Allergic Airway Responses Research Articles

High-dose but not low-dose mainstream cigarette smoke suppresses allergic airway inflammation by inhibiting T cell function

Epidemiological studies have identified childhood exposure to environmental tobacco smoke as a significant risk factor for the onset and exacerbation of asthma, but studies of smoking in adults are less conclusive, and mainstream cigarette smoke (MCS) has been reported to both enhance and attenuate allergic airway inflammation in animal models. We sensitized mice to ovalbumin (OVA) and exposed them to MCS in a well-characterized exposure system. Exposure to MCS (600 mg/m(3) total suspended particulates, TSP) for 1 h/day suppresses the allergic airway response, with reductions in eosinophilia, tissue inflammation, goblet cell metaplasia, IL-4 and IL-5 in bronchoalveolar lavage (BAL) fluid, and OVA-specific antibodies. Suppression is associated with a loss of antigen-specific proliferation and cytokine production by T cells. However, exposure to a lower dose of MCS (77 mg/m(3) TSP) had no effect on the number of BAL eosinophils or OVA-specific antibodies. This is the first report to demonstrate, using identical smoking methodologies, that MCS inhibits immune responses in a dose-dependent manner and may explain the observation that, although smoking provokes a systemic inflammatory response, it also inhibits T cell-mediated responses involved in a number of diseases.

The Tec Family Kinase, IL-2-Inducible T Cell Kinase, Differentially Controls Mast Cell Responses

The Tec family tyrosine kinase, IL-2-inducible T cell kinase (Itk), is expressed in T cells and mast cells. Mice lacking Itk exhibit impaired Th2 cytokine secretion; however, they have increased circulating serum IgE, but exhibit few immunological symptoms of allergic airway responses. We have examined the role of Itk in mast cell function and FcepsilonRI signaling. We report in this study that Itk null mice have reduced allergen/IgE-induced histamine release, as well as early airway hyperresponsiveness in vivo. This is due to the increased levels of IgE in the serum of these mice, because the transfer of Itk null bone marrow-derived cultured mast cells into mast cell-deficient W/W(v) animals is able to fully rescue histamine release in the W/W(v) mice. Further analysis of Itk null bone marrow-derived cultured mast cells in vitro revealed that whereas they have normal degranulation responses, they secrete elevated levels of cytokines, including IL-13 and TNF-alpha, particularly in response to unliganded IgE. Analysis of biochemical events downstream of the FcepsilonRI revealed little difference in overall tyrosine phosphorylation of specific substrates or calcium responses; however, these cells express elevated levels of NFAT, which was largely nuclear. Our results suggest that the reduced mast cell response in vivo in Itk null mice is due to elevated levels of IgE in these mice. Our results also suggest that Itk differentially modulates mast cell degranulation and cytokine production in part by regulating expression and activation of NFAT proteins in these cells.

Strain-specific requirement for eosinophils in the recruitment of T cells to the lung during the development of allergic asthma

Eosinophils have been implicated as playing a major role in allergic airway responses. However, the importance of these cells to the development of this disease has remained ambiguous despite many studies, partly because of lack of appropriate model systems. In this study, using transgenic murine models, we more clearly delineate a role for eosinophils in asthma. We report that, in contrast to results obtained on a BALB/c background, eosinophil-deficient C57BL/6 ΔdblGATA mice (eosinophil-null mice via the ΔDblGATA1 mutation) have reduced airway hyperresponsiveness, and cytokine production of interleukin (IL)-4, -5, and -13 in ovalbumin-induced allergic airway inflammation. This was caused by reduced T cell recruitment into the lung, as these mouse lungs had reduced expression of CCL7/MCP-3, CC11/eotaxin-1, and CCL24/eotaxin-2. Transferring eosinophils into these eosinophil-deficient mice and, more importantly, delivery of CCL11/eotaxin-1 into the lung during the development of this disease rescued lung T cell infiltration and airway inflammation when delivered together with allergen. These studies indicate that on the C57BL/6 background, eosinophils are integral to the development of airway allergic responses by modulating chemokine and/or cytokine production in the lung, leading to T cell recruitment.

A Helminth Immunomodulator Reduces Allergic and Inflammatory Responses by Induction of IL-10-Producing Macrophages

The coincidence between infections with parasitic worms and the reduced prevalence of allergic disease in humans and in animal models has prompted the search for helminth molecules with antiallergic and antiinflammatory potential. We report herein that filarial cystatin, a secreted protease inhibitor of filarial nematodes, suppresses Th2-related inflammation and the ensuing asthmatic disease in a murine model of OVA-induced allergic airway responsiveness. Treatment with recombinant filarial cystatin inhibited eosinophil recruitment, reduced levels of OVA-specific and total IgE, down-regulated IL-4 production, and suppressed allergic airway hyperreactivity when applied during or after sensitization and before challenge with the allergen. Depletion of macrophages by clodronate-containing liposomes prevented the curative effects and restored the levels of infiltrating cells, IgE, and allergic airway reactivity. Blocking of IL-10 by application of anti-IL-10 receptor Abs restored the reduced number of infiltrating cells and the levels of OVA-specific IgE. In contrast, depletion of regulatory T cells by anti-CD25 Abs had only limited effects. Cystatin also modulated macrophage-mediated inflammation in a murine model of dextran sulfate sodium-induced colitis, leading to reduction of inflammatory infiltrations and epithelial damage. Our data demonstrate that treatment with a single helminth protein can exert the antiallergic effects of helminth infections.

Dexamethasone and The Long Acting β 2-Adrenergic Receptor Agonist, R,R-formoterrol (RF), Upregulate CCAAT Enhancer Binding Protein (C/EBP) Gene Expression and Induce Production of The Immunoprotective Surfactant Protein D (SP-D) in Alveolar Epithelial Cells

The innate immune molecule SP-D is thought to exert an essential protective function during the allergic airway response. The regulation of this lung collectin is unclear but there are indications that corticosteroids and cAMP are necessary for expression of SP-D. We hypothesized that the immunosuppressive actions of dexamethasone and the long acting β2-adrenergic receptor agonist, RF, are mediated at least partly, by induction of SP-D production in the airways.

Effect of Intranasal vs. Intraperitoneal Sensitization of Mice with House Dust Mite Antigen on Airway Hyperresponsiveness (AHR) and SOCS expression in the Lung

Increased density of Th2 cells with the secretion of IL-4, IL-5, IL-9, and IL-13 in the airways are hallmarks of allergic asthma, and induce AHR, airway inflammation, mucus hypersecretion, and goblet cell hyperplasia. These cytokines are modulated by a family of proteins called suppressor of cytokine signaling (SOCS). In this study, we investigated whether or not the route of sensitization with house dust mite induces different effects on the development of AHR and on SOCS expression in the lungs. Mice were sensitized either by intranasal injection (IN) or by intraperiotneal injection (IP) with house dust mite antigen (10 μg). Alum was used as an adjuvant only in IP sensitization. AHR to methacholine was determined by BUXCO. Staining of the lungs with H&E and tri-chrome was used to compare morphology and collagen deposition, respectively. Periodic Acid Schiff (PAS) staining was done to visualize goblet cell hyperplasia and mucus deposition and immunohistochemistry (IHC) was performed to visualize the expression of SOCS 1, 3, and 5. IN route of antigen sensitization induced a significantly increased AHR, airway inflammation, goblet cell hyperplasia, collagen deposition, and expression of SOCS1, 3, and 5 in the mouse lung compared to the IP route of antigen administration. Intranasal administration of the house dust mite antigen causes significantly increased induction of allergic airway response than by intraperitoneal route. This could serve as a better model to investigate the effects of potential therapeutic agents in allergic asthma.

IL-21 Receptor Signaling Partially Mediates Th2-mediated Allergic Airway Responses

IL-21 Receptor Signaling Partially Mediates Th2-mediated Allergic Airway Responses

A Key Role for CC Chemokine Receptor 1 in T-Cell-Mediated Respiratory Inflammation

CC chemokine receptor 1 (CCR1) is found on a variety of cells in the immune system and has been shown to play an important role in the host response to pathogens. These studies used a murine model of virus-induced exacerbation of allergic airway disease to examine the role of CCR1 on T cells associated with immune responses taking place in the lung. Lungs of virally exacerbated allergic animals contained elevated levels of interferon-gamma and interleukin-13 and increased levels of CCR1 ligands CCL3 and CCL5. CCR1 expression on T cells was increased in virally exacerbated allergic animals over the level observed in mice sensitized to allergen or exposed to viral infection alone. Using mice deficient for CCR1, we observed decreased airway hyperreactivity and Th2 cytokine production from CD4(+) T cells when this receptor was absent. Transfer studies demonstrated that neither CD4(+) nor CD8(+) T cells from CCR1(-/-) mice migrated to the lymph node as efficiently as wild-type T cells. Intracellular cytokine staining in wild-type mice revealed that CCR1(+) CD4(+) and CD8(+) T cells are associated with interleukin-13 production. Thus, these studies identify CCR1 as a potential target for alleviating T-cell accumulation during exacerbation of asthmatic disease.

Severe Vitamin E deficiency modulates airway allergic inflammatory responses in the murine asthma model

Allergic asthma is a complex immunologically mediated disease associated with increased oxidative stress and altered antioxidant defenses. It was hypothesized that α-tocopherol (α-T) decreases oxidative stress and therefore its absence may influence allergic inflammatory process, a pathobiology known to be accompanied by oxidative stress. Therefore, selected parameters of allergic asthma sensitization and inflammation were evaluated following ovalbumin sensitization and re-challenge of α-T transfer protein (TTP) knock-out mice (TTP−/−) that have greatly reduced lung α-T levels (e.g.<5%) compared to their litter mate controls (TTP+/+). Results showed that severe α-T deficiency result in a blunted lung expression of IL-5 mRNA and IL-5 protein and plasma IgE levels compared with TTP+/+ mice following immune sensitization and rechallenge, although lung lavage eosinophil levels were comparable in both genomic strains. It is concluded that the initial stimulation of immune responses by the TTP−/− mice were generally blunted compared to the TTP+/+ mice, thus diminishing some aspects of subsequent allergic inflammatory processes.

OR.12. Perinatal Exposure to Environmental Estrogen, Bisphenol A (BPA), Promotes Allergic Sensitization and Airway Responsiveness in an Animal Model of Asthma

OR.12. Perinatal Exposure to Environmental Estrogen, Bisphenol A (BPA), Promotes Allergic Sensitization and Airway Responsiveness in an Animal Model of Asthma

Effects of Gasoline Engine Emissions on Preexisting Allergic Airway Responses in Mice

Gasoline-powered vehicle emissions contribute significantly to ambient air pollution. We hypothesized that exposure to gasoline engine emissions (GEE) may exacerbate preexisting allergic airway responses. Male BALB/c mice were sensitized by injection with ovalbumin (OVA) and then received a 10-min aerosolized OVA challenge. Parallel groups were sham-sensitized with saline. Mice were exposed 6 h/day to air (control, C) or GEE containing particulate matter (PM) at low (L), medium (M), or high (H) concentrations, or to the H level with PM removed by filtration (high-filtered, HF). Immediately after GEE exposure mice received another 10-min aerosol OVA challenge (pre-OVA protocol). In a second (post-OVA) protocol, mice were similarly sensitized but only challenged to OVA before air or GEE exposure. Measurements of airway hyperresponsiveness (AHR), bronchoalveolar lavage (BAL), and blood collection were performed ∼24 h after the last exposure. In both protocols, M, H, and HF GEE exposure significantly decreased BAL neutrophils from nonsensitized mice but had no significant effect on BAL cells from OVA-sensitized mice. In the pre-OVA protocol, GEE exposure increased OVA-specific IgG1 but had no effect on BAL interleukin (IL)-2, IL-4, IL-13, or interferon (IFN)-γ in OVA-sensitized mice. Nonsensitized GEE-exposed mice had increased OVA-specific IgG2a, IgE, and IL-2, but decreased total IgE. In the post-OVA protocol, GEE exposure reduced BAL IL-4, IL-5, and IFN-γ in nonsensitized mice but had no effect on sensitized mice. These results suggest acute exposure to the gas–vapor phase of GEE suppressed inflammatory cells and cytokines from nonsensitized mice but did not substantially exacerbate allergic responses.

Lentiviral-mediated GATA-3 RNAi Decreases Allergic Airway Inflammation and Hyperresponsiveness

GATA-3 is the key transcriptional factor for Th2 commitment in T cells and is strongly associated with asthma and allergic disease. We studied the silencing of the GATA-3 gene expression using RNA interference (RNAi) delivered by a lentiviral vector, to evaluate the therapeutic role of GATA-3 short hairpin RNAs (shRNAs) in a murine model of asthma. Mice were sensitized with OVA and instilled intratracheally (IT) with GATA-3 shRNAs lentiviral vector (Lenti-si-GATA-3) once, 48 hours before challenge. After three challenges with the OVA antigen, the mice were assessed for airway hyperresponsiveness (AHR) and inflammation. With infection of Lenti-si-GATA-3 in EL-4 cells, GATA-3 gene expression was abrogated and downstream Th2 cytokines, such as interleukin-4 (IL-4) and IL-5, were also significantly inhibited. IT delivery of Lenti-si-GATA-3 in OVA-immunized mice resulted in a strong inhibition of local GATA-3 gene expression. Treatment with Lenti-si-GATA-3 successfully alleviated OVA-induced airway eosinophilia and Th2 cytokine release. While evaluating AHR by means of enhanced pause (Penh) and pulmonary resistance (R(L)) using body plethysmography, it was found that the administration of Lenti-si-GATA-3 had significantly decreased AHR in OVA-immunized mice. These results suggest that inhibition of GATA-3 gene expression by shRNAs lentiviral vectors strongly attenuates antigen-induced airway inflammation and hyper-responsiveness in mice.

A small-molecule compound targeting CCR5 and CXCR3 prevents airway hyperresponsiveness and inflammation

Asthma is associated with increased numbers of T-cells in the lung. CC chemokine receptor (CCR)5 and CXC chemokine receptor (CXCR)3 have been reported to play important roles in the lung T-cell homing pathway, and may be potential targets for asthma therapy. The aim of the present study was to investigate the role of CCR5 and CXCR3 in allergen-induced acute asthma and to determine whether a novel small-molecule compound, TAK-779, targeting CCR5 and CXCR3 can attenuate allergic airway responses. Mice were sensitised with ovalbumin (OVA). mRNA expression of chemokine receptors in the lung were measured after the challenge with either aerosolised phosphate-buffered saline or OVA. OVA-sensitised mice were also treated with TAK-779. Respiratory function was measured, bronchoalveolar lavage was performed, and blood and lung samples were obtained. OVA challenge increased CCR3, CCR5 and CXCR3 expression in the lung. Treatment with TAK-779 significantly attenuated altered respiratory function and pulmonary allergic inflammation. The beneficial effect was associated with reduced expression of CCR5 and CXCR3 in the lung. These data demonstrate that blockade of CC chemokine receptor 5 and CXC chemokine receptor 3 using TAK-779, a synthetic nonpeptide compound, can prevent the development of asthma features in a mouse model. Thus, CC chemokine receptor 5 and CXC chemokine receptor 3 may be potential targets for asthma therapy.

A potential immunotherapy approach: Mucosal immunization with an IL-13 peptide-based virus-like particle vaccine in a mouse asthma model

Interleukin (IL)-13 is critical in asthma pathogenesis. Previously, we have developed an IL-13 peptide-based vaccine and confirmed that subcutaneous immunization with the vaccine suppressed airway allergic inflammatory responses in a mouse asthma model. In the present study, we sought to test if mucosal immunization with the vaccine could be a potential approach, by inducing specific autoantibodies of both local IgA in the airway and systemic IgG in serum, to provide an overall suppression of redundant IL-13 effects. The results show that intranasal vaccination induces IL-13-specific IgA responses in multiple mucosal tissues and higher titers of IgG in serum than subcutaneous vaccination. This approach leads to a more effective suppression of ovalbumin-driven Th2 patterns of antibody responses and airway IL-13 and eosinophil accumulation than subcutaneous immunization, even when the induced IL-13 IgG responses were at a similar level. In conclusion, mucosal vaccination may be an innovative potential approach in the treatment of asthma.

Allergic airway responses in obese mice.

Epidemiologic data indicate an increased incidence of asthma in the obese. To determine whether obese mice exhibit augmented pulmonary responses after allergen sensitization and challenge. Lean, wild-type (C57BL/6), obese ob/ob, and obese db/db mice were sensitized to ovalbumin (OVA), and then challenged with aerosolized OVA or phosphate-buffered saline (PBS). Changes in total pulmonary resistance (Rl) induced by intravenous methacholine were measured by forced oscillation. Blood was collected, bronchoalveolar lavage (BAL) was performed, and lungs were harvested for measurement of cytokine expression by real-time reverse transcription-polymerase chain reaction. OVA challenge increased baseline Rl in ob/ob, but not wild-type, mice, and airway responsiveness was greater in ob/ob than wild-type mice, regardless of the challenge. Compared with PBS, OVA challenge caused an increase in the number of BAL fluid (BALF) cells, an increase in lung Th2 cytokine expression, and an increase in serum IgE. Significantly fewer BALF cells were recovered from OVA-challenged ob/ob versus wild-type mice, whereas serum IgE levels were elevated significantly more in ob/ob versus wild-type mice. BALF and lung Th2 cytokine expression was not different in ob/ob versus wild-type mice. Airway responsiveness was greater in db/db versus wild-type mice, regardless of the challenge, and OVA caused airway hyperresponsiveness in db/db but not wild-type mice, despite reduced BALF cells in OVA-challenged db/db versus wild-type mice. These results demonstrate that obesity enhances OVA-induced changes in pulmonary resistance and serum IgE and that these changes are not the result of increased Th2 type airway inflammation.

Lipoxin A4stable analogs reduce allergic airway responsesviamechanisms distinct from CysLT1 receptor antagonism

Cellular recruitment during inflammatory/immune responses is tightly regulated. The ability to dampen inflammation is imperative for prevention of chronic immune responses, as in asthma. Here we investigated the ability of lipoxin A4 (LXA4) stable analogs to regulate airway responses in two allergen-driven models of inflammation. A 15-epi-LXA4 analog (ATLa) and a 3-oxa-15-epi-LXA4 analog (ZK-994) prevented excessive eosinophil and T lymphocyte accumulation and activation after mice were sensitized and aerosol-challenged with ovalbumin. At <0.5 mg/kg, these LXA4 analogs reduced leukocyte trafficking into the lung by >50% and to a greater extent than equivalent doses of the CysLT1 receptor antagonist montelukast. Distinct from montelukast, ATLa treatment led to marked reductions in cysteinyl leukotrienes, interleukin-4 (IL-4), and IL-10, and both ATLa and ZK-994 inhibited levels of IL-13. In cockroach allergen-induced airway responses, both intraperitoneal and oral administration of ZK-994 significantly reduced parameters of airway inflammation and hyper-responsiveness in a dose-dependent manner. ZK-994 also significantly changed the balance of Th1/Th2-specific cytokine levels. Thus, the ATLa/LXA4 analog actions are distinct from CysLT1 antagonism and potently block both allergic airway inflammation and hyper-reactivity. Moreover, these results demonstrate these analogs' therapeutic potential as new agonists for the resolution of inflammation.

Novel Recombinant Interleukin-13 Peptide-based Vaccine Reduces Airway Allergic Inflammatory Responses in Mice

Interleukin (IL)-13 plays a pivotal role in the pathogenesis of allergic asthma. Passive administration of its monoclonal antibody or soluble receptor to block overproduced IL-13 has been proven to be effective in controlling airway allergic responses in animal models, but these approaches have disadvantages of short half-lives, high costs, and possible adverse effects. We sought to develop a novel therapeutic strategy through constructing an IL-13 peptide-based vaccine for blocking IL-13 on a persistent effect basis and to evaluate its in vivo effects using a murine model. To break self-tolerance, truncated hepatitis B core antigen was used as a carrier. Vaccine was prepared by inserting a peptide derived from the receptor binding site of mouse IL-13 into the immunodominant epitope region of the carrier using gene recombination methods. Mice received vaccine subcutaneously three times, and then subjected to intraperitoneal sensitization and intranasal challenge with ovalbumin. Control animals received carrier or saline in place of vaccine. The vaccine presented as virus-like particles and induced sustained and high titered IL-13-specific IgG without the use of conventional adjuvant. Vaccination significantly suppressed ovalbumin-induced inflammatory cell number, and IL-13 and IL-5 levels in bronchoalveolar lavage fluids. Serum total and ovalbumin-specific IgE were also significantly inhibited. Moreover, allergen-induced goblet cell hyperplasia, lung tissue inflammatory cell infiltration, and pulmonary hyperresponsiveness to inhaled methacholine were significantly suppressed in vaccinated mice. Our data indicate that IL-13 peptide-based vaccines could be an effective therapeutic approach in the treatment of asthma.

Novel cytokine peptide‐based vaccines: an interleukin‐4 vaccine suppresses airway allergic responses in mice

Monoclonal antibodies or soluble receptors have been used to block over-produced endogenous cytokines. However, they have disadvantages of short half-lives, high costs, and possible adverse effects. Using interleukin (IL)-4 as a model target, we sought to develop a novel therapeutic strategy by constructing an IL-4 peptide-based vaccine for blocking IL-4 on a persistent basis, and to evaluate its efficacy in a mouse model of asthma. A peptide was selected by antigenic prediction and structure analysis of IL-4/receptor complex. The vaccine was constructed by employing truncated hepatitis B core antigen as carrier with the peptide inserted using gene engineering methods. It was then expressed, purified and identified. Prior to intraperitoneal sensitization and intranasal challenge with ovalbumin, mice were subcutaneously immunized three times with the vaccine, or the carrier or saline as controls. Serum antibodies, inflammatory cells in bronchoalveolar lavage fluids (BALF), lung histology, and responsiveness to inhaled methacholine were analyzed. The vaccine presented as virus-like particles and reacted to polyclonal anti-IL-4 in Western blotting. Vaccinated mice produced high titers of IgG to IL-4. Serum ovalbumin-specific IgE, eosinophil accumulation in BALF, goblet cell hyperplasia, tissue inflammation and methacoline-induced respiratory responses were markedly suppressed in vaccinated mice with statistical significance, as compared with those in the control groups. Administration of this novel IL-4 vaccine led to an overall decrease in the development of airway allergic inflammatory responses. The results indicate that cytokine peptide-based vaccines hold potential for treatment of asthma and, by extension, other diseases where over-expressed cytokines play a pivotal role in pathogenesis.

Interferon‐γ‐dependent inhibition of late allergic airway responses and eosinophilia by CD8+γδ T cells

We have previously shown that CD8(+)gammadelta T cells decrease late allergic airway responses, airway eosinophilia, T helper 2 cytokine expression and increase interferon-gamma (IFN-gamma) expression. We hypothesized that the effects of CD8(+)gammadelta T cells were IFN-gamma mediated. Brown Norway rats were sensitized to ovalbumin on day 1. Cervical lymph node CD8(+)gammadelta T cells from sensitized animals were treated with antisense oligodeoxynucleotide (5 micromol/l) to inhibit IFN-gamma synthesis or control oligodeoxynucleotide and 3.5 x 10(4) CD8(+)gammadelta T cells were injected intraperitoneally into sensitized recipients on day 13. Rats were challenged with aerosolized ovalbumin on day 15 and lung resistance was monitored over an 8 hr period, after which bronchoalveolar lavage was performed. Control oligodeoxynucleotide treated gammadelta T cells decreased late airway responses and eosinophilia in bronchoalveolar lavage. There was a complete recovery of late airway responses and a partial recovery of airway eosinophilia in recipients of antisense oligodeoxynucleotide treated cells. Macrophage ingestion of eosinophils was frequent in rats administered gammadeltaT cells but reduced in recipients of antisense oligodeoxynucleotide treated cells. These results indicate that CD8(+)gammadelta T cells inhibit late airway responses and airway eosinophilia through the secretion of IFN-gamma. Defective or altered gammadelta T-cell function may account for some forms of allergic asthma.

Increased Neurotrophin Production in a Penicillium chrysogenum-Induced Allergic Asthma Model in Mice

Neurotrophins, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin (NT)-3, have been implicated in the pathogenesis of many features and symptoms of asthma. The role of neurotrophins in fungal allergic asthma, however, is unknown. Repeated pulmonary challenge with Penicillium chrysogenum extract (PCE) induces dose-dependent allergic asthma-like responses in mice. The aim of this study was to investigate whether neurotrophins are involved in the PCE-induced allergic airway response in mice. Mice were exposed to 10, 20, 50, or 70 μg PCE by involuntary aspiration 4 times over 1 mo. Bronchial alveolar lavage fluid (BALF) was collected immediately before and after the final exposure. The levels of NGF, NT-3, and NT-4 were determined by enzyme-linked immunosorbent assay (ELISA). The lungs were fixed and processed for immunohistochemical examination of NGF production. PCE-exposed mice had dose-dependent increases in NGF, NT-3, and NT-4 in both BALF and sera. Exposures to PCE produced elevation in positive immunohistochemical staining for NGF in the airway epithelium and smooth muscle cells, in addition to infiltrated cells such as mononuclear cells, eosinophils, and macrophages. Taken together, this is the first study to link fungal allergic asthma in an experimental model with enhanced production of neurotrophins in the airways, and suggests that neurotrophins may play a role in the etiology of mold-induced asthma in humans.