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From the Merck Research Laboratories, Rahway, New Jersey 07065

We have recently shown that monomeric bacterial LPS is rapidly delivered from the plasma membrane to an intracellular site and that agents that block vesicular transport block responses of neutrophils to lipopolysaccharide (LPS) (Detmers, P.A., N. Thiéblemont, T. Vasselon, R. Pironkova, D.S. Miller, and S.D. Wright. 1996. J. Immunol. 157:5589–5596). To examine further the connection between intracellular transport of LPS and signaling, we observed internalization of fluorescently labeled LPS in cells from LPS-hyporesponsive (Lps^d) mice. Binding of fluorescent LPS from LPS–soluble CD14 (sCD14) complexes by peritoneal macrophages from Lps^d and control (Lpsⁿ) mice was quantitatively similar, and confocal images obtained from these cells exhibited an identical appearance immediately after labeling. Incubation of labeled Lpsⁿ macrophages at 37°C caused movement of the fluorescence from the cell perimeter in one or two spots in the perinuclear region. However, in Lps^d cells the fluorescence remained dispersed, suggesting a defect in vesicular transport. LPS resembles ceramide, and Lps^d mice fail to respond to ceramide. As with LPS, we found that binding of fluorescent ceramide by Lps^d and Lpsⁿ macrophages was quantitatively similar, and the label moved rapidly to one to two spots in the perinuclear region in Lpsⁿ mice. However, in Lps^d macrophages the fluorescence remained dispersed. These results show that cells deficient in responses to LPS exhibit defective vesicular transport of LPS and ceramide and point to a role for vesicular transport in responses to these mediators.

¹Abbreviations used in this paper: AOAH, acyloxyacyl hydrolase; BODIPY, boron dipyrromethane; BODIPY–ceramide, BODIPY-labeled C5-ceramide; BODIPY–LPS, BODIPY-labeled LPS; DF-BSA, defatted BSA; EGF, epidermal growth factor; ERK1, mitogen-activated protein kinase 1; ERK2, mitogen-activated protein kinase 2; HAP, Dulbecco's PBS with 0.5 U/ml aprotinin, 0.05% human serum albumin, 3 mM D-glucose; HSA, human serum albumin; JNK, c-Jun NH₂-terminal kinase; LBP, LPS-binding protein; Lps^d, LPS hyporesponsive; Lpsⁿ, LPS responsive; PI3K, phosphatidylinositol 3-kinase; sCD14, soluble CD14.

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