Recombinant soluble forms of extracellular TLR4 domain and MD-2 inhibit lipopolysaccharide binding on cell surface and dampen lipopolysaccharide-induced pulmonary inflammation in mice
Recombinant soluble forms of extracellular TLR4 domain and MD-2 inhibit lipopolysaccharide binding on cell surface and dampen lipopolysaccharide-induced pulmonary inflammation in mice
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DOI：
10.4049/jimmunol.177.11.8133
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发表时间：
2006-12-01
影响因子：
4.4
通讯作者：
Kuroki, Yoshio
中科院分区：
文献类型：
--
作者：
Mitsuzawa, Hiroaki;Nishitani, Chiaki;Kuroki, Yoshio
在这项研究中，我们寻求一种新的治疗策略的可能性，以抑制内毒素诱导的炎症，利用细胞外可溶形式的rTLR4结构域(STLR4)和可溶形式的RMD-2(SMD-2)。与sTLR4或SMD-2单独作用相比，sTLR4与SMD-2联合应用可显著抑制脂多糖诱导的U937细胞IL-8释放和细胞内核转录因子-kappaB的活化。因此，我们研究了胞外TLR4结构域在脂质A与MD-2相互作用中的作用。生物素化的sTLR4在与链霉亲和素-琼脂糖共沉淀时不能共沉淀[H-3]类脂A，表明胞外TLR4结构域本身并不直接结合类脂A。当sTLR4与sTLR4共同孵育时，与SMD-2共沉淀的脂质A的量显著增加，并且sTLR4增加了脂质A与SMD-2结合的亲和力。可溶性CD14是sTLR4刺激脂质A与SMD-2结合增加所必需的。我们还发现，加入sTLR4和SMD-2抑制了AlexA结合的内毒素与表达TLR4和MD-2的细胞的结合。接受sTLR4加SMD-2和脂多糖的小鼠肺没有发现任何间质水肿、中性粒细胞流动和出血的迹象。联合应用sTLR4和SMD-2，但不单独应用sTLR4或SMD-2，可显著降低脂多糖诱导的小鼠支气管肺泡灌洗液中中性粒细胞的浸润和肿瘤坏死因子-α的水平。本研究提供了sTLR4和SMD-2作为拮抗剂对抗内毒素诱导的肺部炎症的新用途。
In this study, we sought the possibility of a new therapeutic strategy for dampening endotoxin-induced inflammation using soluble form of extracellular rTLR4 domain (sTLR4) and soluble form of rMD-2 (sMD-2). Addition of sTLR4 plus sMD-2 was significantly effective in inhibiting LPS-elicited IL-8 release from U937 cells and NF-kappa B activation in the cells transfected with TLR4 and MD-2 when compared with a single treatment with sTLR4 or sMD-2. Thus, we investigated the role of the extracellular TLR4 domain in interaction of lipid A with MD-2. Biotinylated sTLR4 failed to coprecipitate [H-3]lipid A when it was sedimented with streptavidin-agarose, demonstrating that the extracellular TLR4 domain does not directly bind lipid A by itself. The amounts of lipid A coprecipitated with sMD-2 significantly increased when coincubated with sTLR4, and sTLR4 increased the affinity of lipid A for the binding to sMD-2. Soluble CD14 is required for the sTLR4-stimulated increase of lipid A binding to sMD-2. We also found that addition of sTLR4 plus sMD-2 inhibited the binding of Alexa-conjugated LPS to the cells expressing TLR4 and MD-2. Murine lungs that had received sTLR4 plus sMD-2 with LPS did not show any findings indicative of interstitial edema, neutrophil flux, and hemorrhage. Coinstillation of sTLR4 plus sMD-2, but not sTLR4 or sMD-2 alone, significantly decreased neutrophil infiltration and TNF-alpha levels in bronchoalveolar lavage fluids from LPS-treated mice. This study provides novel usage of sTLR4 and sMD-2 as an antagonist against endotoxin-induced pulmonary inflammation.