Disciplina de Infectologia, Universidade Federal de São Paulo, SP, Brasil.
Braz J Med Biol Res. 2010 Sep;43(9):853-8. doi: 10.1590/s0100-879x2010007500078. Epub 2010 Aug 16.
Lipopolysaccharide (LPS) activates neutrophils and monocytes, inducing a wide array of biological activities. LPS rough (R) and smooth (S) forms signal through Toll-like receptor 4 (TLR4), but differ in their requirement for CD14. Since the R-form LPS can interact with TLR4 independent of CD14 and the differential expression of CD14 on neutrophils and monocytes, we used the S-form LPS from Salmonella abortus equi and the R-form LPS from Salmonella minnesota mutants to evaluate LPS-induced activation of human neutrophils and monocytes in whole blood from healthy volunteers. Expression of cell surface receptors and reactive oxygen species (ROS) and nitric oxide (NO) generation were measured by flow cytometry in whole blood monocytes and neutrophils. The oxidative burst was quantified by measuring the oxidation of 2',7'-dichlorofluorescein diacetate and the NO production was quantified by measuring the oxidation of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. A small increase of TLR4 expression by monocytes was observed after 6 h of LPS stimulation. Monocyte CD14 modulation by LPS was biphasic, with an initial 30% increase followed by a 40% decrease in expression after 6 h of incubation. Expression of CD11b was rapidly up-regulated, doubling after 5 min on monocytes, while down-regulation of CXCR2 was observed on neutrophils, reaching a 50% reduction after 6 h. LPS induced low production of ROS and NO. This study shows a complex LPS-induced cell surface receptor modulation on human monocytes and neutrophils, with up- and down-regulation depending on the receptor. R- and S-form LPS activate human neutrophils similarly, despite the low CD14 expression, if the stimulation occurs in whole blood.
脂多糖 (LPS) 激活中性粒细胞和单核细胞，诱导广泛的生物学活性。LPS 粗糙 (R) 和光滑 (S) 形式通过 Toll 样受体 4 (TLR4) 信号转导，但对 CD14 的需求不同。由于 R 形式的 LPS 可以与 TLR4 独立于 CD14 相互作用，并且 CD14 在中性粒细胞和单核细胞上的表达不同，因此我们使用来自流产沙门氏菌 equi 的 S 形式 LPS 和来自明尼苏达沙门氏菌突变体的 R 形式 LPS 来评估 LPS 诱导的健康志愿者全血中人类中性粒细胞和单核细胞的激活。通过流式细胞术测量全血单核细胞和中性粒细胞中细胞表面受体的表达和活性氧 (ROS) 和一氧化氮 (NO) 的生成。通过测量 2'，7'-二氯荧光素二乙酸酯的氧化来量化氧化爆发，通过测量 4-氨基-5-甲基氨基-2'，7'-二氟荧光素二乙酸酯的氧化来量化 NO 产生。在 LPS 刺激后 6 小时观察到单核细胞 TLR4 表达略有增加。LPS 对单核细胞 CD14 的调节呈双相，孵育 6 小时后表达最初增加 30%，然后减少 40%。CD11b 的表达迅速上调，在单核细胞上 5 分钟后增加一倍，而在中性粒细胞上观察到 CXCR2 的下调，在 6 小时后达到 50%的减少。LPS 诱导低 ROS 和 NO 的产生。这项研究表明，LPS 诱导人单核细胞和中性粒细胞表面受体的复杂调节，根据受体的不同存在上调和下调。尽管 CD14 表达水平较低，但在全血中刺激时，R 和 S 形式的 LPS 可类似地激活人类中性粒细胞。
