The Role Of Cytokines In The Developing Immune System

有人认为，小岛屿发展中国家可能是在发育中的免疫系统更容易受到炎症介质影响的时候，由微生物产物和/或其他环境因素(如环境烟草烟雾)的组合引起的病理生理反应。我们已经开发了一种模拟双重感染的动物模型，使用了一种非致命性的甲型流感病毒株和亚致死量的内毒素。我们假设，在细菌感染之前暴露于甲型流感病毒可以模拟SID的病理，并改变细胞因子介导的T辅助细胞反应。更具体地说，我们假设，在免疫系统发育的关键窗口，暴露于甲型流感病毒通过改变表达动力学和增加IL-6、IL-10、TNFa和其他炎性细胞因子的产生来改变对细菌产品的免疫反应的调节，这些炎性细胞因子通常促进保护性反应。不成熟的免疫系统要么无法容忍这些变化，要么无法充分协调导致死亡的反应。我们正在使用这个模型来检验导致婴儿不明原因猝死的环境风险因素之间的关系，包括年龄、倾向于睡眠和外源性因素，如ETS或真菌毒素。 本实验用甲型流感病毒(A/Port-Chalmers/1/73，H3N_2)发展而来的大鼠甲型流感病毒(RAIV)作为感染性病毒。使用与免疫学发展的临界点和人类小岛屿发展中国家的高峰年龄(2-6个月)相对应的时间点，进行了初步研究，以确定剂量和内毒素水平之间的最佳时机，以模拟小岛屿发展中国家的死亡率和病理学。这些研究表明，在10日龄接种RAIV，然后在RAIV后2天接种内毒素0.2 mg/kg，致死率最低。这些元素成为模型的定义参数。只有在满足特定标准时才会发生死亡，例如在感染侮辱和幼崽发育年龄之间的时间间隔，而在该模型中，年龄是与死亡率相关的关键风险因素。成年大鼠在类似的挑战后进行了检查，没有观察到死亡。 为了研究这种死亡率的机制，在内毒素注射后2-8小时进行了一系列实验来检查病理和评估免疫参数。对脾、血和支气管肺泡灌洗液中分离的细胞进行免疫表型分析。组织学检查SIDS样病理和炎症介质基因表达的组织学证据。同时检测血清和灌洗液中炎症介质的水平。这些研究的数据表明，死亡是由于快速的全身休克事件，而不是特定于肺的损害。尸检发现的大体病理结果，如心脏周围有肺部瘀点和液体血液，与婴儿死于小婴儿呼吸窘迫综合征时所见的一致。组织病理学损害，包括心内膜下出血和轻微的胸腺细胞坏死，在双重挑战的动物中被发现更严重和更频繁。肝和脾的血管充血与内毒素暴露以及可能导致循环衰竭的炎性细胞因子和反应性氮物种(RNS)的释放一致。与年龄匹配的内毒素对照组相比，内毒素诱导病毒感染的动物立即释放IL-6和IL-10。在死亡时，接种RAIV的动物的脾中巨噬细胞数量显著增加。在接受内毒素和RAIV的动物中，观察到血清干扰素-γ水平有统计学意义的下降。RAIV感染动物肺、肝组织中细胞因子基因表达异常。我们已经定义了人类婴儿SID病理与该模型中所见的相似之处，并检查了与双重挑战相关的基本免疫因素。这些研究表明，发育中的免疫系统可以做好准备，以一种夸张的方式对导致意外死亡的第二次免疫挑战做出反应。 已有文献表明，自身免疫性疾病的易感性在很大程度上取决于特定的主要组织相容性复合体(MHC)基因类型的存在，然而，疾病症状的发展似乎也需要关键的环境因素，如细菌或病毒感染或化学暴露。MRL/LPR小鼠表现出与人类系统性红斑狼疮患者相似的严重肾坏死(肾小球肾炎)，雌性通常在大约14-16周龄时出现针对核蛋白颗粒的循环自身抗体，随后出现肾脏补体-抗体免疫复合体，而雄性在20-22周龄。新生儿暴露在环境污染物2，3，7，8-四氯二苯并-对二恶英(TCDD)中，已被证明会改变成年动物的胸腺细胞成熟并显著损害免疫功能。我们假设，出生前暴露于TCDD可能会增加或加剧遗传倾向的MRL/LPR小鼠出生后自身免疫性疾病的发展。我们观察到，与对照组相比，暴露于TCDD的雌性MRL/LPR小鼠在6周、10周和12周时，抗ssDNA抗体、尿蛋白和肾脏异常均显著增加，且呈剂量依赖关系。在接触TCDD的8周、10周和12周大的雄性中也注意到了类似的结果。

It has been suggested that SIDS may be a patho-physiological response elicited by combinations of microbial products and/or other environmental factors such as environmental tobacco smoke (ETS) at a time when the developing immune system is more vulnerable to the effects of inflammatory mediators. We have developed an animal model to mimic dual infection, using a non-lethal strain of Influenza A virus and a sub-lethal dose of endotoxin. We hypothesized that exposure to Influenza A virus prior to bacterial infection can mimic the pathology of SIDS and alter cytokine-mediated T-helper cell responses. More specifically, we postulate that at critical windows of immune system development exposure to Influenza A virus changes the regulation of the immune response to bacterial products by altering the kinetics of expression and increasing the production of IL-6, IL-10, TNFa and other inflammatory cytokines that normally promote a protective response. The immature immune system is either unable to tolerate these changes and/or unable to adequately coordinate the response resulting in death. We are using this model to examine the relationship between environmental risk factors for sudden unexplained death in infants including age, prone sleeping and exogenous factors such as ETS or mycotoxins. Rat adapted Influenza A virus (RAIV), developed from Influenza A/Port-Chalmers/1/73 (H3N2) virus was used for infectivity in these experiments. Using time points that correspond to critical points in immunological development and the peak age for SIDS in humans (2-6 months), preliminary studies were conducted to ascertain optimal timing between doses and endotoxin levels to model the mortality and pathology of SIDS. These studies established that inoculation with RAIV at 10 days of age followed by endotoxin 0.2 mg/kg 2 days post RAIV caused mortality with the lowest morbidity. These elements became the defining parameters of the model. Mortality only occurred when specific criteria such as timing between infectious insults and developmental age of the pup were met and age was a key risk factor related to mortality in the model. Adult rats were examined following similar challenges and no mortality was observed. To examine the mechanisms underlying this mortality, a series of experiments were conducted to examine pathology and evaluate immune parameters 2-8 hours following endotoxin administration. Immunophenotyping was performed on cells isolated from the spleen, blood and bronchoalveolar lavage fluid. Tissues were examined for histologic evidence of SIDS-like pathology and inflammatory mediator gene expression. Levels of inflammatory mediators in serum and lavage fluid were also measured. The data from these studies suggests that mortality is due to a rapid systemic shock event rather than lung specific damage. Gross pathologic findings such as lung petechiae and liquid blood around the heart on necropsy were consistent with those seen in infants dying of SIDS. Histopathologic lesions including sub-endocardial hemorrhage and mild cortical thymocyte necrosis were found with greater severity and frequency in dually challenged animals. Vascular congestion in the liver and spleen was consistent with endotoxin exposure and with the release of inflammatory cytokines and reactive nitrogen species (RNS) that may have led to circulatory collapse. Endotoxin induced an immediate release of IL-6 and IL-10 in virally infected animals, as compared to age-matched endotoxin only controls. Macrophage numbers in RAIV-inoculated animals were significantly elevated in the spleen at time of death. A statistically significant decrease in serum IFN-gamma levels was observed in animals that received endotoxin and RAIV. Cytokine mRNA expression was dysregulated in the lungs and livers of the RAIV infected animals. We have defined similarities of SIDS pathology in human infants and that seen in this model and examined basic immune factors associat ed with dual challenge. These studies suggest that the developing immune system can be primed to respond in an exaggerated way to a second immune challenge resulting in unexpected death. It is well documented that autoimmune disease susceptibility is largely dependent upon the presence of particular major histocompatibility complex (MHC) genotypes, however, development of disease symptoms also appears to require critical environmental factors, such as bacterial or viral infections or chemical exposure. MRL/lpr mice exhibit severe renal necrosis (glomerulonephritis) similar to that observed in human patients with systemic lupus erythematosus, with females normally presenting circulating autoantibodies against nucleoprotein particles and subsequent renal complement-antibody immune complexes at approximately 14-16 weeks of age vs. males at 20-22 weeks. Neonatal exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to alter thymocyte maturation and significantly impair immune function in adult animals. We hypothesized that prenatal exposure to TCDD may increase or exacerbate post-natal development of autoimmune disease in genetically-prone MRL/lpr mice. We have observed significant, dose-dependent increases in anti-ssDNA, urinary protein, and renal abnormalities in TCDD-exposed female MRL/lpr mice as compared to controls at six, ten, and 12 weeks of age, respectively. Similar results were noted in TCDD-exposed males at eight, ten and 12 weeks of age.