Antigen-specific airway IL-33 production depends on FcγR-mediated incorporation of the antigen by alveolar macrophages in sensitized mice.

Although interleukin (IL)-33 is a candidate for the aggravation of asthma, the mechanisms underlying antigen-specific IL-33 production in the lung are unclear. Therefore, we analysed the mechanisms in mice. Intra-tracheal administration of ovalbumin (OVA) evoked increases in IL-33 and IL-33 mRNA in the lungs of both non-sensitized and OVA-sensitized mice, and the increases in the sensitized mice were significantly higher than in the non-sensitized mice. However, intra-tracheal administration of bovine serum albumin did not increase the IL-33 level in the OVA-sensitized mice. Depletion of neither mast cells/basophils nor CD4+ cells abolished the OVA-induced IL-33 production in sensitized mice, suggesting that the antigen recognition leading to the IL-33 production was not related with either antigen-specific IgE-bearing mast cells/basophils or memory CD4+ Th2 cells. When a fluorogenic substrate-labelled OVA (DQ-OVA) was intra-tracheally administered, the lung cells of sensitized mice incorporated more DQ-OVA than those of non-sensitized mice. The lung cells incorporating DQ-OVA included B-cells and alveolar macrophages. The allergic IL-33 production was significantly reduced by treatment with anti-FcγRII/III mAb. Depletion of alveolar macrophages by clodronate liposomes significantly suppressed the allergic IL-33 production, whereas depletion of B-cells by anti-CD20 mAb did not. These results suggest that the administered OVA in the lung bound antigen-specific IgG Ab, and then alveolar macrophages incorporated the immune complex through FcγRII/III on the cell surface, resulting in IL-33 production in sensitized mice. The mechanisms underlying the antigen-specific IL-33 production may aid in development of new pharmacotherapies.

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.

IgE/antigen-mediated enhancement of IgE production is a mechanism underlying the exacerbation of airway inflammation and remodelling in mice.

IgE is known to enhance some antibody responses to specific antigens, but whether this contributes to allergic asthma remains unclear. We have previously found that repeated antigen challenges in mice sensitized with antigen-specific IgE monoclonal antibody (mAb) exacerbated airway inflammation and remodelling accompanied by increased levels of endogenous antigen-specific IgE and IgG1. Here, we investigated whether IgE/antigen-mediated enhancement of endogenous IgE production contributes to the exacerbation of airway inflammation and remodelling. BALB/c mice passively sensitized with ovalbumin (OVA) -specific IgE mAb were challenged with OVA intratracheally seven times; anti-IgE mAb was intraperitoneally administered 1 day before the fourth challenge. Treatment with anti-IgE mAb inhibited the increased level of endogenous OVA-specific IgE in serum, but not OVA-specific IgG1, and a biphasic increase in airway resistance at the fourth challenge. Furthermore, a biphasic increase in airway resistance, airway hyper-responsiveness to methacholine, OVA-specific IgE and IgG1 production, and infiltrations by neutrophils and eosinophils in the lungs at the seventh challenge were suppressed by treatment; airway remodelling, such as goblet cell hyperplasia and sub-epithelial fibrosis, was also reduced. In addition, the production of interleukin-17A, interleukin-33 and CXCL1 in the lungs related to these IgE-mediated responses was decreased by treatment. Collectively, we found that the mechanism leading to the exacerbation of allergic asthma is closely related to IgE/antigen-mediated enhancement of IgE production, suggesting that this may create a vicious circle leading to the chronic status in asthmatic patients having levels of antigen-specific IgE ready to form complexes with antigen.

Thymic stromal lymphopoietin-induced interleukin-17A is involved in the development of IgE-mediated atopic dermatitis-like skin lesions in mice.

Atopic dermatitis (AD) is a chronic inflammatory skin disease associated with elevated levels of allergen-specific IgE. Although thymic stromal lymphopoietin (TSLP) and interleukin-17A (IL-17A) have been considered as important factors in allergic diseases, their relationships in AD have not been fully defined. Here, we show the contribution of TSLP-induced IL-17A responses to IgE-mediated AD-like skin lesions. BALB/c mice passively sensitized by intraperitoneal injections of ovalbumin (OVA)-specific IgE monoclonal antibody (mAb) were challenged with OVA applied to the skin six times. Treatment with anti-TSLP mAb during the second to sixth challenges inhibited IgE-mediated AD-like skin lesions and IL-17A production in lymph nodes. Furthermore, the increased number of IL-17A-producing CD4(+) and γδ T cells in lymph nodes and neutrophilic inflammation in the skin were reduced by anti-TSLP mAb. These findings prompted us to examine the roles of IL-17A. Treatment with anti-IL-17A mAb suppressed the AD-like skin lesions and neutrophilic inflammation; anti-Gr-1 mAb also inhibited them. Furthermore, treatment with CXCR2 antagonist reduced the AD-like skin lesions and neutrophilic inflammation accompanied by the reduction of IL-17A production; the increased CXCR2 expression in the epidermal cells was suppressed by anti-TSLP mAb. Meanwhile, these treatments, except for anti-Gr-1 mAb, inhibited the increased mast cell accumulation in the skin. Collectively, the mechanism of IgE mediating IL-17A-producing CD4(+) and γδ T cells through TSLP by repeated antigen challenges is involved in AD-like skin lesions associated with skin inflammation, such as neutrophil and mast cell accumulation; TSLP may regulate CXCR2 signalling-induced IL-17A production.

Longitudinal study of effects of oral dosage of Bifidobacterium bifidum G9-1 on Japanese cedar pollen-induced allergic nasal symptoms in guinea pigs.

Previous studies using experimental animal models have reported the beneficial effects of probiotics on allergic responses; however, their long-term effects on allergic nasal symptoms in clinical settings have not yet been elucidated in detail. In the present study, a guinea pig allergic rhinitis model involving repeated inhalation challenges with a natural allergen, Japanese cedar pollen, was used to examine the longitudinal effects of Bifidobacterium bifidum G9-1 (BBG9-1) on allergic nasal symptoms. BBG9-1 was administered orally once a day. Amelioration of nasal blockage was consistently observed throughout the experimental period in the BBG9-1-treated group. Although challenge-induced sneezing was not significantly inhibited in the BBG9-1-treated group, prolonged treatment with BBG9-1 slightly reduced the frequency of sneezing. Antigen-specific IgE antibody production was also not inhibited in the BBG9-1-treated group. Increases in the numbers of eosinophils and neutrophils in nasal cavity lavage fluid collected after pollen challenge were almost completely suppressed by BBG9-1 treatment, whereas those in mast cell mediators, histamine and cysteinyl leukotrienes were not. In contrast, increases in the levels of nitric oxide metabolites were potently suppressed. Furthermore, prolonged BBG9-1 treatment markedly suppressed exogenous leukotriene D4 -induced nasal blockage. Thus, prolonged oral administration of BBG9-1 suppresses Japanese cedar pollen-induced allergic nasal symptoms. The inhibitory mechanisms responsible may involve reductions in the responsiveness of target organs, such as endothelial cells in nasal mucosal blood vessels, to chemical mediators.

Intratracheal exposure to Fab fragments of an allergen-specific monoclonal antibody regulates asthmatic responses in mice.

Fab fragments (Fabs) maintain the ability to bind to specific antigens but lack effector functions due to the absence of the Fc portion. In the present study, we tested whether Fabs of an allergen-specific monoclonal antibody (mAb) were able to regulate asthmatic responses in mice. Asthmatic responses were induced in BALB/c mice by passive sensitization with anti-ovalbumin (OVA) polyclonal antibodies (pAbs) (day 0) and by active sensitization with OVA (days 0 and 14), followed by intratracheal (i.t.) challenge with OVA on day 1 and days 28, 29, 30 and 35. Fabs prepared by the digestion of an anti-OVA IgG1 (O1-10) mAb with papain were i.t. administered only once 30 min before antigenic challenge on day 1 or day 35. The results showed that i.t. administration of O1-10 Fabs with OVA markedly suppressed the early and/or late phases of asthmatic responses caused by passive and active sensitization. Similar results were obtained when Fabs of anti-OVA IgG2b mAb (O2B-3) were i.t. administered. In contrast, neither i.t. injection of intact 01-10/O2B-3 nor systemic injection of O1-10 Fabs suppressed the asthmatic responses. In vitro studies revealed that the capture of OVA by O1-10 Fabs prevented the subsequent binding of intact anti-OVA pAbs to the captured OVA. These results suggest that asthmatic responses may be down-regulated by the i.t. exposure to Fabs of an allergen-specific mAb via a mechanism involving the capture of allergen by Fabs in the respiratory tract before the interaction of intact antibody and allergen essential for the induction of asthmatic responses.

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.

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.

Efficient induction of arthritis in mice by an arthritogenic 4-clone cocktail of anti-type II collagen monoclonal antibodies recognizing different epitopes of the antigen.

We previously reported that a combination of 4 monoclonal antibodies (mAbs) (cocktail A) to type II collagen (CII), including immunoglobulin G (IgG)2b (C2B-9 and C2B-14) and IgG2a (C2A-7 and C2A-12), induced arthritis in DBA/1J mice. In this study, we found that C2B-9 and C2A-7 as well as C2B-14 and C2A-12 recognized the same or similar epitopes on CII. Based on these data, we hypothesized that the combination of more than 3 mAbs recognizing different epitopes on CII may more efficiently induce arthritis. Therefore, in addition to C2B-9 and C2B-14, which show high binding activity to CII compared with C2A-7 and C2A-12, we developed two more mAbs including IgG2b (C2B-17) and IgG2a (C2A-16), to make a new cocktail (cocktail B) consisting of these 4 mAbs. To compare the ability of cocktail B to induce arthritis with cocktail A, DBA/1J mice were injected with these cocktails. The results showed that cocktail B was able to induce more severe arthritis than cocktail A, especially more markedly affecting rear paws. Histologically, there was more marked proliferation of synovial tissues, massive infiltration by inflammatory cells, and severe destruction of cartilage and bone in mice treated with cocktail B than with cocktail A. Collectively, the new combination of 4 mAbs recognizing different respective epitopes appears to effectively induce arthritis in mice. Thus, the results may provide insights into the selection of mAbs associated with the development of arthritis.

Exposure to multiwalled carbon nanotubes and allergen promotes early- and late-phase increases in airway resistance in mice.

The facilitating effects of multiwalled carbon nanotubes (MWCNT) on allergic asthma have not been sufficiently examined, although MWCNT appear to significantly increase the risk of health problems from occupational or environmental exposure. In this study, we examined whether sensitization by the combination of MWCNT with ovalbumin (OVA) promotes allergic asthmatic responses. BALB/c mice administered vehicle, MWCNT, OVA, or MWCNT+OVA through an intranasal route were challenged with OVA intratracheally four times. In the MWCNT+OVA group, the fourth challenge caused not only early- but also late-phase increases in airway resistance, although these responses were not observed in the vehicle, MWCNT, or OVA group; furthermore, the extents of the early and late responses were comparable to those in mice systemically sensitized with OVA+alum. Sensitization with MWCNT and OVA promoted airway inflammation and goblet cell hyperplasia in the lung compared with the vehicle, MWCNT or OVA group. In addition, adjuvant activity for OVA-specific immunoglobulin E (IgE), IgG1, and IgG2a production in serum and increased levels of interleukin-4 (IL-4), IL-5, IL-13, and IL-17 in the lung tissue were observed. In conclusion, these results suggest that exposure to MWCNT and antigen can induce a biphasic increase in airway resistance, airway inflammation, goblet cell hyperplasia, and the production of antigen-specific antibodies. This study highlights the risk of exposure to a combination of MWCNT with antigen.

Effect of Ganoderma lucidum on pollen-induced biphasic nasal blockage in a guinea pig model of allergic rhinitis.

Ganoderma lucidum (GL), an oriental medical mushroom, has been used in Asia for the prevention and treatment of a variety of diseases. However, the effect of GL on allergic rhinitis has not been well defined. The current study describes the inhibitory effect of GL on the biphasic nasal blockage and nasal hyperresponsiveness induced by repeated antigen challenge in a guinea pig model of allergic rhinitis. Intranasally sensitized guinea pigs were repeatedly challenged by inhalation of Japanese cedar pollen once every week. Ganoderma lucidum was orally administered once daily for 8 weeks from the time before the first challenge. The treatment with GL dose-dependently inhibited the early and late phase nasal blockage at the fifth to ninth antigen challenges. Furthermore, nasal hyperresponsiveness to intranasally applied leukotriene D4 on 2 days after the eighth antigen challenge was also inhibited by the treatment with GL. However, Cry j 1-specific IgE antibody production was not affected by the treatment. In conclusion, we demonstrated that the pollen-induced biphasic nasal blockage and nasal hyperresponsiveness were suppressed by the daily treatment with GL in the guinea pig model of allergic rhinitis. These results suggest that GL may be a useful therapeutic drug for treating patients with allergic rhinitis.

Inhibition of hematopoietic prostaglandin D synthase improves allergic nasal blockage in guinea pigs.

Although it has been suggested that prostaglandin (PG) D(2) is involved in the pathogenesis of allergic rhinitis, whether the inhibition of hematopoietic PGD(2) synthase (H-PGDS) shows beneficial effects on allergic rhinitis has been unclear. We evaluated the effects of a selective H-PGDS inhibitor, TFC-007, on nasal symptoms on Japanese cedar pollen-induced allergic rhinitis of guinea pigs. Sensitized animals were challenged with the pollen once a week. TFC-007 (30mg/kg, p.o.) given once before a challenge almost completely suppressed PGD(2) production in the nasal tissue early and late after the challenge. Although pre-treatment did not affect the incidences of sneezing and early phase nasal blockage, late phase nasal blockage was partially but significantly attenuated; however, nasal eosinophilia was not suppressed. In contrast, when TFC-007 was given once 1.5h after the challenge, the late phase response was not affected. Collectively, PGD(2) produced by H-PGDS early after an antigen challenge can participate in the induction of late phase nasal blockage, although the mechanism may be independent of eosinophil infilatration. The strategy for H-PGDS inhibition may be beneficial for allergic rhinitis therapy.

Complete dependence on CD4+ cells in late asthmatic response, but limited contribution of the cells to airway remodeling in sensitized mice.

It is known that the late asthmatic response (LAR), a characteristic feature of asthma, is closely associated with CD4+ Th2 cell-mediated allergic inflammation. Airway remodeling is also a pathogenesis of asthma, but literature reporting roles of CD4+ cells in the remodeling is controversial. There has been no study that simultaneously assessed the roles of CD4+ cells in both LAR and airway remodeling. Sensitized mice were intratracheally challenged with ovalbumin 4 times. Treatment with an anti-CD4 monoclonal antibody (mAb) before the 1st challenge almost completely abolished increase in CD4+ cells in the tissues after the 4th challenge. The late phase increase in airway resistance after the 4th challenge was also completely inhibited by anti-CD4 mAb. Parameters of airway remodeling, subepithelial fibrosis and epithelial thickening were attenuated by treatment, whereas the inhibition was only 30% - 40%. Bronchial smooth muscle thickening was not affected. Because interleukin (IL)-5 production as well as eosinophilia was effectively suppressed by anti-CD4 mAb, the effect of anti-IL-5 mAb was also examined, resulting in no inhibition of airway remodeling. Collectively, although the LAR was completely dependent on CD4+ cell activation, airway remodeling was only partially dependent on the cell.

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-1beta in the lung in this model, and the intratracheal administration of IL-1beta 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-1beta induced by C3a.

Effect of the C3a-receptor antagonist SB 290157 on anti-OVA polyclonal antibody-induced arthritis.

It was investigated whether the C3a-receptor antagonist (C3aRA) SB 290157 was involved in the suppression of anti-OVA pAb-induced arthritis because it is well known that anaphylatoxin C3a plays a crucial role in the development of an effective inflammatory response during complement activation. Anti-OVA pAb-induced arthritis was induced in DBA/1J mice by administration of anti-OVA pAb 0.5 h prior to intra-articular (i.a.) injection of OVA (0 h). Two peaks of joint swelling were observed at 0.5 and 3 h. The role of C3aRA in arthritis was investigated by injection of SB 290157 at concentrations of 10 and 30 mg/kg at 0 and 2 h. The antagonist was able to reduce joint swelling only at 3 h, and about 50% inhibition of joint swelling was observed with the concentration of 30 mg/kg. The C3 level was significantly decreased at 3 h compared with naïve mice showing complement consumption. Furthermore, the C3 activation was observed and increased corresponding to the graded concentration of anti-OVA pAb. The results also revealed that the C3aRA was able to reduce the expression of IL-1beta in synovial tissue. Taken together, the results suggested that C3aRA may be effective in the inhibition of arthritis.

Intratracheal sensitization/challenge-induced biphasic asthmatic response and airway hyperresponsiveness in guinea pigs.

In most experimental model of asthma using guinea pigs, the animals are made to inhale an aerosolized antigen which passes through the nasal cavity. In the present study, we attempted to create an animal model of asthma showing a biphasic asthmatic response and airway hyperresponsiveness, in which the allergic responses are restricted to the lung. Guinea pigs were sensitized by the intratracheal instillation of ovalbumin (OVA)+Al(OH)3 once a day for 7 d, and then intratracheally challenged with OVA 12 d after the last sensitization. The change in specific airway resistance (sRaw) and airway responsiveness to histamine were measured. Pranlukast (100 mg/kg), theophylline (50 mg/kg), and dexamethasone (10 mg/kg) were orally administered 18 and 2 h before the antigen challenge. The challenge caused a marked biphasic elevation of sRaw with peaks at 5 min and 4 h. At 24 h, airway hyperresponsiveness to histamine was observed. Pranlukast, theophylline, and dexamethasone suppressed the late asthmatic response and airway hyperresponsiveness. The early asthmatic response was inhibited by theophylline and dexamethasone. In conclusion, the intratracheal sensitization and challenge caused a biphasic asthmatic response and airway hyperresponsiveness in guinea pigs. This model may be useful for the evaluation of anti-asthma drugs.

CD8+ T cells regulated by CD4+CD25+ regulatory T cells in the early stage exacerbate the development of Dermatophagoides farinae-induced skin lesions via increasing mast cell infiltration in mice.

Atopic dermatitis is a chronic inflammatory skin disease associated with CD4+ Th2 cell-shifted immune responses. Although the infiltration of skin lesions by CD8+ T cells has been recognized, their roles have not been fully defined. In this study, we examined the relationship between CD4+ and CD8+ cells in antigen-induced skin lesions of mice. BALB/c mice were repeatedly challenged with Dermatophagoides farinae (Der f) applied to the right ear nine times. Pre-treatment with anti-CD4 monoclonal antibody (mAb) during the third to sixth challenges, but not the post-treatment during the sixth to ninth challenges, exacerbated the development of Der f-induced ear swelling; pre-treatment with anti-CD25 mAb, which depletes regulatory T cells (Tregs), also exacerbated the lesions. Furthermore, the number of CD8+ T cells in lymph nodes was augmented by these pre-treatments. These findings prompted us to examine the effect of anti-CD8 mAb. Pre-treatment with anti-CD8 mAb, but not post-treatment, strongly inhibited the development of Der f-induced ear swelling; additionally, the epidermal hyperplasia and infiltration of mast cells were inhibited by the pre-treatment. Collectively, we revealed that CD8+ T cells regulated by CD4+CD25+ Tregs in the early stage are key contributors to the development of Der f-induced skin lesions via increasing mast cell infiltration, indicating that CD8+ T and Tregs could be potential therapeutic targets for atopic dermatitis.

Pulmonary emphysema induced by cigarette smoke solution and lipopolysaccharide in guinea pigs.

Exposure of animals to cigarette smoke for longer than 3 months leads to the development of chronic obstructive pulmonary disease (COPD) showing pulmonary emphysema. We attempted to create a COPD model with emphysema that could be established in a shorter period of time. Guinea pigs were intratracheally treated once a day on days 0-3, 5-8, 10-13 and 15-18 with a cigarette smoke solution (CSS), which was prepared by bubbling a stream of smoke into saline. Additionally, lipopolysaccharide (LPS) was administered intratracheally as an exacerbation factor on days 4, 9 and 14. By day 19, there was a gradual elevation of specific airway resistance (sRaw). In addition, both residual volume and functional residual capacity were found to be significantly higher on day 19. In the lungs, there was a marked increase in leukocytes, especially neutrophils. Histologically, we observed epithelial hyperplasia and emphysema. On the other hand, daily oral administration of theophylline during the administration of CSS and LPS suppressed the sRaw increase and the epithelial hyperplasia, but not other functional structural changes. In conclusion, we established an experimental COPD model in guinea pigs by using intratracheal instillations of CSS and LPS over a considerably shorter term than has been reported for other models.

Absence of nasal blockage in a Japanese cedar pollen-induced allergic rhinitis model mouse.

BACKGROUND: Japanese cedar pollen-induced allergic rhinitis in a guinea pig model clearly induced not only sneezing but also biphasic nasal blockage. To date, there have only been a few reports on models of murine allergic rhinitis which clearly show nasal blockage. Therefore, in order to try and develop such a model, we administered multiple dosages of intranasal pollen or purified antigen protein Cry j 1. METHODS: B10.S mice were sensitized by intranasal instillations of either pollen extract or Cry j 1 twice a day for 7 days, which was adsorbed on Al(OH)(3). Subsequently, once a week, the mice were given multiple intranasal instillation challenges of either the pollen suspension or Cry j 1 and the frequency of sneezing was observed after respective challenges were made. Specific airway resistance (sRaw) was measured as an indicator for nasal blockage. Cry j 1-specific IgE levels were measured using an enzyme-linked immunosorbent assay. RESULTS: The serum Cry j 1-specific IgE level showed clear elevation only in the group sensitized by Cry j 1 + Al(OH)(3) and then challenged by Cry j 1. No elevations were seen in the groups sensitized by pollen extract + Al(OH)(3) followed by a pollen suspension challenge. There was an immediate increase in sneezing after challenges in all of the sensitized-challenged groups. Nevertheless, no increases in sRaw in any of the groups were detected at any of the time points during the 8 hours following the challenges. CONCLUSIONS: Cry j 1 may be more effective than crude antigens for efficient sensitization/challenge in mice. No increase in sRaw occurred, even in mice that possessed high amounts of Cry j 1-specific IgE and that exhibited sneezing.

Involvement of peroxynitrite in pollen-induced nasal blockage in guinea pigs.

Nitric oxide (NO) has been implicated in early and late phase nasal blockage in a Japanese cedar pollen-induced experimental allergic rhinitis guinea pig model. In this study, we investigated the role of peroxynitrite, which is formed by a rapid reaction of NO with superoxide anion, in the antigen-induced biphasic nasal blockage. Sensitized guinea pigs were repeatedly challenged by pollen inhalation once every week. The peroxynitrite scavenger, ebselen (30 mg/kg), or the xanthine oxidase inhibitor, allopurinol (50 mg/kg), was intraperitoneally administered 30 min before the antigen challenge. The late phase nasal blockage induced 4 h after the challenge was largely suppressed by ebselen (57% inhibition; P<0.05) and allopurinol (47% inhibition; P<0.05), but neither ebselen nor allopurinol influenced the early phase response. On the other hand, the intranasal instillation of peroxynitrite (10(-3) and 10(-2) M, 10 microl/nostril) caused a remarkable dose-dependent nasal blockage in the sensitized guinea pig. These results suggest that peroxynitrite plays a major role in the late phase nasal blockage induced by the antigen challenge in sensitized guinea pigs.