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The ability of corticosteroids to reduce airway inflammation and improve lung function is significantly reduced in asthmatics who are tobacco smokers compared with asthmatics who are nonsmokers. As not only high levels of tobacco smoke exposure in active smokers, but also significantly lower levels of tobacco smoke exposure from passive environmental tobacco smoke (ETS) exposure in nonsmokers can increase both asthma symptoms and the frequency of asthma exacerbations, we utilized a mouse model to determine whether corticosteroids can reduce levels of airway inflammation, airway remodeling, and airway hyperreactivity in mice exposed to the combination of chronic ETS and ovalbumin (OVA) allergen. Chronic ETS exposure alone did not induce increases in eosinophilic airway inflammation, airway remodeling, or airway hyperreactivity. Mice exposed to chronic OVA allergen had significantly increased levels of peribronchial fibrosis, increased thickening of the smooth muscle layer, increased mucus, and increased airway hyperreactivity, which was significantly enhanced by coexposure to the combination of chronic ETS and chronic OVA allergen. Administration of corticosteroids to mice exposed to chronic ETS and OVA allergen significantly reduced levels of eosinophilic airway inflammation, mucus production, peribronchial smooth muscle thickness, airway hyperreactivity, and the number of peribronchial TGF-[beta]1 + cells. Overall, this study demonstrates that corticosteroids can significantly reduce levels of eosinophilic inflammation, mucus expression, airway remodeling, and airway hyperreactivity in chronic ETS-exposed mice challenged with allergen. allergy; eosinophils

Although anticholinergic therapy inhibits bronchoconstriction in asthmatic patients and antigen-challenged animals, administration of atropine 1 h before antigen challenge significantly potentiates airway hyperreactivity and eosinophil activation measured 24 h later. This potentiation in airway hyperreactivity is related to increased eosinophil activation and is mediated at the level of the airway nerves. Since eosinophils produce nerve growth factor (NGF), which is known to play a role in antigeninduced airway hyperreactivity, we tested whether NGF mediates atropine-enhanced, antigen challenge-induced hyperreactivity. Antibody to NGF (Ab NGF) was administered to sensitized guinea pigs with and without atropine pretreatment (1 mg/kg iv) 1 h before challenge. At 24 h after challenge, animals were anesthetized, vagotomized, paralyzed, and ventilated. Electrical stimulation of both vagus nerves caused bronchoconstriction that was increased in challenged animals. Atropine pretreatment potentiated antigen challenge-induced hyperreactivity. Ab NGF did not affect eosinophils or inflammatory cells in any group, nor did it prevent hyperreactivity in challenged animals that were not pretreated with atropine. However, Ab NGF did prevent atropine-enhanced, antigen challenge-induced hyperreactivity and eosinophil activation (assessed by immunohistochemistry). This effect was specific to NGF, since animals given control IgG remained hyperreactive. These data suggest that anticholinergic therapy amplifies eosinophil interactions with airway nerves via NGF. Therefore, therapeutic strategies that target both eosinophil activation and NGF-mediated inflammatory processes in allergic asthma are likely to be beneficial. anticholinergic; asthma; muscarinic receptors; parasympathetic nerves; neurotrophins

Eosinophil counts in the bronchoalveolar lavage fluid of wild-type (WT) mice increased after ovalbumin (OVA) challenge, a response that was diminished in comparably challenged low-expressing coagulation factor VII ([FVII.sup.tTA/tTA]) mice. Levels of T helper type 2 (Th2) cytokines, IL-4, IL-5, and IL-13, and eosinophil-attracting chemokines, eotaxin and RANTES, were also lower in the OVA-challenged [FVII.sup.tTA/tTA] mice. Eosinophils purified from low-FVII mice underwent apoptosis at a faster rate compared with WT eosinophils, and eosinophil migration in response to eotaxin was reduced in eosinophils obtained from [FVII.sup.tTA/tTA] mice. Airway hyperresponsiveness and mucous layer thickness were reduced in OVA-treated [FVII.sup.tTA/tTA] mice, and addition of exogenous coagulation factor X (FX) enhanced mucin production in human epithelial NCI-H292 cells. Correspondingly, incubation of FX with NCI-H292 cells resulted in activated (a) FX production, suggesting that the components required for FX activation were present on NCI-H292 cells. These results demonstrate that FVIIa functions in the asthmatic response to an allergen by stimulating lung eosinophilia, airway hyperresponsiveness, and mucin production, this latter effect through its ability to activate FX in conjunction with tissue factor. coagulation factor X; eosinophilia; T helper type 2 cytokines; chemokines; airway

Asthma is one of the leading causes of childhood hospitalization, and its incidence is on the rise throughout the world. Currently, the standard treatment for asthma is the use of corticosteroids to try to suppress the inflammatory reaction taking place in the bronchial tree. Using a murine model of atopic allergic asthma employing a methacholine-hyperresponsive (A/J) as well as a hyporesponsive (C57BL/6) strain of mice sensitized and challenged with ovalbumin, we show that treatment with a synthetic Toll-like receptor 7 (TLR7) ligand (S-28463, a member of the imidazoquinoline family) prevents development of the asthmatic phenotype. Treatment with S-28463 resulted in a reduction of airway resistance and elastance following ovalbumin sensitization and challenge. This was accompanied by a dramatic reduction in infiltration of leukocytes, especially eosinophils, into the lungs of both C57BL/6 and A/J mice following OVA challenge. Treatment with S-28463 also abolished both the elevation in serum IgE level as well as the induction of IL-4, IL-5, and IL-13 by OVA challenge. The protective effects of S-28463 were also observed in MK2 knockout, but not MYD88 knockout, mice. We did not observe a switch in cytokine profile from [T.sub.H]2 to [T.sub.H]1, as both 1L-12p70 and IFN-[gamma] levels were reduced following S-28463 treatment. These results clearly demonstrate the anti-inflammatory effect of imidazoquinolines in an allergic asthma model as well as the clinical potential of TLR7 ligands in the treatment of allergic diseases. Toll-like receptors; asthma; eosinophils; lung; inflammation; mitogen-activated protein kinase-activated protein-2

Mucosal eosinophils increase in a number of gastrointestinal diseases that are often associated with altered epithelial barrier function, including food allergic enteropathies and inflammatory bowel diseases. Although eosinophils are known to secrete biologically active mediators including granule proteins, their role in gastrointestinal diseases is uncertain. The aim of this study was to determine the impact of eosinophils on intestinal barrier function. Epithelial barrier function was determined in a coculture of eosinophils and T84 epithelial cells and in a routine model of T helper (Th) type 2-mediated colitis. Coculture conditions resulted in decreased transepithelial resistance (TER) and increased transepithelial flux. Cell-free coculture supematants contained a [greater than or equal to]5-kDa soluble factor that also diminished TER; these supernatants contained the eosinophil-granule proteins major basic protein (MBP) and eosinophil-derived neurotoxin (EDN). T84 barrier function decreased significantly when basolateral surfaces were exposed to native human MBP but not EDN. Additional studies identified downregulation of the tight junctional molecule occludin as at least one mechanism for MBP action. MBP-null mice were protected from inflammation associated with oxazolone colitis compared with wild-type mice. In conclusion, MBP decreases epithelial barrier function and in this manner contributes to the pathogenesis of inflammatory bowel diseases. tight junction; eosinophilic gastroenteritis; food allergy

Ozone hyperreactivity over 24 h is mediated by blockade of inhibitory [M.sub.2] muscarinic autoreceptors by eosinophil major basic protein. Because eosinophil populations in the lungs fluctuate following ozone, the contribution of eosinophils to [M.sub.2] dysfunction and airway hyperreactivity was measured over several days. After one exposure to ozone, [M.sub.2] function, vagal reactivity, smooth muscle responsiveness, and inflammation were measured in anesthetized guinea pigs. Ozone-induced hyperreactivity to vagal stimulation persisted over 3 days. Although hyperreactivity one day after ozone is mediated by eosinophils, AbVLA-4 did not inhibit either eosinophil accumulation in the lungs or around the nerves or prevent hyperreactivity at this time point. Two days after ozone, eosinophils in BAL, around airway nerves and in lungs, were decreased, and neuronal [M.sub.2] receptor function was normal, although animals were still hyperreactive to vagal stimulation. Depleting eosinophils with AbIL-5 prevented hyperreactivity, thus eosinophils contribute to vagal hyperreactivity by mechanisms separate from [M.sub.2] receptor blockade. Three days after ozone, vagal hyperreactivity persisted, eosinophils were again elevated in BAL in lungs and around nerves, and [M.sub.2] receptors were again dysfunctional. At this point, airway smooth muscle was also hyperresponsive to methacholine. Eosinophil depletion with AbIL-5, AbVLA-4, or cyclophosphamide protected [M.sub.2] function 3 days after ozone and prevented smooth muscle hyperreactivity. However, vagal hyperreactivity was significandy potentiated by eosinophil depletion. The site of hyperreactivity, muscle or nerve, changes over 3 days after a single exposure to ozone. Additionally, the role of eosinophils is complex; they mediate hyperreactivity acutely while chronically may be involved in repair. [M.sub.2] muscarinic receptors; vagus nerves

One immunological component of asthma is believed to be the interaction of eosinophils with parasympathetic cholinergic nerves and a consequent inhibition of acetylcholine muscarinic [M.sub.2] receptor activity, leading to enhanced acetylcholine release and bronchoconstriction. Here we have used an in vitro model of cholinergic nerve function, the human IMR32 cell line, to study this interaction. IMR32 cells, differentiated in culture for 7 days, expressed M2 receptors. Cells were radiolabeled with [[sup.3]H]choline and electrically stimulated. The stimulation-induced release of acetylcholine was prevented by the removal of [Ca.sup.2+]. The muscarinic [M.sub.1]/[M.sub.2] receptor agonist arecaidine reduced the release of acetylcholine after stimulation (to 82 [+ or -] 2% of control at [10.sup.-7] M), and the [M.sub.2] receptor antagonist AF-DX 116 increased it (to 175 [+ or -] 23% of control at [10.sup.-5] M), indicating the presence of a functional [M.sub.2] receptor that modulated acetylcholine release. When human eosinophils were added to IMR32 cells, they enhanced acetylcholine release by 36 [+ or -] 10%. This effect was prevented by inhibitors of adhesion of the eosinophils to the IMR32 cells. Pretreatment of IMR32 cells with 10 mM carbachol, to desensitize acetylcholine receptors, prevented the potentiation of acetylcholine release by eosinophils or AF-DX 116. Acetylcholine release was similarly potentiated (by up to 45 [+ or -] 7%) by degranulation products from eosinophils that had been treated with N-formyl-methionyl-leucyl-phenylalanine or that had been in contact with IMR32 cells. Contact between eosinophils and IMR32 cells led to an initial increase in expression of [M.sub.2] receptors, whereas prolonged exposure reduced [M.sub.2] receptor expression. adhesion molecule; [M.sub.2] receptor; asthma

The adhesion of eosinophils to nerve cells and the subsequent release of eosinophil products may contribute to the pathogenesis of conditions such as asthma and inflammatory bowel disease. In this study we have separately examined the consequences of eosinophil adhesion and degranulation for nerve cell morphology and development. Eosinophils induced neurite retraction of cultured guinea pig parasympathetic nerves and differentiated IMR32 cholinergic neuroblastoma cells. Inhibition of eosinophil adhesion to IMR32 cells attenuated this retraction. Eosinophil adhesion to IMR32 cells led to tyrosine phosphorylation of a number of nerve cell proteins, activation of p38 MAP kinase, and generation of neuronal reactive oxygen species (ROS). Inhibition of tyrosine kinases with genistein prevented both the generation of ROS in the nerve cells and neurite retraction. The p38 MAP kinase inhibitor SB-239063 prevented neurite retraction but had no effect on the induction of ROS. Thus eosinophils induced neurite retraction via two distinct pathways: by generation of tyrosine kinase-dependent ROS and by p38 MAP kinase. Eosinophils also prevented neurite outgrowth during differentiation of IMR32 cells. In contrast to their effect on neurite retraction, this effect was mimicked by medium containing products released from eosinophils and by eosinophil major basic protein. These results indicate that eosinophils modify the morphology of nerve cells by distinct mechanisms that involve adhesion and released proteins. inflammation; cell adhesion molecule; cholinergic nerve; major basic protein

Eosinophils are known to play a vital role in allergic conditions such as asthma. Their action is linked to the release of cytotoxic proteins and other mediators that trigger hyperreactivity in respiratory muscle cells. Eosinophils have also been shown to increase phosphatidylcholine (PC) secretion in primary cultures of rat type II pneumocytes. Further examination of the role of eosinophil major basic protein (MBP) shows that MBP induces PC secretion and provides mechanical stability in rat type II pneumocytes, thereby helping to protect against lung atelectasis.

Eosinophil major basic protein (MBP) contracts tracheal smooth muscles (TSM) in guinea pigs through the stimulation of the parasympathetic nervous system and activation of the cyclooxygenase pathway. The synthetic cationic proteins poly-l-lysine (PL) and poly-l-arginine (PA) are unable to cause TSM contraction, though their molecular weight and charge are similar to that of MBP. The cationic charge of MBP appears not to mediate all its effects on airway muscles. Atropine inhibits the response to MBP but not to PL or PA, while indomethacin inhibits the response to MBP and PL but not to PA.

A new method for real-time measurement of variations in luminal area in microexplants of airways is developed. The variation occur during the physiological and pharmacological interventions in the lungs. The technique is based on the concentration-response characteristics and spontaneous tone similarities.

An analysis of the calcium regulation on chemotaxis in new eosinophils showed that reducing the availability of Ca ions by treatment hampered cell polarization and movement. Cells exhibiting chemotaxis in the presence of a soluble factor of proteinaceous origin showed gradients of calcium ion concentrations. Cells displayed increased calcium ion concentration when they underwent a change in their direction of migration. Cells undergoing migration showed calcium ion concentration gradient along the long axis, the front of the cell having less calcium ion concentration than the rear.

Trifilieff, Alexandre, Ahmed El-Hashim, and Claude Bertrand. Time course of inflammatory and remodeling events in a murine model of asthma: effect of steroid treatment. Am J Physiol Lung Cell Mol Physiol 279: L1120-L1128, 2000.--The kinetics of airway inflammation and remodeling processes following ovalbumin aerosol challenge in sensitized BALB/c mice was studied. Mice were exposed to either single or five ovalbumin challenges over 5 days. In both protocols, time-dependent increases in bronchoalveolar lavage (BAL) cellular fibronectin, neutrophils and eosinophils were observed. The kinetics of these events were similar in both protocols; however, the magnitude of the response was much greater following repeated challenges. BAL protein levels and lymphocyte numbers were increased only following repeated challenges, whereas interleukin (IL)-5 and IL-4 were increased in both protocols. Histological analysis revealed a time-dependent increase in epithelial cell proliferation and in mucus-producing epithelial cells. Proliferation of alveolar cells was observed only following repeated challenges. Airway hyperreactivity was observed in both protocols but was much greater following repeated challenges. Pretreatment with dexamethasone fully inhibited the inflammatory response and airway hyperreactivity but only partially inhibited the remodeling process. These data suggest that glucocorticoids, although potent anti-inflammatory agents, may not be potent in reducing the lung remodeling process associated with asthma. airway hyperreactivity; dexamethasone; eosinophils

Munoz, Nilda M., Gijs A. van Seventer, Roshanak T. Semnani, and Alan R. Leff. Augmentation of [LTC.sub.4] synthesis in human eosinophils caused by CD3-stimulated Th2-like cells in vitro. Am J Physiol Lung Cell Mol Physiol 278: L1172-L1179, 2000.--We assessed the effect of anti-CD3-stimulated secretion of cultured human Th1- and Th2-like cells on leukotriene [C.sub.4] ([LTC.sub.4]) secretion in isolated human eosinophils. T helper (Th) cell subsets were generated from human naive [CD4.sup.+] T cells cocultured with irradiated human transformed B cells and either recombinant human interleukin (rhIL)-1[Beta] plus rhIL-6 plus rhIL-12 for Th1-like cells or rhIL-1[Beta] plus rhIL-6 plus rhIL-4 for Th2-like cells. Coincubation of eosinophils with 1:5 dilution of Th2-supernatant (Sup) caused an increase in [LTC.sub.4] secretion caused by 0.1 [micro]M formyl-Met-Leu-Phe and 5 [micro]g/ml cytochalasin B from 921 [+ or -] 238 to 3,067 [+ or -] 1,462 pg/[10.sup.6] eosinophils (P [is less than] 0.01). Th1-Sup at the same dilution had no augmenting effect on stimulated secretion of [LTC.sub.4] in eosinophils despite substantial concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the supernatant. Dilution of Th1-Sup caused increased [LTC.sub.4] that returned to baseline after immunoabsorption of GM-CSF, suggesting the presence of a possible inhibitory factor. We demonstrate that pretreatment of eosinophils with 1:5 dilution of Th2-Sup but not of Th1-Sup causes substantial augmentation of [LTC.sub.4] secretion in vitro and establishes that human Th2 cells cause direct augmentation of [LTC.sub.4] secretion within 15-30 min of exposure. T helper cells; cytokine; T helper type 1-like cells; immunodepletion; leukotriene [C.sub.4]

Munoz, Nilda M., Kimm J. Hamann, Klaus F. Rabe, Hiroyuki Sano, Xiangdong Zhu, and Alan R. Left. Augmentation of eosinophil degranulation and [LTC.sub.4] secretion by integrin-mediated endothelial cell adhesion. Am. J. Physiol. 277 (Lung Cell. Mol. Physiol. 21): L802-L810, 1999.--We examined the effect of eosinophil ligation to cultured human umbilical vein endothelial cells (HUVECs) in augmenting the stimulated secretion of leukotriene (LT) [C.sub.4] and eosinophil peroxidase (EPO). The effects of adhesion were compared before and after specific blockade with monoclonal antibodies directed against eosinophil surface integrins or endothelial counterligands. Adhesion to HUVECs augmented EPO release caused by formyl-methionyl-leucyl-phenylalanine plus cytochalasin B from 403 [+ or -] 15.3 (BSA control) to 778 [+ or -] 225 ng/[10.sup.6] cells for eosinophils exposed to interleukin-l[Alpha]-treated HUVECs (P [is less than] 0.05) and also caused a twofold increase in stimulated [LTC.sub.4] secretion (P [is less than] 0.05). To determine whether augmented secretion resulted directly from adhesive ligation, studies were also performed with paraformaldehyde-treated HUVECs; stimulated secretion of [LTC.sub.4] from eosinophils was comparable to that for living HUVECs. Our study is the first demonstration that adhesion to HUVECs through ligation to [[Alpha].sub.4]- or [[Beta].sub.2]-integrin on the eosinophil surface causes augmentation of stimulated secretion of both EPO and [LTC.sub.4] and that blockade of adhesion molecules on either eosinophils or HUVECs prevents the priming effect on eosinophil secretion. eosinophils; adhesion molecules; leukotriene [C.sub.4]; eosinophil peroxidase