Mast cells in a murine lung ischemia-reperfusion model of primary graft dysfunction

Primary graft dysfunction (PGD), as characterized by pulmonary infiltrates and high oxygen requirements shortly after reperfusion, is the major cause of early morbidity and mortality after lung transplantation. Donor, recipient and allograft-handling factors are thought to contribute, although new insights regarding pathogenesis are needed to guide approaches to prevention and therapy. Mast cells have been implicated in ischemic tissue injury in other model systems and in allograft rejection, leading to the hypothesis that mast cell degranulation contributes to lung injury following reperfusion injury.We tested this hypothesis in a mouse model of PGD involving reversible disruption of blood flow to one lung. Metrics of injury included albumin permeability, plasma extravasation, lung histopathology, and mast cell degranulation. Responses were assessed in wild-type (Kit^+/+) and mast cell-deficient (Kit^W-sh/W-sh) mice. Because mouse lungs have few mast cells compared with human lungs, we also tested responses in mice with lung mastocytosis generated by injecting bone marrow-derived cultured mast cells (BMCMC).We found that ischemic lung responses of mast cell-deficient Kit^W-sh/W-sh mice did not differ from those of Kit^+/+ mice, even after priming for injury using LPS. Degranulated mast cells were more abundant in ischemic than in non-ischemic BMCMC-injected Kit^W-sh/W-sh lungs. However, lung injury in BMCMC-injected Kit^W-sh/W-sh and Kit^+/+ mice did not differ in globally mast cell-deficient, uninjected Kit^W-sh/W-sh mice or in wild-type Kit^+/+ mice relatively deficient in lung mast cells.These findings predict that mast cells, although activated in lungs injured by ischemia and reperfusion, are not necessary for the development of PGD.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0095-0) contains supplementary material, which is available to authorized users.     

Mast cells contribute to double-stranded RNA-induced augmentation of airway eosinophilia in a murine model of asthma

Background

Clinical studies showed the contribution of viral infection to the development of asthma. Although mast cells have multiple roles in the pathogenesis of allergic asthma, their role of in the virus-associated pathogenesis of asthma remains unknown. Most respiratory viruses generate double-stranded (ds) RNA during their replication. dsRNA provokes innate immune responses. We recently showed that an administration of polyinocinic polycytidilic acid (poly IC), a mimetic of viral dsRNA, during allergen sensitization augments airway eosinophilia and hyperresponsiveness in mice via enhanced production of IL-13.

Methods

The effect of poly IC on allergen-induced airway eosinophilia was investigated for mast cell-conserved Kit^+/+ mice and -deficient Kit^W/Kit^W-v mice. The outcome of mast cell reconstitution was further investigated.

Results

Airway eosinophilia and IL-13 production were augmented by poly IC in Kit^+/+ mice but not in Kit^W/Kit^W-v mice. When Kit^W/Kit^W-v mice were reconstituted with bone marrow-derived mast cells (BMMCs), the augmentation was restored. The augmentation was not induced in the mice systemically deficient for TIR domain-containing adaptor-inducing IFN-β (TRIF) or interferon regulatory factor (IRF)-3, both mediate dsRNA-triggered innate immune responses. The augmentation was, however, restored in Kit^W/Kit^W-v mice reconstituted with TRIF-deficient or IRF-3-deficient BMMCs. Although leukotriene B₄ and prostaglandin D₂ are major lipid mediators released from activated mast cells, no their contribution was shown to the dsRNA-induced augmentation of airway eosinophilia.

Conclusions

We conclude that mast cells contribute to dsRNA-induced augmentation of allergic airway inflammation without requiring direct activation of mast cells with dsRNA or involvement of leukotriene B₄ or prostaglandin D₂.     

Spontaneous eosinophilic nasal inflammation in a genetically-mutant mouse: comparative study with an allergic inflammation model

Background

Eosinophilic inflammation is a hallmark of chronic rhinosinusitis with nasal polyps. To model this disease process experimentally, nasal sensitization of mice with ovalbumin or aspergillus has been described. Here, we describe a genetically mutant mouse that develops robust spontaneous nasal eosinophilic inflammation. These mice lack the enzyme SHP-1 that down-regulates the IL-4Rα/stat6 signaling pathway. We compared nasal inflammation and inflammatory mediators in SHP-1 deficient mice (mev) and an ovalbumin-induced nasal allergy model.

Methods

A novel technique of trans-pharyngeal nasal lavage was developed to obtain samples of inflammatory cells from the nasal passages of allergic and mev mice. Total and differential cell counts were performed on cytospin preparations. Expression of tissue mRNA for IL-4, IL-13, and mouse beta-defensin-1 (MBD-1) was determined by quantitative PCR. Eotaxin in the lavage fluid was assessed by ELISA.

Results

Allergic and mev mice had increased total cells and eosinophils compared with controls. Expression of IL-4 was similarly increased in both allergic and mev mice, but expression of IL-13 and eotaxin was significantly greater in the allergic mice than mev mice. Eotaxin was significantly up-regulated in both allergic rhinitis and mev mice. In both models of eosinophilic inflammation, down-regulation of the innate immune marker MBD-1 was observed.

Conclusions

The mev mice display spontaneous chronic nasal eosinophilic inflammation with potential utility for chronic rhinosinusitis with nasal polyps research. The eosinophilic infiltrate is more robust in the mev mice than allergic mice, but Th2 cytokine expression is not as pronounced. Decreased MBD-1 expression in both models supports the concept that Th2-cytokines down-regulate sinonasal innate immunity in humans, and suggests a role for mouse models in investigating the interaction between adaptive and innate immunity in the sinonasal mucosa.     

Topical Application of a Platelet Activating Factor Receptor Agonist Suppresses Phorbol Ester-Induced Acute and Chronic Inflammation and Has Cancer Chemopreventive Activity in Mouse Skin

Platelet activating factor (PAF) has long been associated with acute edema and inflammatory responses. PAF acts by binding to a specific G-protein coupled receptor (PAF-R, Ptafr). However, the role of chronic PAF-R activation on sustained inflammatory responses has been largely ignored. We recently demonstrated that mice lacking the PAF-R (Ptafr-/- mice) exhibit increased cutaneous tumorigenesis in response to a two-stage chemical carcinogenesis protocol. Ptafr-/- mice also exhibited increased chronic inflammation in response to phorbol ester application. In this present study, we demonstrate that topical application of the non-hydrolysable PAF mimetic (carbamoyl-PAF (CPAF)), exerts a potent, dose-dependent, and short-lived edema response in WT mice, but not Ptafr -/- mice or mice deficient in c-Kit (c-Kit ^W-sh/W-sh mice). Using an ear inflammation model, co-administration of topical CPAF treatment resulted in a paradoxical decrease in both acute ear thickness changes associated with a single PMA application, as well as the sustained inflammation associated with chronic repetitive PMA applications. Moreover, mice treated topically with CPAF also exhibited a significant reduction in chemical carcinogenesis. The ability of CPAF to suppress acute and chronic inflammatory changes in response to PMA application(s) was PAF-R dependent, as CPAF had no effect on basal or PMA-induced inflammation in Ptafr-/- mice. Moreover, c-Kit appears to be necessary for the anti-inflammatory effects of CPAF, as CPAF had no observable effect in c-Kit ^W-sh/W-sh mice. These data provide additional evidence that PAF-R activation exerts complex immunomodulatory effects in a model of chronic inflammation that is relevant to neoplastic development.     

Regulation of Nasal Airway Homeostasis and Inflammation in Mice by SHP-1 and Th2/Th1 Signaling Pathways

Allergic rhinitis is a chronic inflammatory disease orchestrated by Th2 lymphocytes. Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 is known to be a negative regulator in the IL-4α/STAT-6 signaling pathway of the lung. However, the role of SHP-1 enzyme and its functional relationship with Th2 and Th1 cytokines are not known in the nasal airway. In this study, we aimed to study the nasal inflammation as a result of SHP-1 deficiency in viable motheaten (mev) mice and to investigate the molecular mechanisms involved. Cytology, histology, and expression of cytokines and chemokines were analyzed to define the nature of the nasal inflammation. Targeted gene depletion of Th1 (IFN-γ) and Th2 (IL-4 and IL-13) cytokines was used to identify the critical pathways involved. Matrix metalloproteinases (MMPs) were studied to demonstrate the clearance mechanism of recruited inflammatory cells into the nasal airway. We showed here that mev mice had a spontaneous allergic rhinitis-like inflammation with eosinophilia, mucus metaplasia, up-regulation of Th2 cytokines (IL-4 and IL-13), chemokines (eotaxin), and MMPs. All of these inflammatory mediators were clearly counter-regulated by Th2 and Th1 cytokines. Deletion of IFN-γ gene induced a strong Th2-skewed inflammation with transepithelial migration of the inflammatory cells. These findings suggest that SHP-1 enzyme and Th2/Th1 paradigm may play a critical role in the maintenance of nasal immune homeostasis and in the regulation of allergic rhinitis.     

Endotoxin Exposure during Sensitization to Blomia tropicalis Allergens Shifts TH2 Immunity Towards a TH17-Mediated Airway Neutrophilic Inflammation: Role of TLR4 and TLR2

Experimental evidence and epidemiological studies indicate that exposure to endotoxin lipopolysaccharide (eLPS) or other TLR agonists prevent asthma. We have previously shown in the OVA-model of asthma that eLPS administration during alum-based allergen sensitization blocked the development of lung TH2 immune responses via MyD88 pathway and IL-12/IFN-γ axis. In the present work we determined the effect of eLPS exposure during sensitization to a natural airborne allergen extract derived from the house dust mite Blomia tropicalis (Bt). Mice were subcutaneously sensitized with Bt allergens co-adsorbed onto alum with or without eLPS and challenged twice intranasally with Bt. Cellular and molecular parameters of allergic lung inflammation were evaluated 24 h after the last Bt challenge. Exposure to eLPS but not to ultrapure LPS (upLPS) preparation during sensitization to Bt allergens decreased the influx of eosinophils and increased the influx of neutrophils to the airways. Inhibition of airway eosinophilia was not observed in IFN-γdeficient mice while airway neutrophilia was not observed in IL-17RA-deficient mice as well in mice lacking MyD88, CD14, TLR4 and, surprisingly, TLR2 molecules. Notably, exposure to a synthetic TLR2 agonist (PamCSK4) also induced airway neutrophilia that was dependent on TLR2 and TLR4 molecules. In the OVA model, exposure to eLPS or PamCSK4 suppressed OVA-induced airway inflammation. Our results suggest that B. tropicalis allergens engage TLR4 that potentiates TLR2 signaling. This dual TLR activation during sensitization results in airway neutrophilic inflammation associated with increased frequency of lung TH17 cells. Our work highlight the complex interplay between bacterial products, house dust mite allergens and TLR signaling in the induction of different phenotypes of airway inflammation.     

TLR4 signaling attenuates ongoing allergic inflammation

The relationship between LPS exposure and allergic asthma is poorly understood. Epidemiologic studies in humans have found that exposure to LPS can protect, have no effect, or exacerbate allergic asthma. Similarly, LPS has had variable effects on allergic pulmonary inflammation in the mouse, depending on the model used. In the present study, we studied the effect of very low doses of LPS in models of both short-term and long-term allergen challenge. When challenged with allergen for short periods, wild-type and tlr4-deficient mice had similar responses. However, when challenged for periods of 1 wk or longer, tlr4-deficient mice developed dramatically increased airway eosinophils, serum IgE, and Th2 cytokines compared with similarly challenged, genetically matched C57BL/6 mice. The relative attenuation of allergic responses seen in C57BL/6 mice was dependent on bone marrow-derived cell-specific expression of tlr4, and was not associated with an increase in Th1 responses. The number of dendritic cells in lungs of challenged tlr4-deficient mice was significantly increased compared with those in challenged C57BL/6 mice. No differences were seen in the abilities of naive C57BL/6 and tlr4-deficient mice to develop allergen-specific tolerance after exposure to similar preparations of OVA, suggesting that tolerance and regulation of existing inflammation develop through different mechanisms. The attenuation of eosinophilic inflammation in C57BL/6 mice was abolished when these mice were challenged with OVA supplemented with additional LPS. Together, these findings show that low doses of endotoxin can have regulatory effects on allergic inflammation, particularly in the setting of ongoing allergen exposure.     

Mast Cells Play No Role in the Pathogenesis of Postoperative Ileus Induced by Intestinal Manipulation

Introduction

Intestinal manipulation (IM) during abdominal surgery results in intestinal inflammation leading to hypomotility or ileus. Mast cell activation is thought to play a crucial role in the pathophysiology of postoperative ileus (POI). However, this conclusion was mainly drawn using mast cell-deficient mouse models with abnormal Kit signaling. These mice also lack interstitial cells of Cajal (ICC) resulting in aberrant gastrointestinal motility even prior to surgery, compromising their use as model to study POI. To avoid these experimental weaknesses we took advantage of a newly developed knock-in mouse model, Cpa3^Cre/+, devoid of mast cells but with intact Kit signaling.

Design

The role of mast cells in the development of POI and intestinal inflammation was evaluated assessing gastrointestinal transit and muscularis externa inflammation after IM in two strains of mice lacking mast cells, i.e. Kit^W-sh/W-sh and Cpa3^Cre/+ mice, and by use of the mast cell stabilizer cromolyn.

Results

Kit^W-sh/W-sh mice lack ICC networks and already revealed significantly delayed gastrointestinal transit even before surgery. IM did not further delay intestinal transit, but induced infiltration of myeloperoxidase positive cells, expression of inflammatory cytokines and recruitment of monocytes and neutrophils into the muscularis externa. On the contrary, Cpa3^Cre/+ mice have a normal network of ICC and normal gastrointestinal. Surprisingly, IM in Cpa3^Cre/+ mice caused delay in gut motility and intestinal inflammation as in wild type littermates mice (Cpa3^+/+). Furthermore, treatment with the mast cell inhibitor cromolyn resulted in an inhibition of mast cells without preventing POI.

Conclusions

Here, we confirm that IM induced mast cell degranulation. However, our data demonstrate that mast cells are not required for the pathogenesis of POI in mice. Although there might be species differences between mouse and human, our results argue against mast cell inhibitors as a therapeutic approach to shorten POI.     

Measuring Local Anaphylaxis in Mice

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. Exposure to an allergen in a sensitized individual can result in clinical symptoms that vary from minor erythema to life threatening anaphylaxis. In the laboratory, various animal models have been developed to understand the mechanisms driving allergic responses. Herein, we describe a detailed method for measuring changes in vascular permeability to quantify localized allergic responses. The local anaphylaxis assay was first reported in the 1920s, and has been adapted from the technique published by Kojima et al. in 2007¹. In this assay, mice sensitized to OVA are challenged in the left ear with vehicle and in the right ear with OVA. This is followed by an intravenous injection of Evans Blue dye. Ten min after injecting Evans Blue, the animal is euthanized and the dye that has extravasated into the ears is extracted overnight in formamide. The absorbance of the extracted dye is then quantified with a spectrophotometer. This method reliably results in a visual and quantifiable manifestation of a local allergic response.     

Bacterial lipopolysaccharide signaling through Toll-like receptor 4 suppresses asthma-like responses via nitric oxide synthase 2 activity

Asthma results from an intrapulmonary allergen-driven Th2 response and is characterized by intermittent airway obstruction, airway hyperreactivity, and airway inflammation. An inverse association between allergic asthma and microbial infections has been observed. Microbial infections could prevent allergic responses by inducing the secretion of the type 1 cytokines, IL-12 and IFN-gamma. In this study, we examined whether administration of bacterial LPS, a prototypic bacterial product that activates innate immune cells via the Toll-like receptor 4 (TLR4) could suppress early and late allergic responses in a murine model of asthma. We report that LPS administration suppresses the IgE-mediated and mast cell-dependent passive cutaneous anaphylaxis, pulmonary inflammation, airway eosinophilia, mucus production, and airway hyperactivity. The suppression of asthma-like responses was not due to Th1 shift as it persisted in IL-12(-/-) or IFN-gamma(-/-) mice. However, the suppressive effect of LPS was not observed in TLR4- or NO synthase 2-deficient mice. Our findings demonstrate, for the first time, that LPS suppresses Th2 responses in vivo via the TLR4-dependent pathway that triggers NO synthase 2 activity.     

A strain of Lactobacillus casei inhibits the effector phase of immune inflammation

Some nonpathogenic bacteria were found to have protective effects in mouse models of allergic and autoimmune diseases. These "probiotics" are thought to interact with dendritic cells during Ag presentation, at the initiation of adaptive immune responses. Many other myeloid cells are the effector cells of immune responses. They are responsible for inflammation that accounts for symptoms in allergic and autoimmune diseases. We investigated in this study whether probiotics might affect allergic and autoimmune inflammation by acting at the effector phase of adaptive immune responses. The effects of one strain of Lactobacillus casei were investigated in vivo on IgE-induced passive systemic anaphylaxis and IgG-induced passive arthritis, two murine models of acute allergic and autoimmune inflammation, respectively, which bypass the induction phase of immune responses, in vitro on IgE- and IgG-induced mouse mast cell activation and ex vivo on IgE-dependent human basophil activation. L. casei protected from anaphylaxis and arthritis, and inhibited mouse mast cell and human basophil activation. Inhibition required contact between mast cells and bacteria, was reversible, and selectively affected the Lyn/Syk/linker for activation of T cells pathway induced on engagement of IgE receptors, leading to decreased MAPK activation, Ca(2+) mobilization, degranulation, and cytokine secretion. Also, adoptive anaphylaxis induced on Ag challenge in mice injected with IgE-sensitized mast cells was abrogated in mice injected with IgE-sensitized mast cells exposed to bacteria. These results demonstrate that probiotics can influence the effector phase of adaptive immunity in allergic and autoimmune diseases. They might, therefore, prevent inflammation in patients who have already synthesized specific IgE or autoantibodies.     

Impact of Histone H1 on the Progression of Allergic Rhinitis and Its Suppression by Neutralizing Antibody in Mice

Nuclear antigens are known to trigger off innate and adaptive immune responses. Recent studies have found that the complex of nucleic acids and core histones that are derived from damaged cells may regulate allergic responses. However, no fundamental study has been performed concerning the role of linker histone H1 in mast cell-mediated type I hyperreactivity. In this study, we explored the impact of histone H1 on mast cell-mediated allergic responses both in vitro and in vivo. In the course of a bona-fide experimental allergen sensitization model upon co-injection with alum adjuvant, ovalbumin (OVA), but not PBS, induced elevated levels of circulating histone H1. Intranasal challenge with histone H1 to OVA/alum- (but not PBS/alum)-sensitized mice induced significantly severer symptoms of allergic rhinitis than those in mice sensitized and challenged with OVA. A monoclonal antibody against histone H1 not only suppressed mast cell degranulation, but also ameliorated OVA-induced nasal hyperreactivity and IgE-mediated passive cutaneous anaphylaxis. Our present data suggest that nuclear histone H1 represents an alarmin-like endogenous mediator acting on mast cells, and that its blockage has a therapeutic potential for mast cell-mediated type I hyperreactivity.     

Cigarette smoke enhances Th-2 driven airway inflammation and delays inhalational tolerance

Background

Active smoking increases asthma severity and is related to diminished treatment efficacy. Animal models in which inhalation of both allergen and mainstream cigarette smoke are combined can help us to understand the complex interaction between both agents. We have recently shown that, in allergic mice, the airway inflammation can be cleared by repeated allergen challenge, resulting in the establishment of a state of inhalational tolerance.

Methods

In this study, we assessed in vivo the impact of cigarette smoke on the efficacy and time course of this form of tolerance induction. We exposed sensitized mice to concurrent mainstream cigarette smoke and allergen (Ovalbumin- OVA) and measured the airway inflammation at different time points.

Results

We first confirmed that aerosolized OVA administered for a prolonged time period (4–8 weeks) resulted in the establishment of tolerance. Concurrent OVA and smoke exposure for 2 weeks showed that tobacco smoke enhanced the Th-2 driven airway inflammation in the acute phase. In addition, the induction of the tolerance by repeated inhalational OVA challenge was delayed significantly by the tobacco smoke, since 4 weeks of concurrent exposure resulted in a more persistent eosinophilic airway inflammation, paralleled by a more mature dendritic cell phenotype. However, smoke exposure could not prevent the establishment of tolerance after 8 weeks of antigen exposure as shown by both histopathology (disappearance of the Th-2 driven inflammation) and by in vivo functional experiments. In these tolerized mice, some of the inflammatory responses to the smoke were even attenuated.

Conclusion

Cigarette smoke enhances acute allergic inflammation and delays, but does not abrogate the development of tolerance due to prolonged challenge with inhaled antigen in experimental asthma.     

Role of mast cells and basophils in IgE responses and in allergic airway hyperresponsiveness

We established a diphtheria toxin (DT)-based conditional deletion system using Il4 enhancer elements previously shown to be specific for IL-4 production in mast cells (MCs) or basophils (Mas-TRECK and Bas-TRECK mice). DT treatment of Bas-TRECK mice resulted in specific deletion of basophils, whereas both MCs and basophils were deleted in Mas-TRECK mice. DT-treated Mas-TRECK mice had impaired passive cutaneous anaphylaxis, IgE-mediated passive systemic anaphylaxis, and IgE-mediated chronic allergic inflammation, whereas DT-treated Bas-TRECK mice had impaired IgE-mediated chronic allergic inflammation. Using these mice, we also sought to tease out the role of MCs and basophils in airway hyperresponsiveness (AHR). Although MC deletion resulted in a slight increase in basal Ag-specific IgE levels and significant increases in basal IgE levels, we found that this deletion markedly impaired the AHR effector phase and was accompanied by decreased histamine levels. By contrast, basophil deletion had no effect on the AHR effector phase or on IgE production induced by systemic OVA immunization. Our results, using these newly established Mas-TRECK and Bas-TRECK models, demonstrated an indispensable role for MCs as effector cells in AHR.     

Nitrogen dioxide promotes allergic sensitization to inhaled antigen

Allergen sensitization and allergic airway disease are likely to come about through the inhalation of Ag with immunostimulatory molecules. However, environmental pollutants, including nitrogen dioxide (NO2), may promote adaptive immune responses to innocuous Ags that are not by themselves immunostimulatory. We tested in C57BL/6 mice whether exposure to NO2, followed by inhalation of the innocuous protein Ag, OVA, would result in allergen sensitization and the subsequent development of allergic airway disease. Following challenge with aerosolized OVA alone, mice previously exposed via inhalation to NO2 and OVA developed eosinophilic inflammation and mucus cell metaplasia in the lungs, as well as OVA-specific IgE and IgG1, and Th2-type cytokine responses. One hour of exposure to 10 parts per million NO2 increased bronchoalveolar lavage fluid levels of total protein, lactate dehydrogenase activity, and heat shock protein 70; promoted the activation of NF-kappaB by airway epithelial cells; and stimulated the subsequent allergic response to Ag challenge. Furthermore, features of allergic airway disease were not induced in allergen-challenged TLR2-/- and MyD88-/- mice exposed to NO2 and aerosolized OVA during sensitization. These findings offer a mechanism whereby allergen sensitization and asthma may result under conditions of high ambient or endogenous NO2 levels.     

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.     

Aspergillus antigen induces robust Th2 cytokine production,inflammation, airway hyperreactivity and fibrosis in the absence of MCP-1 or CCR2

BackgroundAsthma is characterized by type 2 T-helper cell (Th2) inflammation, goblet cell hyperplasia, airway hyperreactivity, and airway fibrosis. Monocyte chemoattractant protein-1 (MCP-1 or CCL2) and its receptor, CCR2, have been shown to play important roles in the development of Th2 inflammation. CCR2-deficient mice have been found to have altered inflammatory and physiologic responses in some models of experimental allergic asthma, but the role of CCR2 in contributing to inflammation and airway hyperreactivity appears to vary considerably between models. Furthermore, MCP-1-deficient mice have not previously been studied in models of experimental allergic asthma.MethodsTo test whether MCP-1 and CCR2 are each required for the development of experimental allergic asthma, we applied an Aspergillus antigen-induced model of Th2 cytokine-driven allergic asthma associated with airway fibrosis to mice deficient in either MCP-1 or CCR2. Previous studies with live Aspergillus conidia instilled into the lung revealed that MCP-1 and CCR2 play a role in anti-fungal responses; in contrast, we used a non-viable Aspergillus antigen preparation known to induce a robust eosinophilic inflammatory response.ResultsWe found that wild-type C57BL/6 mice developed eosinophilic airway inflammation, goblet cell hyperplasia, airway hyperreactivity, elevations in serum IgE, and airway fibrosis in response to airway challenge with Aspergillus antigen. Surprisingly, mice deficient in either MCP-1 or CCR2 had responses to Aspergillus antigen similar to those seen in wild-type mice, including production of Th2 cytokines.ConclusionWe conclude that robust Th2-mediated lung pathology can occur even in the complete absence of MCP-1 or CCR2.     

IgE immune complexes stimulate an increase in lung mast cell progenitors in a mouse model of allergic airway inflammation

Mast cell numbers and allergen specific IgE are increased in the lungs of patients with allergic asthma and this can be reproduced in mouse models. The increased number of mast cells is likely due to recruitment of mast cell progenitors that mature in situ. We hypothesized that formation of IgE immune complexes in the lungs of sensitized mice increase the migration of mast cell progenitors to this organ. To study this, a model of allergic airway inflammation where mice were immunized with ovalbumin (OVA) in alum twice followed by three daily intranasal challenges of either OVA coupled to trinitrophenyl (TNP) alone or as immune complexes with IgE-anti-TNP, was used. Mast cell progenitors were quantified by a limiting dilution assay. IgE immune complex challenge of sensitized mice elicited three times more mast cell progenitors per lung than challenge with the same dose of antigen alone. This dose of antigen challenge alone did not increase the levels of mast cell progenitors compared to unchallenged mice. IgE immune complex challenge of sensitized mice also enhanced the frequency of mast cell progenitors per 10(6) mononuclear cells by 2.1-fold. The enhancement of lung mast cell progenitors by IgE immune complex challenge was lost in FcRγ deficient mice but not in CD23 deficient mice. Our data show that IgE immune complex challenge enhances the number of mast cell progenitors in the lung through activation of an Fc receptor associated with the FcRγ chain. This most likely takes place via activation of FcεRI, although activation via FcγRIV or a combination of the two receptors cannot be excluded. IgE immune complex-mediated enhancement of lung MCp numbers is a new reason to target IgE in therapies against allergic asthma.     

Scrophularia buergeriana attenuates allergic inflammation by reducing NF-κB activation

BackgroundScrophularia buergeriana Miq. (Scrophulariaceae) (SB) has been used as an oriental medicine for the treatment of inflammatory diseases, such as neuritis and pharyngolaryngitis.PurposeWe explored the therapeutic effects of S. buergeriana ethanol extract (SBE) on airway inflammation in ovalbumin (OVA)-induced asthmatic mice and lipopolysaccharide (LPS)-stimulated RAW264.7 cells.MethodsMice were intraperitoneally injected with OVA on days 0 and 14 to elevate the immune response. On days 21 to 23, the mice were challenged with OVA solution and SBE (20 and 40 mg/kg) was administered daily by oral gavage from days 18 to 23. RAW264.7 cells were pretreated with SBE 1 h before LPS stimulation.ResultsSBE administration effectively suppressed inflammatory cell infiltration, the expression of interleukin (IL)-5, IL-13, and IL-17, immunoglobulin E, and airway hyperresponsiveness in an OVA-induced allergic asthma model. A reduction in histological alterations, including airway inflammation and mucus hypersecretion, was observed. These effects of SBE were accompanied by a decrease in matrix metalloproteinase-9 (MMP-9) expression and nuclear factor kappa B (NF-κB) phosphorylation. These responses were observed in LPS-stimulated RAW264.7 cells. SBE treatment reduced the mRNA expression of tumor necrosis factor (TNF)-α, IL-6, and MMP-9, and NF-κB phosphorylation, in LPS-stimulated RAW264.7 cells.ConclusionOur results indicated that SBE effectively attenuated airway inflammation in an OVA-induced allergic asthma model. These properties of SBE were thought to be involved in the suppression of NF-κB phosphorylation, suggesting that the material has the potential to regulate the development of allergic asthma.     

Early phase bronchoconstriction in the mouse requires allergen-specific IgG

Allergen provocation of allergic asthma patients is often characterized by an initial period of bronchoconstriction, or early phase reaction (EPR), that leads to maximal airway narrowing within 15-30 min, followed by a recovery period returning airway function to baseline within 1-2 h. In this study, we used a defined OVA provocation model and mice deficient for specific leukocyte populations to investigate the cellular/molecular origins of the EPR. OVA-sensitized/challenged wild-type (C57BL/6J) mice displayed an EPR following OVA provocation. However, this response was absent in gene knockout animals deficient of either B or T cells. Moreover, transfer of OVA-specific IgG, but not IgE, before the OVA provocation, was capable of inducing the EPR in both strains of lymphocyte-deficient mice. Interestingly, an EPR was also observed in sensitized/challenged mast cell-deficient mice following an OVA provocation. These data show that the EPR in the mouse is an immunologically based pathophysiological response that requires allergen-specific IgG but occurs independent of mast cell activities. Thus, in the mouse the initial period of bronchoconstriction following allergen exposure may involve neither mast cells nor IgE-mediated events.