Garn Holger, Siese Anette, Stumpf Sabine, Wensing Anka, Renz Harald, Gemsa Diethard
Department of Clinical Chemistry and Molecular Diagnostics, Philipps University of Marburg, Biomedical Research Center, Hans-Meerwein-Str., 35043 Marburg, Germany.
Respir Res. 2006 Jan 6;7(1):4. doi: 10.1186/1465-9921-7-4.
Alveolar macrophages (AM) are known to play an important role in the regulation of inflammatory reactions in the lung, e.g. during the development of chronic lung diseases. Exposure of rats to NO2 has recently been shown to induce a shift in the activation type of AM that is characterized by reduced TNF-alpha and increased IL-10 production. So far it is unclear, whether a functional shift in the already present AM population or the occurrence of a new, phenotypically different AM population is responsible for these observations.
AM from rat and mice were analyzed by flow cytometry for surface marker expression and in vivo staining with PKH26 was applied to characterize newly recruited macrophages. Following magnetic bead separation, AM subpopulations were further analyzed for cytokine, inducible NO synthase (iNOS) and matrix metalloproteinase (MMP) mRNA expression using quantitative RT-PCR. Following in vitro stimulation, cytokines were quantitated in the culture supernatants by ELISA.
In untreated rats the majority of AM showed a low expression of the surface antigen ED7 (CD11b) and a high ED9 (CD172) expression (ED7-/ED9high). In contrast, NO2 exposure induced the occurrence of a subpopulation characterized by the marker combination ED7+/ED9low. Comparable changes were observed in mice and by in vivo labeling of resident AM using the dye PKH26 we could demonstrate that CD11b positive cells mainly comprise newly recruited AM. Subsequent functional analyses of separated AM subpopulations of the rat revealed that ED7+ cells showed an increased expression and production of the antiinflammatory cytokine IL-10 whereas TNF-alpha production was lower compared to ED7- AM. However, iNOS and IL-12 expression were also increased in the ED7+ subpopulation. In addition, these cells showed a significantly higher mRNA expression for the matrix metalloproteinases MMP-7, -8, -9, and -12.
NO2 exposure induces the infiltration of an AM subpopulation that, on the one hand may exert antiinflammatory functions by the production of high amounts of IL-10 but on the other hand may contribute to the pathology of NO2-induced lung damage by selective expression of certain matrix metalloproteinases.
已知肺泡巨噬细胞（AM）在肺部炎症反应的调节中发挥重要作用，例如在慢性肺部疾病的发展过程中。最近研究表明，将大鼠暴露于二氧化氮（NO₂）会导致AM的激活类型发生转变，其特征是肿瘤坏死因子-α（TNF-α）产生减少，白细胞介素-10（IL-10）产生增加。到目前为止，尚不清楚这些观察结果是由已存在的AM群体的功能转变还是新出现的、表型不同的AM群体的出现所导致。
通过流式细胞术分析大鼠和小鼠的AM表面标志物表达情况，并应用PKH26进行体内染色以表征新招募的巨噬细胞。经过磁珠分离后，使用定量逆转录聚合酶链反应（RT-PCR）进一步分析AM亚群的细胞因子、诱导型一氧化氮合酶（iNOS）和基质金属蛋白酶（MMP）mRNA表达。体外刺激后，通过酶联免疫吸附测定（ELISA）对培养上清液中的细胞因子进行定量分析。
在未处理的大鼠中，大多数AM表面抗原ED7（CD11b）表达较低，而ED9（CD172）表达较高（ED7⁻/ED9高）。相反，暴露于NO₂会诱导出现一个以标志物组合ED7⁺/ED9低为特征的亚群。在小鼠中也观察到了类似的变化，并且通过使用染料PKH26对驻留AM进行体内标记，我们可以证明CD11b阳性细胞主要包括新招募的AM。随后对大鼠分离出的AM亚群进行功能分析发现，ED7⁺细胞抗炎细胞因子IL-10的表达和产生增加，而与ED7⁻ AM相比，TNF-α的产生较低。然而，ED7⁺亚群中iNOS和IL-12的表达也增加。此外，这些细胞中基质金属蛋白酶MMP-7、-8、-9和-12的mRNA表达明显更高。
暴露于NO₂会诱导AM亚群的浸润，一方面，该亚群可能通过产生大量IL-10发挥抗炎功能，但另一方面，可能通过某些基质金属蛋白酶的选择性表达促进NO₂诱导的肺损伤病理过程。
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