Effect of waterpipe tobacco smoking on airway inflammation in murine model of asthma

Objective: There has been an increase in the popularity of waterpipe tobacco smoking (WTS) worldwide, especially in the younger population, including asthma patients. In this study, we investigated the effects of waterpipe smoking on airway inflammation, cytokine levels and oxidative stress markers in an antigen-driven murine model of asthma.

Materials and methods: Balb/c mice were divided into four groups; (1) control (received fresh air, ovalbumin sensitization and saline challenge), (2) WTS (received WTS, ovalbumin sensitization and saline challenge), (3) Ova S/C (received fresh air, ovalbumin sensitization and ovalbumin challenge) and (4) simultaneous WTS and Ova S/C (received WTS, ovalbumin sensitization and ovalbumin challenge). Airway inflammatory cells were evaluated in the broncho-alveolar lavage fluid. Cytokines [interleukin (IL)-13, 10 and 18] and oxidative stress markers [superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx)] were evaluated in the lung homogenates.

Results: Chronic exposure to WTS significantly increased the number of airway inflammatory cells in mice, specifically: eosinophils, neutrophils, macrophages and lymphocytes. The level of IL-13 in the lungs was increased and the level of IL-10 was reduced (p?Chronic WTS potentiated the increase in inflammatory cells induced by Ova S/C (p?p?Conclusions: Chronic WTS exposure induced airway inflammation in control mice and enhanced airway inflammation in murine model of asthma.     

变应性鼻炎大鼠模型建造

目的建立稳定的实验性变应性鼻炎（AR）SD大鼠模型。方法将SD大鼠雌雄各60只,随机分为实验组（n=60）和对照组（n=60）。实验组经腹腔隔日1次注射卵清蛋白（OVA）变应原7次后,鼻腔滴入OVA鼻内激发;对照组以生理盐水替代OVA。结果实验组SD大鼠均出现鼻痒、喷嚏、流清涕等变应性鼻炎的临床特征（评分〉5分）,鼻黏膜见大量的嗜酸性粒细胞、淋巴细胞浸润,粘膜水肿增厚、腺体增生、分泌旺盛,鼻黏膜表面纤毛破坏等组织形态学变化。对照组SD大鼠仅轻度抓鼻,且喷嚏少（评分〈5分）,鼻黏膜组织形态学均无实验组上述变化。结论本实验成功建立OVA变应原致敏的变应性鼻炎鼠类模型,具有操作简单、重复性好的优点,为今后变应性鼻炎的病变研究提供了有效的方法及组织形态学依据。     

Murine autoimmune hearing loss mediated by CD4+ T cells specific for β-tubulin

Autoimmune inner ear disease is described as progressive, bilateral although asymmetric, sensorineural hearing loss and can be improved by immunosuppressive therapy. We showed that the inner ear autoantigen β-tubulin is capable of inducing experimental autoimmune hearing loss (EAHL) in mice. Immunization of BALB/c mice with β-tubulin resulted in hair cell loss and hearing loss, effects that were not seen in animals immunized with control peptide. Moreover, the EAHL model showed that β-tubulin responsiveness involved CD4(+) T cells producing IFN-γ, and T cell mediation of EAHL was determined by significantly increased auditory brainstem response after adoptive transfer of β-tubulin-activated CD4(+) T cells into naive BALB/c recipients. The potential mechanisms responsible for the observed pathology of EAHL can be attributed to decreased frequency and impaired suppressive function of regulatory T cells. Our study suggests that EAHL may be a T cell-mediated organ-specific autoimmune disorder of the inner ear.     

IL-10 controls Th2-type cytokine production and eosinophil infiltration in a mouse model of allergic airway inflammation

Interleukin-10 was originally described as a factor that inhibits cytokine production by murine Th1 clones. Recent studies have since shown that IL-10 can also downregulate Th2 clones and their production of IL-4 and IL-5. Because of its immuno-suppressive properties, IL-10 has been suggested as a potential therapy for allergic inflammation and asthma. However, the pathophysiological role of IL-10 in vivo has not been clearly elucidated. We investigated the effects of IL-10 administration on the production of IgE, cytokine and allergen-induced Th2 cytokine production as well as its effects on eosinophilic inflammation. We established GATA-3/TCR double transgenic (GATA-3/TCR-Tg) mice by crossing GATA-3 transgenic mice with ovalbumin (OVA)-specific TCR transgenic mice; these mice were then sensitized using an intraperitoneal injection of OVA adsorbed to alum and challenged with the intratracheal instillation of an allergen. When GATA-3/TCR-Tg mice sensitized with OVA and alum were injected with C57-IL-10 cells before OVA inhalation, the levels of IL-5, IL-13, and IL-4 decreased by 40-85% and number of eosinophils decreased by 70% (P < 0.03) in the murine bronchoalveolar lavage fluid (BALF). These results suggest that IL-10 plays an important role downstream of the inflammatory cascade in the Th2 response to antigens and in the development of BALF eosinophilia and cytokine production in a murine model of asthma. These immunosuppressive properties in animal models indicate that IL-10 could be a potential clinical therapy for the treatment of allergic inflammation.     

Antigen-specific CD4 T cell clonal expansion and differentiation in the aged lymphoid microenvironment. I. The primary T cell response is unaffected

Aging is associated with changes in the immune system that lead to decreased immunity in the elderly. Prior studies from humans and mice have shown that aged T cells exhibit numerous defects, including decreased proliferation following in vitro stimulation, suggesting that intrinsic defects exist within aged T cells, leading to defective T cell activation and clonal expansion. In vivo, however, cellular and soluble factors in the lymphoid microenvironment influence T cell function. To investigate the effects of the aged lymphoid microenvironment on T cell function, we monitored the immune response of CD4 T cells from DO11.10 TCR transgenic mice following adoptive transfer into young and aged hosts. After immunization with specific antigen similar rates of donor DO11.10 T cell division were observed in the two host types. However, at the peak of the response, greater numbers of DO11.10 T cells were found in the aged hosts. Regardless of the age of the host, the donor DO11.10 T cell population differentiated into functional effector cells. Despite the increased CD4 T cell growth in aged hosts, similar numbers of memory DO11.10 T cells were found in young and in aged hosts. As CD4 T cell clonal expansion and differentiation is not impaired in the aged microenvironment, our data suggest that diminished T cell immunity during aging is largely due to intrinsic T cell defects, rather than to extrinsic influences associated with the aged lymphoid microenvironment.     

Tolerogenic microenvironment in neonatal period induced by maternal immunization with ovalbumin

Maternal immunization with allergens, such as ovalbumin (OVA), can inhibit the development of an allergic response in offspring. The regulatory mechanisms seem to be mediated by maternal antibodies (MatAbs) and factors generated by the maternal–fetal interface. The aim of this study was to verify the pathways of inhibitory Ab transference after maternal immunization with OVA and the effect of the offspring's dendritic cells (DCs) on the generation of regulatory T (Treg) cells. We verified that preconceptional OVA immunization induces high levels of proinflammatory and regulatory cytokines in the amniotic fluid, allowing the transference of high levels of anti-OVA IgG1 Abs to the offspring. Using an adoptive nursing protocol, we verified that maternal immunization leads to MatAb transference by the placental route and by breastfeeding contribute to the inhibition of anaphylactic IgE and IgG1 Ab responses in immunized offspring. We observed that maternal immunization decreased eosinophil numbers in recovered bronchoalveolar lavage fluid and in the lung tissue, whereas with a lack of control of airway responsiveness to methacholine. Maternal immunization induced in young offspring a decreased percentage of CD11c+ DCs expressing MHC class II and CD40 molecules. Moreover, DCs from both groups of offspring when pulsed with OVA, were able to induce Treg cells in vitro. Similarly, OVA immunization at the neonatal stage increased the frequency of Treg cells, regardless of the mother's immunization status. These findings emphasize that maternal immunization leads to a complex interaction of regulatory factors, with MatAbs, DCs and Treg cells affecting the tolerance of offspring during an allergic response.     

Intranasal delivery of whole influenza vaccine prevents subsequent allergen-induced sensitization and airway hyper-reactivity in mice

BACKGROUND: Infection with influenza virus has been associated with seemingly opposing effects on the development of asthma. However, there are no data about the effects of mucosal vaccination with inactivated influenza on the inception of allergic asthma. OBJECTIVE: To assess the immunological effects of inhaled inactivated influenza vaccine, using two different types of flu vaccines, on the inception of allergic sensitization and allergen-mediated airway disease in a mouse model. METHODS: BALB/c mice were intranasally or intratracheally vaccinated with whole or split influenza virus vaccine (days -1 or -1, 27) before systemic sensitization with ovalbumin (OVA) (days 1, 14) and repeated airway allergen challenges (days 28-30). Allergen sensitization (IgE serum levels), airway inflammation (differential cells in bronchoalveolar lavage fluid) and airway hyper-reactivity (AHR) (in vivo lung function) were analysed. RESULTS: The intranasal instillation of whole influenza vaccine before allergen sensitization significantly reduced the serum levels of total and OVA-specific IgE as well as allergen-induced AHR. Prevention was due to an allergen-specific shift from a predominant T helper (Th)2- towards a Th1-immune response. Application of split influenza vaccine did not show the same preventive effect. CONCLUSION: Intranasal administration of inactivated whole influenza vaccine reduced subsequent allergen sensitization and prevented allergen-induced AHR. Our results show that the composition of the influenza vaccine has a major influence on subsequent development of allergen-induced sensitization and AHR, and suggest that mucosal inactivated whole influenza vaccination may represent a step towards the development of a preventive strategy for atopic asthma.     

Antibodies with specificity for native and denatured forms of ovalbumin differ in reactivity between enzyme‐linked immunosorbent assays

In this study, polyclonal and monoclonal antibodies to native and denatured chicken ovalbumin (OVA) were produced to compare their dependency on continuous and three‐dimensional epitopes. These antibodies were characterized with respect to reactivity to native and denatured OVA by enzyme‐linked immunosorbent assay (ELISA) employing surface‐bound OVA and streptavidin‐capture ELISA to determine whether effects of different coating influence antibody specificity and with respect to epitope specificity by peptide ELISA, using overlapping peptides, covering the complete OVA sequence. Polyclonal antibodies to native OVA reacted strongly with native and denatured OVA in both assays, but did not react with the overlapping peptides. Polyclonal antibodies to denatured OVA reacted strongly with both OVA forms and with several of the overlapping peptides. Monoclonal antibodies to native OVA reacted preferentially with three‐dimensional epitopes on native OVA and not with denatured OVA. Monoclonal antibodies to denatured OVA showed reactivity to both OVA forms. Two of these monoclonal antibodies, HYB 94‐06 and 94‐07, showed reactivity to overlapping peptides and their epitopes were identified as flexible structures constituting amino acids 130–135 and 136–141, respectively. Moreover, comparison of antibody reactivity to N OVA revealed that in the streptavidin‐capture ELISA, antibody reactivity was notably reduced compared to ELISA employing surface‐bound OVA. Collectively, immunization with native OVA preferentially generates highly specific antibodies reacting with three‐dimensional epitopes, whereas immunization with denatured OVA generates antibodies occasionally reacting with continuous epitopes. Moreover, as differences in monoclonal antibody reactivity was found between the two assays, monoclonal antibodies always should be selected by an assay mimicking the desired use of the final antibodies as closely as possible.     

Increased expression of cytosolic phospholipase A2, 5-lipoxygenase and 5-lipoxygenase-activating protein in rat peritoneal macrophages during ovalbumin-induced sensitization

BACKGROUND: Macrophages are involved in immediate hypersensitivity reactions by their ability to release leukotrienes involved in the symptomatology of allergy. To date it is unknown whether this ability to secrete leukotrienes has been favoured by modifications, occurring during the sensitization phase, of the enzymes involved in leukotriene metabolism. OBJECTIVE: We used ovalbumin-sensitized rats to study the expression of cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) in peritoneal macrophages during active sensitization. We compared basal and challenged (PMA, A23187 and allergen) arachidonic acid (AA) metabolism of macrophages from control (cPM) and sensitized (sPM) rats. Then we tested, in cultured cPM, whether IL-4, the predominant cytokine of sensitization process, could reproduce the enzymatic modifications occurring in macrophages during sensitization. METHODS: cPLA2, 5-LO and FLAP expression was assessed by Western blotting. The arachidonic acid (AA) metabolism study was performed after incorporation of tritiated AA in macrophages and analysis of secreted tritiated eicosanoids. RESULTS: Ovalbumin-sensitization of rats increased cPLA2, 5-LO and FLAP expression in peritoneal macrophages. These increased expressions were not paralleled by modifications of basal and PMA- or A23187-stimulated AA metabolism of sPM. However, when macrophages encountered the specific allergen for a second time, sPM secreted higher levels of leukotrienes than cPM. IL-4 induced FLAP expression in cPM but had no effect on cPLA2 and 5-LO expression. CONCLUSION: Active sensitization of rats induces an increase, in peritoneal macrophages, of the enzymes involved in leukotriene metabolism. The increased leukotriene secretion of sPM in response to ovalbumin challenge may be favoured by this increased expression of cPLA2, 5-LO and FLAP that, however, is not able to lead to modifications of macrophage AA metabolism in any circumstance. Our results also suggest that IL-4 is not the major element originating the enzymatic modification induced by sensitization in peritoneal macrophages.