CD14-dependent Endotoxin Internalization via a Macropinocytic Pathway

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.

Streptococci Engage TLR13 on Myeloid Cells in a Site-Specific Fashion

“…2E). In contrast, the LPS response was only modestly affected, which is in accordance with published data (31). Therefore, phagocytosis is required for the GBS-induced cytokine response in monocytes, mice were infected with WT or lgt-deficient GBS (multiplicity of infection [MOI] = 10) for 2 h and then 250 mg/ml gentamicin was added to the culture.…”

Transcriptional Inhibition of Interleukin-12 Promoter Activity in Leishmania Spp.–Infected Macrophages

“…parasites to determine if only cell contact was required for IL-12 inhibition. In both cases, the inhibitors alone suppressed LPS-induced luciferase expression and endogenous IL-12 transcription, suggesting that contrary to previous reports (Poussin et al, 1998) LPS-dependent activation requires LPS internalization. The inhibition was selective, however, as TNF-α and IL-10 production were enhanced in the presence of the phagocytosis inhibitors.…”

Functional CD1a is stabilized by exogenous lipids

“…Amongst its effects, BFA blocks protein egression from the transGolgi network, thus affecting surface expression of de novo synthesized proteins. CHD inhibits fluid-phase, receptor-or caveolae-mediated uptake of extracellular and transmembrane molecules [11][12][13]. In the presence of BFA or CHD, CD1a continued to be altered after 24 h of culture in LS (Table 1).…”