Ward M D, Madison S L, Andrews D L, Sailstad D M, Gavett S H, Selgrade M J
National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711, USA.
Toxicology. 2000 Jun 8;147(2):133-45. doi: 10.1016/s0300-483x(00)00191-8.
Metarhizium anisopliae, an entomopathogenic fungus, is a prototypic microbial pesticide licensed for indoor control of cockroaches, a major source of allergens. We have previously demonstrated allergy and asthma-like responses in BALB/c mice intraperitoneally (IP) sensitized in the presence of adjuvant and intratracheally (IT) challenged with the soluble factors from M. anisopliae crude antigen (MACA) (Ward et al., 1998, 2000). This protocol has been used frequently to establish animal models of allergenicity. However, the sensitization protocol is artificial and not representative of an environmental exposure. Concern has been raised that this protocol might produce allergic responses that would not occur under normal environmental exposure conditions. The objective of this study was to compare responses in mice to MACA by two exposure protocols: (1) exclusive respiratory exposures without adjuvant (representative of environmental exposures) and (2) intraperitoneal sensitization in the presence of adjuvant followed by IT challenge (the traditional approach). The intratracheal protocol consisted of four IT exposures of 10 microg MACA in 50 microl HBSS each over a 4-week period. A vehicle control group of mice was exposed IT to HBSS. The intraperitoneal protocol consisted of IP sensitization with 25 microg MACA in 0.2 ml of 1.3% alhydrogel (aluminum hydroxide) followed 14 days later with an IT challenge (10 microg MACA/50 microl HBSS). Airway reactivity responsiveness to methacholine was assessed, serum and bronchoalveolar lavage fluid (BALF) samples were obtained, and the lungs were fixed for histopathology at 1, 3, and 8 days following the last MACA IT challenge. Both groups exhibited immune and pulmonary responses typical of allergic asthma. In general, local responses in the lung, including inflammatory responses (eosinophils, lymphocytes, and macrophages), BALF IgE, and functional responses to methacholine were greater in the IT sensitized group compared to the IP sensitized group, whereas the systemic IgE response was greater in the IP sensitized group. The BALF IL-5 cytokine levels were elevated before and throughout the eosinophil influx. IL-4 was detected in the BALF of IP sensitized, but not IT sensitized mice. Histopathologic changes in the two groups were similar in nature but more severe in the IT mice. The results suggest that the IP sensitization protocol does not induce the level of respiratory responsiveness that results from sensitization by a physiologically relevant route of exposure. Thus total serum IgE levels, which were greater following IP sensitization, may not be the best indicator of allergen potency, at least with respect to respiratory responses.
绿僵菌是一种昆虫病原真菌，是一种已获许可用于室内控制蟑螂（主要过敏原来源）的典型微生物杀虫剂。我们之前已经证明，在佐剂存在下腹腔注射（IP）致敏并经气管内（IT）给予绿僵菌粗抗原（MACA）的可溶性因子攻击后，BALB/c小鼠会出现过敏和哮喘样反应（Ward等人，1998年、2000年）。该方案经常被用于建立致敏性动物模型。然而，致敏方案是人为的，并不代表环境暴露情况。有人担心该方案可能会产生在正常环境暴露条件下不会出现的过敏反应。本研究的目的是通过两种暴露方案比较小鼠对MACA的反应：（1）无佐剂的单纯呼吸道暴露（代表环境暴露）和（2）在佐剂存在下腹腔致敏，随后进行IT攻击（传统方法）。气管内方案包括在4周内分4次IT给予10微克MACA，每次溶于50微升HBSS中。一组小鼠作为溶媒对照组，IT给予HBSS。腹腔方案包括用25微克MACA溶于0.2毫升1.3%氢氧化铝佐剂中进行IP致敏，14天后进行IT攻击（10微克MACA/50微升HBSS）。评估对乙酰甲胆碱的气道反应性，采集血清和支气管肺泡灌洗液（BALF）样本，并在最后一次MACA IT攻击后的第1天、第3天和第8天固定肺组织进行组织病理学检查。两组均表现出过敏性哮喘典型的免疫和肺部反应。一般来说，与IP致敏组相比，IT致敏组肺部的局部反应，包括炎症反应（嗜酸性粒细胞、淋巴细胞和巨噬细胞）、BALF IgE以及对乙酰甲胆碱的功能反应更强，而IP致敏组的全身IgE反应更强。在嗜酸性粒细胞流入之前及整个过程中，BALF IL-5细胞因子水平均升高。在IP致敏小鼠的BALF中检测到IL-4，但在IT致敏小鼠中未检测到。两组的组织病理学变化本质相似，但IT组小鼠的变化更严重。结果表明，IP致敏方案不会诱导通过生理相关暴露途径致敏所导致的呼吸道反应水平。因此，IP致敏后更高的总血清IgE水平可能不是过敏原效力的最佳指标，至少就呼吸道反应而言。
