Kita, Hirohito

Abstract Tissue eosinophilia probably plays important roles in the pathophysiology of bronchial asthma and allergic disorders; however, this concept was challenged recently by controversial results in mouse models of bronchial asthma treated with anti-IL-5 Ab and the failure of anti-IL-5 therapy in humans. We have now used a unique model, IL-5 transgenic (TG) mice, to address a fundamental question: is airway eosinophilia beneficial or detrimental in the allergic response? After sensitization and challenge with OVA, IL-5 TG mice showed a marked airway eosinophilia. Surprisingly, these IL-5 TG mice showed lower airway reactivity to methacholine. Immunohistochemical analysis of the lungs revealed a marked peribronchial infiltration of eosinophils, but no eosinophil degranulation. In vitro, mouse eosinophils from peritoneal lavage fluids did not produce superoxide anion, but did produce an anti-inflammatory and fibrotic cytokine, TGF-β1. Indeed, the TGF-β1 levels in bronchoalveolar lavage fluid specimens from IL-5 TG mice directly correlated with airway eosinophilia (r = 0.755). Furthermore, anti-IL-5 treatment of IL-5 TG mice decreased both airway eosinophilia and TGF-β1 levels in bronchoalveolar lavage fluids and increased airway reactivity. Thus, in mice, marked eosinophilia prevents the development of airway hyper-reactivity during an allergic response. Overall, the roles of eosinophils in asthma and in animal models need to be addressed carefully for their potentially detrimental and beneficial effects.

Abstract Protease-activated receptors (PARs) are a unique class of G protein-coupled receptors, which are activated by proteolytic cleavage of the amino terminus of the receptor itself. PARs are most likely involved in various biological responses, such as hemostasis and regulation of muscle tone; however, the roles of PARs in the functions of inflammatory and immune cells are poorly understood. Because eosinophils are most likely involved in allergic inflammation and are exposed to a variety of proteases derived from allergens and other inflammatory cells, we investigated whether PARs regulate effector functions of eosinophils. Human eosinophils constitutively transcribe mRNA for PAR2 and PAR3, but not those for PAR1 and PAR4. The expression of PAR2 protein was confirmed by flow cytometry. When trypsin, an agonist for PAR2, was incubated with eosinophils, it potently induced superoxide anion production and degranulation; 5 nM trypsin induced responses that were 50∼70% of those induced by 100 nM platelet-activating factor, a positive control. In contrast, thrombin, an activator for PAR1, PAR3, and PAR4, showed minimal effects. The stimulatory effect of trypsin was dependent on its serine protease activity and was blocked 59% by anti-PAR2 Ab. Furthermore, a specific tethered peptide ligand for PAR2 potently induced superoxide production and degranulation; the effects of peptide ligands for PAR1, PAR3, and PAR4 were negligible. These findings suggest that human eosinophils express functional PAR2, and serine proteases at the inflammation site may play important roles in regulating effector functions of human eosinophils. The expression and functional relevance of other PARs still need to be determined.

Abstract We present a novel immunotherapeutic strategy using a human B7-DC cross-linking Ab that prevents lung inflammation, airway obstruction, and hyperreactivity to allergen in a mouse model of allergic inflammatory airway disease. Dendritic cells (DC) have the ability to skew the immune response toward a Th1 or Th2 polarity. The sHIgM12 Ab functions in vitro by cross-linking the costimulatory family molecule B7-DC (PD-L2) on DC up-regulating IL-12 production, homing to lymph nodes, and T cell-activating potential of these APCs. Using chicken OVA as a model Ag, the administration of sHIgM12 Ab to BALB/c mice blocked lung inflammation, airway pathology, and responsiveness to methacholine, even after animals were presensitized and a Th2-polarized immune response was established. This therapeutic strategy was ineffective in STAT4-deficient animals, indicating that IL-12 production is critical in this system. Moreover, the polarity of the immune response upon in vitro restimulation with Ag is changed in wild-type mice, with a resulting decrease in Th2 cytokines IL-4 and IL-5 and an increase in the immunoregulatory cytokine IL-10. These studies demonstrate that the immune response of hypersensitized responders can be modulated using B7-DC cross-linking Abs, preventing allergic airway disease upon re-exposure to allergen.

Abstract IL-12p70, a heterodimer composed of p35 and p40 subunits, is a key polarizing cytokine produced by maturing dendritic cells (DCs). We report that cigarette smoke extract (CSE), an extract of soluble cigarette smoke components, suppresses both p35 and p40 production by LPS or CD40L-matured DCs. Suppression of IL-12p70 production from maturing DCs was not observed in the presence of nicotine concentrations achievable in CSE or in the circulation of smokers. The suppressed IL-12p70 protein production by CSE-conditioned DCs was restored by pretreatment of DCs or CSE with the antioxidants N-acetylcysteine and catalase. Inhibition of DC IL-12p70 by CSE required activation of ERK-dependent pathways, since inhibition of ERK abrogated the suppressive effect of CSE on IL-12 secretion. Oxidative stress and sustained ERK phosphorylation by CSE enhanced nuclear levels of the p40 transcriptional repressor c-fos in both immature and maturing DCs. Suppression of the p40 subunit by CSE also resulted in diminished production of IL-23 protein by maturing DCs. Using a murine model of chronic cigarette smoke exposure, we observed that systemic and lung DCs from mice “smokers” produced significantly less IL-12p70 and p40 protein upon maturation. This inhibitory effect was selective, since production of TNF-α during DC maturation was enhanced in the smokers. These data imply that oxidative stress generated by cigarette smoke exposure suppresses the generation of key cytokines by maturing DCs through the activation of ERK-dependent pathways. Some of the cigarette smoke-induced inhibitory effects on DC function may be mitigated by antioxidants.

Abstract Eosinophils and their products are likely important in the pathophysiology of allergic diseases, such as bronchial asthma, and in host immunity to parasitic organisms. However, the mechanisms for proinflammatory mediator release by eosinophils are poorly understood. CD66b (CEACAM8, CGM6, NCA-95) is a single chain, GPI-anchored, highly glycosylated protein belonging to the carcinoembryonic Ag supergene family. CD66b is an activation marker for human granulocytes; however, its biological functions are largely unknown in eosinophils. We found that CD66b is highly expressed on the surface of human peripheral blood eosinophils isolated from healthy individuals. Engagement of CD66b, but not CD66a, by mAb or a natural ligand, galectin-3, activated a Src kinase family molecule, hemopoietic cell kinase (Hck), and induced cellular adhesion, superoxide production, and degranulation of eosinophils. CD66b molecules were localized in lipid rafts, and disruption of lipid rafts or removal of the GPI anchor inhibited the adhesion and activation of eosinophils. Importantly, CD66b was constitutively and physically associated with a β2 integrin, CD11b, and cross-linking of CD66b induced a striking clustering of CD11b molecules. Thus, CD66b molecules are involved in regulating adhesion and activation of eosinophils, possibly through their localization in lipid rafts and interaction with other cell surface molecules, such as CD11b. Binding of exogenous or endogenous carbohydrate ligands(s) to CD66b may be important in the release of proinflammatory mediators by human eosinophils.

Abstract Eosinophils produce and release various proinflammatory mediators and also show immunomodulatory and tissue remodeling functions; thus, eosinophils may be involved in the pathophysiology of asthma and other eosinophilic disorders as well as host defense. Several major questions still remain. For example, how do human eosinophils become activated in diseased tissues or at the site of an immune response? What types of host immunity might potentially involve eosinophils? Herein, we found that human eosinophils react vigorously to a common environmental fungus, Alternaria alternata, which is implicated in the development and/or exacerbation of human asthma. Eosinophils release their cytotoxic granule proteins, such as eosinophil-derived neurotoxin and major basic protein, into the extracellular milieu and onto the surface of fungal organisms and kill the fungus in a contact-dependent manner. Eosinophils use their versatile β2 integrin molecule, CD11b, to adhere to a major cell wall component, β-glucan, but eosinophils do not express other common fungal receptors, such as dectin-1 and lactosylceramide. The I-domain of CD11b is distinctively involved in the eosinophils’ interaction with β-glucan. Eosinophils do not react with another fungal cell wall component, chitin. Because human eosinophils respond to and kill certain fungal organisms, our findings identify a previously unrecognized innate immune function for eosinophils. This immune response by eosinophils may benefit the host, but, in turn, it may also play a role in the development and/or exacerbation of eosinophil-related allergic human diseases, such as asthma.

Abstract Although type 2 immune responses to environmental Ags are thought to play pivotal roles in asthma and allergic airway diseases, the immunological mechanisms that initiate the responses are largely unknown. Many allergens have biologic activities, including enzymatic activities and abilities to engage innate pattern-recognition receptors such as TLR4. In this article, we report that IL-33 and thymic stromal lymphopoietin were produced quickly in the lungs of naive mice exposed to cysteine proteases, such as bromelain and papain, as a model for allergens. IL-33 and thymic stromal lymphopoietin sensitized naive animals to an innocuous airway Ag OVA, which resulted in production of type 2 cytokines and IgE Ab, and eosinophilic airway inflammation when mice were challenged with the same Ag. Importantly, upon exposure to proteases, uric acid (UA) was rapidly released into the airway lumen, and removal of this endogenous UA by uricase prevented type 2 immune responses. UA promoted secretion of IL-33 by airway epithelial cells in vitro, and administration of UA into the airways of naive animals induced extracellular release of IL-33, followed by both innate and adaptive type 2 immune responses in vivo. Finally, a potent UA synthesis inhibitor, febuxostat, mitigated asthma phenotypes that were caused by repeated exposure to natural airborne allergens. These findings provide mechanistic insights into the development of type 2 immunity to airborne allergens and recognize airway UA as a key player that regulates the process in respiratory mucosa.

ObjectivesA truly objective method of measuring disease severity in chronic rhinosinusitis (CRS) has only recently existed. We evaluated computed tomography (CT) scans of CRS patients using this novel objective 3D computerized system and compared results with a novel 2D computerized analysis of a single coronal slice through the osteomeatal complex (OMC) and subjective methods including Lund‐Mackay and Zinreich's modified Lund‐Mackay.Study DesignProspective multicenter study.SettingTwo academic tertiary referral centers.Subjects and MethodsForty‐six adults with a diagnosis of CRS underwent CT examination and received an intramuscular triamcinolone injection, dosage weight dependent, followed by CT scan 4 to 5 weeks later. Recruitment lasted 21 months. Scans were evaluated with all 4 scoring methods over 5 months.ResultsThe Lin's concordance class correlation (CCC) of the OMC method revealed the best correlation to the 3D volumetric computerized values (0.915), followed by the Zinreich (0.904) and Lund‐Mackay methods (0.824). Posttreatment results demonstrated that both the OMC (0.824) and Zinreich's (0.778) methods had strong agreement with the 3D volumetric methods and were very sensitive to change, whereas the Lund‐Mackay (0.545) had only moderate agreement.ConclusionComputerized CT analysis provides the most comprehensive, objective, and reproducible method of measuring disease severity and is very sensitive to change induced by treatment intervention. A 2D coronal image through the OMC provides a valid, user‐friendly method of assessing CRS and is representative of CRS severity in all sinuses. Zinreich's subjective method correlated well overall, but the Lund‐Mackay method lagged behind in disease representation and sensitivity to change.

BackgroundWe aimed to test the hypothesis that three‐dimensional (3D) volume‐based scoring of computed tomography (CT) images of the paranasal sinuses was superior to Lund‐Mackay CT scoring of disease severity in chronic rhinosinusitis (CRS). We determined correlation between changes in CT scores (using each scoring system) with changes in other measures of disease severity (symptoms, endoscopic scoring, and quality of life) in patients with CRS treated with triamcinolone.MethodsThe study group comprised 48 adult subjects with CRS. Baseline symptoms and quality of life were assessed. Endoscopy and CT scans were performed. Patients received a single systemic dose of intramuscular triamcinolone and were reevaluated 1 month later. Strengths of the correlations between changes in CT scores and changes in CRS signs and symptoms and quality of life were determined.ResultsWe observed some variability in degree of improvement for the different symptom, endoscopic, and quality‐of‐life parameters after treatment. Improvement of parameters was significantly correlated with improvement in CT disease score using both CT scoring methods. However, volumetric CT scoring had greater correlation with these parameters than Lund‐Mackay scoring.ConclusionVolumetric scoring exhibited higher degree of correlation than Lund‐Mackay scoring when comparing improvement in CT score with improvement in score for symptoms, endoscopic exam, and quality of life in this group of patients who received beneficial medical treatment for CRS.