Abstract
Gram-negative bacterial endotoxin (a lipopolysaccharide (LPS)) specifically binds to CD14, a glycosylphosphatidyl inositol (GPI)-anchored surface myeloid glycoprotein. This interaction leads to cell activation, but it also promotes LPS internalization and detoxification. In this work, we investigated the route of LPS and CD14 internalization and the relevance of CD14 GPI anchor in the endocytic pathway. In promonocytic THP-1 cells transfected with a GPI or a chimeric integral form of CD14, we showed by differential buoyancy in sucrose density gradients that these two forms of CD14 were sorted to different plasma membrane subdomains. However, both forms of CD14 associated preferentially with the same surface microfilament-enriched microvilli or ruffles. Electron microscopic studies indicated that CD14 internalized via macropinocytosis, a process resembling that of phagocytosis, different from "classical" receptor-mediated endocytic pathways, such as clathrin-coated pits or caveolae. With cell warming, the CD14-enriched ruffles fused and formed large vesicles. Later, these vacuoles made stacks and condensed into phago-lysosomes. CD14 was specifically associated with all of these structures. Radiolabeled LPS internalization paralleled CD14 internalization. Confocal microscopic studies confirmed the co-localization of LPS and CD14 both at the cell surface and in endosomal compartments. The microfilament-disrupting, macropinocytosis blocking agent cytochalasin D inhibited LPS and CD14 internalization but did not prevent LPS-dependent activation, indicating that these two processes are dissociated.
Highlights
Clearance of bacteria and bacterial molecules, predominantly performed by macrophages, is an important feature of bacterial immunity and requires specific recognition and endocytic pathways [1, 2]
Membrane Targeting of CD14 and Surface Events—Using sucrose density fractionation of Triton X-100 cell extracts, we observed that the GPI form of CD14 and the transmembrane CD14 chimera were targeted to different membrane subdomains, as indicated by their differential buoyancy in the sucrose gradient
Nondetergent isolation of low-density GPIrich domains [29] showed that CD14 was enriched at least 100 times in these domains as compared with transmembrane CD14 chimera (tmCD14), as determined by serial dilutions of GPI-rich domains of wt- and tmCD14-THP1 cells in the dot-blot assay. This latter experiment indicated that the separation by buoyancy of wt- and tmCD14 was not an artifact due to the presence of detergents. These experiments demonstrated that the GPI anchoring was responsible for targeting CD14 to GPI-rich, lipid-rich plasma membrane subdomains, whereas tmCD14 was sorted into other membrane domains, where most integral proteins are found
Summary
Clearance of bacteria and bacterial molecules, predominantly performed by macrophages, is an important feature of bacterial immunity and requires specific recognition and endocytic pathways [1, 2]. Apart from cell activation, membrane-bound CD14 plays other important roles: it mediates LPS internalization [7, 13, 14], as well as Gram-negative bacteria and mycobacteria phagocytosis [15, 16]. Glycosylphosphatidyl-inositol (GPI)-linked molecules are sorted to plasma membrane subdomains, rich in sphyngomyelin, cholesterol, receptors, and other signaling molecules (18 – 20). In epithelial cells, these plasma membrane domains correspond morphologically to noncoated pits and vesicles, known as caveolae [19]. The modification of the GPI-anchored CD14 into an integral protein did not significantly affect rates and pathways of receptor-ligand internalization, and CD14 endocytosis did not seem to be regulated by its ligand, LPS. Cytochalasin D inhibited LPS endocytosis but did not prevent LPS-dependent cell activation
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