in the small intestine of dogs with inflammatory bowel disease (IBD) by use of a monoclonal antibody (mAb) against eosinophil peroxidase (EPX). ANIMALS 11 untreated dogs with IBD, 5 dogs with IBD treated with prednisolone, and 8 control dogs with no clinical evidence of gastrointestinal tract disease and no immunosuppressive treatment. PROCEDURES 4-µm-thick sections of paraffin-embedded tissues from necropsy specimens were immunostained with EPX mAb. Stained intact and degranulated eosinophils in consecutive microscopic fields (400X magnification) of the upper (villus tips) and lower (between the muscularis mucosae and crypts) regions of the lamina propria of the jejunum were manually counted. RESULTS Compared with control and treated IBD dogs, untreated IBD dogs had a significantly higher number of degranulated eosinophils in the lower region of the lamina propria. However, no significant differences were detected in the number of intact eosinophils in this region among groups. In the upper region of the lamina propria, untreated IBD dogs had a significantly higher number of degranulated and intact eosinophils, compared with control and treated IBD dogs. Number of degranulated and intact eosinophils did not differ significantly between control and treated IBD dogs. CONCLUSIONS AND CLINICAL RELEVANCE Immunohistologic analysis with EPX mAb yielded prominent granule staining that allowed reliable morphological identification of degranulated and intact eosinophils, which may provide a strategy for quantitative and selective evaluation of eosinophils in gastrointestinal biopsy specimens and a potential method to diagnose IBD and evaluate treatment outcome.

High-fat diet promotes lung fibrosis and attenuates airway eosinophilia after exposure to cockroach allergen in mice.

Obesity is an important risk factor for asthma but the mechanistic basis for this association is not well understood. In the current study, the impact of obesity on lung inflammatory responses after allergen exposure was investigated. C57BL/6 mice maintained on a high-fat diet (HFD) or a normal diet (ND) after weaning were sensitized and challenged with cockroach allergen (CRA). Airway inflammation was assessed based on inflammatory cell recruitment, measurement of lung Th1-Th2 cytokines, chemokines, eicosanoids, and other proinflammatory mediators as well as airway hyperresponsiveness (AHR). CRA-challenged mice fed a HFD exhibited significantly decreased allergen-induced airway eosinophilia along with reduced lung IL-5, IL-13, LTC4, CCL11, and CCL2 levels as well as reduced mucus secretion and smooth muscle mass compared to ND fed mice. However, allergen-challenged HFD fed mice demonstrated significantly increased PAI-1 and reduced PGE2 levels in the lung relative to corresponding ND fed mice. Interestingly, saline-exposed HFD fed mice demonstrated elevated baseline levels of TGF-ß1, arginase-1, hypoxia-inducible factor-1α, and lung collagen expression associated with decreased lung function compared to corresponding ND fed mice. These studies indicate that a HFD inhibits airway eosinophilia while altering levels of PAI-1 and PGE2 in response to CRA in mice. Further, a HFD can lead to the development of lung fibrosis even in the absence of allergen exposure which could be due to innate elevated levels of specific profibrotic factors, potentially affecting lung function during asthma.

Regulation of serotonin-induced trafficking and migration of eosinophils.

Association of the neurotransmitter serotonin (5-HT) with the pathogenesis of allergic asthma is well recognized and its role as a chemoattractant for eosinophils (Eos) in vitro and in vivo has been previously demonstrated. Here we have examined the regulation of 5-HT-induced human and murine Eos trafficking and migration at a cellular and molecular level. Eos from allergic donors and bone marrow-derived murine Eos (BM-Eos) were found to predominantly express the 5-HT2A receptor. Exposure to 5-HT or 2,5-dimethoxy-4-iodoamphetamine (DOI), a 5-HT2A/C selective agonist, induced rolling of human Eos and AML14.3D10 human Eos-like cells on vascular cell adhesion molecule (VCAM)-1 under conditions of flow in vitro coupled with distinct cytoskeletal and cell shape changes as well as phosphorylation of MAPK. Blockade of 5-HT2A or of ROCK MAPK, PI3K, PKC and calmodulin, but not G(αi)-proteins, with specific inhibitors inhibited DOI-induced rolling, actin polymerization and changes in morphology of VCAM-1-adherent AML14.3D10 cells. More extensive studies with murine BM-Eos demonstrated the role of 5-HT in promoting rolling in vivo within inflamed post-capillary venules of the mouse cremaster microcirculation and confirmed that down-stream signaling of 5-HT2A activation involves ROCK, MAPK, PI3K, PKC and calmodulin similar to AML14.3D10 cells. DOI-induced migration of BM-Eos is also dependent on these signaling molecules and requires Ca(2+). Further, activation of 5-HT2A with DOI led to an increase in intracellular Ca(2+) levels in murine BM-Eos. Overall, these data demonstrate that 5-HT (or DOI)/5-HT2A interaction regulates Eos trafficking and migration by promoting actin polymerization associated with changes in cell shape/morphology that favor cellular trafficking and recruitment via activation of specific intracellular signaling molecules (ROCK, MAPK, PI3K and the PKC-calmodulin pathway).

The p110δ subunit of PI3K regulates bone marrow-derived eosinophil trafficking and airway eosinophilia in allergen-challenged mice.

Trafficking and recruitment of eosinophils during allergic airway inflammation is mediated by the phosphatidylinositol 3-kinase (PI3K) family of signaling molecules. The role played by the p110δ subunit of PI3K (PI3K p110δ) in regulating eosinophil trafficking and recruitment was investigated using a selective pharmacological inhibitor (IC87114). Treatment with the PI3K p110δ inhibitor significantly reduced murine bone marrow-derived eosinophil (BM-Eos) adhesion to VCAM-1 as well as ICAM-1 and inhibited activation-induced changes in cell morphology associated with reduced Mac-1 expression and aberrant cell surface localization/distribution of Mac-1 and α4. Infused BM-Eos demonstrated significantly decreased rolling and adhesion in inflamed cremaster muscle microvessels of mice treated with IC87114 compared with vehicle-treated mice. Furthermore, inhibition of PI3K p110δ significantly attenuated eotaxin-1-induced BM-Eos migration and prevented eotaxin-1-induced changes in the cytoskeleton and cell morphology. Knockdown of PI3K p110δ with siRNA in BM-Eos resulted in reduced rolling, adhesion, and migration, as well as inhibition of activation-induced changes in cell morphology, validating its role in regulating trafficking and migration. Finally, in a mouse model of cockroach antigen-induced allergic airway inflammation, oral administration of the PI3K p110δ inhibitor significantly inhibited airway eosinophil recruitment, resulting in attenuation of airway hyperresponsiveness in response to methacholine, reduced mucus secretion, and expression of proinflammatory molecules (found in inflammatory zone-1 and intelectin-1). Overall, these findings indicate the important role played by PI3K p110δ in mediating BM-Eos trafficking and migration by regulating adhesion molecule expression and localization/distribution as well as promoting changes in cell morphology that favor recruitment during inflammation.

Chronic allergen challenge induces pulmonary extramedullary hematopoiesis.

Allergic inflammation is associated with increased generation and trafficking of inflammatory cells, especially eosinophils, to sites of inflammation. The effect of acute versus chronic airway allergen challenge on hematopoietic activity in the bone marrow (BM) and lungs was investigated using murine models of allergic airway inflammation. Acute allergen challenge induced proliferation of BM cells and significantly increased generation of eosinophil, but not multipotent, granulocyte-macrophage (GM), or B-lymphocyte progenitor cells. However, no hematopoietic activity was observed in the lungs. With chronic challenge, BM cells failed to proliferate, but exhibited increased capacity to generate multipotent as well as eosinophil, GM, and B-lymphocyte progenitors. In addition, increased generation of eosinophil- and GM-specific progenitors was observed in the lungs. Although no differences were observed in their ability to roll on BM endothelium in vitro or in vivo, CD34-enriched hematopoietic/stem progenitor cells (HSPCs) from chronic-, but not acute-, challenged mice demonstrated reduced migration across BM endothelial cells associated with decreased CXCR4 expression. Overall, these studies demonstrate that chronic allergen exposure can alter BM homing due to decreased transendothelial migration enabling noninteracting HSPCs to egress out of the BM and recruit to sites of inflammation such as the airways, resulting in extramedullary hematopoiesis.

Allergen-induced airway remodeling is impaired in galectin-3-deficient mice.

The role played by the beta-galactoside-binding lectin galectin-3 (Gal-3) in airway remodeling, a characteristic feature of asthma that leads to airway dysfunction and poor clinical outcome in humans, was investigated in a murine model of chronic allergic airway inflammation. Wild-type (WT) and Gal-3 knockout (KO) mice were subjected to repetitive allergen challenge with OVA up to 12 wk, and bronchoalveolar lavage fluid (BALF) and lung tissue collected after the last challenge were evaluated for cellular features associated with airway remodeling. Compared to WT mice, chronic OVA challenge in Gal-3 KO mice resulted in diminished remodeling of the airways with significantly reduced mucus secretion, subepithelial fibrosis, smooth muscle thickness, and peribronchial angiogenesis. The higher degree of airway remodeling in WT mice was associated with higher Gal-3 expression in the BALF as well as lung tissue. Cell counts in BALF and lung immunohistology demonstrated that eosinophil infiltration in OVA-challenged Gal-3 KO mice was significantly reduced compared with that WT mice. Evaluation of cellular mediators associated with eosinophil recruitment and airway remodeling revealed that levels of eotaxin-1, IL-5, IL-13, found in inflammatory zone 1, and TGF-beta were substantially lower in Gal-3 KO mice. Finally, leukocytes from Gal-3 KO mice demonstrated decreased trafficking (rolling) on vascular endothelial adhesion molecules compared with that of WT cells. Overall, these studies demonstrate that Gal-3 is an important lectin that promotes airway remodeling via airway recruitment of inflammatory cells, specifically eosinophils, and the development of a Th2 phenotype as well as increased expression of eosinophil-specific chemokines and profibrogenic and angiogenic mediators.

Roflumilast increases Clara cell secretory protein in cigarette smoke-exposed mice.

Decreased Clara cell secretory protein (CCSP) levels have been found in smokers and chronic obstructive pulmonary disease (COPD) patients, which may be related to the development of COPD. A phosphodiesterase-4 (PDE4) inhibitor, roflumilast, appears to have therapeutic value for COPD. However, its effect on CCSP in cigarette smoke (CS)-exposed lungs has not been investigated. AKR/J mice were treated as follows: air control, CS, roflumilast plus CS, and roflumilast. Mice underwent four weeks of air or CS exposure. Roflumilast was administrated at 5mg/kg via gavage once daily for the duration of the study. CCSP levels in bronchoalveolar lavage (BAL) fluid and ERK1/2 activation in lungs were examined. CS exposure tended to decrease CCSP levels in BAL fluid compared to air controls. Treatment with roflumilast significantly reversed CS-induced downward trend of CCSP in BAL fluid. Roflumilast significantly inhibited CS-induced upward trend of ERK1/2 activation in lungs, and the levels of activated ERK1/2 in lungs negatively correlated with CCSP levels of BAL fluid in CS, and CS plus roflumilast groups. Our results demonstrate that one of the therapeutic mechanisms of roflumilast is to reverse CS-induced downward trend in CCSP levels of BAL fluid, which may be mediated by down-regulating ERK1/2 activity.

[Effects of cigarette smoke extract on the proliferation and the expression of TGF-beta1 and EGF in human embryonic lung fibroblasts].

OBJECTIVE: To observe the effects of cigarette smoke extract (CSE) on the proliferation and the expression of TGF-beta(1) and EGF in human embryonic lung fibroblasts (HELF). METHODS: The cultures of HELF were incubated with CSE (1:50, 1:25 and 1:10) for examining the effects of CSE on the proliferation and the proliferating cell nuclear antigen (PCNA) levels in HELF. The total RNA was extracted from the cells incubated with CSE at different doses. The expression levels of TGF-beta(1) mRNA were evaluated by reverse transcription-polymerase chain reaction (RT-PCR). The mRNA levels of TGF-beta(1) were corrected by GAPDH transcripts. TGF-beta(1) and EGF protein levels were determined by immunocytochemistry. RESULTS: The proliferation of HELF was inhibited by CSE in a dose-dependent manner. CSE also decreased the PCNA levels of HELF at all doses (P < 0.05). CSE stimulated TGF-beta(1) mRNA and protein expression of HELF at lower concentrations (1:50 and 1:25, P < 0.05). TGF-beta(1) mRNA and protein production were not increased by CSE at a higher concentration (1:10, P > 0.05). Decreased expression of EGF protein was observed in HELF treated by CSE (1:25 and 1:10, P < 0.05). CONCLUSIONS: CSE can inhibit the proliferation of HELF and the effect may involve the abnormal expression of TGF-beta(1) and EGF in HELF.

[Changes of the actin and transforming growth factor-beta1 expression in the small airways of the rat with chronic obstructive lung disease].

OBJECTIVE: To study the roles of actin and transforming growth factor (TGF)-beta(1) in the injury repair and the development of emphysema. METHODS: Wistar rats were randomly divided into two groups: the smoking and infection group (group SI) and the control group (group C). The rats of group SI received smoking irritation accompanying with repeated intranasal infection. Subgroups of the experimental animals were killed in the 2nd, 4th, 8th and 16th weeks respectively. The morphological changes of lungs were compared and PaO(2), PaCO(2) as well as the right ventricular systolic pressure (RVSP) were analysed. The lung sections were stained with immunohistochemistry for actin and TGF-beta(1). RESULTS: In comparison with animals of group C, thickening of the bronchiolar walls, narrowing of bronchiolar lumens, and area of emphysema were much severe in animals of group SI (P < 0.05). The muscularization of intra-alveolar arteries in group SI in the 16th week was apparent in comparing with that in group C (P < 0.05). PaO(2) values in group SI were significantly decreased, and RVSP values in group SI were significantly increased in the 8th and 16th week (P < 0.05). Actin expression was increased in animals of group SI in the 4th and 8th week (0.24 +/- 0.06 and 0.25 +/- 0.05) in comparing with that of group C (0.09 +/- 0.03) (P < 0.05). Animals of group SI showed a significant increase of TGF-beta(1) in lung tissue in different periods as mentioned in above (33.33 +/- 12.11, 45.71 +/- 15.12, 71.43 +/- 16.76 and 86.25 +/- 20.66 respectively). CONCLUSIONS: The increased expression of actin and TGF-beta(1) protein in small airways induced by smoking irritation and Klebsiella Pneumoniae may interfere with the repair response, and contributes to the development of emphysema.