Chronic allergen challenge induces bronchial mast cell accumulation in BALB/c but not C57BL/6 mice and is independent of IL-9

As genetically engineered mutant mice deficient in single genes are usually generated on a C57BL/6 background, to study mast cell trafficking in mutant mice, we initially investigated whether mast cells accumulated in bronchi in C57BL/6 mice challenged with OVA allergen acutely or chronically for 1 to 3 months. The total number of bronchial mast cells were quantitated using toluidine blue staining in airways of different sizes, i.e. , small (<90 μm), medium (90–155 μm), or large (>150 μm) airways. Non-OVA challenged and acute OVA challenged mice (C57BL/6 and BALB/c) had no detectable bronchial mast cells. Chronic OVA challenge in BALB/c mice for 1 or 3 months induced a significant increase in the number of bronchial mast cells in small-, medium-, and large-sized airways but minimal change in the number of bronchial mast cells in C57BL/6 mice. Both BALB/c and C57BL/6 mice developed significant lung eosinophilia following acute or chronic OVA challenge. Studies of IL-9-deficient mice on a BALB/c background demonstrated a significant increase in the number of bronchial mast cells in IL-9-deficient mice suggesting that IL-9 was not required for the bronchial accumulation of mast cells. Overall, these studies demonstrate that the chronic OVA challenge protocol we have utilized in BALB/c mice provides a model to study the mechanism of bronchial mast cell accumulation and that bronchial mast cell accumulation in chronic OVA challenged mice is independent of IL-9 in this model.     

Overexpression of IL-15 in vivo enhances Tc1 response, which inhibits allergic inflammation in a murine model of asthma

IL-15, a pleiotropic cytokine, is involved in the inflammatory responses in various infectious and autoimmune diseases. We have recently constructed IL-15-transgenic (Tg) mice, which have an increased number of memory-type CD8+ T cells in the peripheral lymphoid tissues. In the present study, we found that eosinophilia and Th2-type cytokine production in the airway were severely attenuated in OVA-sensitized IL-15-Tg mice following OVA inhalation. IL-15-Tg mice preferentially developed Tc1 responses mediated by CD8+ T cells after OVA sensitization, and in vivo depletion of CD8+ T cells by anti-CD8 mAb aggravated the allergic airway inflammation in IL-15-Tg mice following OVA inhalation. Adoptive transfer of CD8+ T cells from OVA-sensitized IL-15-Tg mice into normal mice before OVA sensitization suppressed Th2 response to OVA in the normal mice. These results suggest that overexpression of IL-15 in vivo suppresses Th2-mediated-allergic airway response via induction of CD8+ T cell-mediated Tc1 response.     

Comparative functional characterization of mouse bone marrow-derived mast cells and peritoneal mast cells in response to non-immunological stimuli

The cultured mouse mast cells that are dependent on spleen-derived factor for their proliferation and maintenance and have been shown to be similar to mucosal mast cells in terms of their T-cell dependence and histochemical staining characteristics. Mast cell heterogeneity has been confirmed by functional characterization of mouse bone marrow-derived mast cells (MBMMC) and mouse peritoneal mast cells (MPMCs). MPMCs released around 30% of histamine when stimulated with compound 48/80 whereas MBMMC were almost unresponsive to the same stimulus. Calcium Ionophore A23187 on the other hand, released histamine in dose-dependent manner from MBMMC. The study was undertaken to investigate the effect of antiallergic drug, disodium cromoglycate (DSCG), a synthetic cromone and quercetin, a plant-derived flavonoid on Ca ionophore A23187 induced histamine release from MBMMC. MBMMCs were almost unresponsive to DSCG whereas Ca Ionophore induced histamine release was blocked by Quercetin. The results indicate that response of mast cells at one anatomic site to a given stimulus does not necessarily predict the response of mast cells at a different anatomic location to the same stimulus. It shows functional heterogeneity within a single species. So, it cannot be assumed that antiallergic compounds stabilizing mast cells in one tissue site or organ will be equally efficacious against mast cells in other sites.     

Selective blockade of NF-kappa B activity in airway immune cells inhibits the effector phase of experimental asthma

Knockout mice studies have revealed that NF-kappaB plays a critical role in Th2 cell differentiation and is therefore required for induction of allergic airway inflammation. However, the questions of whether NF-kappaB also plays a role in the effector phase of airway allergy and whether inhibiting NF-kappaB could have therapeutic value in the treatment of established asthma remain unanswered. To address these issues, we have assessed in OVA-sensitized wild-type mice the effects of selectively antagonizing NF-kappaB activity in the lungs during OVA challenge. Intratracheal administration of NF-kappaB decoy oligodeoxynucleotides to OVA-sensitized mice led to efficient nuclear transfection of airway immune cells, but not constitutive lung cells and draining lymph node cells, associated with abrogation of NF-kappaB activity in the airways upon OVA provocation. NF-kappaB inhibition was associated with strong attenuation of allergic lung inflammation, airway hyperresponsiveness, and local production of mucus, IL-5, IL-13, and eotaxin. IL-4 and OVA-specific IgE and IgG1 production was not reduced. This study demonstrates for the first time that activation of NF-kappaB in local immune cells is critically involved in the effector phase of allergic airway disease and that specific NF-kappaB inhibition in the lungs has therapeutic potential in the control of pulmonary allergy.     

Activation of mast cells is essential for development of house dust mite Dermatophagoides farinae-induced allergic airway inflammation in mice

In this study, we demonstrate that Dermatophagoides farinae (Der f), a major source of airborne allergens, but not OVA, could rapidly activate mast cells in mice. This was indicated by an elevation of serum mouse mast cell protease 1, a mast cell-specific proteinase, as early as 30 min after intratracheal challenge. Administration of sodium cromoglycate (40 mg/kg, i.p., 1 h before Der f instillation), a mast cell stabilizer, not only suppressed acute mouse mast cell protease 1 production but also attenuated the allergic airway inflammation provoked by repetitive Der f challenge in mice (five times at 1-wk interval). Der f induced the expression of mRNA for TNF-alpha, IL-1beta, IL-4, IL-6, IL-9, and IL-13 in mastocytoma P815 cells and stimulated both P815 cells and bone marrow-derived mast cells to produce IL-4, IL-6, and TNF-alpha in a dose- and time-dependent manner. Cycloheximide as well as sodium cromoglycate blocked the Der f-induced IL-4 production, indicating a de novo protein synthesis process. Supernatants of Der f-stimulated mast cells chemoattracted monocytes and T lymphocytes; they up-regulated the expression of costimulatory B7 molecules, eotaxin, RANTES, monocyte chemoattractant protein 1, and IFN-inducible protein 10 mRNA of alveolar macrophages; they supported PHA-induced T cell proliferation; and they promoted Th2 cell development. Our data indicate that mast cells may be an important cell type during the initiation of Der f sensitization in the airway by modulating the function of alveolar macrophages and T cells.     

IL-9 promotes but is not necessary for systemic anaphylaxis

Anaphylaxis represents an extreme form of allergic reaction, consisting of a sensitization phase during which allergen-specific IgE are produced and an acute effector phase triggered by allergen-induced degranulation of mast cells. We studied the role of IL-9, a Th2 cytokine implicated in asthma, in different models of murine anaphylaxis. Using a passive model of systemic anaphylaxis, in which anti-DNP IgE Abs were administered before challenge with DNP-BSA, we found that IL-9-transgenic mice or wild-type mice treated with IL-9 for 5 days were highly sensitive to fatal anaphylaxis. This effect was reproduced in both anaphylaxis-susceptible and -resistant backgrounds (FVB/N or [FVB/N x BALB/c] F(1) mice, respectively) and correlated with increased serum concentrations of mouse mast cell protease-1 level, a protein released upon mast cells degranulation. By contrast, IL-9 did not increase the susceptibility to passive cutaneous anaphylaxis. IL-9 expression also increased the susceptibility to fatal anaphylaxis when mice were sensitized by immunization against OVA before challenge with the same Ag. In this model, serum from sensitized, IL-9-transgenic mice was more potent in transferring susceptibility to OVA challenge into naive mice, indicating that IL-9 also promotes the sensitization stage. Finally, using IL-9R-deficient mice, we found that despite its anaphylaxis-promoting activity, IL-9 is dispensable for development of both passive and active anaphylaxis, at least in the C57BL/6 mouse background. Taken together, the data reported in this study indicate that IL-9 promotes systemic anaphylaxis reactions, acting at both the sensitization and effector stages, but is not absolutely required for this process.     

Modulation of the induction of lung and airway allergy in the offspring of IFN-gamma-treated mother mice

Recent studies have highlighted the influence of fetal/maternal interactions on the development of asthma. Because IFN-gamma reduces Th2-mediated allergic responses, we assessed its capacity to modulate asthma in the offspring when injected into mothers during pregnancy. IFN-gamma was injected in CD1 female mice on day 6.5 of gestation. Immediately after birth, male newborns were housed in cages with interchanged mothers: the offspring from IFN-gamma-treated mothers were breastfed by normal mothers (IFN/nor), and those from normal mothers were breastfed by IFN-gamma-treated (Nor/IFN) or normal mothers (Nor/nor). Immediately after weaning, the spleen cells from IFN/nor and Nor/IFN mice produced less IL-4 and more IFN-gamma than Nor/nor mice when stimulated with Con A. At the age of 6-7 wk, mice were immunized with OVA on days 0 and 7. From day 14 to 16, they were exposed to aerosolized OVA. The bronchoalveolar lavage fluid from Nor/nor mice showed eosinophilia, a large number of these cells being present in perivascular and peribronchial regions of lung tissues. IFN/nor or Nor/IFN mice showed greatly reduced eosinophil numbers in bronchoalveolar lavage fluid. In addition, lung sections from IFN/nor, but not Nor/IFN mice showed almost normal histology. In OVA-sensitized IFN/nor and Nor/IFN mice, the production of IFN-gamma, IL-4, and IL-5 by spleen cells was significantly reduced as compared with cells from the OVA-sensitized Nor/nor group. IgE and anaphylactic IgG1 were also reduced in plasma of IFN/nor mice. In conclusion, the presence of IFN-gamma during pregnancy confers to the fetus a protection against allergenic provocations in the adult life.     

Mast cells and pulmonary fibrosis. Identification of a histamine releasing factor in bronchoalveolar lavage fluid.

As elevated bronchoalveolar lavage (BAL) fluid histamine levels are noted in patients with pulmonary fibrosis (PF), we assayed BAL fluid from 16 patients with PF for the presence of a histamine releasing factor (HRF). HRF activity was assayed by measuring release of the preformed mast cell-derived mediators, histamine, or beta-hexosaminidase (beta-hex) from a purified population of IL-3 dependent mouse bone marrow derived mast cells (MBMMC) or human blood basophils. Mean BAL cell free histamine levels in the patients with PF was 1226 +/- 1349 pg/ml, whereas BAL histamine levels in a comparison group of six non-PF patients was 118 +/- 60 pg/ml. HRF was significantly elevated in BAL fluid of patients with PF (mean beta-hex release 24.5 +/- 12.9%; range 6.8 to 52.4%) compared to the non-PF group of patients (mean beta-hex release 7.9 +/- 7.7%; range 1.8 to 20.7%). The PF HRF not only degranulated MBMMC, but also induced the generation of the arachidonic acid metabolite leukotriene C4 from MBMMC (24.6 +/- 4.2 ng leukotriene C4/10(6) MBMMC). The PF HRF did not appear to be a cytokine previously identified in BAL fluid of patients with PF (i.e., platelet derived growth factor or insulin growth factor-1) or a human cytokine able to degranulate human basophils (i.e., IL-1, or granulocyte-macrophage-CSF) as these recombinant human cytokines did not induce MBMMC beta-hex release. Physicochemical characterization of the HRF revealed that it was relatively heat stable, pronase sensitive and on Sephadex G-75 and G-200 column chromatography had an apparent molecular mass of 30 to 50 kDa. The ability of PF BAL to induce beta-hex release from MBMMC was not dependent on IgE as unsensitized or lactic acid treated MBMMC release similar amounts of beta-hex compared to MBMMC sensitized with IgE. Thus, BAL fluid of patients with PF contains an HRF that induces beta-hex release from MBMMC via an IgE-independent mechanism. The presence of the HRF could explain elevated BAL histamine levels in patients with PF.     

Transforming growth factor-beta 1 selectively inhibits IL-3-dependent mast cell proliferation without affecting mast cell function or differentiation

Transforming growth factor-beta 1 (TGF-beta 1) is an important regulator of cell growth, differentiation, and function. We show that TGF-beta 1 selectively inhibits IL-3-dependent mouse bone marrow derived mast cell (MBMMC) proliferation without affecting MBMMC function or differentiation. TGF-beta 1 significantly decreased [3H]thymidine uptake by IL-3-dependent MBMMC in a dose-dependent manner with 50% inhibition of proliferation occurring with a TGF-beta 1 concentration of 0.1 ng/ml. A brief (i.e., 30 min) incubation of MBMMC with TGF-beta 1 is sufficient to inhibit IL-3-induced proliferation of MBMMC (cultured in the absence of TGF-beta 1) for 24 to 48 h. The inhibitory effect of TGF-beta 1 on the IL-3-dependent proliferation of MBMMC is not cytotoxic as evident from the absence of MBMMC trypan blue staining, the retained functional characteristics of the MBMMC cultured in TGF-beta 1, and the reversibility of the TGF-beta 1 induced inhibition of IL-3 dependent MBMMC proliferation. MBMMC grown in TGF-beta 1 acutely (24 to 48 h) or chronically (7 to 14 days) do not exhibit functional differences in performed or newly generated mediator secretion (Ag/IgE or calcium ionophore A23187 induced MBMMC beta-hexosaminidase or leukotriene C4 release) from MBMMC grown in the absence of TGF-beta 1. In addition, MBMMC cultured for 2 wk in TGF-beta 1 do not show evidence of differentiation as assessed by cellular histamine content or Alcian blue/safranin staining. Thus, TGF-beta 1 is an important negative regulator of IL-3-dependent mast cell proliferation in vitro, selectively inhibiting IL-3-dependent MBMMC proliferation without affecting MBMMC function or differentiation.     

Inhibition of allergen-induced airway remodeling in Smad 3-deficient mice

Intracellular signaling pathways that converge on Smad 3 are used by both TGF-beta and activin A, key cytokines implicated in the process of fibrogenesis. To determine the role of Smad 3 in allergen-induced airway remodeling, Smad 3-deficient and wild-type (WT) mice were sensitized to OVA and challenged by repetitive administration of OVA for 1 mo. Increased levels of activin A and increased numbers of peribronchial TGF-beta1(+) cells were detected in WT and Smad 3-deficient mice following repetitive OVA challenge. Smad 3-deficient mice challenged with OVA had significantly less peribronchial fibrosis (total lung collagen content and trichrome staining), reduced thickness of the peribronchial smooth muscle layer, and reduced epithelial mucus production compared with WT mice. As TGF-beta and Smad 3 signaling are hypothesized to mediate differentiation of fibroblasts to myofibroblasts in vivo, we determined the number of peribronchial myofibroblasts (Col-1(+) and alpha-smooth muscle actin(+)) as assessed by double-label immunofluorescence microscopy. Although the number of peribronchial myofibroblasts increased significantly in WT mice following OVA challenge, there was a significant reduction in the number of peribronchial myofibroblasts in OVA-challenged Smad 3-deficient mice. There was no difference in levels of eosinophilic airway inflammation or airway responsiveness in Smad 3-deficient compared with WT mice. These results suggest that Smad 3 signaling is required for allergen-induced airway remodeling, as well as allergen-induced accumulation of myofibroblasts in the airway. However, Smad 3 signaling does not contribute significantly to airway responsiveness.     

Mast cells promote atherosclerosis by releasing proinflammatory cytokines

Mast cells contribute importantly to allergic and innate immune responses by releasing various preformed and newly synthesized mediators. Previous studies have shown mast cell accumulation in human atherosclerotic lesions. This report establishes the direct participation of mast cells in atherogenesis in low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice. Atheromata from compound mutant Ldlr(-/-) Kit(W-sh)(/W-sh) mice showed decreased lesion size, lipid deposition, T-cell and macrophage numbers, cell proliferation and apoptosis, but increased collagen content and fibrous cap development. In vivo, adoptive transfer of syngeneic wild-type or tumor necrosis factor (TNF)-alpha-deficient mast cells restored atherogenesis to Ldlr(-/-)Kit(W-sh/W-sh) mice. Notably, neither interleukin (IL)-6- nor interferon (IFN)-gamma-deficient mast cells did so, indicating that the inhibition of atherogenesis in Ldlr(-/-)Kit(W-sh/W-sh) mice resulted from the absence of mast cells and mast cell-derived IL-6 and IFN-gamma. Compared with wild-type or TNF-alpha-deficient mast cells, those lacking IL-6 or IFN-gamma did not induce expression of proatherogenic cysteine proteinase cathepsins from vascular cells in vitro or affect cathepsin and matrix metalloproteinase activities in atherosclerotic lesions, implying that mast cell-derived IL-6 and IFN-gamma promote atherogenesis by augmenting the expression of matrix-degrading proteases. These observations establish direct participation of mast cells and mast cell-derived IL-6 and IFN-gamma in mouse atherogenesis and provide new mechanistic insight into the pathogenesis of this common disease.     

Decreased expression of uteroglobin-related protein 1 in inflamed mouse airways is mediated by IL-9

Uteroglobin-related protein 1 (UGRP1) is a secretory protein, highly expressed in epithelial cells of airways. Although an involvement of UGRP1 in the pathogenesis of asthma has been suggested, its function in airways remains unclear. In the present study, a relationship between airway inflammation, UGRP1 expression, and interleukin-9 (IL-9), an asthma candidate gene, was evaluated by using a murine model of allergic bronchial asthma. A severe airway inflammation accompanied by airway eosinophilia and elevation of IL-9 in bronchoalveolar lavage (BAL) fluids was observed after ovalbumin (OVA) challenge to OVA-sensitized mice. In this animal model of airway inflammation, lung Ugrp1 mRNA expression was greatly decreased compared with control mice. A significant inverse correlation between lung Ugrp1 mRNA levels and IL-9 levels in BAL fluid was demonstrated by regression analysis (r = 0.616, P = 0.023). Immunohistochemical analysis revealed a distinct localization of UGRP1 in airway epithelial cells of control mice, whereas UGRP1 staining was patchy and faint in inflamed airways. Intranasal administration of IL-9 to naive mice decreased the level of Ugrp1 expression in lungs. These findings suggest that UGRP1 is downregulated in inflamed airways, such as allergic asthmatics, and IL-9 might be an important mediator for modulating UGRP1 expression.     

Chronic OVA allergen challenged Siglec-F deficient mice have increased mucus,remodeling, and epithelial Siglec-F ligands which are up-regulated by IL-4 and IL-13

Background

In this study we examined the role of Siglec-F, a receptor highly expressed on eosinophils, in contributing to mucus expression, airway remodeling, and Siglec-F ligand expression utilizing Siglec-F deficient mice exposed to chronic allergen challenge.

Methods

Wild type (WT) and Siglec-F deficient mice were sensitized and challenged chronically with OVA for one month. Levels of airway inflammation (eosinophils), Siglec-F ligand expresion and remodeling (mucus, fibrosis, smooth muscle thickness, extracellular matrix protein deposition) were assessed in lung sections by image analysis and immunohistology. Airway hyperreactivity to methacholine was assessed in intubated and ventilated mice.

Results

Siglec-F deficient mice challenged with OVA for one month had significantly increased numbers of BAL and peribronchial eosinophils compared to WT mice which was associated with a significant increase in mucus expression as assessed by the number of periodic acid Schiff positive airway epithelial cells. In addition, OVA challenged Siglec-F deficient mice had significantly increased levels of peribronchial fibrosis (total lung collagen, area of peribronchial trichrome staining), as well as increased numbers of peribronchial TGF-β1+ cells, and increased levels of expression of the extracellular matrix protein fibronectin compared to OVA challenged WT mice. Lung sections immunostained with a Siglec-Fc to detect Siglec-F ligand expression demonstrated higher levels of expression of the Siglec-F ligand in the peribronchial region in OVA challenged Siglec-F deficient mice compared to WT mice. WT and Siglec-F deficient mice challenged intranasally with IL-4 or IL-13 had significantly increased levels of airway epithelial Siglec-F ligand expression, whereas this was not observed in WT or Siglec-F deficient mice challenged with TNF-α. There was a significant increase in the thickness of the peribronchial smooth muscle layer in OVA challenged Siglec-F deficient mice, but this was not associated with significant increased airway hyperreactivity compared to WT mice.

Conclusions

Overall, this study demonstrates an important role for Siglec-F in modulating levels of chronic eosinophilic airway inflammation, peribronchial fibrosis, thickness of the smooth muscle layer, mucus expression, fibronectin, and levels of peribronchial Siglec-F ligands suggesting that Siglec-F may normally function to limit levels of chronic eosinophilic inflammation and remodeling. In addition, IL-4 and IL-13 are important regulators of Siglec-F ligand expression by airway epithelium.     

Modulation of Th2 responses by peptide analogues in a murine model of allergic asthma: amelioration or deterioration of the disease process depends on the Th1 or Th2 skewing characteristics of the therapeutic peptide

Allergen-specific CD4+ Th2 cells play an important role in the immunological processes of allergic asthma. Previously we have shown that, by using the immunodominant epitope OVA323-339, peptide immunotherapy in a murine model of OVA induced allergic asthma, stimulated OVA-specific Th2 cells, and deteriorated airway hyperresponsiveness and eosinophilia. In the present study, we defined four modulatory peptide analogues of OVA323-339 with comparable MHC class II binding affinity. These peptide analogues were used for immunotherapy by s.c. injection in OVA-sensitized mice before OVA challenge. Compared with vehicle-treated mice, treatment with the Th2-skewing wild-type peptide and a Th2-skewing partial agonistic peptide (335N-A) dramatically increased airway eosinophilia upon OVA challenge. In contrast, treatment with a Th1-skewing peptide analogue (336E-A) resulted in a significant decrease in airway eosinophilia and OVA-specific IL-4 and IL-5 production. Our data show for the first time that a Th1-skewing peptide analogue of a dominant allergen epitope can modulate allergen-specific Th2 effector cells in an allergic response in vivo. Furthermore, these data suggest that the use of Th1-skewing peptides instead of wild-type peptide may improve peptide immunotherapy and may contribute to the development of a successful and safe immunotherapy for allergic patients.     

Mesenchymal stem cells combined with traditional Chinese medicine (qi-fang-bi-min-tang) alleviates rodent allergic rhinitis

Mesenchymal stem cells (MSCs) have been proved to exert anti-inflammatory effects and regulate immune reactions. Traditional Chinese medicine (TCM), qi-fang-bi-min-tang, is effective for some patients with allergic diseases. However, it remains unclear whether MSCs combined with TCM could benefit the treatment of allergic rhinitis (AR). In this study, we reported an additional effect of TCM (qi-fang-bi-min-tang) on the therapy of AR under MSCs treatment. Intriguingly, we observed that TCM-treated MSCs significantly inhibited the symptoms of AR and reduced the pathological changes of nasal mucosa in ovalbumin (OVA)-induced rats. The expression levels of interferon γ (IFN-γ), interleukin-17 (IL-17), and IL-4 were significantly decreased in the plasma of AR rats after injection of TCM-treated MSCs. TCM-treated MSCs reduced the levels of histamine secreted by mast cells and immunoglobulin E (IgE) secreted by plasma cells. In addition, we found that MSCs combined with TCM had a better therapeutic effect than TCM alone on AR in an OVA-induced mouse model. After OVA induction, MSCs combined with TCM significantly reduced the ratio of T helper type 1 (Th1), Th2, and Th17, but increased the proportion of Treg in the spleen of mice. Consistently, the expression levels of IFN-γ, IL-4, and IL-17 were significantly decreased, but transforming growth factor-β1 was significantly increased in the plasma of AR mice after treated with TCM and MSCs. Our results from both rats and mice indicated that the effects of TCM combined with MSCs on the AR might be through regulating the secretion of Th1, Th2, and Th17 cytokines. This study suggested that TCM (qi-fang-bi-min-tang)-treated MSCs could be used in the clinical therapy of AR.     

Tryptase inhibition blocks airway inflammation in a mouse asthma model

Release of human lung mast cell tryptase may be important in the pathophysiology of asthma. We examined the effect of the reversible, nonelectrophilic tryptase inhibitor MOL 6131 on airway inflammation and hyper-reactivity in a murine model of asthma. MOL 6131 is a potent selective nonpeptide inhibitor of human lung mast cell tryptase based upon a beta-strand template (K(i) = 45 nM) that does not inhibit trypsin (K(i) = 1,061 nM), thrombin (K(i) = 23, 640 nM), or other serine proteases. BALB/c mice after i.p. OVA sensitization (day 0) were challenged intratracheally with OVA on days 8, 15, 18, and 21. MOL 6131, administered days 18-21, blocked the airway inflammatory response to OVA assessed 24 h after the last OVA challenge on day 22; intranasal delivery (10 mg/kg) had a greater anti-inflammatory effect than oral delivery (10 or 25 mg/kg) of MOL 6131. MOL 6131 reduced total cells and eosinophils in bronchoalveolar lavage fluid, airway tissue eosinophilia, goblet cell hyperplasia, mucus secretion, and peribronchial edema and also inhibited the release of IL-4 and IL-13 in bronchoalveolar lavage fluid. However, tryptase inhibition did not alter airway hyper-reactivity to methacholine in vivo. These results support tryptase as a therapeutic target in asthma and indicate that selective tryptase inhibitors can reduce allergic airway inflammation.     

The roles of IL-4, IL-5 and mast cells in the accumulation of eosinophils during allergic cutaneous late phase reaction in mice

Late phase allergic response has been implicated in the pathogenesis of allergic diseases. In the current study, we investigated the role of IL-4, IL-5 and mast cells in the development of cutaneous late phase reaction (LPR) in mice. Antigenic challenge of ears of ovalbumin (OVA)-immunized BALB/c mice caused a biphasic ear swelling peaking at 1 hr (immediate phase reaction; IPR) and 24 hr (LPR). Ear swelling in LPR was significantly suppressed by the treatment with anti-IL-4 monoclonal antibody (mAb) before antigen challenge. Local eosinophil accumulation during LPR, however, was not inhibited by anti-IL-4 mAb. Moreover, anti-IL-5 mAb had no effect on the swelling response though it significantly suppressed the local accumulation of eosinophils. Interestingly, mast cell-deficient mice (WBB6F1-W/Wv) developed LPR without exhibiting IPR, while the magnitude of ear swelling and local eosinophilia was significantly lower than in normal congenic mice (+/+ mice). The present findings show that IL-4 and IL-5 differently regulate the development of LPR, and that IgE-mediated mast cell activation is required for full response.     

Corticosteroids prevent myofibroblast accumulation and airway remodeling in mice

At present there are conflicting results from studies investigating the role of corticosteroids in inhibiting airway remodeling in asthma. We have used a mouse model to determine whether administration of corticosteroids prevents the development of allergen-induced structural features of airway remodeling. Mice treated with corticosteroids were subjected to repetitive ovalbumin (OVA) challenge for 3 mo, at which time levels of peribronchial fibrosis and the thickness of the peribronchial smooth muscle layer were assessed by immunohistology, levels of transforming growth factor (TGF)-beta1 by ELISA, and the number of alpha-smooth muscle actin+/Col-1+ peribronchial myofibroblasts by immunohistochemistry. Corticosteroids significantly reduced allergen-induced increases in peribronchial collagen deposition and levels of total lung collagen but did not reduce allergen-induced increases in the thickness of the peribronchial smooth muscle layer. Levels of lung TGF-beta1 were significantly reduced in mice treated with systemic corticosteroids, and this was associated with a significant decrease in the number of peribronchial inflammatory cells that expressed TGF-beta1, including eosinophils and mononuclear cells. Corticosteroids also significantly reduced the number of peribronchial myofibroblasts. Overall, these studies demonstrate that administration of corticosteroids significantly reduces levels of allergen-induced peribronchial fibrosis. The reduction in peribronchial fibrosis mediated by corticosteroids is likely to be due to several mechanisms including inhibition of expression of TGF-beta1, a reduction in the number of peribronchial inflammatory cells expressing TGF-beta1 (eosinophils, macrophages), as well as by corticosteroids reducing the accumulation of peribronchial myofibroblasts that contribute to collagen expression.     

1alpha,25-dihydroxyvitamin D3 potentiates the beneficial effects of allergen immunotherapy in a mouse model of allergic asthma: role for IL-10 and TGF-beta

1alpha,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), a potent inhibitor of NF-kappaB expression, can prevent the maturation of dendritic cells in vitro leading to tolerogenic dendritic cells with increased potential to induce regulatory T cells. Herein, we investigated whether the combination of allergen immunotherapy with 1,25(OH)(2)D(3) potentiates the suppressive effects of immunotherapy and whether the immunoregulatory cytokines IL-10 and TGF-beta are involved in the effector phase. OVA-sensitized and challenged BALB/c mice displayed airway hyperresponsiveness (AHR) and increased serum OVA-specific IgE levels, bronchoalveolar lavage eosinophilia, and Th2 cytokine levels. In this model, the dose response of allergen immunotherapy 10 days before OVA inhalation challenge shows strong suppression of asthma manifestations at 1 mg of OVA, but partial suppression of bronchoalveolar lavage eosinophilia, IgE up-regulation, and no reduction of AHR at 100 microg. Interestingly, coadministration of 10 ng of 1,25(OH)(2)D(3) with 100 microg of OVA immunotherapy significantly inhibited AHR and potentiated the reduction of serum OVA-specific IgE levels, airway eosinophilia, and Th2-related cytokines concomitant with increased IL-10 levels in lung tissues and TGF-beta and OVA-specific IgA levels in serum. Similar effects on suboptimal immunotherapy were observed by inhibition of the NF-kappaB pathway using the selective IkappaB kinase 2 inhibitor PS-1145. The suppressive effects of this combined immunotherapy were partially reversed by treatment with mAb to either IL-10R or TGF-beta before OVA inhalation challenge but completely abrogated when both Abs were given. These data demonstrate that 1,25(OH)(2)D(3) potentiates the efficacy of immunotherapy and that the regulatory cytokines IL-10 and TGF-beta play a crucial role in the effector phase of this mouse model.