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The effect of antigen challenge on the function of neuronal M2-muscarinic autoreceptors in the lungs was studied in anesthetized guinea pigs. Guinea pigs were injected intraperitoneally with saline (control group) or ovalbumin (10 mg/kg) on days 1, 3, and 5. One group of sensitized animals was challenged on days 20–25 with aerosolized ovalbumin for 5 min/day (challenged group), while another group of the sensitized animals was not challenged (sensitized group). On day 26 the animals were anesthetized, paralyzed, tracheostomized, and artificially ventilated. Pulmonary inflation pressure (Ppi), tidal volume, blood pressure, and heart rate were recorded. Both vagus nerves were cut, and electrical stimulation of the distal portions caused bronchoconstriction (measured as an increase in Ppi) and bradycardia. In the control group, pilocarpine (1–100 micrograms/kg iv) attenuated vagally induced bronchoconstriction by stimulating inhibitory M2-muscarinic receptors on parasympathetic nerves in the lungs. Conversely, blockade of these receptors with the antagonist gallamine (0.1–10 mg/kg iv) produced a marked potentiation of vagally induced bronchoconstriction. These results confirm previous findings. In the challenged guinea pigs, pilocarpine did not inhibit vagally induced bronchoconstriction. Furthermore, gallamine did not potentiate vagally induced bronchoconstriction to the same degree as in the controls. In the group of animals that was sensitized but not challenged, the potentiation of vagally induced bronchoconstriction by gallamine was identical to the controls. There was no increase in baseline Ppi in the sensitized or challenged animals compared with the controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Unraveling the exact signaling events mediating the distinct functions of the T cell-derived cytokines interleukin-4 (IL-4) and IL-13 has been challenging because they are structurally similar and share a functional signaling receptor chain. A study now proposes a potential molecular mechanism to explain the functional differences between IL-4 and IL-13 that involves the ability of IL-4, but not IL-13, to effectively activate the insulin receptor substrate-2 (IRS-2) signaling cascade through binding to its receptor. A better understanding of the interactions of IL-4 and IL-13 with their cognate receptors may facilitate the development of therapies without unintended side effects.

Maladaptive, Th2-polarized inflammatory responses are integral to the pathogenesis of allergic asthma. As regulators of T cell activation, dendritic cells (DCs) are important mediators of allergic asthma, yet the precise signals which render endogenous DCs “pro-asthmatic”, and the extent to which these signals are regulated by the pulmonary environment and host genetics, remains unclear. Comparative phenotypic and functional analysis of pulmonary DC populations in mice susceptible (A/J), or resistant (C3H) to experimental asthma, revealed that susceptibility to airway hyperresponsiveness is associated with preferential myeloid DC (mDC) allergen uptake, and production of Th17-skewing cytokines (IL-6, IL-23), whereas resistance is associated with increased allergen uptake by plasmacytoid DCs. Surprisingly, adoptive transfer of syngeneic HDM-pulsed bone marrow derived mDCs (BMDCs) to the lungs of C3H mice markedly enhanced lung IL-17A production, and rendered them susceptible to allergen-driven airway hyperresponsiveness. Characterization of these BMDCs revealed levels of antigen uptake, and Th17 promoting cytokine production similar to that observed in pulmonary mDCs from susceptible A/J mice. Collectively these data demonstrate that the lung environment present in asthma-resistant mice promotes robust pDC allergen uptake, activation, and limits Th17-skewing cytokine production responsible for driving pathologic T cell responses central to the development of allergen-induced airway hyperresponsiveness.

Background Allergic asthma results from inappropriate TH2-mediated inflammation. Both IL-4 and IL-13 contribute to asthma pathogenesis, but IL-4 predominantly drives TH2 induction, whereas IL-13 is necessary and sufficient for allergen-induced airway hyperresponsiveness and goblet cell hyperplasia. Although these 2 cytokines share signaling components, the molecular mechanisms by which they mediate different phases of the allergic asthmatic response remain elusive. Objective We sought to clarify the role or roles of IL-4 and IL-13 in asthma-pathogenesis. Methods We used DNA Affymetrix microarrays to profile pulmonary gene expression in BALB/c mice inoculated intratracheally with ragweed pollen, house dust mite, IL-4, IL-13, or both cytokines. IL-13 dependence was confirmed by comparing pulmonary gene expression in house dust mite–inoculated wild-type and IL-13 knockout mice. Results A signature gene expression profile consisting of 23 genes was commonly induced by means of inoculation with house dust mite, ragweed pollen, or IL-4 plus IL-13. Although rIL-4 and rIL-13 treatment induced an overlapping set of genes, IL-4 uniquely induced 21 genes, half of which were interferon response genes and half of which were genes important in immunoregulation. IL-13 uniquely induced 8 genes, most of which encode proteins produced by epithelial cells. Conclusions IL-4 and IL-13 together account for most allergen-induced pulmonary genes. Selective IL-4 induction of IFN-γ response genes and other genes that might negatively regulate allergic inflammation could partially explain the greater importance of IL-13 in the effector phase of allergic airway disease.

It is becoming increasingly clear that innate immune mediators play a role in regulating adaptive immune responses in asthma pathogenesis. Cockroach exposure is a major risk factor for the development of asthma. In this study we asked whether German cockroach (GC) feces (frass) could initiate an innate immune response. Naive BALB/c mice were challenged with a single intratracheal inhalation of GC frass. Proinflammatory cytokines were significantly increased in the bronchoalveolar lavage fluid at 3 h and were maintained at higher than baseline levels for at least 24 h. Neutrophil migration into the airways was evident as early as 3 h but was maximal between 6 and 24 h postinhalation. The early increase in cytokine expression was independent of TLR2 or TLR4. Newly infiltrated airway neutrophils were responsible for maintaining high levels of cytokines in the airways. Using neutrophils as an early marker of the innate immune response, we show that show that neutrophils isolated from the airways following GC frass inhalation express TLR2 and release cytokines. GC frass directly affected neutrophil cytokine production via TLR2, but not TLR4, as evidenced by the use of TLR-neutralizing Abs and neutrophils from TLR-deficient mice. Activation of cytokine expression occurred via GC frass-induced NF-κB translocation and DNA binding. These data show that GC frass contains a TLR2 agonist and, to our knowledge, this is the first report of an allergen directly activating cells of the innate immune system via TLR2 and suggests an important link between innate and adaptive immunity.

Background Cockroach exposure is a major risk factor for the development of asthma. Inhalation of fecal remnants (frass) is the likely sensitizing agent; however isolated frass has not been tested for its ability to induce experimental asthma in mice. Methods Mice (Balb/c or C57Bl/6) were sensitized and challenged with GC frass or GC frass devoid of proteases and measurements of airway inflammation and hyperresponsiveness were performed (interleukin (IL)-5, -13, and interferon gamma (IFNγ) levels in bronchoalveolar lavage fluid, serum IgE levels, airway hyperresponsiveness, cellular infiltration, and mucin production). Results Sensitization and challenge of Balb/c mice with GC frass resulted in increased airway inflammation and hyperresponsiveness. C57Bl/6 mice were not susceptible to this model of sensitization; however they were sensitized to GC frass using a more aggressive sensitization and challenge protocol. In mice that were sensitized by inhalation, the active serine proteases in GC frass played a role in airway hyperresponsiveness as these mice had less airway hyperresponsiveness to acetylcholine and less mucin production. Proteases did not play a role in mediating the allergic inflammation in mice sensitized via intraperitoneal injection. Conclusion While both strains of mice were able to induce experimental asthma following GC frass sensitization and challenge, the active serine proteases in GC frass only play a role in airway hyperresponsiveness in Balb/c mice that were susceptible to sensitization via inhalation. The differences in the method of sensitization suggest genetic differences between strains of mice.

IL-13 receptor alpha2 (IL-13R alpha 2) is a high-affinity receptor for IL-13, a central mediator of allergic asthma. It acts predominantly as a decoy receptor but can also contribute to IL-13 responses under certain conditions. IL-13R alpha 2 exists in soluble and membrane forms, which can both bind IL-13 and modulate its activity. Yet the proteolytic processes that contribute to the generation of soluble IL-13R alpha 2 are largely unknown.We sought to investigate the role of matrix metalloproteinases (MMPs) in the generation of soluble IL-13R alpha 2.Acellular cleavage assays by MMPs were performed by using glutathione-S-transferase fusion proteins of murine or human IL-13R alpha 2. IL-13R alpha 2 stable-transfected cells were used for analysis of surface expression and release of soluble IL-13R alpha 2. Wild-type and MMP-8-deficient mice were used for analysis of allergen-induced airway hyperresponsiveness and solubilization of IL-13R alpha 2.Among several MMPs tested, only MMP-8 cleaved IL-13R alpha 2. Treatment of transfected human or murine cells expressing high levels of surface IL-13R alpha 2 with MMP-8 resulted in release of soluble IL-13R alpha 2 into the supernatants, with a concomitant decrease in surface IL-13R alpha 2 levels. The IL-13R alpha 2 solubilized by MMP-8 retained IL-13 binding activity. In an asthma model MMP-8-deficient mice displayed increased airway hyperresponsiveness and decreased soluble IL-13R alpha 2 protein levels in bronchoalveolar lavage fluid compared with those seen in wild-type mice after house dust mite challenge.MMP-8 cleaves IL-13R alpha 2 in vitro and contributes to the solubilization of IL-13R alpha 2 in vivo.

Gata3 is a positional candidate gene for allergic asthma. We determined allergen-induced GATA-3 mRNA and protein expression in asthma susceptible and resistant mice and generated Gata3 sequence data. Our data indicate that the Gata3 gene in isolation is not a causative agent of asthma susceptibility in our model.

Pulmonary exposure to innocuous aeroallergens is a common event leading to inhalation tolerance. Distinct subsets of pulmonary dendritic cells (DC) and regulatory T cells (T(Reg)) play critical roles in mediating and maintaining such tolerance. In asthmatics, the same aeroallergens drive a maladaptive, Th2-biased immune response resulting in airway inflammation and airway hyper-reactivity. The mechanisms underlying the breakdown of inhalation tolerance, leading to the Th2-driven inflammation in rising numbers of asthmatic patients from industrialized countries remain elusive. The recent resurgence of interest in the role of the innate immune mediators in regulating adaptive immune response has sparked studies aimed at identifying the role of complement in allergic asthma. In this context, an unexpected role for the anaphylatoxin C5a receptor in allergic sensitization has been found. In models of experimental allergic asthma, ablation of C5aR signaling during initial allergen exposure either induced or enhanced Th2 sensitization. Mechanistically, C5aR signaling directly affected the function of distinct pulmonary DC subsets that induce or control allergen-induced adaptive immune responses. Signaling pathways downstream of C5 may also impact the function of T(Reg), as T(Reg) from C5 sufficient, but not from C5 deficient mice, suppress DC activation and subsequent development of Th2-driven inflammation. The emerging paradigm is that constitutive local generation of C5a and C5aR signaling in airway DCs controls inhalation tolerance directly as well as indirectly through sensitization of airway DCs for T(Reg)-mediated immunosuppression.

Few data exist on the modulation of cytokine receptor signaling by the actin or tubulin cytoskeleton. Therefore, we studied interleukin-2 receptor (IL-2R) signaling in phytohemagglutinine (PHA)-pretreated human T cells in the context of alterations in the cytoskeletal system induced by cytochalasin D (CyD), jasplaklinolide (Jas), taxol (Tax), or colchicine (Col). We found that changes in cytoskeletal tubulin polymerization altered the strength of several IL-2-triggered signals. Moreover, Tax-induced tubulin hyperpolymerization augmented the surface expression of the IL-2R ss -chain and enhanced the association of the IL-2R beta -chain with cytoskeletal tubulin. The IL-2R beta-chain, in turn, was constitutively associated with tubulin and, more weakly, actin. To exclude the possibility that these associations are artifacts caused by PHA, we confirmed them in T cells from TCR-transgenic DO 11.10 mice stimulated with their nominal antigen. We conclude that altered polymerization of cytoskeletal components, especially tubulin, is accompanied by modulation of IL-2 signaling at the receptor level.

Background The related TH2 cytokines IL-4 and IL-13 are produced during allergic responses, signal through receptors that contain IL-4 receptor (IL-4R) α, and promote allergic inflammation by activating signal transducer and activator of transcription 6. IL-4 promotes TH2 response induction, and IL-13 is necessary and sufficient to induce airways hyperresponsiveness (AHR) and goblet cell hyperplasia in some mouse models of asthma. The nonredundant role of IL-13 could reflect unique IL-13 activation of a signaling pathway, inhibitory effects induced by IL-4 but not IL-13, or greater production-potency of IL-13 than IL-4. Objectives We sought to distinguish among these possibilities by determining whether IL-4 inhalation can induce acute allergic airways disease in the absence of IL-13. Methods Mice were inoculated intratracheally with IL-13 or a long-acting formulation of IL-4. Responses of IL-13–deficient and IL-13–sufficient mice were compared, as were responses in mice treated with a potent IL-13 antagonist, anti–IL-4Rα antibody, or control reagents. Results IL-4 inhalation stimulated bronchoalveolar lavage fluid eosinophilia, AHR, and goblet cell hyperplasia. These responses were similar in IL-13–deficient and IL-13–sufficient mice and were not inhibited by an IL-13 antagonist but were blocked by anti–IL-4Rα antibody. Conclusion IL-4 can induce IL-13–independent AHR and goblet cell hyperplasia. Thus the greater role for IL-13 than IL-4 in the induction of these acute allergy-related changes reflects increased production, potency, or both of IL-13 relative to IL-4 rather than a unique IL-13–signaling pathway or a suppressive effect of IL-4. Clinical implications Dual IL-4/IL-13 inhibition might be more effective than selective IL-13 inhibition at suppressing allergic inflammation in some circumstances.

Complement has a long-recognized role as a lytic effector system that protects against microbial pathogens as well as a mediator of acute and chronic inflammatory responses. Many of the inflammatory properties related to complement activation can be related to the complement cleavage fragments C3a and C5a, the so-called anaphylatoxins. Cloning and subsequent gene targeting of their corresponding receptors, as well as generation of specific C3a and CSa inhibitors, have fueled new interest in studies aimed at defining the roles of the anaphylatoxins in inflammatory diseases. Traditionally, the anaphylatoxins have been considered mediators of end-stage effector mechanisms. However, recent data from animal models of allergic asthma suggest that C3a and C5a provide a critical link between innate and adaptive immunity. Further, the anaphylatoxins appear to form a sophisticated regulatory network together with immunoglobulin G Fc receptors that links regulatory events with effector activities in autoimmune disease. In this review, we will focus exclusively on the role of C3a and C5a in allergic asthma and autoimmune disease.

Although interleukin 13 (IL-13) is an important mediator of asthma and allergic diseases, the molecular mechanisms regulating IL-13 gene expression are not well understood. This study was designed to define the molecular mechanisms governing IL-13 gene expression in T cells. IL-13 expression was examined in human peripheral blood T cells and in the EL-4 T-cell line by enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction. An IL-13 promoter deletion analysis was performed using luciferase-based reporter plasmids transiently transfected into EL-4 cells by electroporation. DNA binding factors were investigated using electrophoretic mobility shift assays. In contrast to IL-4 expression, which required concomitant activation of calcium- and protein kinase C- (PKC-) dependent signalling pathways, PKC activation alone was sufficient for IL-13 protein secretion in mitogen-primed (but not resting) peripheral blood T cells, and for IL-13 mRNA expression and promoter activity in EL-4 T cells. Promoter deletion analysis localized a phorbol 12-myristate 13-acetate (PMA) -sensitive element to a proximal promoter region between −109 and −79 base pairs upstream from the IL-13 transcription start site. This promoter region supported the binding of both constitutive and PMA-inducible nuclear factors in gel shift assays.

Accumulating evidence in animal models and human asthma support a central role for IL-13 signaling in disease pathogenesis. In order to identify asthma and therapy associated genes, global transcriptional changes were monitored in mouse lung following antigen challenge (ovalbumin (OVA)), either alone or in the presence of a soluble IL-13 antagonist. Changes in whole lung gene expression after instillation of mIL-13 were also measured both in wild type and STAT6 deficient mice. A striking overlap in the gene expression profiles induced by either OVA challenge or mIL-13 was observed, further strengthening the relationship of IL-13 signaling to asthma. Consistent with results from functional studies, a subset of the OVA-induced gene expression was significantly inhibited by a soluble IL-13 antagonist while IL-13-modulated gene expression was completely attenuated in the absence of STAT6-mediated signaling. Results from these experiments greatly expand our understanding of asthma and provide novel molecular targets for therapy and potential biomarkers of IL-13 antagonism.

This study compared production of IL-2, IFN-γ, IL-4, IL-13, IL-5 and IL-10 in peripheral blood mononuclear cells from 20 children with autism spectrum disorder to those from matched controls. Levels of all Th2 cytokines were significantly higher in cases after incubation in media alone, but the IFN-γ/IL-13 ratio was not significantly different between cases and controls. Cases had significantly higher IL-13/IL-10 and IFN-γ/IL-10 than controls. Conclusion: Children with ASD had increased activation of both Th2 and Th1 arms of the adaptive immune response, with a Th2 predominance, and without the compensatory increase in the regulatory cytokine IL-10.

Despite extensive inquiry, the mechanisms underlying the pathophysiology of allergic diseases remain unknown. Recently, there has been a resurgence of interest in the role of the innate immune mediators of the complement pathway in asthma pathogenesis, particularly the anaphylatoxins (C3a, C5a). The emerging paradigm is that C3a production at the airway surface serves as a common pathway for the induction of airway hyperresponsiveness to a variety of asthma triggers (ie, allergens, viral infections, particulate matter, ozone, smoke), whereas C5/C5a plays a dual immunoregulatory role by protecting against Th2-mediated immune responses during initiation of responses and a proinflammatory role once immune responses are established. Support for a causal role for altered anaphylatoxin production in human disease comes from reports of exaggerated complement production in the lungs of asthmatics as well as the association of asthma with polymorphisms in C3/C3aR genes. Herein, we explore our current understanding of the role of complement activation in asthma pathogenesis and highlight the potential of targeting complement pathways for therapeutic drug development.

Background The gene encoding acyloxyacyl hydroxylase (AOAH), an enzyme that hydrolyzes secondary fatty acyl chains of LPS, is localized on chromosome 7p14-p12, where evidence for linkage to total IgE (tIgE) concentrations and asthma has been previously reported. Objective We hypothesized that variants in AOAH are associated with asthma and related phenotypes. Because both AOAH and soluble CD14 respond to LPS, we tested for gene-gene interaction. Methods We investigated the association between 28 single nucleotide polymorphisms throughout the AOAH gene and asthma, concentrations of tIgE, the ratio of IL-13/IFN-γ, and soluble CD14 levels among 125 African Caribbean, multiplex asthmatic pedigrees (n = 834). Real-time PCR was used to assess whether AOAH cDNA expression differed with AOAH genotype. Results Significant effects were observed for all 4 phenotypes and AOAH markers in 3 distinct regions (promoter, introns 1-6, and the intron 12/exon 13 boundary/intron 13 region) by means of single-marker and haplotype analyses, with the strongest evidence for a 2-single-nucleotide-polymorphism haplotype and log[tIgE] ( P = .006). There was no difference in AOAH expression levels by AOAH genotype for any of the markers. Comparing genotypic distributions at both the AOAH marker rs2727831 and CD14 (−260)C>T raises the possibility of gene-gene interaction ( P = .006-.036). Conclusion Our results indicate that polymorphisms in markers within the AOAH gene are associated with risk of asthma and associated quantitative traits (IgE and cytokine levels) among asthmatic subjects and their families in Barbados, and there is an interactive effect on tIgE and asthma concentrations between an AOAH marker and the functional CD14 (−260)C>T polymorphism. Clinical implications AOAH is a novel innate immunity candidate gene associated with asthma and related phenotypes in an African ancestry population.

Asthma is a chronic disease of the lung that has been increasing at an alarming rate in industrialized countries around the world over the last few decades. Although considerable progress has been made in our understanding of the underlying pathogenesis of the disease, the exact causes of the increasing prevalence are unknown. Studies suggest that most asthma develops in early childhood and that environmental factors present early in life may be crucial in the development of disease. One potential explanation for the recent epidemic referred to as the "hygiene hypothesis" postulates that factors that have resulted in a reduction in exposure to microbial products and/or infections in the western world may be contributing to this rise in disease prevalence. As early life influences are known to play an important role in establishment of asthma, studies have focused on the interface between mother and child that occurs during gestation and through breastfeeding. In this regard, the body of evidence regarding the relationship between breastfeeding and asthma indicates benefit but with the potential for risk. While providing population-level protection from infections and atopy in infancy and early childhood, breastfeeding might also pose an increased risk of atopic asthma among children with asthmatic mothers. In order to put this controversy in context, we discuss our current understanding of asthma pathogenesis, current theories on the factors driving the rising prevalence of asthma, and then discuss the potential influence of breastfeeding on asthma pathogenesis.

The complement system has long been recognized for its role as a lytic effector system that protects against microbial pathogens, as well as for its role in mediating acute and chronic inflammatory responses. Many of the inflammatory sequelae of complement activation can be related to the complement cleavage fragments C3a and C5a, the so-called anaphylatoxins (ATs). Cloning and subsequent gene targeting of their corresponding receptors, as well as generation of specific C3a and C5a inhibitors, have fueled new interest in studies aimed at defining the roles of the anaphylatoxins in inflammatory diseases. Traditionally, the anaphylatoxins have been considered mediators of end-stage effector mechanisms. However, recent data from animal models of allergic asthma suggest that C3a and C5a provide a critical link between innate and adaptive immunity. This review is aimed at outlining our current knowledge of when and where anaphylatoxins contribute to and control the development of allergic asthma. The accumulated data suggest a model in which C3a and C5a play important but opposing roles during allergen-induced T-cell polarization: C3a promotes Th2 responses, whereas C5a prevents Th2 polarization. During the effector phase, both anaphylatoxins trigger the inflammatory response and contribute to bronchoconstriction.

Asthma is a chronic disease of the lung that has been increasing at an alarming rate in industrialized countries around the world over the last few decades. Although considerable progress has been made in our understanding of the underlying pathogenesis of the disease, the exact causes of the increasing prevalence are unknown. Studies suggest that most asthma develops in early childhood and that environmental factors present early in life may be crucial in the development of disease. One potential explanation for the recent epidemic referred to as the "hygiene hypothesis" postulates that factors that have resulted in a reduction in exposure to microbial products and/or infections in the western world may be contributing to this rise in disease prevalence. As early life influences are known to play an important role in establishment of asthma, studies have focused on the interface between mother and child that occurs during gestation and through breastfeeding. In this regard, the body of evidence regarding the relationship between breastfeeding and asthma indicates benefit but with the potential for risk. While providing population-level protection from infections and atopy in infancy and early childhood, breastfeeding might also pose an increased risk of atopic asthma among children with asthmatic mothers. In order to put this controversy in context, we discuss our current understanding of asthma pathogenesis, current theories on the factors driving the rising prevalence of asthma, and then discuss the potential influence of breastfeeding on asthma pathogenesis.

Some time ago, the Th2 cytokine, interleukin (IL)-13, was identified as a critical regulator of the allergic response. Initial studies in animal models of disease provided compelling evidence that IL-13, independent from other Th2 cytokines, was necessary and sufficient to induce all features of allergic asthma. This contention was supported in human disease when strong associations between IL-13 levels and genetic polymorphisms in the IL-13 gene and disease correlates were found. With the preponderance of evidence continuing to support the importance of IL-13 in allergic disorders, attention is now turned towards understanding the mechanisms by which this cytokine may mediate the pathophysiologic features of allergic disease. The emerging paradigm is that IL-13 induces features of the allergic response via its actions on epithelial and smooth muscle cells not through traditional effector pathways involving eosinophils and IgE-mediated events. In light of recent developments, this review will explore our current understanding of the role of IL-13 in the pathogenesis of allergy and asthma with a particular focus on new insights into the mechanisms by which IL-13 induces the features of asthma.

Rising rates of allergic disease accompany the healthier benefits of a contemporary westernized lifestyle, such as low infant mortality. It is likely that these twinned phenomena are causally related. The hygiene hypothesis states that allergy and increased longevity are both consequences of reducing infectious stressors during early childhood for millennia. Mechanistic explanations for the hygiene hypothesis have typically invoked the T-helper-type 1/2 (T(H)1/T(H)2) model. Here, we discuss why we favour a broader 'counter-regulatory' model--one that might also explain the increasing incidence of autoimmune disease in westernized countries.

Background: Recently, it has been demonstrated that immunostimulatory DNA sequences (ISS) containing CpG motifs prevent the development of allergic airway responses in murine models of disease. However, few studies have addressed the issue of whether these agents will reverse established Tm H 2-driven allergic airway responses. Objective: The aim of this study was to determine whether intradermal delivery of an immunogenic protein of ragweed pollen linked to an immunostimulatory DNA sequence could reverse an established allergic response in the mouse lung. Methods: Mice sensitized and challenged with ragweed pollen extract were treated intradermally twice at 1-week intervals with an ISS chemically linked to Amb a 1 (Amb a 1-ISS). One week after the Amb a 1-ISS treatment, mice were rechallenged intratracheally with ragweed extract, and airway responses were assessed. Results: Amb a 1-ISS treatment of ragweed-sensitized and ragweed-challenged mice significantly reversed allergen-induced airway hyperresponsiveness and suppressed the total number of eosinophils in bronchoalveolar lavage fluid. The inhibitory effect of Amb a 1-ISS was associated with a marked increase in IFN-γ levels by Amb a 1-stimulated splenocytes and a shift in the antibody profile from a T H 2-directed IgG1 response to a T H 1-directed IgG2a response. Interestingly, the inhibitory effect of Amb a 1-ISS on allergen-driven airway hyperresponsiveness was independent of suppression of T H 2 cytokine production. Conclusion: These results demonstrate that intradermal delivery of allergen-specific DNA conjugates can reverse established allergic responses in the murine lung, supporting their potential use in the treatment of human asthma. (J Allergy Clin Immunol 2002;109:455-62.)

Interleukin 12 (IL-12) is central to the orchestration of cell-mediated immune responses in the innate as well as the adaptive immune system. Recent studies of the pathogenesis of diseases as disparate as measles and asthma have suggested that the complement system, itself at the interface of innate and adaptive immunity, is a biologically relevant regulator of IL-12 production. These data are reviewed here.

Dysregulated expression of the T helper 2 cytokine interleukin (IL)-4 is thought to play a fundamental role in the pathogenesis of allergic asthma. The molecular basis for dysregulated IL-4 production is not well understood. We analyzed in detail the molecular factors involved in regulating IL-4 transcription in a well-characterized mouse model. In this model, A/J mice developed allergen-induced IL-4 cytokine gene expression, airway inflammation, and hyperresponsiveness, whereas C3H/HeJ (C3H) mice did not. Here we report that isolated splenocytes from A/J and C3H mice stimulated ex vivo with concanavalin A reproduced the cytokine phenotype observed in the airway after antigen challenge. We hypothesized that differences in splenocyte IL-4 production involved either polymorphisms in regulatory IL-4 promoter regions, or the expression and activation of transcription factors necessary for promoter transactivation in a strain-dependent manner. To address these questions, we first sequenced ~ 700 base pairs containing well-characterized IL-4 promoter regulatory elements using genomic DNA obtained from C3H and A/J mice. Next, we used electrophoretic mobility shift assays with relevant IL-4 promoter sequences to screen nuclear extracts isolated from A/J and C3H splenocytes for functional transcriptional factor complexes. Here we show that susceptibility to antigen-induced airway hyperresponsiveness is not due to polymorphisms in the IL-4 promoter, but is associated with preferential expression of nuclear factor of activated T cells c in splenocyte nuclear extracts obtained from A/J mice. In conclusion, our data link dysregulated activation of a specific transcription factor with susceptibility to allergic airway inflammation.

Allergic airway diseases are associated with skewed T(H)2 cytokine production, although the underlying cause of this aberrant immune response is not well understood. Recently, 2 double-digit cytokines, IL-12 and IL-13, have been proposed to play pivotal roles in the T(H)2-polarized immune response to inhaled allergens. IL-12 is a critical determinant of T(H)1-mediated immune responses, and it has been shown that deficiency in this cytokine can lead to T(H)2-polarized immune responses. IL-13, on the other hand, has recently been shown to be a critical mediator of the effector arm of the allergic response. Overproduction of this cytokine has been shown to induce many common features of the allergic diathesis, such as airway hyperresponsiveness, eosinophilic inflammation, IgE production, mucus hypersecretion, and subepithelial fibrosis. Substantial evidence suggests that an imbalance in the production of these 2 critical immunoregulatory cytokines occurs in the lungs of atopic and asthmatic individuals, such that IL-13 is overproduced, and IL-12 production is impaired. As a result of this imbalance, IL-13 production may go unchecked and induce the classical allergic phenotype. Although it is not entirely clear what tips the balance between these 2 cytokines, genetic studies suggest that this could indeed be a primary event. Interestingly, the genes encoding both of these cytokines reside within the chromosomal regions on 5q, which have been associated with asthma in many populations. Although no evidence exists to date to support an association between asthma and polymorphisms in the IL12 gene, evidence is accumulating that supports an association between genetic alterations in the IL13 gene and atopy and asthma. From these observations, we can conclude that an imbalance in the IL-12/IL-13 axis may contribute to the development of allergic diseases, such as asthma. Studies to more fully examine the yin-yang relationship between these 2 critical immunoregulatory cytokines are clearly warranted.

Asthma is a chronic inflammatory disease that has been on the rise in recent years despite increased use of medication. Nevertheless, the fundamental mechanisms that underlie the development and perturbation of the asthmatic state remain elusive. Asthma is characterized by variable airflow obstruction, airway Ž . hyperresponsiveness AHR and airway inflammation. The inflammatory response in the asthmatic lung is characterized by infiltration of the airway wall with mast cells, lymphocytes and eosinophils. Although asthma is multifactorial in origin, recent advances suggest that asthma is an immune disease with a prominent role for T lymphocytes in the pathogenesis. In particular, CD4 T cells producing Ž Ž . a Th2 pattern of cytokines interleukin IL -4, IL-5, . IL-13, IL-9 have been hypothesized to play a piv

▪ Abstract The incidence, morbidity, and mortality of asthma has increased worldwide over the last two decades. Asthma is a complex inflammatory disease of the lung characterized by variable airflow obstruction, airway hyperresponsiveness (AHR), and airway inflammation. The inflammatory response in the asthmatic lung is characterized by infiltration of the airway wall with mast cells, lymphocytes, and eosinophils. Although asthma is multifactorial in origin, the inflammatory process in the most common form of the disease (extrinsic asthma) is believed to be a result of inappropriate immune responses to common aero-allergens in genetically susceptible individuals. As such, it has been hypothesized that CD4+T cells that produce a Th2 pattern of cytokines play a pivotal role in the pathogenesis of this disease. Through the release of cytokines such as IL-4, IL-13, and IL-5, these cells orchestrate the recruitment and activation of the primary effector cells of the allergic response, the mast cell and the eosinophil. Activation of these cells results in the release of a plethora of inflammatory mediators that individually or in concert induce changes in airway wall geometry and produce the symptoms of the disease. The aim of this review is to discuss our current understanding of the pathophysiologic mechanisms by which Th2 cytokines induce airway disease, and the factors that predispose to the generation of these pathogenic cells in response to inhalation of ubiquitous aero-allergens. Elucidation of the exact immunological basis for allergic asthma may yield immunotherapeutic strategies to reverse the development of pathogenic Th2-mediated immune responses and reduce the morbidity and mortality associated with this disease.

Allergic asthma is a chronic, often debilitating disease which has been increasing in both prevalence and mortality worldwide, despite increased use of currently available medications. Although extensive research has been devoted to the study of this disease, the fundamental mechanisms which underlie the development and perturbation of the asthmatic state remain elusive.

The worldwide incidence, morbidity, and mortality of allergic asthma are increasing. The pathophysiological features of allergic asthma are thought to result from the aberrant expansion of CD4+T cells producing the type 2 cytokines interleukin-4 (IL-4) and IL-5, although a necessary role for these cytokines in allergic asthma has not been demonstrable. The type 2 cytokine IL-13, which shares a receptor component and signaling pathways with IL-4, was found to be necessary and sufficient for the expression of allergic asthma. IL-13 induces the pathophysiological features of asthma in a manner that is independent of immunoglobulin E and eosinophils. Thus, IL-13 is critical to allergen-induced asthma but operates through mechanisms other than those that are classically implicated in allergic responses.

We have demonstrated previously that susceptibility of murine strains to the development of allergic airway responses is associated with a type 2 cytokine pattern. In the present study, we examine the in vivo role of IL-12 in the immune response to allergen exposure in susceptible (A/J) and resistant (C3H/HeJ, C3H) strains of mice. OVA sensitization and challenge induced significant increases in airway reactivity in A/J mice as compared with their PBS-challenged controls, while no increases in airway reactivity were observed in OVA-challenged C3H mice. OVA exposure of A/J mice resulted in marked increases in the Th2 cytokines, IL-4 and IL-10, in the bronchoalveolar lavage fluid, whereas increases in IFN-γ were observed in C3H mice. Strikingly, anti-IL-12 mAb (1 mg/mouse) treatment resulted in threefold increases in airway reactivity in OVA-challenged resistant C3H mice, concomitant with significant increases in bronchoalveolar lavage levels of Th2 cytokines and decreases in IFN-γ. IL-12 depletion of C3H mice also suppressed OVA-specific serum IgG2a levels and increased both serum OVA-specific IgG1 and IgE levels. Blockade of endogenous IL-12 levels in susceptible A/J mice resulted in further augmentation of type 2 immune responses. These results demonstrate that endogenous production of IL-12 is essential for resistance to Ag-induced airway hyperresponsiveness, and furthermore, that dysregulation of IL-12 production may lead to the development of deleterious type 2 immune responses to inhaled allergens.

Asthma is a chronic inflammatory disease of the airways, driven by a Th2 immune response to allergens like house dust mite (HDM). While Th2 immune responses (IL-13 in particular) alone can induce allergic asthma, Th17 cytokines (IL-17) can exacerbate the disease. Although, the components of HDM that drive asthma remain unclear, HDM is a complex mixture including immunogenic structural moieties like carbohydrates. Such carbohydrate moieties can be recognized by pattern recognition receptors on dendritic cells (DCs) like C-type lectin receptors (CLRs). Recently, CLRs like Dectin-1 & Dectin-2 have been implicated in driving Th2 & Th17 responses against various infections. However, the role of carbohydrate recognition in modulation of HDM-induced allergic asthma remains unclear. Therefore we hypothesized that Dectin-1 & Dectin-2 induce Th2 & Th17 responses following HDM exposure. Surprisingly, Dectin-1 KO mice had exacerbated Th2 immune responses leading to enhance airway resistance. In contrast, mice in which Dectin-2 was blocked (using monoclonal antibody) had diminished HDM-induced Th2 immune responses and airway reactivity. Interestingly, neither Dectin-1 nor Dectin-2 modulated Th17 responses. Overall, our data suggests that while Dectin-1 protects against allergic asthma by negatively regulating Th2 immune responses, Dectin-2 promotes asthma by enhancing Th2 immune responses.

Certain families of allergens contain domains that bear structural and functional similarity to homologs from pathogens enabling them to sabotage host immunity by hijacking crucial innate immune pathways. We focused on members of the Bet v 1-like superfamily that share a remarkably conserved architecture, the so called Bet v 1-fold, with the aim to identify structural mimicry as one possible cause of allergenicity. Here we show that Bet v 1, the major birch pollen (BP) allergen, has functional homology with a bacterial homolog, namely TTHA0849 from Thermus thermophilus. TTHA0849 and Bet v 1 competed for the same uptake mechanism by monocyte-derived DCs (MoDCs) of BP allergic and normal donors as visualized by fluorescence microscopy. Using a pre-designed antibacterial response PCR array, we could reveal striking similarities in gene expression patterns induced by Bet v 1 in MoDCs of both donor groups to those induced by TTHA0849. Importantly, Bet v 1 and its bacterial homolog also triggered the transcription of the Th2 signature cytokines IL-4 and IL-13, as well as EGR-3, a marker of early cell activation, in MoDCs from BP allergic but not normal donors. These findings propose that conserved structural features are present on both Bet v 1 and TTHA0849 targeting the same pathways common to allergic and normal individuals. Altered signal processing and translation in predisposed allergic individuals might be a mechanism underlying the phenomenon of allergic sensitization.

Allergic diseases are increasingly prevalent in Westernized environments. Recent data indicate that the molecular substrates of allergenicity are a function of direct interactions with pathways of innate immune activation that evolved to sense microbial infection. After lipid-binding proteins exemplified by Der p 2, proteases represent the next most common class of allergens. The relevant mechanisms by which proteases lead to innate immune activation remain under-defined. α2-macroglobulin (α2M) is a plasma protein that inhibits extracellular proteases, targeting them for internalization by LDL receptor-related protein 1 (LRP1). In antigen presenting cells (APC), LRP1 directs α2M-associated proteins for antigen presentation. We hypothesized that protease adjuvanticity and allergenicity result from interactions with α2M and LRP1. Thus, we intratracheally challenged mice with papain, an occupational allergen and cysteine protease. In C57Bl/6 mice, this induced eosinophil and lymphocyte recruitment to the lung and increases in total and papain-specific IgE. While α2M-deficient mice exhibited no defects in the generation of allergic responses to papain, deletion of LRP-1 on APCs resulted in increased allergic inflammatory responses after challenge. These data suggest a role for α2M/LRP-1 in the clearance of protease allergens and suppression of excessive immune activation.

Although allergic asthma is a chronic inflammatory disease of the lung mediated by Th2 cells, the pathways leading to this response are not clear. Recent studies have highlighted influence of pulmonary dendritic cells (DCs) on T-cell differentiation. Rapid DC recruitment into the bronchial mucosa has been well documented in animals and humans in response to allergen. Central to early DC recruitment, CCL20 is the only known ligand for CCR6, a receptor preferentially expressed on immature DCs. The potential importance of CCL20/CCR6 in allergic sensitization was demonstrated in a cockroach model of allergic asthma in which Ccr6-/- mice were protected against asthma. At present, the mechanisms by which allergen induces secretion of DC-attractant chemokines are unknown. We previously showed that bronchial epithelial cells release CCL20 relatively rapidly after house dust mite (HDM) (Nathan, 2009). This induction was not prevented by the inhibiting protein synthesis (cycloheximide) suggesting release through a transcriptionally independent mechanism. These data suggested that HDM may induced the release of preformed stores of CCL20

Airway inflammation and airway hyperreactivity are consistent features of allergic asthma. The inflammatory response is characterized chiefly by an increase in eosinophils and T cells in bronchial mucosa and bronchoalveolar lavage fluids(1–5). Furthermore, as therapeutic interventions that reduce airway inflammation ameliorate airway hyperreactivity, inflammation is hypothesized to play a key role in the development of asthma-associated airway hyperreactivity(6,7). As a primary regulator of the inflammatory cascade, the T lymphocyte has been implicated in the pathogenesis of asthma(8). Considerable circumstantial evidence has been accumulating to support this hypothesis. For example, increased numbers of activated T cells are found in the bronchial mucosa of asthmatics as compared with control subjects(3). The degree of peripheral blood T lymphocyte activation correlates with the severity of asthmatic symptoms(9). In patients with allergic asthma, which is characterized as such by elevated serum IgE levels, allergen challenge induces a selective recruitment of CD4+ T cells into the airways(5). Moreover, the degree of activation of CD4+ lymphocytes correlates with the number of eosinophils in BAL fluid, with the degree of bronchial hyperreactivity, and with disease severity(10). CD4+ T cells isolated from bronchoalveolar fluids (BAL) of allergic asthmatics express increased levels of markers of acute and chronic activation(11). Such lymphocytes demonstrate a specific pattern of cytokine expression, namely, elevated levels of IL-4 and IL-5 mRNA, a pattern consistent with a T-helper 2 (TH2) pattern of differentiation(12) A possible immunopathogenic role for Th2 CD4+ T lymphocytes in asthma is suggested by the eosinophilia that characterizes asthma in general and the elevated IgE levels associated with allergic asthma.

Airways smooth muscle contractility changes over the course of life. In general, airway responses to stimulatory agents are greatest in infants and children and decline progressively thereafter. Although our understanding of the numerous factors that control airways smooth muscle tone in the adult has advanced considerably in the last decade, very little is understood about the way these factors may vary with ageing.