Tapping R I, Orr S L, Lawson E M, Soldau K, Tobias P S
Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037, USA.
J Immunol. 1999 May 1;162(9):5483-9.
Inflammatory responses of myeloid cells to LPS are mediated through CD14, a glycosylphosphatidylinositol-anchored receptor that binds LPS. Since CD14 does not traverse the plasma membrane and alternatively anchored forms of CD14 still enable LPS-induced cellular activation, the precise role of CD14 in mediating these responses remains unknown. To address this, we created a transmembrane and a glycosylphosphatidylinositol-anchored form of LPS-binding protein (LBP), a component of serum that binds and transfers LPS to other molecules. Stably transfected Chinese hamster ovary (CHO) fibroblast and U373 astrocytoma cell lines expressing membrane-anchored LBP (mLBP), as well as separate CHO and U373 cell lines expressing membrane CD14 (mCD14), were subsequently generated. Under serum-free conditions, CHO and U373 cells expressing mCD14 responded to as little as 0.1 ng/ml of LPS, as measured by NF-kappaB activation as well as ICAM and IL-6 production. Conversely, the vector control and mLBP-expressing cell lines did not respond under serum-free conditions even in the presence of more than 100 ng/ml of LPS. All the cell lines exhibited responses to less than 1 ng/ml of LPS in the presence of the soluble form of CD14, demonstrating that they are still capable of LPS-induced activation. Taken together, these results demonstrate that mLBP, a protein that brings LPS to the cell surface, does not mediate cellular responses to LPS independently of CD14. These findings suggest that CD14 performs a more specific role in mediating responses to LPS than that of simply bringing LPS to the cell surface.
髓样细胞对脂多糖（LPS）的炎症反应是通过CD14介导的，CD14是一种糖基磷脂酰肌醇锚定受体，可结合LPS。由于CD14不穿过质膜，且CD14的替代锚定形式仍能使LPS诱导细胞活化，因此CD14在介导这些反应中的精确作用仍不清楚。为了解决这个问题，我们构建了一种跨膜形式和一种糖基磷脂酰肌醇锚定形式的脂多糖结合蛋白（LBP），LBP是血清中的一种成分，可结合LPS并将其转移至其他分子。随后产生了稳定转染的表达膜锚定LBP（mLBP）的中国仓鼠卵巢（CHO）成纤维细胞系和U373星形细胞瘤细胞系，以及单独表达膜CD14（mCD14）的CHO和U373细胞系。在无血清条件下，通过NF-κB激活以及细胞间黏附分子（ICAM）和白细胞介素-6（IL-6）的产生来测定，表达mCD14的CHO和U373细胞对低至0.1 ng/ml的LPS有反应。相反，载体对照和表达mLBP的细胞系在无血清条件下即使存在超过100 ng/ml的LPS也无反应。在存在可溶性CD14的情况下，所有细胞系对低于1 ng/ml的LPS均有反应，表明它们仍能被LPS诱导活化。综上所述，这些结果表明，将LPS带到细胞表面的蛋白mLBP不能独立于CD14介导细胞对LPS的反应。这些发现表明，CD14在介导对LPS的反应中所起的作用比仅仅将LPS带到细胞表面更为特殊。
