Airway eosinophils from actively sensitized guinea pigs exhibit enhanced superoxide anion release in response to antigen challenge.

Antigen challenge of actively sensitized guinea pigs produces airway eosinophilia, airway hyperreactivity, and late-phase bronchoconstriction. The recruited eosinophils are thought to be important cells in the development of the airway hyperreactivity and the late-phase bronchoconstriction. However, the functional abilities of these eosinophils have not been determined in response to antigen challenge. The purpose of this study was to describe the characteristics of superoxide anion release from airway eosinophils obtained 24 h after ovalbumin challenge of actively sensitized guinea pigs. Eosinophils were collected by bronchoalveolar lavage. The total bronchoalveolar lavage eosinophil count was 17- to 27-fold greater in sensitized, ovalbumin-challenged guinea pigs (9.30 +/- 0.11 x 10(6)/guinea pig) than in unsensitized guinea pigs (0.35 +/- 0.07 x 10(6)/guinea pig) or sensitized, saline-challenged guinea pigs (0.56 x 10(6)/guinea pig; n = 2). The increase in eosinophils was due to increased lavage leukocyte count and increased eosinophil differential. Eosinophils were isolated on a Percoll-plasma discontinuous gradient. Two populations of eosinophils were collected, one at the 1.093 g/ml gradient step and one at the 1.107 g/ml gradient step. Unstimulated or phorbol myristate acetate (PMA)-stimulated superoxide anion release was measured by the reduction of ferricytochrome c. Unstimulated superoxide anion release from both eosinophil populations of challenged guinea pigs (4.50 +/- 2.37 and 4.07 +/- 1.48 nmol from 1.093 and 1.107 g/ml eosinophils, respectively) was 6- to 7-fold greater than superoxide anion release from eosinophils of control guinea pigs (0.74 +/- 0.43 and 0.56 +/- 025 nmol from 1.093 and 1.107 g/ml eosinophils, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Anti-inflammatory actions of interleukin-13: suppression of tumor necrosis factor-alpha and antigen-induced leukocyte accumulation in the guinea pig lung

The Th2 cytokine interleukin (IL)-13 is believed to play an important role in the development of allergy, although it has also been ascribed anti-inflammatory roles in several experimental models. In this study, we have examined the effects of human recombinant IL-13 on eosinophilic lung inflammation in the guinea pig. IL-13 (1 to 100 ng, given by intratracheal instillation) did not elicit airway eosinophil recruitment. A pronounced accumulation of eosinophils, as well as monocyte/macrophages, was elicited by intratracheal instillation of guinea pig tumor necrosis factor alpha (gpTNF-alpha). Intratracheal administration of IL-13 (1 to 100 ng) given immediately prior to exposure to gpTNF-alpha resulted in a dose-related suppression of eosinophil and monocyte/macrophage accumulation in the airways, as assessed by bronchoalveolar lavage (BAL) and eosinophil peroxidase activity in whole-lung homogenates. IL-13 treatment also reduced BAL fluid (BALF) leukocyte accumulation induced by subsequent aerosol antigen challenge of sensitized guinea pigs. Antigen challenge also resulted in elevated levels of immunoreactive eotaxin and eosinophil-stimulating activity in BALF, although only the latter was reduced significantly by IL-13 instillation prior to challenge. In contrast to the suppressive effects of IL-13, instillation of human recombinant IL-4 (100 ng) alone elicited an increase in BALF monocyte/macrophage numbers, and IL-4 was unable to inhibit gpTNF-alpha-induced leukocyte accumulation. Hence, IL-13 (but not human IL-4) exhibits an anti-inflammatory action in the airways of gpTNF-alpha- or antigen-challenged guinea pigs, by mechanisms that may involve the decreased generation of eosinophil-stimulating activity in the airways.     

Kinetics of eosinophilia and eosinophil activation in the development of non-allergic bronchial hyperresponsiveness in guinea pigs injected with Sephadex beads

Eosinophils are believed to play a crucial role in the pathogenesis of airway hyperresponsiveness (AHR). In the present study, the involvement of blood and pulmonary eosinophilia as well as the eosinophil activation in the onset of non-allergic AHR caused by the injection of G-50 Sephadex beads in guinea pigs was investigated. Reactivity of the isolated lower bronchus to histamine was measured ex vivo in a bioassay system. The increase of reactivity of the isolated lower bronchus of Sephadex-injected animals to histamine was observed as early as 3 h after the Sephadex injection and was maximal between 6–24 h. Sephadex-induced blood eosinophilia was characterized by two successive increases of blood eosinophil counts peaking at 3 and 12 h respectively. The recruitment of inflammatory cells into the lungs, as measured in bronchoalveolar lavage fluid (BALF) have shown that the neutrophils were initially increased at 3 h whereas the number of eosinophils increased only 6 h after the bead injection; both cell populations were maximal 24 h later. Eosinophil peroxidase (EPO) activity was used as a marker for the apparent number of eosinophils in airways and the degree of activation of eosinophils recovered in BALF. Results have shown that EPO activity in the lower bronchus of Sephadex-injected animals increased at 6 h, decreased at 12 h and was maximal 24 h later. The EPO activity recovered in BALF was maximal between 6 to 24 h after the bead injection in guinea pigs. Correlation between the number of eosinophils and the EPO activity in BALF suggests that BALF eosinophils have been activated and have degranulated in airways. Correlation studies also indicated that both Sephadex-induced blood eosinophilia and eosinophil activation were associated to the development of AHR. In contrast, the increase of EPO activity in the lower bronchus and BALF eosinophilia were not correlated to the development of AHR in our model. In conclusion, our results suggest that Sephadex-induced non-allergic AHR in guinea pigs could be related, at least in part, to blood eosinophilia and eosinophil activation. Whether blood, airway and BALF eosinophilia as well as eosinophil activation are relevant factors to determine the potential role of eosinophils in the pathogenesis of AHR is discussed.     

卡介苗对豚鼠实验性哮喘的预防性治疗作用

目的:观察卡介苗(BCG)对哮喘豚鼠的预防治疗作用。方法:采用31只豚鼠,分为3组进行处理,分别为对照组、卵蛋白(OVA)致敏组和BCG处理组。用OVA(Ⅲ级)致敏豚鼠复制豚鼠哮喘模型。结果:本模型采用10%的OVA致敏,1%的OVA激发,所有动物都表现有不同程度的过敏反应症状。实验动物在接受BCG注射后,表现为以下特点:一是外周血淋巴细胞和单核细胞增加;二是BALF中细胞分类的变化,支气管肺泡灌洗液(BALF)中以淋巴细胞的增加最为明显。 经过OVA致敏的动物BALF和肺组织中嗜酸性粒细胞(EOS)明显增加,BCG不同程度地降低肺组织EOS的气道浸润及减轻OVA致敏豚鼠的气道反应。结论:[HTSS]使用本实验体系BCG可以减轻实验性哮喘的气道炎症反应。     

Synergistic effects of interleukin-4 or interleukin-13 and tumor necrosis factor-alpha on eosinophil activation in vitro

Increased concentrations of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-4, and IL-13 have been measured in bronchoalveolar lavage fluid (BALF) of patients with asthma following allergen provocation. In addition, these cytokines have also been reported to activate eosinophils in vitro. Although cytokine interactions have been postulated in the activation of eosinophils, the combined effects of cytokines on eosinophil activation remain poorly understood. Because activation of eosinophils has been regarded as a crucial event in the pathogenesis of asthmatic inflammation, we tested the hypothesis that IL-4 and IL-13 could enhance the effects of TNF-alpha on eosinophil activation. For this purpose, eosinophils from normal donors were purified and cultured in the presence of IL-4 or IL-13 and TNF-alpha. Eosinophil survival and surface expression of CD69 were assessed by flow cytometry. There was a concentration- and time-dependent upregulation in CD69 expression as well as eosinophil survival when eosinophils were incubated with IL-13, IL-4, or TNF-alpha. However, eosinophil viability and CD69 expression increased synergistically when eosinophils were incubated with IL-13 or IL-4 in the presence of TNF-alpha. This synergistic effect of IL-4 and IL-13 on CD69 expression was not limited to TNF-alpha but was also observed with IL-5. Our study provides evidence that IL-4 can activate eosinophils in a similar fashion as does IL-13. Furthermore, this study shows that the addition of IL-4 or IL-13 to TNF-alpha or IL-5 has synergistic effects on eosinophil activation, suggesting that the combined effects of different cytokines present in BALF following allergen provocation can enhance eosinophil activation in vitro.     

Effects of xiao-qing-long-tang (XQLT) on bronchoconstriction and airway eosinophil infiltration in ovalbumin-sensitized guinea pigs: in vivo and in vitro studies

BACKGROUND: Xiao-qing-long-tang (XQLT sho-seiru-to), a traditional Chinese medicine, has been used to treat patients with bronchial asthma in Oriental countries for several centuries. However, the therapeutic mechanisms of this Chinese medicine remain a matter of considerable debate. Therefore, a series of experiments using ovalbumin-sensitized guinea pigs was performed to elucidate the possible antiasthmatic effect of XQLT. METHODS: The effect of XQLT on ovalbumin-induced airway inflammation in a guinea pig model of allergic asthma was examined, and early and late asthmatic responses were measured in terms of airway resistance and extent of eosinophil infiltration. Furthermore, the bronchorelaxing effect of XQLT was measured in isolated guinea pig trachea. RESULTS: XQLT significantly inhibited the antigen-induced immediate asthmatic response (IAR) and late asthmatic response (LAR) in actively sensitized guinea pigs. Cumulative administration of XQLT caused concentration-dependent relaxation of the carbachol-precontracted guinea pig trachea. The bronchorelaxing effect of XQLT was reversed by ICI-118551, a selective beta2-adrenoceptor antagonist. Furthermore, examination of bronchoalveolar lavage fluid (BALF) revealed that XQLT significantly suppressed the increase in eosinophils (24 h after antigen challenge) in the airway. In addition, XQLT significantly attenuated the increase in eosinophils at 1, 6, 24, 48, and 72 h after antigen challenge when it was administered once daily from the day of sensitization to the day of challenge. Histopathologic examination results showed that XQLT suppressed eosinophil infiltration into lung tissue. CONCLUSIONS: These results demonstrate that the antiasthmatic effects of XQLT appear to be partly mediated by stimulation of beta2-adrenoceptors, leading to bronchorelaxation, and that XQLT inhibits the infiltration of eosinophils into the airway. Thus, XQLT may be useful for the prevention or treatment of asthma.     

Expression and regulation of intercellular adhesion molecule-1 on airway parasympathetic nerves

BACKGROUND: Eosinophils cluster along airway nerves in patients with asthma and release eosinophil major basic protein, an antagonist of inhibitory M2 muscarinic receptors on nerves. Blocking M2 function increases bronchoconstriction, leading to airway hyperreactivity. Intercellular adhesion molecule-1 (ICAM-1) mediates eosinophil adhesion to nerves. OBJECTIVE: We investigated mechanisms of ICAM-1 expression by parasympathetic nerves. METHODS: ICAM-1 expression was examined by immunocytochemistry of lung sections from ovalbumin-sensitized and challenged guinea pigs. ICAM-1 was measured in parasympathetic nerves isolated from subjects and guinea pigs and in human neuroblastoma cells by real-time RT-PCR, immunocytochemistry, and Western blot. RESULTS: ICAM-1 was not detected in control airway parasympatheric nerves in vivo or in cultured cells. ICAM-1 was expressed throughout antigen-challenged guinea pig lung tissue and was selectively decreased by dexamethasone only in nerves. ICAM-1 was induced in human and guinea pig parasympathetic nerves by TNF-alpha and IFN-gamma and was inhibited by dexamethasone and by an inhibitor of nuclear factor-kappaB (NF-kappaB). In neuroblastoma cell lines TNF-alpha and IFN-gamma-induced ICAM-1 was blocked by an inhibitor of NF-kappaB but not by inhibitors of mitogen-activated protein kinases. Dexamethasone did not inhibit ICAM-1 expression in neuroblastoma cells. CONCLUSIONS: ICAM-1 induced in nerves by antigen challenge and proinflammatory cytokines is sensitive to dexamethasone. ICAM-1 expression is also sensitive to inhibitors of NF-kappaB. Neuroblastoma cells mimic many, but not all, characteristics of ICAM-1 expression in parasympathetic nerves. CLINICAL IMPLICATIONS: Dexamethasone and NF-kappaB inhibitors could prevent eosinophils from adhering to nerves by blocking ICAM-1 expression on parasympathetic nerves, thus protecting inhibitory M2 muscarinic receptors and making this pathway a potential target for asthma treatment.     

Effects of Ding-Chuan-Tang on Bronchoconstriction and Airway Leucocyte Infiltration in Sensitized Guinea Pigs

Ding-Chuan-Tang (DCT), a traditional Chinese medicine, has been used in treatment of the bronchial asthma for several centuries. However, the therapeutic mechanism of these Chinese medicine are still far from clear. To understand the mechanism of antiasthmatic property of DCT. A guinea pig model of allergic asthma was used to investigate the effects of DCT on ovalbumin-induced early and late asthmatic responses and airway inflammation, particularly the extent of eosinophil infiltration, and examine it direct β2-adrenoceptor agonist activity in guinea-pig isolated trachea. We had used three different protocals in ovalbumin sensitized guinea pigs by administrating 10 g/kg of DCT extracts to sensitized guinea pigs 30 min before antigen challenge (group I), 5 hr after antigen challenge (group II) and 2.5 g/kg once daily from the day of sensitization to the day of challenge. Our result showed that administration of DCT singificantly inhibited the antigen induced immediate asthmatic responses (IAR) in group I and inhibited both IRA and late asthmatic responses (LAR) in actively sensitized guinea pig in group III. DCT caused concentration-dependent relaxations in strips of guinea pig trachea contracted with carbachol, however ICI-118551, a selective β2-adrenoceptor antagonist, didn't significantly competitively inhibit the relaxations caused by DCT. Furthermore, examination of bronchoalveolar lavage fluid (BALF) revealed that DCT significantly inhibited the increase in percent of eosinophils in the airway after antigen challenge in three group. Histopathologic examination showed DCT suppressed the eosinophil infiltration into lung tissue. These results suggest that the antiasthmatic effect of DCT is mainly due to its bronchodilatation effect and its ability to inhibit the eosinophil into the airway and there is prophylactic effect of DCT on allergen-induced airway inflammation.     

Effects of Ding-Chuan-Tang on bronchoconstriction and airway leucocyte infiltration in sensitized guinea pigs

Ding-Chuan-Tang (DCT), a traditional Chinese medicine, has been used in treatment of the bronchial asthma for several centuries. However, the therapeutic mechanism of these Chinese medicine are still far from clear. To understand the mechanism of antiasthmatic property of DCT. A guinea pig model of allergic asthma was used to investigate the effects of DCT on ovalbumin-induced early and late asthmatic responses and airway inflammation, particularly the extent of eosinophil infiltration, and examine it direct beta2-adrenoceptor agonist activity in guinea-pig isolated trachea. We had used three different protocals in ovalbumin sensitized guinea pigs by administrating 10 g/kg of DCT extracts to sensitized guinea pigs 30 min before antigen challenge (group I), 5 hr after antigen challenge (group II) and 2.5 g/kg once daily from the day of sensitization to the day of challenge. Our result showed that administration of DCT singificantly inhibited the antigen induced immediate asthmatic responses (IAR) in group I and inhibited both IRA and late asthmatic responses (LAR) in actively sensitized guinea pig in group III. DCT caused concentration-dependent relaxations in strips of guinea pig trachea contracted with carbachol, however ICI-118551, a selective beta2-adrenoceptor antagonist, didn't significantly competitively inhibit the relaxations caused by DCT. Furthermore, examination of bronchoalveolar lavage fluid (BALF) revealed that DCT significantly inhibited the increase in percent of eosinophils in the airway after antigen challenge in three group. Histopathologic examination showed DCT suppressed the eosinophil infiltration into lung tissue. These results suggest that the antiasthmatic effect of DCT is mainly due to its bronchodilatation effect and its ability to inhibit the eosinophil into the airway and there is prophylactic effect of DCT on allergen-induced airway inflammation.     

Human eosinophils induce histamine release from antigen-activated rat peritoneal mast cells: a possible role for mast cells in late-phase allergic reactions

BACKGROUND: Mast cells and eosinophils are believed to interact during the late and the chronic stages of allergic inflammation. OBJECTIVE: In this study we investigated whether eosinophils can cause activation and consequent histamine release of already challenged mast cells, a situation likely to take place during the allergic late-phase reaction. METHODS: Rat peritoneal mast cells presensitized with IgE anti-dinitrophenol-human serum albumin and challenged by dinitrophenol-human serum albumin or compound 48/80 were incubated with either eosinophil sonicate or major basic protein (MBP). Eosinophils were purified from the peripheral (>98%) blood of mildly allergic patients. Heparin and pertussis toxin and different extracellular Ca(2+) concentrations were used to modulate mast cell reactivation by MBP. Histamine release was assessed as a marker of mast cell activation. RESULTS: IgE-challenged mast cells were sensitive to reactivation induced by eosinophil sonicate and MBP. Reactivation was not cytotoxic for the mast cells. Mast cells previously challenged with compound 48/80 did not respond to subsequent MBP activation. Furthermore, heparin and pertussis toxin both inhibited mast cell reactivation induced by MBP. The ability of eosinophil sonicate and MBP to activate mast cells was not significantly affected at the different Ca(2+) concentrations. CONCLUSIONS: In summary, we have shown a direct activating activity of eosinophils, partially due to MBP, toward IgE-challenged and immunologically desensitized mast cells. This suggests that in vivo mast cells can be reactivated during a late-phase reaction to release histamine by a non-IgE-dependent mechanism.     

Eotaxin levels and eosinophils in guinea pig broncho-alveolar lavage fluid are increased at the onset of a viral respiratory infection

In previous studies we found that guinea pigs demonstrate an increase in airway reactivity and eosinophil numbers 4 days after a respiratory infection with parainfluenza-3 (PI3) virus. Clinical data support the possible involvement of eosinophils in virus-induced airway hyperresponsiveness. Eotaxin, a newly discovered chemokine, could be involved in eosinophil migration to the airways. In this study, eosinophil numbers were counted in blood and bronchoalveolar lavage (BAL) fluid and related with eotaxin concentrations in BAL fluid 1, 2, 3, and 4 days after intratracheal PI3 virus administration. On day 1, blood eosinophils increased by more than 200% ( P  < 0.01). The number of eosinophils were only slightly enhanced from day 2 to day 4 (40%–70%). BAL fluid eosinophils were not increased on day 1 but were significantly elevated on day 2 (180%) and remained high on days 3–4 (>300%, P  < 0.05). This increase in lung eosinophils correlated well with eotaxin levels measured in BAL fluid. There was no significant increase in eotaxin on day 1 following PI3 infection; however, on days 2–4 eotaxin levels in BAL fluid were significantly elevated (four–sixfold increase) when compared with medium inoculated controls. Eotaxin appears to play an important role in eosinophil accumulation in guinea pig lung following PI3 infection.     

Eosinophil degranulation in the allergic lung of mice primarily occurs in the airway lumen

Eosinophil degranulation is thought to play a pivotal role in the pathogenesis of allergic disorders. Although mouse models of allergic disorders have been used extensively to identify the contribution of eosinophils to disease, ultrastructural evidence of active granule disassembly has not been reported. In this investigation, we characterized the degree of eosinophil activation in the bone marrow, blood, lung tissue, and airways lumen [bronchoalveolar lavage fluid (BALF)] of ovalbumin-sensitized and aero-challenged wild-type and interleukin-5 transgenic mice. Degranulation was most prominent in and primarily compartmentalized to the airways lumen. Eosinophils released granule proteins by the process of piecemeal degranulation (PMD). Accordingly, recruitment and activation of eosinophils in the lung correlated with the detection of cell-free eosinophil peroxidase in BALF and with the induction of airways hyper-reactivity. As in previous studies with human eosinophils, degranulation of isolated mouse cells did not occur until after adherence to extracellular matrix. However, higher concentrations of exogenous stimuli appear to be required to trigger adherence and degranulation (piecemeal) of mouse eosinophils when compared with values reported for studies of human eosinophils. Thus, mouse eosinophils undergo PMD during allergic inflammation, and in turn, this process may contribute to pathogenesis. However, the degranulation process in the allergic lung of mice is primarily compartmentalized to the airway lumen. Understanding the mechanism of eosinophil degranulation in the airway lumen may provide important insights into how this process occurs in human respiratory diseases.     

Guinea pig lung eosinophil: purification and prostaglandin production

Guinea pig lung eosinophils have been purified from bronchoalveolar lavage (BAL) fluid, and the production of prostaglandin E2 (PGE2), thromboxane B2 (TXB2), and 6-keto-PGF1 alpha was measured after stimulation with phorbol myristate acetate (PMA), platelet-activating factor (PAF), and formyl-methionyl-leucyl-phenylalanine (fMLP). A combination of plating and discontinuous Percoll gradient centrifugation was used to purify eosinophils. The purity of eosinophils in fraction E (interface between Percoll density 1.057-1.068) was around 96%, and the viability was 99%, with a mean yield of 2.7 x 10(6) cells per guinea pig. Similarly, in fraction D (interface between Percoll density 1.047-1.057), the mean purity of eosinophils was 76% and the viability of cells was 99%, with a mean yield of 1.1 x 10(6) cells per guinea pig. Purified eosinophils produced TXB2 predominantly after stimulation with PMA, fMLP, and PAF. The 6-keto-PGF1 alpha was slightly increased in cell supernatants after stimulation with PMA and fMLP, but PGE2 was not elevated with any stimulus. The highly purified eosinophils of fraction E generated amounts of TXB2 and 6-keto-PGF1 alpha similar to the amount generated by eosinophils contaminated with some macrophages (fraction D). These results suggest a role for eosinophils in the production of TXA2 by guinea pig lung.     

Identification of interleukin-2 in human peripheral blood eosinophils.

Interleukin-2 (IL-2) is an essential growth factor for T cells. Previous studies have shown that human peripheral eosinophils respond to IL-2 in chemotaxis and express the IL-2 receptor (CD25). In addition, eosinophils have been shown to transcribe messenger RNA for IL-2. The aim of the present study was to determine whether eosinophils translate mRNA for IL-2 and to determine the site of intracellular localization. By immunocytochemistry, an average of 9% of cells showed cytoplasmic staining for IL-2 in freshly isolated unstimulated blood eosinophils obtained from asthmatic subjects who were not receiving oral corticosteroid treatment (n = 5). Freshly isolated, disrupted, highly purified eosinophils (> 99%, by CD16- immunomagnetic selection) contained an average of 6 pg/10(6) cells of IL-2 measured by a specific enzyme linked immunosorbent assay (ELISA) (n = 7). Purified eosinophil incubated with serum-coated Sephadex beads showed an increase in the amount of intracellularly-retained IL-2 (26.2 +/- 7.2 pg/10(6) cells) with some evidence for release of this cytokine but only in three out of six eosinophil preparations (range 1.3-5.8 pg/10(6) cells). The intracellular localization of IL-2 was determined by fractionation of the cells on a linear (0-45%) Nycodenz gradient in sucrose buffer followed by detection of IL-2 in the fractions using an IL-2-specific ELISA and dot blotting. The majority of the IL-2 detected co-eluted with known eosinophil granule markers (i.e. major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil peroxidase (EPO) and beta-hexosaminidase) but small quantities were also detected in the cytosolic (lactate dehydrogenase-(LDH) associated) and membrane (CD9+) fractions. Immunogold labelling of intact eosinophils using an anti-IL-2 monoclonal antibody confirmed IL-2 immunoreactivity in association with the eosinophil crystalline granule cores. These data are consistent with the hypothesis that eosinophils synthesize, release and store IL-2 largely within cystalloid granules. This stored IL-2 may serve as a reservoir for rapid release of IL-2 in inflammatory reactions associated with eosinophilia.     

IL-27对哮喘小鼠气道炎症的影响

目的研究IL-27对卵白蛋白(OVA)激发哮喘小鼠气道炎症的影响。方法 24只雌性BALB/c小鼠随机分为生理盐水组、哮喘组及IL-27组,每组8只。应用OVA建立哮喘模型,IL-27组小鼠应用1μgIL-27(溶于50μlPBS中)滴鼻给药,观察3组小鼠肺组织病理改变,计数支气管肺泡灌洗液(BALF)中嗜酸性粒细胞;ELISA法测定小鼠BALF中IL-4和IFN-γ浓度,RT-PCR测定肺组织T-bet mRNA的表达量。结果 IL-27组小鼠肺组织炎症反应明显轻于哮喘组小鼠;IL-27组小鼠BALF中嗜酸性粒细胞计数为(2.21±0.33)×107/L明显低于哮喘组的(12.82±2.17)×107/L(P0.01);IL-27组小鼠BALF中IL-4浓度为(20.4±3.2)μg/L,明显低于哮喘组的(61.3±13.1)μg/L(P0.05);IL-27组小鼠BALF中IFN-γ浓度为(50.3±6.3)μg/L,明显高于哮喘组的(11.1±3.3)μg/L(P0.05);IL-27组小鼠肺组织T-bet mRNA表达量(吸光度积分比值)为(0.268±0.048),明显高于哮喘组的(0.130±0.012)(P0.05)。结论 IL-27可能通过增强T-bet mRNA的表达增强Th1反应,减少BALF中嗜酸性粒细胞数量,进而减轻了哮喘小鼠肺组织炎症反应。     

活菌卡介苗对哮喘小鼠IL-17的调节作用

目的初步探讨活菌卡介苗（BCG）对哮喘小鼠IL-17的调节作用。方法 4周龄雌性Balb/c小鼠30只随机分为A组（对照组）、B组（哮喘组）和C组（BCG干预组）,每组各10只。B、C两组予OVA腹腔注射致敏、OVA雾化诱发哮喘,C组在致敏前14d接种BCG。HE染色观察小鼠肺部病理改变,计数支气管肺泡灌洗液（bronchoalveolar lavage fluid,BALF）中细胞总数并分类,酶联免疫吸附试验（ELISA）检测血清及BALF中白介素-17（IL-17）含量。结果肺组织病理观察显示A组气管周围基本无炎症细胞浸润,B组支气管周围大量炎症细胞浸润,杯状细胞增生,C组炎症较B组减轻。B、C组BALF中细胞总数和嗜酸性粒细胞、淋巴细胞百分率均显著高于对照组（P〈0.01）,而C组嗜酸性粒细胞比例则明显低于B组（P〈0.01）。B、C组小鼠血清、BALF中IL-17含量高于A组（P〈0.01）,而C组则明显低于B组（P〈0.01）。各组小鼠血清及BALF中IL-17水平与BALF中嗜酸性粒细胞呈正相关（P〈0.01）。结论 BCG接种可抑制IL-17的产生,减轻哮喘小鼠气道炎症反应。     

A sensitive high throughput ELISA for human eosinophil peroxidase: A specific assay to quantify eosinophil degranulation from patient-derived sources

Quantitative high throughput assays of eosinophil-mediated activities in fluid samples from patients in a clinical setting have been limited to ELISA assessments for the presence of the prominent granule ribonucleases, ECP and EDN. However, the demonstration that these ribonucleases are expressed by leukocytes other than eosinophils, as well as cells of non-hematopoietic origin, limits the usefulness of these assays. Two novel monoclonal antibodies recognizing eosinophil peroxidase (EPX) were used to develop an eosinophil-specific and sensitive sandwich ELISA. The sensitivity of this EPX-based ELISA was shown to be similar to that of the commercially available ELISA kits for ECP and EDN. More importantly, evidence is also presented confirming that among these granule protein detection options, EPX-based ELISA is the only eosinophil-specific assay. The utility of this high throughput assay to detect released EPX was shown in ex vivo degranulation studies with isolated human eosinophils. In addition, EPX-based ELISA was used to detect and quantify eosinophil degranulation in several in vivo patient settings, including bronchoalveolar lavage fluid obtained following segmental allergen challenge of subjects with allergic asthma, induced sputum derived from respiratory subjects following hypotonic saline inhalation, and nasal lavage of chronic rhinosinusitis patients. This unique EPX-based ELISA thus provides an eosinophil-specific assay that is sensitive, reproducible, and quantitative. In addition, this assay is adaptable to high throughput formats (e.g., automated assays utilizing microtiter plates) using the diverse patient fluid samples typically available in research and clinical settings.     

A novel antagonist of CRTH2 blocks eosinophil release from bone marrow, chemotaxis and respiratory burst

BACKGROUND: Chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) has been revealed to be a novel receptor for prostaglandin (PG) D(2), which is a major mast cell product released during the allergic response. The aim of this study was to analyze the effects of a newly developed small molecule antagonist of CRTH2, Cay10471, on eosinophil function with respect to recruitment, respiratory burst and degranulation. METHODS: Chemotaxis of guinea pig bone marrow eosinophils and human peripheral blood eosinophils were determined using microBoyden chambers. Eosinophil release from bone marrow was investigated in the in situ perfused guinea pig hind limb preparation. Respiratory burst and degranulation were measured by flow cytometry. RESULTS: Cay10471 bound with high affinity to recombinant human and guinea pig CRTH2, but not DP, receptors. The antagonist prevented the PGD(2)-induced release of eosinophils from guinea pig bone marrow, and inhibited the chemotaxis of guinea pig bone marrow eosinophils and human peripheral blood eosinophils. Pretreatment with PGD(2) primed eosinophils for chemotaxis towards eotaxin, and this effect was prevented by Cay10471. In contrast, PGD(2) inhibited the C5a-induced up-regulation of CD63, a cellular marker of degranulation, in a Cay10471-sensitive manner. Finally, Cay10471 abolished the respiratory burst of eosinophils upon stimulation by PGD(2). CONCLUSION: These data further emphasize the importance of CRTH2 in eosinophil function and show that Cay10471 is a highly potent and selective antagonist of PGD(2)-induced eosinophil responses. Cay10471 might hence be a useful compound for the treatment of allergic diseases.     

卵清蛋白雾化吸入诱导肠道菌群失调小鼠肺部过敏反应的发生

目的:在抗生素所致肠道菌群失调小鼠模型上采用卵清蛋白(OVA)雾化吸入激发,探讨气道变应性反应与肠道菌群失调的关系.方法:112只BALB/c小鼠分为6组:菌群失调Ⅰ组、对照Ⅰ组、菌群失调Ⅱ组、菌群失调加激发组、激发组、对照Ⅱ组.前2组和后4组分别于第6天和第14天时取盲肠内容物做细菌培养计数,后4组同时行支气管肺泡灌洗液(BALF)细胞分类计数、BALF和血清中OVA特异性IgE(OVA-sIgE)检测以及肺部病理观察;采用流式细胞术检测肺组织中Th1以及Th2细胞水平.结果:应用抗生素的小鼠出现肠道菌群失调,菌群失调加激发组小鼠肺部出现以嗜酸粒细胞和淋巴细胞为主的炎症细胞浸润,肺部黏液分泌明显增高,BALF中细胞总数、淋巴细胞、嗜酸粒细胞和中性粒细胞增加,OVA-sIgE水平显著增高,肺部Th2细胞水平增高,Th1细胞与对照组无差异.结论:抗生素致肠道菌群失调小鼠经过OVA雾化吸入后,可产生Th2细胞优势的变应性气道反应,提示抗生素所致肠道菌群失调是诱发哮喘等变应性疾病的危险因素之一.