Kramer A A, Salhab K F, Shafii A E, Norman J, Carey L C, Mendez C
Department of Surgery, University of South Florida, Tampa, Florida 33612, USA.
J Surg Res. 1999 May 15;83(2):89-94. doi: 10.1006/jsre.1999.5571.
Tolerance to hemorrhagic or endotoxic shock can be induced by prior sublethal hemorrhage (SLH). The purpose of this study was to explore whether alterations in signal transduction pathways involving NF-kappaB occur in macrophages (Mphi) following induction of tolerance by SLH.
Using a model of SLH previously shown in our lab to impart a survival benefit to subsequent hemorrhagic or endotoxic shock, rats (n = 30) were conditioned by SLH. Peritoneal Mphi were harvested 24 h after conditioning and stimulated with lipopolysaccharide (LPS) (10 microg/mL). Nuclear and cytosolic proteins were isolated 1 h later for determination of NF-kappaB activation by gel-shift assay and IkappaB-alpha by Western blot. TNF mRNA gene expression was measured 4 h after LPS stimulation by reverse transcription/polymerase chain reaction (RT/PCR). TNF protein levels were measured in cellular supernatants by enzyme-linked immunosorbent assay (ELISA) 18 h after LPS. RESULTS. LPS stimulation of sham Mphi increased NF-kappaB activation with corresponding loss of its inhibitor IkappaB-alpha. In contrast, IkappaB-alpha was not detectable following conditioning, and conditioned Mphi had NF-kappaB activation at baseline which increased minimally with LPS stimulation. LPS increased TNF gene expression and significantly increased protein production by both sham and conditioned Mphi, but this increase was greater in the sham-conditioned group.
The ability of Mphi from animals made tolerant by SLH to produce TNF in vitro is conserved. Nevertheless, these same Mphi exhibit alterations in TNF gene induction and expression as well as signal transduction, specifically, changes in IkappaB-alpha and NF-kappaB activation. This suggests a role for activation of NF-kappaB in the induction of tolerance.
预先的亚致死性出血（SLH）可诱导对出血性或内毒素性休克的耐受性。本研究的目的是探讨在通过SLH诱导耐受性后，巨噬细胞（Mphi）中涉及核因子-κB（NF-κB）的信号转导通路是否发生改变。
使用我们实验室先前展示的一种SLH模型，该模型可使动物对随后的出血性或内毒素性休克具有生存优势，对30只大鼠进行SLH预处理。预处理24小时后收集腹腔巨噬细胞，并用脂多糖（LPS）（10微克/毫升）刺激。1小时后分离细胞核和细胞质蛋白，通过凝胶迁移试验测定NF-κB的活化情况，通过蛋白质印迹法测定IκB-α。在LPS刺激4小时后，通过逆转录/聚合酶链反应（RT/PCR）测量肿瘤坏死因子（TNF）mRNA基因表达。在LPS刺激18小时后，通过酶联免疫吸附测定（ELISA）测量细胞上清液中的TNF蛋白水平。结果：LPS刺激假处理的巨噬细胞增加了NF-κB的活化，同时其抑制剂IκB-α相应减少。相比之下，预处理后未检测到IκB-α，且预处理的巨噬细胞在基线时具有NF-κB活化，LPS刺激后其增加幅度最小。LPS增加了假处理和预处理巨噬细胞的TNF基因表达，并显著增加了蛋白产生，但假处理组的增加幅度更大。
通过SLH诱导产生耐受性的动物的巨噬细胞在体外产生TNF的能力得以保留。然而，这些相同的巨噬细胞在TNF基因诱导和表达以及信号转导方面表现出改变，具体而言，IκB-α和NF-κB活化发生变化。这表明NF-κB的活化在耐受性诱导中起作用。
