Blondin C, Le Dur A, Cholley B, Caroff M, Haeffner-Cavaillon N
INSERM U430, Hôpital Broussais, Paris, France.
Eur J Immunol. 1997 Dec;27(12):3303-9. doi: 10.1002/eji.1830271229.
Using flow cytometry we have compared the binding of Neisseria meningitidis lipopolysaccharide labeled with fluorescein isothiocyanate (FITC-LPS) to normal human monocytes in whole blood with the binding to chinese hamster ovary (CHO) cells transfected with human CD14 gene (hCD14-CHO cells). Binding of FITC-LPS to cells was dose dependent, saturable and enhanced in the presence of increasing concentrations of serum. Blockade of membrane CD14 with saturating concentrations of anti-CD14 monoclonal antibody (mAb) My4 inhibited 50% of the binding of FITC-LPS to monocytes and 100% to hCD14-CHO cells. Similarly, removal of membrane CD14 by phosphatidylinositol phospholipase C (PI-PLC) treatment of the cells partially decreased the binding of FITC-LPS to monocytes but totally inhibited the binding to hCD14-CHO-transfected cells. These results suggest that binding of FITC-LPS to monocytes is not only mediated by membrane CD14. Using two-color flow cytometry, we observed that FITC-LPS binds to My4-saturated monocytes in association with soluble (s)CD14 present in serum as revealed by staining with rhodamine-labeled My4 mAb. The binding of FITC-LPS/sCD14 complexes to monocytes treated with saturating amounts of unlabeled My4 prior to addition of the complexes was completely inhibited by anti-CD14 mAb 10G33. When cells were first saturated with a mixture of My4 and 10G33 mAb, washed and further incubated with FITC-LPS/sCD14, inhibition of the binding of LPS was similar to that observed with cells saturated with My4 alone, showing that the binding of FITC-LPS is not mediated by the 10G33 epitope present on mCD14. These results suggest that either the 10G33 epitope on sCD14 is involved in the binding of LPS/sCD14 complexes to the cells, or that 10G33 mAb inhibits the binding of FITC-LPS to sCD14. Taken together, these data indicate that sCD14 which is present in normal serum, in addition to membrane CD14, enables LPS to bind monocytes through an as yet unidentified molecule and that sCD14 does not simply serve as a shuttle for transfer of LPS to membrane CD14.
我们运用流式细胞术，比较了用异硫氰酸荧光素标记的脑膜炎奈瑟菌脂多糖（FITC-LPS）与全血中正常人单核细胞的结合情况，以及与转染了人CD14基因的中国仓鼠卵巢（CHO）细胞（hCD14-CHO细胞）的结合情况。FITC-LPS与细胞的结合呈剂量依赖性、可饱和性，且在血清浓度增加时增强。用饱和浓度的抗CD14单克隆抗体（mAb）My4阻断膜CD14，可抑制50%的FITC-LPS与单核细胞的结合以及100%与hCD14-CHO细胞的结合。同样，用磷脂酰肌醇磷脂酶C（PI-PLC）处理细胞以去除膜CD14，可部分降低FITC-LPS与单核细胞的结合，但完全抑制其与hCD14-CHO转染细胞的结合。这些结果表明，FITC-LPS与单核细胞的结合不仅由膜CD14介导。运用双色流式细胞术，我们观察到，如用罗丹明标记的My4 mAb染色所示，FITC-LPS与My4饱和的单核细胞结合，并与血清中存在的可溶性（s）CD14相关联。在用未标记的My4饱和量处理细胞后再加入FITC-LPS/sCD14复合物，FITC-LPS/sCD14复合物与单核细胞的结合被抗CD14 mAb 10G33完全抑制。当细胞先用My4和10G33 mAb的混合物饱和，洗涤后再与FITC-LPS/sCD14进一步孵育时，LPS结合的抑制情况与仅用My4饱和的细胞相似，表明FITC-LPS的结合不是由mCD14上存在的10G33表位介导的。这些结果表明，要么sCD14上 的10G33表位参与LPS/sCD复合物与细胞的结合，要么10G33 mAb抑制FITC-LPS与sCD14的结合。综上所述，这些数据表明，除了膜CD14外，正常血清中存在的sCD14能使LPS通过一种尚未明确的分子与单核细胞结合，且sCD14并非仅仅作为将LPS转移至膜CD14的载体。
