Alkan S S, Akdiş A C, Feuerlein D, Grüninger M
Department of Asthma/Allergy Pharmaceuticals Research Division, Ciba-Geigy, Ltd., Basle, Switzerland.
Ann N Y Acad Sci. 1996 Oct 31;796:82-90. doi: 10.1111/j.1749-6632.1996.tb32569.x.
A mouse in vivo model has been developed in which cytokines produced within the regional lymph nodes and other organs after an antigenic challenge were measured directly without the need of an in vitro reculturing step. Mice were subcutaneously (s.c.) immunized with antigens such as keyhole limpet hemocyanin (KLH) in aluminium hydroxide (alum) at the base of the tail. After defined periods of time lymph nodes, spleen, liver, and lung were collected and frozen. The organs were then homogenized (or sonicated), centrifuged, and the cytokine levels, for example, IFN-gamma, IL-4, IL-5, and IL-6 were determined by ELISA. It was found that injection of alum alone caused measurable cytokine production in draining lymph nodes, but inoculation of an antigen-alum mixture, such as KLH and dust-mite antigen (DMA), significantly increased in vivo cytokine production over that of alum alone. In the dose range tested there appeared to be an inverse relationship between the dose of antigen and cytokine levels. Thus, 0.1-microgram KLH induced more IL-4 in vivo than 10-micrograms KLH. Kinetic studies showed that for IL-4 and IFN-gamma, peak production occurred around days 5 and 6, respectively. Route of immunization and the dose of antigen were found to be very critical, in that injection of 10-micrograms DMA intraperitoneally induced significant levels of cytokines in the lung, while the same dose of antigen given at the base of tail, s.c., did not induce appreciable levels of cytokines in any organ tested. Cyclosporin A inhibited in vivo production of IFN-gamma, IL-4, and IL-5 by approximately 80%, but not that of IL-6. Surprisingly, dexamethasone enhanced the production of IL-6 while inhibiting all the other cytokines. In conclusion, these results suggest that direct determination of cytokines in the organs may provide an easy readout to assess differential effects of immunoregulatory molecules on the production of Th1- and Th2-type cytokines in vivo.
已经建立了一种小鼠体内模型，在该模型中，抗原攻击后区域淋巴结和其他器官中产生的细胞因子无需体外再培养步骤即可直接测量。将小鼠在尾巴根部皮下（s.c.）用抗原如钥孔戚血蓝蛋白（KLH）与氢氧化铝（明矾）进行免疫。在规定的时间段后，收集淋巴结、脾脏、肝脏和肺并冷冻。然后将器官匀浆（或超声处理）、离心，并通过酶联免疫吸附测定法（ELISA）测定细胞因子水平，例如干扰素-γ（IFN-γ）、白细胞介素-4（IL-4）、白细胞介素-5（IL-5）和白细胞介素-6（IL-6）。发现单独注射明矾会导致引流淋巴结中产生可测量的细胞因子，但接种抗原-明矾混合物，如KLH和尘螨抗原（DMA），会使体内细胞因子产生量比单独使用明矾时显著增加。在所测试的剂量范围内，抗原剂量与细胞因子水平之间似乎存在反比关系。因此，0.1微克KLH在体内诱导产生的IL-4比10微克KLH更多。动力学研究表明，对于IL-4和IFN-γ，峰值产生分别出现在第5天和第6天左右。发现免疫途径和抗原剂量非常关键，因为腹腔内注射10微克DMA会在肺中诱导产生显著水平的细胞因子，而在尾巴根部皮下给予相同剂量的抗原，在任何测试器官中都不会诱导产生可观水平的细胞因子。环孢素A在体内对IFN-γ、IL-4和IL-5产生的抑制作用约为80%，但对IL-6没有抑制作用。令人惊讶的是，地塞米松增强了IL-6的产生，同时抑制了所有其他细胞因子。总之，这些结果表明，直接测定器官中的细胞因子可能为评估免疫调节分子对体内Th1型和Th2型细胞因子产生的不同影响提供一种简便的检测方法。
