Nobuaki Mizutani

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.

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.

Complement C3a-Induced IL-17 Plays a Critical Role in an IgE-Mediated Late-Phase Asthmatic Response and Airway Hyperresponsiveness via Neutrophilic Inflammation in Mice

Allergen-specific IgE plays an essential role in the pathogenesis of allergic asthma. Although there has been increasing evidence suggesting the involvement of IL-17 in the disease, the relationship between IL-17 and IgE-mediated asthmatic responses has not yet been defined. In this study, we attempted to elucidate the contribution of IL-17 to an IgE-mediated late-phase asthmatic response and airway hyperresponsiveness (AHR). BALB/c mice passively sensitized with an OVA-specific IgE mAb were challenged with OVA intratracheally four times. The fourth challenge caused a late-phase increase in airway resistance associated with elevated levels of IL-17+CD4+ cells in the lungs. Multiple treatments with a C3a receptor antagonist or anti-C3a mAb during the challenges inhibited the increase in IL-17+CD4+ cells. Meanwhile, a single treatment with the antagonist or the mAb at the fourth challenge suppressed the late-phase increase in airway resistance, AHR, and infiltration by neutrophils in bronchoalveolar lavage fluid. Because IL-17 production in the lungs was significantly repressed by both treatments, the effect of an anti–IL-17 mAb was examined. The late-phase increase in airway resistance, AHR, and infiltration by neutrophils in bronchoalveolar lavage fluid was inhibited. Furthermore, an anti–Gr-1 mAb had a similar effect. Collectively, we found that IgE mediated the increase of IL-17+CD4+ cells in the lungs caused by repeated Ag challenges via C3a. The mechanisms leading to the IgE-mediated late-phase asthmatic response and AHR are closely associated with neutrophilic inflammation through the production of IL-17 induced by C3a.

Pharmacological characterization of the leukocyte kinetics after intranasal antigen challenge in a guinea pig model of allergic rhinitis.

Abstract. Objective and design: We characterized the leukocyte kinetics after antigen challenge, and 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 this leukocyte response in 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 15 weeks by inhalation of Japanese cedar pollen as the antigen. Dexamethasone and other agents were administered orally 3 and 1 h, respectively, before the 15th challenge.¶Methods: The time-related changes in the numbers of differential leukocytes in nasal cavity lavage fluid (NCLF) and in peripheral blood after pollen inhalation challenge were investigated. The effects of the drugs on the antigen-induced changes in the leukocyte counts were evaluated. In addition, histopathological examination of the nasal mucosa was also performed 5 h after the challenge.¶Results: There was a marked increase in the number of leukocytes in NCLF, especially of eosinophils, which peaked at 5 h, after antigen challenge in this model. This response was also accompanied by the peripheral blood eosinophilia and neutrophilia. Seratrodast (30 mg/kg), pranlukast (30 mg/kg) and dexamethasone (10 mg/kg) inhibited the eosinophilia in all of the blood, the nasal mucosa and NCLF seen 5 h after the antigen challenge. Terfenadine (10 mg/kg) had no apparent effect on the blood and the mucosal eosinophilia, although it tended to suppress the eosinophil accumulation in NCLF.¶Conclusions: These results suggest that the present model is useful for analyzing the mechanisms of antigen-induced eosinophilic inflammation in allergic rhinitis and that both TXA2 and p-LTs, but not histamine, contribute to the antigen-induced eosinophilia in this model of allergic rhinitis.

Involvement of nitric oxide in pollen-induced biphasic nasal blockage in sensitised guinea pigs

We have developed a reproducible allergic rhinitis model showing biphasic nasal blockage on repetitive inhalation challenge with Japanese cedar pollen in sensitised guinea pigs. The role of nitric oxide (NO) in inducing nasal blockage was evaluated with this model. N(omega)-nitro-L-arginine methyl ester (L-NAME), a non-selective NO synthase (NOS) inhibitor, intravenously administered before the challenge, significantly inhibited both early and late nasal blockage by approximately 80% and 50%, respectively. When L-NAME treatment was performed after the challenge, the late response was inhibited by approximately 70%. This inhibition was completely reversed by co-administration of L-arginine. However, aminoguanidine and L-N(6)-(1-iminoethyl)lysine, selective inhibitors of inducible NOS, negligibly influenced the degree of nasal blockage. Meanwhile, the alpha-adrenergic agonist, naphazoline, strongly suppressed both early and late nasal blockage. These results indicate that NO, likely produced by constitutive rather than inducible NOS, plays a major role in the occurrence of biphasic nasal blockage, primarily by inducing vasodilatation.

Regulatory role of antigen-induced interleukin-10, produced by CD4(+) T cells, in airway neutrophilia in a murine model for asthma.

It has been suggested that interleukin (IL)-10 exerts immunosuppressive effects on allergic inflammation, including asthma, mainly through inhibition of Th2 cell-mediated eosinophilic airway inflammation. In a model of experimental asthma utilizing multiple intratracheal antigen challenges in sensitized mice, IL-10 production as well as eosinophilia and neutrophilia in the lung were induced by the multiple challenges. In this study, we set out to reveal the cellular source of endogenously produced IL-10, and the roles of IL-10 in airway leukocyte inflammation using an anti-IL-10 receptor monoclonal antibody. Balb/c mice were sensitized i.p. with ovalbumin+Al(OH)(3), and then challenged by intratracheal administration of ovalbumin 4 times. Flow cytometric analyses revealed that the cellular source of IL-10 was CD4(+) T cells lacking the transcription factor, forkhead box P3. Treatment with anti-IL-10 receptor monoclonal antibody prior to the 4th challenge significantly augmented airway neutrophilia as well as the production of IL-1β, and CXC chemokines, keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-2, but not airway eosinophilia, Th2 cytokine (IL-4 and IL-5) production, or a late-phase increase in specific airway resistance. Approximately 40% of IL-10 receptor(+) cells expressed the macrophage marker F4/80, whereas only 3-4% of the IL-10 receptor(+) cells were granulocyte differentiation antigen (Gr)-1(high) cells (neutrophils). In conclusion, multiple airway antigen challenges induced the proliferation of IL-10-expressing CD4(+) T cells in regulating airway neutrophilia. Systemic blockade of IL-10 function coincided with increases in IL-1β and CXC chemokines. Thus, IL-1β and CXC chemokines may be targets for development of novel pharmacotherapy for neutrophilic asthma.

Establishment and characterization of a murine model for allergic asthma using allergen-specific IgE monoclonal antibody to study pathological roles of IgE

Allergen-specific IgE has long been regarded as a major molecular component of allergic asthma. Although IgE plays a central role in the early asthmatic response, its roles in the chronic phase, such as the late asthmatic response, airway hyperresponsiveness (AHR), and airway remodeling (goblet cell hyperplasia and subepithelial fibrosis) have not yet been defined well. In this study, we investigated the hypothesis that chronic responses could be induced by IgE-dependent mechanisms. BALB/c mice passively sensitized with an ovalbumin (OVA)-specific IgE monoclonal antibody (mAb) were repeatedly challenged with intratracheal administration of OVA. The first challenge induced early phase airway narrowing without any late response, but the fourth challenge caused not only an early but also a late phase response, AHR, and goblet cell hyperplasia. Macrophages, lymphocytes and neutrophils, but not eosinophils, were significantly increased in the lung 24h after the fourth challenge. Interestingly, levels of OVA-specific IgG1 in serum increased by multiple antigen challenges. A C3a receptor antagonist inhibited the late asthmatic response, AHR, and infiltration by neutrophils. In contrast, no late response, goblet cell hyperplasia, inflammatory cells, or production of IgG1 was observed in severe combined immunodeficient mice. On the other hand, seven challenges in BALB/c mice induced subepithelial fibrosis associated with infiltration by eosinophils. In conclusion, the allergic asthmatic responses induced by passive sensitization with IgE mAb can provide a useful model system to study the pathological roles of IgE in acute and chronic phases of allergic asthma.