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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: BODIPY-LPS presented to the transfectants as complexes with soluble CD14 first colocalized with mCD14-EGFP on the cell surface.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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Intracellular mCD14–EGFP is rapidly exocytosed. Adherent U373–CD14–EGFP (□) and U373–EGFP (▵) cells cultured in 96-well plates were incubated with LPS–sCD14 complexes (40 ng/ml LPS) at 37°C for the times indicated. At 0 min, no LPS was added. At the end of the incubation, total fluorescence associated with the cells was measured in a fluorescent plate reader. Trypan blue (200 μg/ml) was added to quench extracellular fluorescence, and the remaining intracellular fluorescence was measured. Intracellular fluorescence was calculated as a percentage of total fluorescence in each well. Results are expressed as the means of triplicate wells ± SEM and are from an experiment performed three times with the same result. *Direct comparison using Student's t test shows that these values are significantly different from the value of cells incubated 0 min with LPS (P < 0.003).
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Figure 8: Intracellular mCD14–EGFP is rapidly exocytosed. Adherent U373–CD14–EGFP (□) and U373–EGFP (▵) cells cultured in 96-well plates were incubated with LPS–sCD14 complexes (40 ng/ml LPS) at 37°C for the times indicated. At 0 min, no LPS was added. At the end of the incubation, total fluorescence associated with the cells was measured in a fluorescent plate reader. Trypan blue (200 μg/ml) was added to quench extracellular fluorescence, and the remaining intracellular fluorescence was measured. Intracellular fluorescence was calculated as a percentage of total fluorescence in each well. Results are expressed as the means of triplicate wells ± SEM and are from an experiment performed three times with the same result. *Direct comparison using Student's t test shows that these values are significantly different from the value of cells incubated 0 min with LPS (P < 0.003).

Mentions: Exocytosis of the mCD14-containing vesicles was confirmed by quantitative measurements of the time course. U373–CD14–EGFP cells were grown in 96-well tissue culture plates, and both total and intracellular fluorescence was measured before and at various times after exposure to LPS–sCD14 complexes (see Materials and Methods). Stimulation with LPS did not induce any change in total fluorescence associated with the cells (data not shown) but did induce a rapid decrease in intracellular mCD14–EGFP (Fig. 8). A decrease of ∼20% in the intracellular fluorescence was observed after a 15-min incubation of U373–CD14–EGFP cells with LPS–sCD14 complexes. A similar decrease in intracellular fluorescence associated with U373–EGFP cells was observed in response to LPS–sCD14 complexes (Fig. 8), indicating that the compartments released contained GPI-anchored proteins in addition to mCD14–EGFP.


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

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

Intracellular mCD14–EGFP is rapidly exocytosed. Adherent U373–CD14–EGFP (□) and U373–EGFP (▵) cells cultured in 96-well plates were incubated with LPS–sCD14 complexes (40 ng/ml LPS) at 37°C for the times indicated. At 0 min, no LPS was added. At the end of the incubation, total fluorescence associated with the cells was measured in a fluorescent plate reader. Trypan blue (200 μg/ml) was added to quench extracellular fluorescence, and the remaining intracellular fluorescence was measured. Intracellular fluorescence was calculated as a percentage of total fluorescence in each well. Results are expressed as the means of triplicate wells ± SEM and are from an experiment performed three times with the same result. *Direct comparison using Student's t test shows that these values are significantly different from the value of cells incubated 0 min with LPS (P < 0.003).
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Related In: Results  -  Collection

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Figure 8: Intracellular mCD14–EGFP is rapidly exocytosed. Adherent U373–CD14–EGFP (□) and U373–EGFP (▵) cells cultured in 96-well plates were incubated with LPS–sCD14 complexes (40 ng/ml LPS) at 37°C for the times indicated. At 0 min, no LPS was added. At the end of the incubation, total fluorescence associated with the cells was measured in a fluorescent plate reader. Trypan blue (200 μg/ml) was added to quench extracellular fluorescence, and the remaining intracellular fluorescence was measured. Intracellular fluorescence was calculated as a percentage of total fluorescence in each well. Results are expressed as the means of triplicate wells ± SEM and are from an experiment performed three times with the same result. *Direct comparison using Student's t test shows that these values are significantly different from the value of cells incubated 0 min with LPS (P < 0.003).
Mentions: Exocytosis of the mCD14-containing vesicles was confirmed by quantitative measurements of the time course. U373–CD14–EGFP cells were grown in 96-well tissue culture plates, and both total and intracellular fluorescence was measured before and at various times after exposure to LPS–sCD14 complexes (see Materials and Methods). Stimulation with LPS did not induce any change in total fluorescence associated with the cells (data not shown) but did induce a rapid decrease in intracellular mCD14–EGFP (Fig. 8). A decrease of ∼20% in the intracellular fluorescence was observed after a 15-min incubation of U373–CD14–EGFP cells with LPS–sCD14 complexes. A similar decrease in intracellular fluorescence associated with U373–EGFP cells was observed in response to LPS–sCD14 complexes (Fig. 8), indicating that the compartments released contained GPI-anchored proteins in addition to mCD14–EGFP.

Bottom Line: BODIPY-LPS presented to the transfectants as complexes with soluble CD14 first colocalized with mCD14-EGFP on the cell surface.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