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Internalization of monomeric lipopolysaccharide occurs after transfer out of cell surface CD14.

Vasselon T, Hailman E, Thieringer R, Detmers PA - J. Exp. Med. (1999)

Bottom Line: Lipopolysaccharide (LPS) fluorescently labeled with boron dipyrromethane (BODIPY) first binds to the plasma membrane of CD14-expressing cells and is subsequently internalized.These results suggest that monomeric LPS is transferred out of mCD14 at the plasma membrane and traffics within the cell independently of mCD14.In contrast, aggregates of LPS were internalized in association with mCD14, suggesting that LPS clearance occurs via a pathway distinct from that which leads to signaling via monomeric LPS.

View Article: PubMed Central - PubMed

Affiliation: Department of Endocrinology and Chemical Biology, Merck Research Laboratories, Rahway, New Jersey 07065, USA.

ABSTRACT
Lipopolysaccharide (LPS) fluorescently labeled with boron dipyrromethane (BODIPY) first binds to the plasma membrane of CD14-expressing cells and is subsequently internalized. Intracellular LPS appears in small vesicles near the cell surface and later in larger, punctate structures identified as the Golgi apparatus. To determine if membrane (m)CD14 directs the movement of LPS to the Golgi apparatus, an mCD14 chimera containing enhanced green fluorescent protein (mCD14-EGFP) was used to follow trafficking of mCD14 and BODIPY-LPS in stable transfectants. The chimera was expressed strongly on the cell surface and also in a Golgi complex-like structure. mCD14-EGFP was functional in mediating binding of and responses to LPS. BODIPY-LPS presented to the transfectants as complexes with soluble CD14 first colocalized with mCD14-EGFP on the cell surface. However, within 5-10 min, the BODIPY-LPS distributed to intracellular vesicles that did not contain mCD14-EGFP, indicating that mCD14 did not accompany LPS during endocytic movement. These results suggest that monomeric LPS is transferred out of mCD14 at the plasma membrane and traffics within the cell independently of mCD14. In contrast, aggregates of LPS were internalized in association with mCD14, suggesting that LPS clearance occurs via a pathway distinct from that which leads to signaling via monomeric LPS.

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CD14 is present in intracellular vesicles that are exocytosed in response to LPS. U373–CD14–EGFP cells were incubated for 30 min at 37°C in HAP buffer in the absence (A and B) or presence (C and D) of preformed LPS–sCD14 complexes (40 ng/ml LPS). Trypan blue (200 μg/ml) was added to quench mCD14–EGFP on the cell surface, and the cells were examined by confocal microscopy. Most of the intracellular mCD14–EGFP was localized to a juxtanuclear reticulum (A and C), presumably the Golgi apparatus. In untreated cells, a pool of mCD14–EGFP was also observed in vesicles located throughout the cell but especially prevalent near the basal aspect (B). Most of the vesicular pool of intracellular mCD14–EGFP was no longer visible after stimulation of the cells with preformed LPS–sCD14 complexes (D). Bars, 10 μm.
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Figure 7: CD14 is present in intracellular vesicles that are exocytosed in response to LPS. U373–CD14–EGFP cells were incubated for 30 min at 37°C in HAP buffer in the absence (A and B) or presence (C and D) of preformed LPS–sCD14 complexes (40 ng/ml LPS). Trypan blue (200 μg/ml) was added to quench mCD14–EGFP on the cell surface, and the cells were examined by confocal microscopy. Most of the intracellular mCD14–EGFP was localized to a juxtanuclear reticulum (A and C), presumably the Golgi apparatus. In untreated cells, a pool of mCD14–EGFP was also observed in vesicles located throughout the cell but especially prevalent near the basal aspect (B). Most of the vesicular pool of intracellular mCD14–EGFP was no longer visible after stimulation of the cells with preformed LPS–sCD14 complexes (D). Bars, 10 μm.

Mentions: Although there was no apparent change in the distribution of mCD14–EGFP in response to LPS in the colocalization studies, the bright cell surface fluorescence prevented observation of any changes in the distribution of intracellular mCD14–EGFP. To better observe the distribution of intracellular mCD14–EGFP, we quenched the cell surface fluorescence on U373–CD14–EGFP cells with trypan blue (Fig. 7A and Fig. B). In addition to the bright fluorescence emanating from the Golgi apparatus area, numerous fluorescent vesicles of ∼50–100-nm average diameter were distributed throughout the cytoplasm. The vesicles were particularly evident near the basal aspects of the cells (Fig. 7 B). A similar intracellular localization of mEGFP in the Golgi complex and cytoplasmic vesicles was also observed (data not shown). Thus, the vesicular compartment may represent either a component of the secretory pathway en route to the cell surface or a recycling compartment for GPI-anchored proteins.


Internalization of monomeric lipopolysaccharide occurs after transfer out of cell surface CD14.

Vasselon T, Hailman E, Thieringer R, Detmers PA - J. Exp. Med. (1999)

CD14 is present in intracellular vesicles that are exocytosed in response to LPS. U373–CD14–EGFP cells were incubated for 30 min at 37°C in HAP buffer in the absence (A and B) or presence (C and D) of preformed LPS–sCD14 complexes (40 ng/ml LPS). Trypan blue (200 μg/ml) was added to quench mCD14–EGFP on the cell surface, and the cells were examined by confocal microscopy. Most of the intracellular mCD14–EGFP was localized to a juxtanuclear reticulum (A and C), presumably the Golgi apparatus. In untreated cells, a pool of mCD14–EGFP was also observed in vesicles located throughout the cell but especially prevalent near the basal aspect (B). Most of the vesicular pool of intracellular mCD14–EGFP was no longer visible after stimulation of the cells with preformed LPS–sCD14 complexes (D). Bars, 10 μm.
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Related In: Results  -  Collection

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Figure 7: CD14 is present in intracellular vesicles that are exocytosed in response to LPS. U373–CD14–EGFP cells were incubated for 30 min at 37°C in HAP buffer in the absence (A and B) or presence (C and D) of preformed LPS–sCD14 complexes (40 ng/ml LPS). Trypan blue (200 μg/ml) was added to quench mCD14–EGFP on the cell surface, and the cells were examined by confocal microscopy. Most of the intracellular mCD14–EGFP was localized to a juxtanuclear reticulum (A and C), presumably the Golgi apparatus. In untreated cells, a pool of mCD14–EGFP was also observed in vesicles located throughout the cell but especially prevalent near the basal aspect (B). Most of the vesicular pool of intracellular mCD14–EGFP was no longer visible after stimulation of the cells with preformed LPS–sCD14 complexes (D). Bars, 10 μm.
Mentions: Although there was no apparent change in the distribution of mCD14–EGFP in response to LPS in the colocalization studies, the bright cell surface fluorescence prevented observation of any changes in the distribution of intracellular mCD14–EGFP. To better observe the distribution of intracellular mCD14–EGFP, we quenched the cell surface fluorescence on U373–CD14–EGFP cells with trypan blue (Fig. 7A and Fig. B). In addition to the bright fluorescence emanating from the Golgi apparatus area, numerous fluorescent vesicles of ∼50–100-nm average diameter were distributed throughout the cytoplasm. The vesicles were particularly evident near the basal aspects of the cells (Fig. 7 B). A similar intracellular localization of mEGFP in the Golgi complex and cytoplasmic vesicles was also observed (data not shown). Thus, the vesicular compartment may represent either a component of the secretory pathway en route to the cell surface or a recycling compartment for GPI-anchored proteins.

Bottom Line: Lipopolysaccharide (LPS) fluorescently labeled with boron dipyrromethane (BODIPY) first binds to the plasma membrane of CD14-expressing cells and is subsequently internalized.These results suggest that monomeric LPS is transferred out of mCD14 at the plasma membrane and traffics within the cell independently of mCD14.In contrast, aggregates of LPS were internalized in association with mCD14, suggesting that LPS clearance occurs via a pathway distinct from that which leads to signaling via monomeric LPS.

View Article: PubMed Central - PubMed

Affiliation: Department of Endocrinology and Chemical Biology, Merck Research Laboratories, Rahway, New Jersey 07065, USA.

ABSTRACT
Lipopolysaccharide (LPS) fluorescently labeled with boron dipyrromethane (BODIPY) first binds to the plasma membrane of CD14-expressing cells and is subsequently internalized. Intracellular LPS appears in small vesicles near the cell surface and later in larger, punctate structures identified as the Golgi apparatus. To determine if membrane (m)CD14 directs the movement of LPS to the Golgi apparatus, an mCD14 chimera containing enhanced green fluorescent protein (mCD14-EGFP) was used to follow trafficking of mCD14 and BODIPY-LPS in stable transfectants. The chimera was expressed strongly on the cell surface and also in a Golgi complex-like structure. mCD14-EGFP was functional in mediating binding of and responses to LPS. BODIPY-LPS presented to the transfectants as complexes with soluble CD14 first colocalized with mCD14-EGFP on the cell surface. However, within 5-10 min, the BODIPY-LPS distributed to intracellular vesicles that did not contain mCD14-EGFP, indicating that mCD14 did not accompany LPS during endocytic movement. These results suggest that monomeric LPS is transferred out of mCD14 at the plasma membrane and traffics within the cell independently of mCD14. In contrast, aggregates of LPS were internalized in association with mCD14, suggesting that LPS clearance occurs via a pathway distinct from that which leads to signaling via monomeric LPS.

Show MeSH
Related in: MedlinePlus