Variation of LPS-binding capacity, epitope expression, and shedding of membrane-bound CD14 during differentiation of...

The myeloid differentiation Ag CD14 is considered to play a critical role in the binding of LPS to monocytes. To determine if differences in LPS-binding capacities of cells could reflect a variability of CD14 molecules, we analyzed the interactions of various reagents with these molecules in human blood monocytes and in promyelocytic (HL60) and monocytic (THP-1) cell lines. The expression of CD14 epitopes was analyzed with the fluorescent anti-CD14 mAbs My4 and LeuM3. Expression of LPS-binding sites (LPS+ molecules) was detected with LPS-FITC. THP-1 cells stimulated with calcitriol (VitD3), as well as the majority of blood monocytes (50-90%) were My4+/LPS+. However, untreated THP-1 cells, and a substantial population (10-50%) of human monocytes from healthy donors, were My4+/LPS-, thus suggesting the existence of CD14 isoforms with different LPS-binding capacities. In line with this assumption, monocytes stimulated with PMA selectively shed LeuM3+ molecules, but almost no My4+ and LPS+ constituents. Analysis of monocytes after treatment with phosphatidylinositol-specific phospholipase C indicated that among CD14 molecules with LPS-binding capacity, some are susceptible and others are resistant to the enzyme, each type being mainly expressed by a different monocyte subset. Studies of uninduced and chemically induced THP-1 cells showed that wheat-germ agglutinin blocked the binding of My4 to constitutive, but not to chemically inducible CD14. The overall results suggest the existence of at least three different forms of CD14, which may reflect different stages of cell maturation.