Nasal epithelium integrity,environmental stressors,and allergic sensitization: A biomarker study in adolescents

Changes in the airways epithelium caused by environmental insults might play a role in the development of allergic rhinitis. We measured albumin and Clara cell protein (CC16) in the nasal lavage fluid (NALF) from 474 adolescents (263 girls and 211 boys). The NALF CC16/albumin ratio, integrating the permeability and cellular integrity of the nasal epithelium, decreased mostly with time spent in chlorinated pools. In boys, a lower CC16/albumin ratio in NALF was associated with an increased risk of house dust mite sensitization. The results suggest that the CC16/albumin ratio in NALF can be used to detect nasal epithelium alterations linked to allergic sensitization.     

Clara cell protein in nasal lavage fluid and nasal nitric oxide - biomarkers with anti-inflammatory properties in allergic rhinitis

Background

Clara cell protein (CC16) is ascribed a protective and anti-inflammatory role in airway inflammation. Lower levels have been observed in asthmatic subjects as well as in subjects with intermittent allergic rhinitis than in healthy controls. Nasal nitric oxide (nNO) is present in high concentrations in the upper airways, and considered a biomarker with beneficial effects, due to inhibition of bacteria and viruses along with stimulation of ciliary motility. The aim of this study was to evaluate the presumed anti-inflammatory effects of nasal CC16 and nNO in subjects with allergic rhinitis.

Methods

The levels of CC16 in nasal lavage fluids, achieved from subjects with persistent allergic rhinitis (n = 13), intermittent allergic rhinitis in an allergen free interval (n = 5) and healthy controls (n = 7), were analyzed by Western blot. The levels of nNO were measured by the subtraction method using NIOX^?. The occurrences of effector cells in allergic inflammation, i.e. metachromatic cells (MC, mast cells and basophiles) and eosinophils (Eos) were analyzed by light microscopy in samples achieved by nasal brushing.

Results

The levels of CC16 correlated with nNO levels (r² = 0.37; p = 0.02) in allergic subjects. The levels of both biomarkers showed inverse relationships with MC occurrence, as higher levels of CC16 (p = 0.03) and nNO (p = 0.05) were found in allergic subjects with no demonstrable MC compared to the levels in subjects with demonstrable MC. Similar relationships, but not reaching significance, were observed between the CC16 and nNO levels and Eos occurrence. The levels of CC16 and nNO did not differ between the allergic and the control groups.

Conclusions

The correlation between nasal CC16 and nNO levels in patients with allergic rhinitis, along with an inverse relationship between their levels and the occurrences of MC in allergic inflammation, may indicate that both biomarkers have anti-inflammatory effects by suppression of cell recruitment. The mechanisms behind these observations warrant further analyses.     

Fructus Amomi extract attenuates nasal inflammation by restoring Th1/Th2 balance and down-regulation of NF-κB phosphorylation in OVA-induced allergic rhinitis

Fructus Amomi Cardamomi (FA) is the mature fruit of Amomum villosum Lour (family Zingiberaceae) and is commonly used in Chinese traditional medicine to treat various gastrointestinal disorders. FA’s possible benefits as an allergic rhinitis (AR) treatment, however, have not been examined. We used an ovalbumin (OVA)-induced AR mouse model to identify any anti-allergic effects associated with the administration of 200 mg/kg FA or dexamethasone (Dex) 2.5 mg/kg by oral administration. The results of our testing confirm that FA ameliorated nasal symptoms and alleviated nasal epithelium swelling, reduced the goblet cell hyperplasia and eosinophil cell infiltration in the nasal epithelium, and inhibited lung tissue inflammation and Dex as well. Significantly decreased Th2 cytokine (interleukin (IL)-1β, IL-4, and IL-5) expression, and a correspondingly significant increase in Th1 cytokine (IL-12, interferon (IFN)-γ) production, was observed in nasal lavage fluid (NALF) taken from mice that received FA or Dex treatment. FA also reduced the presence of OVA-specific immunoglobulin (Ig) E, OVA-specific IgG₁, and histamine levels in serum, and inhibited mast cell degranulation in vitro. In addition, these effects were involved with the reduction in NF-κB phosphorylation. These results suggest that FA restores Th1/Th2 balance and inhibits NF-κB phosphorylation and mast cell degranulation, thereby achieving a notable anti-inflammatory effect. Accordingly, it has the potential to be used as an efficacious therapeutic treatment for AR.     

Association of serum Clara cell protein CC16 with respiratory infections and immune response to respiratory pathogens in elite athletes

Background

Respiratory epithelium integrity impairment caused by intensive exercise may lead to exercise-induced bronchoconstriction. Clara cell protein (CC16) has anti-inflammatory properties and its serum level reflects changes in epithelium integrity and airway inflammation. This study aimed to investigate serum CC16 in elite athletes and to seek associations of CC16 with asthma or allergy, respiratory tract infections (RTIs) and immune response to respiratory pathogens.

Methods

The study was performed in 203 Olympic athletes. Control groups comprised 53 healthy subjects and 49 mild allergic asthmatics. Serum levels of CC16 and IgG against respiratory viruses and Mycoplasma pneumoniae were assessed. Allergy questionnaire for athletes was used to determine symptoms and exercise pattern. Current versions of ARIA and GINA guidelines were used when diagnosing allergic rhinitis and asthma, respectively.

Results

Asthma was diagnosed in 13.3% athletes, of whom 55.6% had concomitant allergic rhinitis. Allergic rhinitis without asthma was diagnosed in 14.8% of athletes. Mean CC16 concentration was significantly lower in athletes versus healthy controls and mild asthmatics. Athletes reporting frequent RTIs had significantly lower serum CC16 and the risk of frequent RTIs was more than 2-fold higher in athletes with low serum CC16 (defined as equal to or less than 4.99 ng/ml). Athletes had significantly higher anti-adenovirus IgG than healthy controls while only non-atopic athletes had anti-parainfluenza virus IgG significantly lower than controls. In all athletes weak correlation of serum CC16 and anti-parainfluenza virus IgG was present (R = 0.20, p < 0.01). In atopic athletes a weak positive correlations of CC16 with IgG specific for respiratory syncytial virus (R = 0.29, p = 0.009), parainfluenza virus (R = 0.31, p = 0.01) and adenovirus (R = 0.27, p = 0.02) were seen as well.

Conclusions

Regular high-load exercise is associated with decrease in serum CC16 levels. Athletes with decreased CC16 are more susceptible to respiratory infections. Atopy may be an additional factor modifying susceptibility to infections in subjects performing regular high-load exercise.     

Club cell protein (CC16) in plasma,bronchial brushes,BAL and urine following an inhaled allergen challenge in allergic asthmatics

Background: Club cell protein (CC16) is a pneumoprotein secreted by epithelial club cells. CC16 possesses anti-inflammatory properties and is a potential biomarker for airway epithelial damage. We studied the effect of inhaled allergen on pulmonary and systemic CC16 levels.

Methods: Thirty-four subjects with allergic asthma underwent an inhaled allergen challenge. Bronchoscopy with bronchoalveolar lavage (BAL) and brushings was performed before and 24?h after the challenge. CC16 was quantified in BAL and CC16 positive cells and CC16 mRNA in bronchial brushings. CC16 was measured in plasma and urine before and repeatedly after the challenge. Thirty subjects performed a mannitol inhalation challenge prior to the allergen challenge.

Results: Compared to baseline, CC16 in plasma was significantly increased in all subjects 0–1?h after the allergen challenge, while CC16 in BAL was only increased in subjects without a late allergic response. Levels of CC16 in plasma and in the alveolar fraction of BAL correlated significantly after the challenge. There was no increase in urinary levels of CC16 post-challenge. Mannitol responsiveness was greater in subjects with lower baseline levels of CC16 in plasma.

Conclusions: The increase in plasma CC16 following inhaled allergen supports the notion of CC16 as a biomarker of epithelial dysfunction.     

Chlamydia pneumoniae infection induced allergic airway sensitization is controlled by regulatory T-cells and plasmacytoid dendritic cells

Chlamydia pneumoniae (CP) is associated with induction and exacerbation of asthma. CP infection can induce allergic airway sensitization in mice in a dose- and time-dependent manner. Allergen exposure 5 days after a low dose (mild-moderate), but not a high dose (severe) CP infection induces antigen sensitization in mice. Innate immune signals play a critical role in controlling CP infection induced allergic airway sensitization, however these mechanisms have not been fully elucidated. Wild-type, TLR2-/-, and TLR4-/- mice were infected intranasally (i.n.) with a low dose of CP, followed by i.n. exposure to human serum albumin (HSA) and challenged with HSA 2 weeks later. Airway inflammation, immunoglobulins, eosinophils, and goblet cells were measured. Low dose CP infection induced allergic sensitization in TLR2-/- mice, but not in TLR4-/- mice, due to differential Treg responses in these genotypes. TLR2-/- mice had reduced numbers of Tregs in the lung during CP infection while TLR4-/- mice had increased numbers. High dose CP infection resulted in an increase in Tregs and pDCs in lungs, which prevented antigen sensitization in WT mice. Depletion of Tregs or pDCs resulted in allergic airway sensitization. We conclude that Tregs and pDCs are critical determinants regulating CP infection-induced allergic sensitization. Furthermore, TLR2 and TLR4 signaling during CP infection may play a regulatory role through the modulation of Tregs.     

CRTH2 plays an essential role in the pathophysiology of Cry j 1-induced pollinosis in mice

PGD(2) is the major prostanoid produced during the acute phase of allergic reactions. Two PGD(2) receptors have been isolated, DP and CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells), but whether they participate in the pathophysiology of allergic diseases remains unclear. We investigated the role of CRTH2 in the initiation of allergic rhinitis in mice. First, we developed a novel murine model of pollinosis, a type of seasonal allergic rhinitis. Additionally, pathophysiological differences in the pollinosis were compared between wild-type and CRTH2 gene-deficient mice. An effect of treatment with ramatroban, a CRTH2/T-prostanoid receptor dual antagonist, was also determined. Repeated intranasal sensitization with Cry j 1, the major allergen of Cryptomeria japonica pollen, in the absence of adjuvants significantly exacerbated nasal hyperresponsive symptoms, Cry j 1-specific IgE and IgG1 production, nasal eosinophilia, and Cry j 1-induced in vitro production of IL-4 and IL-5 by submandibular lymph node cells. Additionally, CRTH2 mRNA in nasal mucosa was significantly elevated in Cry j 1-sensitized mice. Following repeated intranasal sensitization with Cry j 1, CRTH2 gene-deficient mice had significantly weaker Cry j 1-specific IgE/IgG1 production, nasal eosinophilia, and IL-4 production by submandibular lymph node cells than did wild-type mice. Similar results were found in mice treated with ramatroban. These results suggest that the PGD(2)-CRTH2 interaction is elevated following sensitization and plays a proinflammatory role in the pathophysiology of allergic rhinitis, especially pollinosis in mice.     

Interplay of filaggrin loss-of-function variants, allergic sensitization, and eczema in a longitudinal study covering infancy to 18 years of age

Background

Immune specific genes as well as genes regulating the formation of skin barrier are major determinants for eczema manifestation. There is a debate as to whether allergic sensitization and filaggrin gene (FLG) variants lead to eczema or FLG variants and eczema increase the risk of allergic sensitization. To investigate the time-order between eczema and allergic sensitization with respect to FLG variants, data from a large prospective study covering infancy to late adolescence were analyzed.

Methodology/Principal Findings

Repeated measurements of eczema and allergic sensitization (documented by skin prick tests) at ages 1, 2, 4, 10, and 18 years were ascertained in the Isle of Wight birth cohort (n = 1,456). Three transition periods were analyzed: age 1-or-2 to 4, 4 to 10, and 10 to 18 years. FLG variants were genotyped in 1,150 participants. Over the three transition periods, in temporal sequence analyses of initially eczema-free participants, the combined effect of FLG variants and allergic sensitization showed a 2.92-fold (95% CI: 1.47–5.77) increased risk ratio (RR) of eczema in subsequent examinations. This overall risk was more pronounced at a younger age (transition period 1-or-2 to 4, RR = 6.47, 95% CI: 1.96–21.33). In contrast, FLG variants in combination with eczema showed a weaker, but significant, risk ratio for subsequent allergic sensitization only up to 10 years of age.

Conclusions/Significance

Taking the time order into account, this prospective study demonstrates for the first time, that a combination of FLG variants and allergic sensitization increased the risk of eczema in subsequent years. Also FLG variants interacted with eczema and increased the risk of subsequent allergic sensitization, which, was limited to the younger age. Hence, early restoration of defective skin barrier could prevent allergic sensitization and subsequently reduce the risk of eczema development.     

Initial high-dose nasal allergen exposure prevents allergic sensitization to a neoantigen

Primary allergic sensitization--IgE formation after Ag exposure--is fundamental in the development of allergic respiratory disease. With the rising prevalence of asthma and allergic rhinitis, improved understanding of the determining factors for allergic sensitization is needed. Human epidemiologic studies suggest high-dose allergen exposure may paradoxically protect against sensitization. Prospective human studies of allergen dose effect on primary allergic sensitization are lacking. We prospectively examined the effect of respiratory Ag dose exposure on the rate of primary allergic sensitization to a neoantigen, keyhole limpet hemocyanin, using a unique model of human nasal allergic sensitization. Atopic human subjects were exposed to 0.1-, 10-, 1,000-, or 100,000-mug doses of intranasal keyhole limpet hemocyanin in conjunction with adjuvant intranasal diesel exhaust particles. Ag-specific IgE, IgG, and IgG4 were measured in nasal lavage samples at the conclusion of the sensitization protocol. Allergic sensitization rates for the 0.1-, 10-, 1,000-, and 100,000-mug dose groups were 0, 100, 57, and 11%, respectively. All subjects produced Ag-specific IgG with the highest levels observed in the high-dose group. These results provide direct evidence that primary allergic sensitization may be prevented by initial high levels of respiratory Ag exposure through induction of a modified, nonallergic immune response. This Ag dose effect was capable of overcoming the well-established allergic adjuvant effects of diesel exhaust particle exposure. Whether this immune response represents durable allergic tolerance is not yet known. Studies investigating the molecular mechanisms of this non-IgE response may be useful in developing therapy to prevent allergic sensitization.     

Thymic stromal lymphopoietin enhances tight-junction barrier function of human nasal epithelial cells

Epithelial-derived thymic stromal lymphopoietin (TSLP) triggers dendritic cell (DC)-mediated Th2-type inflammatory responses and is a master switch for allergic inflammatory diseases. In the present study, the expression and induction of TSLP and the effects of TSLP on the tight-junctional barrier of human nasal epithelial cells (HNECs) have been investigated in order to elucidate the role of TSLP in allergic rhinitis. We have found high expression of TSLP in the epithelium from patients with allergic rhinitis with recruitment and infiltration of DCs. In vitro, TSLP is significantly produced in HNECs after treatment with a toll-like receptor 2 (TLR2) ligand, Pam₃Cys-Ser-(Lys)₄, and a mixture of interleukin-1β and tumor necrosis factor-α. Treatment with TSLP rapidly enhances the barrier function of cultured HNECs, together with an increase of tight-junction proteins claudin-1, -4, -7, and occludin. The nasal-epithelial-derived TSLP thus not only activates DCs but also preserves the epithelial barrier via the upregulation of tight-junction proteins, thereby regulating antigen sensitization during the early stage of allergic rhinitis.     

Epithelial,dendritic, and CD4 T cell regulation of and by reactive oxygen and nitrogen species in allergic sensitization

Background

While many of the contributing cell types and mediators of allergic asthma are known, less well understood are the factors that induce allergy in the first place. Amongst the mediators speculated to affect initial allergen sensitization and the development of pathogenic allergic responses to innocuous inhaled antigens and allergens are exogenously or endogenously generated reactive oxygen species (ROS) and reactive nitrogen species (RNS).

Scope of review

The interactions between ROS/RNS, dendritic cells (DCs), and CD4⁺ T cells, as well as their modulation by lung epithelium, are of critical importance for the genesis of allergies that later manifest in allergic asthma. Therefore, this review will primarily focus on the initiation of pulmonary allergies and the role that ROS/RNS may play in the steps therein, using examples from our own work on the roles of NO₂ exposure and airway epithelial NF-κB activation.

Major conclusions

Endogenously generated ROS/RNS and those encountered from environmental sources interact with epithelium, DCs, and CD4⁺ T cells to orchestrate allergic sensitization through modulation of the activities of each of these cell types, which quantitiatively and qualitatively dictate the degree and type of the allergic asthma phenotype.

General significance

Knowledge of the effects of ROS/RNS at the molecular and cellular levels has the potential to provide powerful insight into the balance between inhalational tolerance (the typical immunologic response to an innocuous inhaled antigen) and allergy, as well as to potentially provide mechanistic targets for the prevention and treatment of asthma.     

Endogenous attenuation of allergic lung inflammation by syndecan-1

The airway plays a vital role in allergic lung diseases by responding to inhaled allergens and initiating allergic inflammation. Various proinflammatory functions of the airway epithelium have been identified, but, equally important, anti-inflammatory mechanisms must also exist. We show in this study that syndecan-1, the major heparan sulfate proteoglycan of epithelial cells, attenuates allergic lung inflammation. Our results show that syndecan-1-null mice instilled with allergens exhibit exaggerated airway hyperresponsiveness, glycoprotein hypersecretion, eosinophilia, and lung IL-4 responses. However, administration of purified syndecan-1 ectodomains, but not ectodomain core proteins devoid of heparan sulfate, significantly inhibits these inflammatory responses. Furthermore, syndecan-1 ectodomains are shed into the airway when wild-type mice are intranasally instilled with several biochemically distinct inducers of allergic lung inflammation. Our results also show that syndecan-1 ectodomains bind to the CC chemokines (CCL7, CCL11, and CCL17) implicated in allergic diseases, inhibit CC chemokine-mediated T cell migration, and suppress allergen-induced accumulation of Th2 cells in the lung through their heparan sulfate chains. Together, these findings uncover an endogenous anti-inflammatory mechanism of the airway epithelium where syndecan-1 ectodomains attenuate allergic lung inflammation via suppression of CC chemokine-mediated Th2 cell recruitment to the lung.     

Carnosine binding: characterization of steric and charge requirements for ligand recognition.

The steric and charge requirements for binding of l-carnosine (β-alanyl-l-histidine) by bovine serum albumin were investigated with proton magnetic resonance (¹HMR) spectrometry. The histidinyl side chain of the dipeptide is responsible for primary recognition by the binding site. Furthermore, recognition is specific to a particular orientation of the histidinyl side chain that is determined by the other amino acid residue of the dipeptide. It was found that, although salts do not have a great effect on the binding of carnosine to bovine serum albumin, this binding cannot be measured by equilibrium dialysis in the presence of salt because of formation of a complex Donnan equilibrium. Carnosine, which has been postulated to have a role in olfaction, binds to the crude particulate fraction of nasal olfactory epithelium in the same steric orientation as it does to bovine serum albumin. Therefore, we have used the binding of carnosine to bovine serum albumin as a model system to test potential competitive inhibitors of carnosine binding that ultimately could be tested for activity in the olfactory pathway. It was found that the binding of carnosine to bovine serum albumin is a good model of nonspecific binding of carnosine to tissue preparations but not of the specific binding of carnosine to the nasal olfactory epithelium. In addition to requiring the proper conformation of the histidinyl residue, the binding to olfactory epithelium also appears to require recognition of the β-alanyl residue and of substituents on the imidazole ring. Evidence is provided that the carnosine binding by the nasal olfactory epithelium demonstrated by ¹HMR spectroscopy does not occur with the mature olfactory receptor neurons.     

Unlipidated Outer Membrane Protein Omp16 (U-Omp16) from Brucella spp. as Nasal Adjuvant Induces a Th1 Immune Response and Modulates the Th2 Allergic Response to Cow’s Milk Proteins

The discovery of novel mucosal adjuvants will help to develop new formulations to control infectious and allergic diseases. In this work we demonstrate that U-Omp16 from Brucella spp. delivered by the nasal route (i.n.) induced an inflammatory immune response in bronchoalveolar lavage (BAL) and lung tissues. Nasal co-administration of U-Omp16 with the model antigen (Ag) ovalbumin (OVA) increased the amount of Ag in lung tissues and induced OVA-specific systemic IgG and T helper (Th) 1 immune responses. The usefulness of U-Omp16 was also assessed in a mouse model of food allergy. U-Omp16 i.n. administration during sensitization ameliorated the hypersensitivity responses of sensitized mice upon oral exposure to Cow’s Milk Protein (CMP), decreased clinical signs, reduced anti-CMP IgE serum antibodies and modulated the Th2 response in favor of Th1 immunity. Thus, U-Omp16 could be used as a broad Th1 mucosal adjuvant for different Ag formulations.     

Plasma extravasation through neuronal stimulation in human nasal mucosa in the setting of allergic rhinitis

Sanico, Alvin M., Satsuki Atsuta, David Proud, and AlkisTogias. Plasma extravasation through neuronal stimulation in humannasal mucosa in the setting of allergic rhinitis. J. Appl. Physiol. 84(2): 537-543, 1998.We havepreviously shown that capsaicin nasal challenge in subjects withallergic rhinitis produces a dose-dependent increase in the albumincontent of nasal lavage fluids. In the present set of studies, wedetermined whether this observation represents plasma extravasationthat is neuronally mediated. To evaluate whether glandular secretionscontribute to the albumin increase in nasal lavage fluids, volunteerswith allergic rhinitis were pretreated with atropine or placebo before capsaicin challenge. Atropine significantly reduced the volume ofreturned lavage fluids and their lysozyme content but increased theiralbumin and fibrinogen content. To assess the contribution of sensorynerve stimulation, subjects with allergic rhinitis were pretreated in asecond study with lidocaine or placebo before capsaicin challenge.Lidocaine significantly attenuated the capsaicin-induced increases inthe volume of nasal lavage fluids, as well as their lysozyme andalbumin content. To rule out the possibility of a direct effect oflidocaine on blood vessels rather than on nerves, healthy subjects werepretreated in a third study with lidocaine or placebo before bradykininnasal challenge. Lidocaine did not affect the bradykinin-inducedincrease in the albumin content of nasal fluids. We conclude that, inallergic rhinitis, high-dose capsaicin induces plasma extravasation inthe human nose and that this effect is neuronally mediated. Thisprovides more definitive evidence that neurogenic inflammation canoccur in vivo in the human upper airway.

     

Pathogenesis of murine experimental allergic rhinitis: a study of local and systemic consequences of IL-5 deficiency

Recent studies have demonstrated an important role for IL-5-dependent bone marrow eosinophil progenitors in allergic inflammation. However, studies using anti-IL-5 mAbs in human asthmatics have failed to suppress lower airway hyperresponsiveness despite suppression of eosinophilia; therefore, it is critical to examine the role of IL-5 and bone marrow responses in the pathogenesis of allergic airway disease. To do this, we studied the effects of IL-5 deficiency (IL-5(-/-)) on bone marrow function as well as clinical and local events, using an established experimental murine model of allergic rhinitis. Age-matched IL-5(+/+) and IL-5(-/-) BALB/c mice were sensitized to OVA followed by 2 wk of daily OVA intranasal challenge. IL-5(-/-) OVA-sensitized mice had significantly higher nasal mucosal CD4(+) cells and basophilic cell counts as well as nasal symptoms and histamine hyperresponsiveness than the nonsensitized group; however, there was no eosinophilia in either nasal mucosa or bone marrow; significantly lower numbers of eosinophil/basophil CFU and maturing CFU eosinophils in the presence of recombinant mouse IL-5 in vitro; and significantly lower expression of IL-5Ralpha on bone marrow CD34(+)CD45(+) progenitor cells in IL-5(-/-) mice. These findings suggest that IL-5 is required for normal bone marrow eosinophilopoiesis, in response to specific Ag sensitization, during the development of experimental allergic rhinitis. However, the results also suggest that suppression of the IL-5-eosinophil pathway in this model of allergic rhinitis may not completely suppress clinical symptoms or nasal histamine hyperresponsiveness, because of the existence of other cytokine-progenitor pathways that may induce and maintain the presence of other inflammatory cell populations.     

Mucosal allergic sensitization to cockroach allergens is dependent on proteinase activity and proteinase-activated receptor-2 activation

We have shown that proteinase-activated receptor-2 (PAR(2)) activation in the airways leads to allergic sensitization to concomitantly inhaled Ags, thus implicating PAR(2) in the pathogenesis of asthma. Many aeroallergens with proteinase activity activate PAR(2). To study the role of PAR(2) in allergic sensitization to aeroallergens, we developed a murine model of mucosal sensitization to cockroach proteins. We hypothesized that PAR(2) activation in the airways by natural allergens with serine proteinase activity plays an important role in allergic sensitization. Cockroach extract (CE) was administered to BALB/c mice intranasally on five consecutive days (sensitization phase) and a week later for four more days (challenge phase). Airway hyperresponsiveness (AHR) and allergic airway inflammation were assessed after the last challenge. To study the role of PAR(2), mice were exposed intranasally to a receptor-blocking anti-PAR(2) Ab before each administration of CE during the sensitization phase. Mucosal exposure to CE induced eosinophilic airway inflammation, AHR, and cockroach-specific IgG1. Heat-inactivated or soybean trypsin inhibitor-treated CE failed to induce these effects, indicating that proteinase activity plays an important role. The use of an anti-PAR(2) blocking Ab during the sensitization phase completely inhibited airway inflammation and also decreased AHR and the production of cockroach-specific IgG1. PAR(2) activation by CE acts as an adjuvant for allergic sensitization even in the absence of functional TLR4. We conclude that CE induces PAR(2)-dependent allergic airway sensitization in a mouse model of allergic airway inflammation. PAR(2) activation may be a general mechanism used by aeroallergens to induce allergic sensitization.