Effects of erlotinib on pulmonary function and airway remodeling after sensitization and repeated allergen challenge in Brown-Norway rats

Erlotinib, an EGFR tyrosine kinase inhibitor, can inhibit the proliferation and survival of cancer cells. It has been widely used to treat non-small cell lung cancer. This study aimed to evaluate the effects of erlotinib on bronchial hyperresponsiveness, airway inflammation, and airway remodeling in sensitized, ovalbumin-challenged rats. Two experimental groups of Brown-Norway rats were sensitized and repeatedly challenged by breathing aerosolized ovalbumin. Since Day 1, one group was given oral erlotinib (OA-erlotinib group) while the other group was given only oral saline (OA-saline group). The control group was sensitized and challenged using saline. All were anesthetized and paralyzed, and pulmonary function tests conducted at baseline and after provocation with varying doses of acetylcholine. Lung tissues were examined for airway inflammation, airway remodeling, and Th2-related cytokine mRNA expression. Results showed that the OA-erlotinib group had better pulmonary function and less airway inflammation, Th2-related cytokines and their mRNA expression, and airway remodeling compared to the OA-saline group. In conclusion, erlotinib effectively prevents bronchial hyperreactivity, airway inflammation, Th2-related cytokine mRNA expression, and airway remodeling after sensitization and repeated allergen challenge in Brown-Norway rats.     

Fas deficiency delays the resolution of airway hyperresponsiveness after allergen sensitization and challenge

BACKGROUND: In asthma, persistent inflammation might be the result of (1) an impaired ability to clear inflammatory cells from the airways and/or (2) impaired apoptotic responses. OBJECTIVE: In a mouse model, we investigated the regulatory role of Fas (CD95)-induced apoptosis in the development and resolution of airway inflammation and airway hyperresponsiveness (AHR). METHODS: Mice that were either Fas-sufficient (wild-type; WT) or Fas-deficient (lpr ) were sensitized by intraperitoneal injections of ovalbumin (OVA) and challenged once intranasally with OVA (IP-IN mice). Control (IN) mice were challenged only. RESULTS: IP-IN WT mice developed AHR at 48 hours; changes in airway resistance resolved by 96 hours. Airway responsiveness at 48 hours in IP-IN lpr mice was similar to that in IP-IN WT mice. However, in contrast to WT mice, IP-IN lpr mice sustained significant AHR at 96 hours in comparison with IN lpr mice; the AHR resolved by 6 days. Bronchoalveolar lavage fluid cell composition was similar in all of the different groups at 48 hours and 96 hours. Both IP-IN WT mice and lpr mice exhibited similar tissue eosinophilia, whereas IP-IN lpr mice had significantly lower numbers of TdT-mediated dUTP nick end labeling (TUNEL)-positive cells in comparison with IP-IN WT mice at 48 hours. Anti-IL-5 antibody given to IP-IN lpr mice 48 hours and 72 hours after the challenge significantly decreased AHR and eosinophilic inflammation and increased TUNEL-positive cell numbers at 96 hours. CONCLUSION: These results suggest that Fas expression can regulate the onset and resolution of AHR through an increase in eosinophil apoptosis.     

Effects of ketotifen on airway responses to allergen challenge in the actively sensitized Brown Norway rat

The effects of the phorbol ester, phorbol-12-myristate-13-acetate (PMA), and of the calcium ionophore A23187 on DNA synthesis in murine quiescent and mitogen-stimulated lymphocytes have been examined. Neither PMA nor A23187 had any mitogen effect on their own on quiescent lymphocytes. However, stimulation of cells sequentially with A23187 and then PMA resulted in a proliferative response in proportion to the duration of the exposure to A23187, and the sustained simultaneous presence of both agents was necessary for maximum proliferation. On the other hand, while short incubations with A23187 potentiated mitogen-induced DNA synthesis, prolonged exposure inhibited it. Furthermore, on lymphocytes stimulated with two T cell mitogens, the effects of A23187 and PMA depended on the proliferation-inducing mitogens and the responsiveness level induced by them. Therefore, while PMA and short pretreatments with A23187 had no effect on high intensity mitogenic responses, low intensity responses were significantly enhanced. These results demonstrate differential effects of A23187 and PMA on DNA synthesis that should be useful in studies on the mechanisms of activation of lymphocyte proliferation.     

A preceding airway reaction to one allergen may lead to priming of the airway responses to another allergen

This study aimed to determine whether a preceding airway response to one allergen leads to priming of the airway responses to another allergen. Twelve asthmatic children who had positive prick tests to two allergens, Dermatophagoides pteronyssinus (D.p.) and German cockroach (CR), participated in a randomized, placebo-controlled crossover study. We performed two consecutive inhalation challenges, D.p. challenge being followed 48 h later by CR challenge. The effect of initial (D.p.) challenge on the early and late airway responses to the subsequent (CR) challenge (CR2) was examined by comparing the responses with those to CR challenge preceded by sham challenge (CR1). The geometric mean PD²⁰ of CR allergen in the CR2 was 2.8 BU (breath unit) (range of 1 SD; 0.77-10.4), which was 12.0-fold less than that (33.7 BU, 10.8-105.2) in the CR1. The administration of a 6.1–fold less dose (8.9 BU, 2.7-28.8) in the CR2 than hi the CR1 (54.5 BU, 44.1-69.3) provoked a similar degree of late-phase reactions (18.7±7.3% vs 15.8 ± 9.6%). Our data indicated that the early- and late-phase reactions to CR challenge were augmented by the preceding reaction to D.p. This suggests that a preceding airway response to one allergen may lead to priming, with enhancement of the early and late airway responses to the subsequent challenge with another allergen.     

CD8(+) T cells regulate immune responses in a murine model of allergen-induced sensitization and airway inflammation

The role of CD8(+) T cells in the development of allergic airway disease is controversial. On the one hand, CD8(+) T cells are known to inhibit the development of airway hyperreactivity (AHR) in murine models of asthma. In humans, IL-10-producing CD8(+) T cells were shown to act as regulatory cells, inhibiting both proliferation and cytokine secretion of T cells. On the other hand, CD8(+) T cells can promote IL-5-mediated eosinophilic airway inflammation and the development of AHR in animal models. To examine this, we investigated the role of CD8(+) T cells during the induction of allergen-induced AHR and demonstrated a protective effect of CD8(+) T cells. Depletion of CD8(+) T cells prior to the immunization led to increased Th2 responses and increased allergic airway disease. However, after development of AHR, CD8(+) T cells that infiltrated the lungs secreted high levels of IL-4, IL-5 and IL-10, but little IFN-gamma, whereas CD8(+) T cells in the peribronchial lymph nodes or spleen produced high levels of IFN-gamma, but little or no Th2 cytokines. These data demonstrate protective effects of CD8(+)T cells against the induction of immune responses and show a functional diversity of CD8(+) T cells in different compartments of sensitized mice.     

Effects of contact sensitization and delayed hypersensitivity reactions on immune responses to non-related antigens. Modulation of Immune responses.

Guinea pigs were immunized intracutaneously into the ears with sheep red blood cells (SRBC). Application of a sensitizing dose of the contact allergen dinitrochlorobenzene (DNCB) onto the same ears was shown to suppress or enhance the humoral response to SRBC depending on the time of application. When guinea pigs were sensitized to a contact allergen, application of a sensitizing dose of a non-related allergen on the same ears either had no effect or caused a clear enhancement of the development of delayed type hypersensitivity (DTH). Strongest enhancement was found when both sensitizations were performed on the same day. Further experiments on the effects of a concomitant DTH reaction elicited at the site of application of a contact allergen showed a strong potentiation of DTH when B-cell suppression was minimized by pretreatment with cyclophosphamide (CY). It was considered that CY-DTH-immunopotentiation might be a useful tool for achieving a higher level of sensitivity after epicutaneous sensitization.     

Effects on immune responses in rats after neuromanipulation with capsaicin

The effect of capsaicin treatment on the immune response, assessed as antibody formation in vivo and in vitro, was studied in ovalbumin (OA)-immunized rates. Rats were treated with capsaicin at 1-2 days of life or at adult age, before or after immunization. The levels of IgA, IgE and IgG antibodies as well as immunoglobulins were measured in serum and supernatants from cultured lymph node cells, spleen cells and peripheral blood lymphocytes. Capsaicin treatment affected the antibody levels depending on the timing of capsaicin treatment in relation to immunization. Different effects of capsaicin treatment were also observed on the different immunoglobulin isotypes. One of the most striking effects by capsaicin treatment was the reduction of IgA and IgG synthesis in cultured lymphoid cells from aerosol immunized animals treated with capsaicin after immunization. In contrast, in vivo the level of total serum IgA was increased in similarly treated animals. In this study we show that capsaicin treatment, which is known to decrease the levels of neuropeptides of sensory origin, has a time-dependent effect on both antibody levels in vivo as well as the formation of immunoglobulin in vitro. Although the mechanisms responsible for this are not obvious, we conclude a link between depletion of neuropeptides in sensory nerves and the antibody synthesis.     

GM-CSF produced by the airway epithelium is required for sensitization to cockroach allergen

Airway epithelial cells are among the first to encounter inhaled allergens and can initiate allergic responses by producing pro-Th2 innate cytokines. In this study, we investigated the role of epithelial-derived cytokines in sensitization to a clinically relevant allergen, cockroach allergen (CRA). Among the epithelial-derived cytokines, granulocyte macrophage colony-stimulating factor (GM-CSF) had a central role in the initiation of Th2 allergic responses to CRA. We show that initial exposure to CRA directly activated airway epithelial cells through a TLR4-MyD88-dependent pathway and MyD88 signaling in epithelial cells induced upregulation of GM-CSF during sensitization. Epithelial-derived GM-CSF was required for allergic sensitization and selectively restored Th2 responses in the absence of MyD88. Thus, we demonstrate that epithelial-derived GM-CSF is a critical early signal during allergic sensitization to CRA.     

Bioaerosols and innate immune responses in airway diseases

PURPOSE OF REVIEW: We review the role of bioaerosols in the pathogenesis of inflammatory airway disease. The focus is on recent discoveries in innate immune responses induced by common components of bioaerosols. RECENT FINDINGS: Common components of bioaerosols include endotoxin, peptidoglycan and beta-glucan; all of which have been associated with inflammatory airway disease. Endotoxin signaling through toll-like receptor 4 is well characterized and updated. Peptidoglycan is now known to signal through three types of molecules: toll-like receptor 2; peptidoglycan recognition proteins; and nucleotide-binding oligomerization domain molecules. Beta-glucan, a common fungal cell wall component, signals through the newly discovered receptor, dectin-1. Emerging data indicate that genetic polymorphisms influence the response to bioaerosols. SUMMARY: Activation of the innate immune system by bioaerosols is becoming better understood. This knowledge provides an opportunity to better prevent and treat airway diseases that result from environmental exposure.     

Maternal respiratory sensitization and gestational allergen exposure does not affect subsequent pup responses to homologous or heterologous allergen

Evidence suggests that the predisposition towards atopy begins early in life. Maternal allergy has been associated with an increased risk of the development of allergic disease in offspring. Some studies suggest that the development of childhood atopy may also be influenced by prenatal allergen exposure. In this study, a respiratory allergen exposure model was used to determine the impact of maternal sensitization (with or without additional exposures during pregnancy) on subsequent pup responses to homologous or heterologous allergen. Female BALB/c mice received two intratracheal aspiration (IA) exposures to Metarhizium anisopliae crude antigen (MACA) or Hank’s buffered salt solution (HBSS) prior to breeding. Some mice also received three additional exposures during pregnancy. Control mothers did not receive treatment. Young adult offspring received three IA exposures to MACA, house dust mite extract (HDM) or HBSS. Offspring sensitized as young adults to either HDM or MACA developed an airway inflammatory response, including increased bronchoalveolar lavage fluid lactate dehydrogenase activity, total protein and total and differential cell counts compared to offspring exposed to HBSS. Increased airway responsiveness to methacholine was observed in pups treated with HDM but not with MACA. Maternal sensitization status (with or without gestational allergen exposure) had no effect on offspring response to either MACA or HDM. In conclusion, this study demonstrates that IA administration of MACA or HDM extract to young adult BALB/c mice induces the development of an inflammatory airway response. In contrast to previous reports, neither maternal sensitization nor gestational allergen exposure could be demonstrated to have a clear effect on offspring sensitization. This discrepancy may be a function of the respiratory sensitization and exposure protocol used in this study, which mimics natural sensitization more closely than do parenteral routes of exposure.     

Time course study on the development of allergen-induced airway remodeling in mice: the effect of allergen avoidance on established airway remodeling

OBJECTIVE AND DESIGN: We carried out a time course study on the development of allergen-induced airway remodeling in a mouse model of allergic asthma. Moreover, we examined the effect of allergen avoidance on the established airway remodeling. MATERIALS AND METHODS: BALB/c mice were sensitized to ovalbumin (OA) with alum, and exposed daily for 3 weeks to aerosolized OA. At each designated point, bronchial responsiveness was measured, and bronchoalveolar lavage and histological examination were carried out. RESULTS: The numbers of inflammatory leukocytes in the airways and the percentage of goblet cells in the epithelium, Th2 cytokine production, IgE production, collagen deposition beneath the basement membrane and bronchial responsiveness to acetylcholine were all markedly increased after repeated antigen challenge for 1-3 weeks. In contrast, after cessation of antigen exposure, goblet cell hyperplasia, inflammatory infiltrates and bronchial hyperresponsiveness were gradually attenuated and had almost resolved 4 weeks after cessation, but subepithelial fibrosis was still observed at this time point. CONCLUSIONS: The present findings demonstrated that epithelial changes following repeated allergen challenge are rapidly induced and recover after the cessation of exposure, but subepithelial fibrosis has a late onset and relatively irreversible changes, and subepithelial fibrosis in contrast to goblet cells hyperplasia did not appear to contribute to bronchial hyperresponsiveness, at least, in this mouse model.     

Effect of rush immunotherapy on airway inflammation and airway hyperresponsiveness after bronchoprovocation with allergen in asthma

Background: Rush immunotherapy (RIT) has been shown to be effective in allergic asthma. Objective: We investigated the mechanisms of RIT on the basis of cytokine production by T-cell lines and airway inflammation and responsiveness. Methods: Subjects were 8 patients with house dust mite–allergic asthma treated with dust mite extract RIT for 6 months and 6 RIT-untreated control patients. IL-5 production by Dermatophagoides farinae –specific T-cell lines, eosinophil percentages, and eosinophil cationic protein (ECP) in induced sputum and airway responsiveness to allergen and histamine were evaluated before and after treatment. Changes in eosinophil percentages and ECP in induced sputum and responsiveness to histamine 24 hours after allergen inhalation were also studied. Results: After 6 months of RIT, percentages of total eosinophils (43.0% ± 6.90% to 16.8% ± 2.48%; P < .01), percentages of EG2⁺ eosinophils (32.6% ± 6.39% to 19.7% ± 4.68%; P < .01) and ECP (362.7 ± 125.3 ng/mL to 26.2 ± 5.15 ng/mL; P < .05) decreased in induced sputum, and IL-5 production by T-cell lines decreased (617 ± 93.2 pg/mL to 200.0 ± 34.1 pg/mL; P < .01). RIT decreased both early- and late-phase bronchoconstriction (early phase: 33.2% ± 3.46% to 25.4% ± 1.42%; P < .03; late phase: 16.2% ± 3.52% to 6.2% ± 1.96%; P < .03) and suppressed increases in the percentages of total (61.8% ± 4.89% to 42.0% ± 4.67%; P < .01) and EG2-positive eosinophils (55.54% ± 7.21% to 36.5% ± 6.43%; P < .01) and ECP (685.6 ± 217.0 ng/mL to 85.4 ± 23.4 ng/mL; P < .05) in induced sputum after allergen inhalation. RIT also decreased airway responsiveness to dust mite (1:303.7 ± 123.7 wt/vol to 1:65.0 ± 13.2 wt/vol; P < .03) and to histamine before (397.1 ± 206.9 μg/mL to 1391.3 ± 283.3 μg/mL; P < .03) and after allergen inhalation (139.2 ± 36.5 μg/mL to 629.1 ± 196.3 μg/mL; P < .03). Conclusion: RIT decreases airway inflammation and airway hyperresponsiveness before and after bronchial provocation with allergen, possibly by inhibiting both allergen-specific T-cell– and mast cell-dependent pathways. RIT is an effective antiinflammatory treatment in allergic asthma. (J Allergy Clin Immunol 1998;102:927-34.)     

Vagal afferents contribute to exacerbated airway responses following ozone and allergen challenge

Brown-Norway rats (n=113) sensitized and challenged with nDer f 1 allergen were used to examine the contribution of lung sensory nerves to ozone (O(3)) exacerbation of asthma. Prior to their third challenge rats inhaled 1.0ppm O(3) for 8h. There were three groups: (1) control; (2) vagus perineural capsaicin treatment (PCT) with or without hexamethonium; and (3) vagotomy. O(3) inhalation resulted in a significant increase in lung resistance (R(L)) and an exaggerated response to subsequent allergen challenge. PCT abolished the O(3)-induced increase in R(L) and significantly reduced the increase in R(L) induced by a subsequent allergen challenge, while hexamethonium treatment reestablished bronchoconstriction induced by allergen challenge. Vagotomy resulted in a significant increase in the bronchoconstriction induced by O(3) inhalation and subsequent challenge with allergen. In this model of O(3) exacerbation of asthma, vagal C-fibers initiate reflex bronchoconstriction, vagal myelinated fibers initiate reflex bronchodilation, and mediators released within the airway initiate bronchoconstriction.     

Isoforms of the major allergen of birch pollen induce different immune responses after genetic immunization.

BACKGROUND: Recent publications indicate that immunization with plasmid DNA encoding allergens might represent a potential approach in allergen-specific immunotherapy. OBJECTIVE: In the present study we have compared the immune responses induced by plasmid DNA encoding for two isoforms of Bet v 1, the major allergen of birch pollen. METHODS: BALB/c mice were injected intradermally with plasmid DNA encoding for the genes of Bet v 1a (pCMV-Beta) and Bet v 1d (pCMV-Betd). In addition, the effect of immunostimulatory DNA sequences was investigated by appending and/or coinjecting CpG motifs. Antibody responses and IFN-gamma and IL-4 levels were measured by ELISA. Allergen-specific proliferation was determined by incorporation of [(3)H]-thymidine. RESULTS: The two isoforms induced a similar humoral response. The lack of any IgE production and the ratio of IgG1 to IgG2a clearly indicated a Th-1-type response. The antisera against both isoforms were highly cross-reactive, which was supported by the energy plot indicating similar folding of the two protein isoforms. However, determination of IFN-gamma and IL-4 in the serum elicited a strikingly different cytokine profile during the course of the immune response. In contrast to pCMV-Beta, pCMV-Betd caused no significant allergen-specific proliferation and induced only marginal levels of the key cytokines. CONCLUSIONS: Based on the assumption that the induction of a strong Th-1 type response is a prerequisite for successful treatment of allergy, our results favor the use of isoform Bet v 1a in combination with CpG motifs for a novel type of allergen immunotherapy based on plasmid DNA immunization. Additionally, the data also confirm the assumption that the antigen itself can have a marked influence on the immune response after genetic immunization.     

Respiratory syncytial virus infection provokes airway remodelling in allergen-exposed mice in absence of prior allergen sensitization

Background The mechanisms underlying exacerbation of asthma induced by respiratory syncytial virus (RSV) infection have been extensively studied in human and animal models. However, most of these studies focused on acute inflammation and little is known of its long-term consequences on remodelling of the airway tissue.
Objective The aim of the study was to use a murine model of prolonged allergen-induced airway inflammation to investigate the effect of RSV infection on allergic airway inflammation and tissue remodelling.
Methods We subjected mice to RSV infection before or during the chronic phase of airway challenges with OVA and compared parameters of airway inflammation and remodelling at the end-point of the prolonged allergen-induced airway inflammation protocol.
Results RSV infection did not affect the severity of airway inflammation in any of the groups studied. However, RSV infection provoked airway remodelling in non-sensitized, allergen-challenged mice that did not otherwise develop any of the features of allergic airways disease. Increased collagen synthesis in the lung and thickening of the bronchial basal membrane was observed in non-sensitized allergen-challenged mice only after prior RSV infection. In addition, fibroblast growth factor (FGF)-2 but not TGF-β₁ was increased in this group following RSV infection.
Conclusion Our data show for the first time that RSV infection can prime the lung of mice that are not previously systemically sensitized, to develop airway remodelling in response to allergen upon sole exposure via the airways. Moreover, our results implicate RSV-induced FGF-2 in the remodelling process in vivo .     

Estrogen determines sex differences in airway responsiveness after allergen exposure

The female hormone estrogen is an important factor in the regulation of airway function and inflammation, and sex differences in the prevalence of asthma are well described. Using an animal model, we determined how sex differences may underlie the development of altered airway function in response to allergen exposure. We compared sex differences in the development of airway hyperresponsiveness (AHR) after allergen exposure exclusively via the airways. Ovalbumin (OVA) was administered by nebulization on 10 consecutive days in BALB/c mice. After methacholine challenge, significant AHR developed in male mice but not in female mice. Ovariectomized female mice showed significant AHR after 10-day OVA inhalation. ICI182,780, an estrogen antagonist, similarly enhanced airway responsiveness even when administered 1 hour before assay. In contrast, 17beta-estradiol dose-dependently suppressed AHR in male mice. In all cases, airway responsiveness was inhibited by the administration of a neurokinin 1 receptor antagonist. These results demonstrate that sex differences in 10-day OVA-induced AHR are due to endogenous estrogen, which negatively regulates airway responsiveness in female mice. Cumulatively, the results suggest that endogenous estrogen may regulate the neurokinin 1-dependent prejunctional activation of airway smooth muscle in allergen-exposed mice.