Limonene and its ozone-initiated reaction products attenuate allergic lung inflammation in mice

Inhalation of indoor air pollutants may cause airway irritation and inflammation and is suspected to worsen allergic reactions. Inflammation may be due to mucosal damage, upper (sensory) and lower (pulmonary) airway irritation due to activation of the trigeminal and vagal nerves, respectively, and to neurogenic inflammation. The terpene, d-limonene, is used as a fragrance in numerous consumer products. When limonene reacts with the pulmonary irritant ozone, a complex mixture of gas and particle phase products is formed, which causes sensory irritation. This study investigated whether limonene, ozone or the reaction mixture can exacerbate allergic lung inflammation and whether airway irritation is enhanced in allergic BALB/cJ mice. Naïve and allergic (ovalbumin sensitized) mice were exposed via inhalation for three consecutive days to clean air, ozone, limonene or an ozone–limonene reaction mixture. Sensory and pulmonary irritation was investigated in addition to ovalbumin-specific antibodies, inflammatory cells, total protein and surfactant protein D in bronchoalveolar lavage fluid and hemeoxygenase-1 and cytokines in lung tissue. Overall, airway allergy was not exacerbated by any of the exposures. In contrast, it was found that limonene and the ozone–limonene reaction mixture reduced allergic inflammation possibly due to antioxidant properties. Ozone induced sensory irritation in both naïve and allergic mice. However, allergic but not naïve mice were protected from pulmonary irritation induced by ozone. This study showed that irritation responses might be modulated by airway allergy. However, aggravation of allergic symptoms was observed by neither exposure to ozone nor exposure to ozone-initiated limonene reaction products. In contrast, anti-inflammatory properties of the tested limonene-containing pollutants might attenuate airway allergy.     

Deficiency of regulatory B cells increases allergic airway inflammation

Objective: To investigate the effect of the X-linked immunodeficiency (Xid) B cell defect on the response to the cockroach allergen in mice. Methods: Two cockroach allergen immunization and challenge protocols were employed to sensitize CBA/J wild-type and CBA/CaHN-btk(-/-)xid/J (Xid) mice. Blood and tissue samples were collected 24 and 48 hrs after the last intratracheal antigen challenge and were analyzed for several parameters of allergic inflammation. Results: Nearly equivalent amounts of serum IgE were detected in Xid and CBA/J mice after short-term antigen challenge despite the B cell deficiency in Xid mice. A decreased concentration of IgE was detected in CBA/J mice after repeated allergen challenges but not in the Xid mice. Correlating with the discrepancy in serum IgE levels, higher levels of IL-13, IL-5, IL-10 and CCL5 were measured in whole lung homogenates from allergen-challenged Xid mice compared to CBA/J mice. In addition, draining lymph node cells from Xid mice expressed elevated levels of IL-4, IL-5, IL-10 and IFNγ mRNA compared to cells from CBA/J mice after in vitro culture with cockroach antigen. An increase in lung inflammation, interstitial eosinophilia and mucus production was also observed in allergen-challenged Xid mice. CD95L expression increased on B-1a cells following allergen challenge, which was accompanied by an increase in lung CD4⁺ Th cell apoptosis in wild-type CBA/J mice. In contrast, Xid mice did not have an increase in CD4⁺ T cell apoptosis following allergen challenge. Conclusions: These data suggest a regulatory role for B-1a cells in reducing cytokine production, pulmonary inflammation, and CD4⁺ T cell survival during cockroach allergen-induced airway inflammation. Received 10 June 2005; returned for revision 13 September 2005; accepted by M. Parnham 14 September 2005 Funding source: U. S. Government NIH Grant# AI36302     

Epstein-Barr virus, B cell lymphoproliferative disease, and SCID mice: modeling T cell immunotherapy in vivo

Epstein‐Barr virus (EBV)‐associated post‐transplant lymphoproliferative disease (PTLD) arises in up to 10% of organ transplant recipients and is fatal in ～50% of cases. PTLD can be modeled in SCID mice using EBV+ve human B lymphoblastoid cell lines (BLCLs), and the current study investigated intraperitoneal (ip) inoculation of such animals in experiments which assessed the effect of EBV‐specific cytotoxic T lymphocytes (CTLs) and cytokines on PTLD growth. Ip transfer of one dose of autologous CTLs, or CD8‐enriched T cells, into ip BLCL‐inoculated animals significantly delayed tumor development (P = 0.001) and prevented tumor formation in a significant proportion (40%) of mice (P = 0.001). A combination of interleukin (IL)2, 7, and 15 conditioning of CTLs prior to ip injection significantly delayed ip BLCL‐derived tumor formation in vivo when compared to CTLs expanded in vitro using only IL2 (P = 0.04) and prevented tumor outgrowth in a significant proportion (60%) of mice (P = 0.02). Daily ip IL2 dosing of ip CTL‐inoculated mice significantly delayed tumor development in vivo (P = 0.004) and prevented tumor outgrowth in a significant proportion (78%) of mice (P = 0.02) when compared to animals dosed with vehicle only. In SCID mice, autologous CTLs, and CD8‐enriched T cells, have significant capacity to hinder development of PTLD‐like tumors. Whilst studies are needed to delineate the role of cytokine conditioning and CD4‐enriched T cells, the results suggest that IL2 plays a key role in supporting CTL funtion in vivo. J. Med. Virol. 83:1585–1596, 2011. © 2011 Wiley‐Liss, Inc.     

Mice deficient in heparanase exhibit impaired dendritic cell migration and reduced airway inflammation

Heparanase is a β‐d ‐endoglucuronidase that cleaves heparan sulphate, a key component of the ECM and basement membrane. The remodelling of the ECM by heparanase has been proposed to regulate both normal physiological and pathological processes, including wound healing, inflammation, tumour angiogenesis and cell migration. Heparanase is also known to exhibit non‐enzymatic functions by regulating cell adhesion, cell signalling and differentiation. In this study, constitutive heparanase‐deficient (Hpse^?/?) mice were generated on a C57BL/6 background using the Cre/loxP recombination system, with a complete lack of heparanase mRNA, protein and activity. Although heparanase has been implicated in embryogenesis and development, Hpse^?/? mice are anatomically normal and fertile. Interestingly, consistent with the suggested function of heparanase in cell migration, the trafficking of dendritic cells from the skin to the draining lymph nodes was markedly reduced in Hpse^?/? mice. Furthermore, the ability of Hpse^?/? mice to generate an allergic inflammatory response in the airways, a process that requires dendritic cell migration, was also impaired. These findings establish an important role for heparanase in immunity and identify the enzyme as a potential target for regulation of an immune response.     

feG-COOH blunts eosinophilic airway inflammation in a feline model of allergic asthma

Objective and design  This study investigated if feG-COOH would decrease allergen-induced airway inflammation. Materials or subjects  Seven adult cats sensitised to Bermuda grass allergen (BGA) to induce an asthmatic phenotype. Treatment  Cats were randomized to receive either feG-COOH (1 mg/kg, PO) or placebo (saline 1 ml, PO) immediately prior to BGA aerosol challenge in a cross-over design. Methods  Bronchoalveolar lavage fluid (BALF) was collected and airway inflammatory response assessed via inflammatory cell number and type; IL-4, IFN-γ and nitric oxide metabolite concentrations. A paired t test was used to compare parameters with a P < 0.05 considered significant. Results  The BALF eosinophil percentage was significantly lower in asthmatic cats treated with feG compared with placebo (placebo, 35.3 ± 12.2%; feG, 22.4 ± 8.6%; P = 0.002). Treatment with feG did not result in a significant change in any other parameter measured. Conclusions  These data indicate that a single dose of feG-COOH partially attenuates eosinophilic airway inflammation in experimental feline asthma.     

乳胶致小鼠气道炎症的研究

目的：以乳胶为致敏原，建立具有哮喘主要特征的小鼠模型，研究乳胶与气道炎症之间的关系。方法：乳胶腹腔注射与滴鼻诱发BALB／C小鼠气道炎症，在激发后2，4小时行支气管肺泡灌洗液(BALF)细胞计数分类、肺组织病理检查、血清总IgE及乳胶特异性IgE(sIgE)测定并检测IL-5、IFN-γ的表达。结果：乳胶诱发后，实验组小鼠BALF中细胞总数及嗜酸粒细胞百分比升高；肺组织病理切片示支气管上皮增生、脱落，粘液腺分泌现象，支气管痉挛、收缩，炎症细胞浸润；血清总IgE及乳胶sIgE水平升高；BALF及肺组织局部IFN-γ减少，IL-5增高。结论：用乳胶蛋白作为致敏原，采用腹腔致敏与多次滴鼻激发可使BALB/C小鼠发生气道炎症，具有与变应原诱导的人类哮喘迟发反应相一致的主要特征：气道变应性炎症；外周血IgE浓度升高；肺组织中Th2型CK表达占优势。     

CD28 costimulation is critical for experimental allergic asthma in HLA-DQ8 transgenic mice

The objective of this study was to investigate the contribution of the CD28 costimulatory molecules to allergen-induced primary and chronic inflammatory responses. To this end, we have developed and characterized a short ragweed allergen-induced asthma model involving sensitization of HLA-DQ transgenic mice followed by intranasal challenge with allergen. Forty-eight hours after primary challenge, sensitized DQ8 mice developed pulmonary eosinophilic inflammation, airway hyperreactivity, Th2 cytokines, and IgE/IgG1 Ab. This allergic inflammatory response was absent in H-2Abeta(0) and DQ8/CD28(0) mice. Secondary rechallenge with allergen 4 weeks later induced even greater inflammatory changes in the airways of DQ8 mice with eosinophils being the predominant inflammatory cells while only pulmonary lymphocytosis was observed in DQ8/CD28(0) mice. No inflammation was detected in H-2Abeta(0) mice. Proliferation and cytokine profile studies demonstrated that CD28 regulates T-cell activation and effector function. Therefore, CD28 is essential for the extrinsic asthma and can be a target for immunotherapy.     

TLR2胞外段特异性抗体对TLR2激动剂诱导的炎症和促过敏反应的抑制作用

目的:观察小鼠TLR2胞外段抗原肽特异性抗体对TLR2激动剂诱导的炎症和促过敏反应的影响。方法:用小鼠TLR2胞外段单表位抗原肽(T20)免疫动物制备抗体(T20抗体);体外观察T20抗体对PGN和LTA刺激RAW264.7细胞生成TNF-α和IL-6的影响,用ELISA法检测各组TNF-α和IL-6量;体内观察T20抗体对OVA致敏小鼠PGN致死性攻击的影响,记录各组直肠温度变化和死亡率。结果:获得小鼠TLR2特异性T20抗体;该抗体可抑制TLR2激动剂(PGN和LTA)对相应靶细胞的刺激作用,使其生成的TNF-α和IL-6显著降低;体内实验证实T20抗体对PGN加剧的过敏反应具有抑制作用。结论:T20抗体可特异结合小鼠TLR2,抑制TLR2激动剂诱导的炎症及TLR2激动剂加剧的过敏反应。     

Maturation of mast cell progenitors to mucosal mast cells during allergic pulmonary inflammation in mice

In contrast to resident constitutive mast cells (CMCs), mucosal MCs (MMCs) appear in the lung and trachea of sensitized mice only following inhalation challenge. We monitored the influx and maturation of MCs by their expression of Kit, FcɛRI, β7-integrin and side scatter (SSC) by flow cytometry. Influx of MC progenitors (MCps) (FcɛRI^lo, Kit^int, β7^hi, and SSC^lo) peaks 1 day after challenges and subsides to baseline by day 7 after challenge. The mature MMCs appear as a distinct population on day 7 and peak at day 14 with higher SSC and FcɛRI expression, but lower β7 and Kit expression. A distinct transitional population is present between 1 and 7 days after challenge. Maturation occurs more rapidly in the trachea. The resident tracheal CMCs had higher SSC, FcɛRI, and Kit and lower β7-integrin expression than the MMCs. By histology, the MMCs follow similar kinetics to the flow cytometry-identified mature MMCs and are notably persistent for >42 days. Steroid treatment reduced inflammation and MCp influx but had no effect on established MMCs. Thus, changes in SSC, FcɛRI, and Kit together with the expression of αE/α4:β7-integrins characterizes the development of induced MMCs from MCps and distinguishes them from resident CMCs in the trachea and large airways.     

Serum-derived exosomes from antigen-fed mice prevent allergic sensitization in a model of allergic asthma

Oral tolerance is an active process that starts with sampling of luminal antigens by the intestinal epithelial cells (IEC), followed by processing and assembly with major histocompatibility complex class II and subsequently a release of tolerogenic exosomes (tolerosomes) from the IEC. We have previously shown that tolerosomes can be isolated from serum shortly after an antigen feed, and will potently transfer antigen-specific tolerance to naive recipients. Here we study the capacity of the tolerosomes to protect against allergic sensitization in a mouse model of allergic asthma. Serum or isolated serum exosomes from tolerized BALB/c donor mice were transferred to syngeneic recipients followed by sensitization and intranasal exposure to ovalbumin (OVA). Blood, bronchoalveolar lavage (BAL) and lymph nodes were sampled 24 hr after the final exposure. The number of eosinophils was counted in BAL fluid and the levels of immunoglobulin E (IgE) and OVA-specific IgE were measured in serum. Mediastinal and coeliac lymph nodes were analysed by flow cytometry. The animals receiving serum from OVA-fed mice displayed significantly lower numbers of airway eosinophils and lower serum levels of total IgE as well as of OVA-specific IgE compared with controls. Moreover, the tolerant animals showed a significantly higher frequency of activated T cells with a regulatory phenotype in both mediastinal and coeliac lymph nodes. The results show that serum or isolated serum exosomes obtained from OVA-fed mice and administered intraperitoneally to naive recipient mice abrogated allergic sensitization in the recipients.     

In vitro preactivated human T cells engraft in SCID mice and migrate to murine lymphoid tissues.

Mice with severe combined immunodeficiency (SCID) accept grafts of human T and B lymphocytes derived from resting peripheral blood mononuclear cells (PBMC). We wished to determine whether activated human T cells engraft and migrate into lymphoid tissues in SCID mice. PBMC (50 x 10(6)) activated in vitro in a 4-day mixed lymphocyte culture (MLC) were injected into the peritoneum of 12 SCID mice. In 11 of 12 animals killed at 3 or 4 weeks after injection, human cells were detected in cells pooled from lymphoid organs by flow cytometry and by immunohistochemical staining of frozen tissue sections. The percentage of CD45+ cells in the 11 mice ranged from 2% to 45% and the absolute numbers of CD45+ cells recovered from lymphoid organs ranged from 4 x 10(6) to 90 x 10(6). Up to 93% of the human cells expressed the CD3 antigen together with either CD4 or CD8. Human T cells were localized in periarteriolar areas in murine spleens, whereas in the lymph nodes and gut mucosa, the T cells did not show the pattern for T-dependent areas found in human lymphoid tissue. Numerous human plasma cells were detected in the spleen and gut mucosal crypts of engrafted SCID mice. Human IgG was detected in the serum of all 11 engrafted SCID mice. The functional activity of human T cells recovered from murine splenic tissue was very low 3-4 weeks after engraftment.     

Role of nerve growth factor in a mouse model of allergic airway inflammation and asthma

The role of nerve growth factor (NGF), a potent mediator acting in the development and differentitation of both neuronal and immune cells, was examined in a mouse model of allergic asthma. NGF-positive cells were detected in the inflammatory infiltrate of the lung and enhanced levels of NGF were detected in serum and broncho-alveolar lavage fluids. Mononuclear cells in inflamed airway mucosa as well as broncho-alveolar macrophages were identified as one source of NGF production. Splenic mononuclear cells from allergen-sensitized mice produced NGF in response to allergen. They responded to exogenously added NGF with a dose-dependent increase in IL-4 and IL-5 production and augmented IgE and IgG₁ synthesis. In contrast, IFN-γ and IgG_2a levels remained unaffected. The effects were NGF specific, since they could be blocked by an anti-NGF-antibody. Nasal application of anti-NGF to allergen-sensitized mice significantly reduced IL-4 and prevented development of airway hyperreactivity. These results show that allergic airway inflammation is accompanied by enhanced local NGF production that acts as an amplifier for Th2 effector functions and plays an important role in the development of airway hyperreactivity. Therefore it is suggested that NGF may serve as a link between the immune and nerve system.     

Patterns of mast cell alterations and in vivo mediator release in human allergic skin reactions

Patterns of in vivo histamine release in skin sites challenged with ragweed antigen were compared in five human subjects sensitive to this antigen and four nonallergic individuals, using a newly developed skin-chamber technique. These findings were compared with inflammatory cell responses in the reaction sites and patterns of ultramicroscopic mast cell alterations in biopsy specimens of skin tests in the same subjects. Definite mast cell alterations occurred within 15 sec and appeared maximal within 5 to 10 min after antigen injection. Histamine levels in appended chambers increased after a lag of 10 to 30 min and were elevated for at least 60 min after antigen challenge. Eosinophils accumulated only in antigen-induced reaction sites. However, there was no precise quantitative correlation among the degree of change in these three measurements. These appear to be promising approaches to further in vivo studies of human allergic reactions.     

The fate of human peripheral blood lymphocytes after transplantation into SCID mice

Human peripheral blood lymphocytes (hu-PBL) can be adoptively transferred by intraperitoneal injection into mice with severe combined immunodeficiency (SCID). The transplanted lymphocytes can produce immunoglobulin (Ig), respond to antigens, and survive for months in this chimeric model (hu-PBL SCED). However, whether the lymphocytes actually repopulate and reconstitute lymphoid structures and organs has been subject of some debate. To address this question and to characterize the hu-PBL SCID model better, we employed a novel technique for the identification of human cells in xenogeneic mice. We used fluorescence in situ hybridization (FISH) with a biotinylated DNA probe to all human centromeres. We demonstrated that FISH could be used to detect human cells when they accounted for less than 1 % of human/mouse cell mixtures; it could also be employed for the identification and localization of individual human cells in tissue sections. By using FISH, we studied 31 SCID mice injected with 1.5 × 10⁷ ?4 × 10⁷ hu-PBL via intravenous (i.v.) or intraperitoneal (i.p.) routes. In the 6 i.v -injected mice, we found that the human cells were removed from the circulation into the lung within 1 h. In 22 of 25 i.p -injected animals, 90–3716 μg/ml of human IgG was found in the sera at 3 to 13 weeks after transplantation (a.t.). Human cells colonized the peritoneal cavity and persisted for up to 13 weeks a.t. and, in the 12 mice studied, accounted for 4 % to 57 % of the cells in the peritoneal fluid. However, only rare, isolated human cells were found in the spleen, blood, bone marrow, lung or Peyer's patches. In 7 of 19 mice that received hu-PBL i.p. from Epstein-Barr virus-seropositive donors, we found masses of human cells usually beneath the peritoneal lining but sometimes infiltrating normal tissue. We conclude that FISH offers a simple means for accurate identification of human cells in the xenogeneic mouse. Although there is colonization of the peritoneal cavity in most mice, and development of lymphoid masses in some, there is no reconstitution of lymphoid structures and only minimal engraftment of lymphoid organs by human cells in conventionally-prepared hu-PBL SCID constructs.     

A severe combined immunodeficiency disease mouse model of human adenocarcinoma with lepidic-predominant growth

Severe combined immunodeficiency disease (SCID) mice with human lepidic adenocarcinoma were established by the intrabronchial implantation of fresh surgically resected specimens. Human pulmonary adenocarcinoma tissue from 16 different cases was transplanted into SCID mice, and SCID mouse tumors were established from four of these cases (25%). Among the four tumors, the tumor cells of two SCID mice showed replacement lepidic growth of mouse alveolar structures accompanied by multiple intrapulmonary lesions. Human lung carcinoma cell lines showing lepidic growth are rare and the xenograft models using the SCID mouse model developed in the current study will be useful for analyzing the growth and/or progression patterns and clinical behavior of lepidic adenocarcinoma, the major histological subtype of human carcinoma of the lung.     

人骨髓造血细胞经腹腔内和尾静脉注射移植SCID鼠的研究

目的 :比较腹腔内与尾静脉注射对SCID鼠进行异种骨髓移植的体内植入能力及GVHD的发生情况 ,为异种骨髓移植构建银屑病动物模型奠定基础。方法 :密度梯度离心法分离正常人骨髓单个核细胞 (BMMNC) ,以 4× 10 7的剂量分别经尾静脉和腹腔注射移植于经γ射线预照射的SCID鼠 ,移植后观察GVHD反应及外周血白细胞恢复动力学 ,并采用流式细胞术检测小鼠外周血及骨髓中人源性CD4 5 + 细胞比例 ,观察嵌合状态。结果 :单纯尾静脉注射移植组 ,在移植后 2周即出现明显GVHD症状 ,12周仅存活 1例 ;尾静脉注射结合CsA +MTX处理组 ,个别小鼠出现轻度GVHD表现 ,12周存活率 80 % (8 10 ) ;而经腹腔内注射者移植后出现轻度GVHD症状 ,之后逐渐恢复正常 ,12周存活率 70 % (7 10 )。移植后外周血白细胞动力学恢复情况、外周血及骨髓中人CD4 5 + 细胞比例 ,尾静脉注射结合CsA +MTX处理者与经腹膜腔内注射者二组间比较无显著性差异。结论 :人骨髓造血细胞可以顺利地由SCID鼠腹腔归巢到骨髓造血组织 ,并能重建骨髓造血 ,腹腔内注射的移植方式不影响植入能力 ,既可达到骨髓移植的目的 ,又可减少GVHD的发生及反应强度     

Oral tolerance attenuates airway inflammation and remodeling in a model of chronic pulmonary allergic inflammation

We investigated the effects of oral tolerance (OT) in controlling inflammatory response, hyperresponsiveness and airway remodeling in guinea pigs (GP) with chronic allergic inflammation. Animals received seven inhalations of ovalbumin (1-5mg/mL-OVA group) or normal saline (NS group). OT was induced by offering ad libitum ovalbumin 2% in sterile drinking water starting with the 1st ovalbumin inhalation (OT1 group) or after the 4th (OT2 group). The induction of OT in sensitized animals decreased the elastance of respiratory system (Ers) response after both antigen and methacholine challenges, peribronchial edema formation, eosinophilic airway infiltration, eosinophilopoiesis, and airways collagen and elastic fiber content compared to OVA group (P<0.05). The number of mononuclear cells and resistance of respiratory system (Rrs) responses after antigen and methacholine challenges were decreased only in OT2 group compared to OVA group (P<0.05). Concluding, our results show that inducing OT attenuates airway remodeling as well as eosinophilic inflammation and respiratory system mechanics.