Takeshi Nabe

DEVELOPMENT OF POLLEN-INDUCED ALLERGIC RHINITIS WITH EARLY AND LATE PHASE NASAL BLOCKAGE IN GUINEA PIGS

Abstract.Objective and Design: Development of nasal blockage and sneezing during repeated inhalation challenges with Japanese cedar pollens was evaluated in guinea pigs.¶Subjects: Male Hartley guinea pigs.¶Treatment: Guinea pigs were sensitized by intranasal instillation of cedar pollen extracts + Al(OH)3 2 times a day for 7 days. The animal was then forced to inhale the pollens for challenge, which was restrictively trapped in the upper airways, once a week.¶Methods: Change of specific airway resistance (sRaw), sneezing frequency, and titers of anaphylactic antibodies in the serum were measured after each of the 30 challenges.¶Results: At the first challenge, no obvious increase in sRaw was observed. However, the second and third challenges to the animals caused modest biphasic elevations of sRaw, with peaks at the first and the fourth to sixth hour. At the fourth to tenth challenges, marked elevations of sRaw were observed. However, with repetition of the inhalation challenge, the early and the late responses became almost indistinguishable because of partial overlapping as the responses expanded. All guinea pigs sneezed immediately after each pollen inhalation challenge. Apparent increases of both circulating γ1 and IgE antibodies were seen after the seventh challenge.¶Conclusions: These results indicate that the experimental allergic rhinitis established in the present study can be a valuable model for analyzing the pathogenesis of the disease and developing new therapeutic drugs.

IL-17A Promotes the Exacerbation of IL-33–Induced Airway Hyperresponsiveness by Enhancing Neutrophilic Inflammation via CXCR2 Signaling in Mice

Neutrophilic airway inflammation is a hallmark of patients with severe asthma. Although we have reported that both IL-33 and IL-17A contributed to IgE-mediated neutrophilic inflammation in mice, the relationship remains unclear. In this article, we examined how IL-17A modifies IL-33–induced neutrophilic inflammation and airway hyperresponsiveness (AHR). IL-33 was intratracheally administered to BALB/c mice on days 0–2; furthermore, on day 7, the effect of the combination of IL-33 and IL-17A was evaluated. Compared with IL-33 or IL-17A alone, the combination exacerbated neutrophilic inflammation and AHR, associated with more increased levels of lung glutamic acid-leucine-arginine+ CXC chemokines, including CXCL1, CXCL2, and CXCL5, and infiltration by alveolar macrophages expressing CXCR2. Treatment with anti-CXCR2 mAb or depletion of alveolar macrophages repressed neutrophilic inflammation and AHR; in addition, depletion of neutrophils suppressed AHR. These findings prompted us to examine the role of CXCR2 in IgE-sensitized mice; a single treatment with anti-CXCR2 mAb in the seventh Ag challenge inhibited late-phase airway obstruction, AHR, and neutrophilic inflammation. In addition to inhibition, multiple treatments during the fourth to seventh challenge attenuated early-phase airway obstruction, eosinophilic inflammation, and goblet cell hyperplasia associated with the reduction of Th2 cytokine production, including IL-4, IL-5, and IL-13. Collectively, IL-33 cooperated with IL-17A to exacerbate AHR by enhancing neutrophilic inflammation via CXCR2 signaling; furthermore, CXCR2 signaling derived Th2 responses. We thus suggest the underlying mechanisms of IL-33 and IL-17A in allergic asthma and CXCR2 as potential therapeutic targets for the disease.

Endothelin-1 induces release of histamine and leukotriene C4 from mouse bone marrow-derived mast cells.

Whether specific binding sites for endothelin-1 and endothelin-3 exist in mouse bone marrow-derived mast cells (BMMC) and if these endothelins are capable of stimulating chemical mediator release from the cells was investigated. A single component of binding sites for endothelin-1 was found in the cells, but no binding sites for endothelin-3 were observed. Endothelin-1 at 1-100 nM concentration dependently induced release of histamine and immunoreactive leukotriene C4 from BMMC, while endothelin-3 at up to 100 nM did not stimulate the release of either mediator. Time course experiments revealed that the release of histamine and immunoreactive leukotriene C4 induced by endothelin-1 occurred rapidly, reaching near maximal levels within 20 s and 2 min, respectively, after the stimulation, while histamine release induced by antigen, at the concentration which induced an extent of release similar to that induced by 100 nM endothelin-1, required comparatively prolonged incubation (approximately 10 min for submaximal levels). Cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123), a selective antagonist of endothelin ETA receptors, not only dissociated [125I]endothelin-1 specifically bound to BMMC but also inhibited the release of both mediators from endothelin-1-induced cells. These results suggested strongly that BMMC have endothelin ETA receptors on their cell membrane, stimulation of which leads to chemical mediator release, probably via a mechanism different from that involved in the antigen-induced release.

The Effects of Air Pollution on Structures, Proteins and Allergenicity of Pollen Grains

The prevalence of allergic disease has increased world wide during the last decades. Pollen allergy is the most typical form of allergic disease. The increase in its frequency during recent years is the most evident. Environmental factors play an important role in the problem of pollen allergy in large cities. The aim of this research is determination of allergenicity of Canna pollen in polluted and non-polluted conditions, detection of their allergenic proteins and also elucidation of some microscopic effects of air pollutants on pollen structure and proteins. Mature and immature pollen grains of Canna indica were collected from polluted and non-polluted areas. Pollen grains were studied by scanning electron microscopy. Mice were sensitized by injection of pollen extract and an adjuvant for five times. Allergy potency of different pollen extracts were compared by means of skin test, blood eosinophills number and IgE levels in sensitized and treated animals. Pollen proteins were studied by SDS-PGE and allergenic proteins were detected by immunoblotting techniques. Scanning electron microscope study of the pollen grains showed that in polluted areas, air born particles accumulated on the surface of pollen and changed both pollens shape and pollens tectum. Also many vesicles were released out of polluted pollen and the pollen material agglomerated on the surface of pollen. SDS-PAGE showed that different proteins exist in mature and immature pollen. In pollen collected from polluted area, some of protein bands between 22 and 45 kDa were disappeared . Also in all polluted pollen grains, protein content of pollen decreased in response to air pollution causing the release of pollen proteins. According to our experiments and regarding induction of allergic symptoms, the polluted pollen is more effective than non-polluted one, and mature pollen has more allergy potency than immature one.

Interleukin-33 and alveolar macrophages contribute to the mechanisms underlying the exacerbation of IgE-mediated airway inflammation and remodelling in mice.

Allergen‐specific IgE has long been regarded as a major molecular component of allergic asthma. Additionally, there is increasing evidence of the important roles of interleukin‐33 (IL‐33) in the disease. Here, we show that IL‐33 and alveolar macrophages play essential roles in the exacerbation of IgE‐mediated airway inflammation and remodelling. BALB/c mice passively sensitized with ovalbumin (OVA)‐specific IgE monoclonal antibody (mAb) were challenged with OVA seven times intratracheally. The seventh challenge exacerbated airway inflammation and remodelling compared with the fourth challenge; furthermore, markedly increased expression of IL‐33 in the lungs was observed at the fourth and seventh challenges. When anti‐IL‐33 or anti‐ST2 antibody was administered during the fourth to seventh challenge, airway inflammation and remodelling were significantly inhibited at the seventh challenge. Because increases of IL‐33+ and ST2+ alveolar macrophages and ST2+ CD4+ T cells in the lungs were observed at the fourth challenge, the roles of macrophages and CD4+ cells were investigated. Depletion of macrophages by 2‐chloroadenosine during the fourth to seventh challenge suppressed airway inflammation and remodelling, and IL‐33 production in the lung at the seventh challenge; additionally, anti‐CD4 mAb inhibited airway inflammation, but not airway remodelling and IL‐33 production. Meanwhile, treatment with 2‐chloroadenosine or anti‐CD4 mAb decreased IL‐33‐induced airway inflammation in normal mice; airway remodelling was repressed only by 2‐chloroadenosine. These results illustrate that macrophage‐derived IL‐33 contributes to the exacerbation of IgE‐mediated airway inflammation by mechanisms associated with macrophages and CD4+ cells, and airway remodelling through the activation of macrophages.

Complement C3a Regulates Late Asthmatic Response and Airway Hyperresponsiveness in Mice

Allergic asthma is a chronic inflammatory disorder of the airways characterized by biphasic airway obstruction and airway hyperresponsiveness. In this study, we attempted to elucidate the contribution of the complement C3a to these asthmatic symptoms. BALB/c mice sensitized by i.p. injections of OVA plus alum were challenged with OVA intratracheally four times. The fourth challenge caused a biphasic asthmatic response peaking at 10 min and 3–4 h, as well as airway hyperresponsiveness to methacholine. Histological examination revealed increased expression of C3a receptors in the lung on the fourth challenge. Additionally, the C3 level in serum 4 h after the fourth challenge was significantly reduced compared with that before the challenge. When a C3a receptor antagonist, SB290157, was administered i.p. 30 min before the fourth challenge, the late-phase asthmatic response and airway hyperresponsivness induced by the fourth challenge were significantly inhibited, although the early-phase response was not influenced. In bronchoalveolar lavage fluid, neutrophil infiltration 24 h after the fourth challenge was reduced by the treatment. On the other hand, SB290157 suppressed the increased expression of IL-1β in the lung in this model, and the intratracheal administration of IL-1β induced airway obstruction, airway hyperresponsiveness, and neutrophil infiltration in normal mice. These results illustrate that C3a is involved in the development of the late asthmatic response and airway hyperresponsiveness. The mechanism leading to the development of these symptoms may correlate with the recruitment of neutrophils and/or the production of IL-1β induced by C3a.

Involvement of thromboxane A2 and peptide leukotrienes in early and late phase nasal blockage in a guinea pig model of allergic rhinitis.

Abstract. Objective and design: We investigated the effects of the thromboxane (TX) A2 antagonist seratrodast, the peptide leukotriene (p-LT) antagonist pranlukast, the antihistaminic drug terfenadine and the glucocorticoid dexamethasone on antigen-induced sneezing, biphasic nasal blockage and nasal hyperresponsiveness to histamine using a guinea pig model of allergic rhinitis.¶Subjects: Male Hartley guinea pigs were used.¶Treatment: Intranasally sensitized guinea pigs were challenged once every week for 13 weeks by inhalation of Japanese cedar pollen as the antigen. Dexamethasone and other agents were administered orally 3 and 1 h, respectively, before the 4th, 6th and 13th challenge.¶Methods: Sneezing frequency and the change in specific airway resistance (sRaw) were measured at these challenges. Two days after the 13th challenge, nasal responsiveness to histamine was evaluated by measuring sRaw after intranasal instillation of increasing doses of histamine. Moreover, the levels of TXB2, p-LTs and histamine were estimated in nasal cavity lavage fluid (NCLF) collected at the 13th challenge.¶Results: Only terfenadine (10 mg/kg) significantly inhibited sneezing at any challenge time. Seratrodast (3 and 10 mg/ kg), pranlukast (30 mg/kg) and dexamethasone (10 mg/kg), but not terfenadine, suppressed both the early and late phase elevation of sRaw (biphasic nasal blockage), although the degree of inhibition on the early phase response varied with the challenge time. In contrast, the development of nasal hyperresponsiveness to histamine was inhibited by only dexamethasone. Furthermore, biphasic increases in TXB2, p-LTs and histamine in NCLF were observed after the challenge in sensitized animals.¶Conclusions: These results suggest that TXA2 and p-LTs, but not histamine, play important roles in both the early and the late phase nasal blockage in this model of allergic rhinitis.

Nasal hyperresponsiveness to histamine induced by repetitive exposure to cedar pollen in guinea-pigs

Nasal hyperresponsiveness is one of the characteristic features of the pathogenesis of allergic rhinitis. This study examined whether repetitive inhalation of antigen (Japanese cedar pollen) led to the development of nasal hyperresponsiveness to histamine in sensitized conscious guinea-pigs. Guinea-pigs were repeatedly challenged by pollen inhalation once every week following sensitization by means of intranasal application of pollen extract plus aluminium hydroxide. The upper airways obstruction (increase in specific airway resistance (sRaw)) in response to intranasally instilled histamine was measured as an index of nasal (hyper)responsiveness. The hyperresponsiveness to histamine gradually developed with repeated pollen inhalation challenge, and the airway response at the 20th and 24th challenges was three to four orders of magnitude higher than that in nonsensitized animals. Similar degrees of hyperresponsiveness were observed at 10 h and 2 days after a pollen inhalation challenge, but the hyperresponsiveness had almost disappeared by day 7. The increased responsiveness was suppressed by pretreatment with mepyramine but not with atropine. The maximum sRaw, which was observed 10 min after histamine instillation, was largely blocked by naphazoline. Hyperresponsiveness was hardly observed on methacholine instillation. The present allergic rhinitis model, showing marked nasal hyperresponsiveness to histamine after repeated intranasal allergen challenge in guinea pigs, should be useful for investigating the pathogenesis of allergic rhinitis.

Important role of neutrophils in the late asthmatic response in mice

AIMS Neutrophils have been found increasingly in the lungs of patients with severe asthma; however, it is unclear whether the neutrophils contribute to the induction of the airway obstruction. We determined using a murine model whether neutrophils are involved in the late asthmatic response (LAR), and analyzed mechanisms underlying the antigen-induced airway neutrophilia. MAIN METHODS BALB/c mice sensitized by ovalbumin (OVA)+Al(OH)(3) were challenged 4 times by intratracheal administration of OVA. Airway mechanics were measured as specific airway resistance. KEY FINDINGS Induction of the LAR after the 4th challenge coincided with airway neutrophilia. In contrast, eosinophil infiltration was established prior to the 4th challenge. A treatment with an anti-Gr-1 monoclonal antibody (mAb) before the 4th challenge selectively suppressed increases in the neutrophil number and myeloperoxidase (MPO) level in bronchoalveolar lavage fluid (BALF), and attenuated the magnitude of LAR by 60-70%. Selective suppression of eosinophilia by anti-IL-5 mAb had little effect on the LAR. The increases in neutrophil number and MPO level were partially inhibited by an anti-CD4 mAb treatment. The CD4(+) cell depletion also significantly inhibited increases in neutrophil chemoattractants, IL-17A, keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-2 in BALF. However, blockade of FcγRII/III failed to suppress the neutrophilia. SIGNIFICANCE These data suggest that neutrophils are key inducers of the LAR, and that the antigen-induced neutrophilia is partially dependent on activated CD4(+) cells that are involved in the production of IL-17A, KC and MIP-2.

Atopic dermatitis‐like pruritic skin inflammation caused by feeding a special diet to HR‐1 hairless mice

Abstract:  Dry skin/barrier dysfunction is considered to be one of the characteristic features of atopic dermatitis (AD). When HR‐1 hairless mice are fed a special diet, HR‐AD, dry red skin is induced. We examined whether HR‐AD–fed mouse could be used as a model for AD by showing itch‐associated scratching behaviour and by analysing the immunological change. HR‐1 mice were fed HR‐AD from 4 weeks old. HR‐AD–fed mice showed severe dry skin symptoms accompanied by a decrease in dermal water content and an increase in transepidermal water loss and prolonged scratching bout duration on day 14 or 28. These symptoms became gradually worse until day 56. Marked epidermal hyperplasia and slight increase in CD4+ cells in the skin were observed from day 28. In contrast, increases in circulating T cells and serum immunoglobulin E were seen from day 41. Other skin‐infiltrating inflammatory cells, such as eosinophils and mast cells, were increased on day 56 but not on day 28. Though daily oral treatment with dexamethasone reduced the increased numbers of these cells, it did not affect the dry skin symptoms or the prolonged scratching episodes. In contrast, the development of dry skin was inhibited by feeding with 10% normal diet‐containing HR‐AD. The skin barrier dysfunction in HR‐AD–fed mice is closely associated with the development of AD‐like pruritus. Changes in the immunological parameters observed may be the consequence of skin barrier dysfunction. Our findings suggest that HR‐AD–fed mouse could be used as a dry skin‐based experimental model for AD.