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Analysis of protease activity in live antigen-presenting cells shows regulation of the phagosomal proteolytic contents during dendritic cell activation.

Lennon-Duménil AM, Bakker AH, Maehr R, Fiebiger E, Overkleeft HS, Rosemblatt M, Ploegh HL, Lagaudrière-Gesbert C - J. Exp. Med. (2002)

Bottom Line: Furthermore, the delivery of active proteases to the phagosome is significantly reduced after the activation of DCs with lipopolysaccharide.This observation is in agreement with the notion that DCs prevent the premature destruction of antigenic determinants to optimize T cell activation.Phagosomal maturation is therefore a tightly regulated process that varies according to the type and differentiation stage of the phagocyte.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA.

ABSTRACT
Here, we describe a new approach designed to monitor the proteolytic activity of maturing phagosomes in live antigen-presenting cells. We find that an ingested particle sequentially encounters distinct protease activities during phagosomal maturation. Incorporation of active proteases into the phagosome of the macrophage cell line J774 indicates that phagosome maturation involves progressive fusion with early and late endocytic compartments. In contrast, phagosome biogenesis in bone marrow-derived dendritic cells (DCs) and macrophages preferentially involves endocytic compartments enriched in cathepsin S. Kinetics of phagosomal maturation is faster in macrophages than in DCs. Furthermore, the delivery of active proteases to the phagosome is significantly reduced after the activation of DCs with lipopolysaccharide. This observation is in agreement with the notion that DCs prevent the premature destruction of antigenic determinants to optimize T cell activation. Phagosomal maturation is therefore a tightly regulated process that varies according to the type and differentiation stage of the phagocyte.

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LPS delays phagosome maturation in APCs. (A, B, and D) Labeled proteases were analyzed by SDS-PAGE on 12.5% reducing gel followed by streptavidin blotting. (A) Analysis of proteases incorporated into the phagosome of DCs (CD11c+) upon activation. Bone marrow cells cultured in GM-CSF for 6 d were incubated for 5 min at 37°C with fluorescent yellow beads coupled to DCG-04 in the presence or absence of 0.1 μg/ml LPS. Excess beads were removed and CD11c+ and CD11c− cells were separated. CD11c+ cells were additionally chased at 37°C. After chase, cells were lysed in reducing sample buffer containing 100 μM JPM-565. (B) Analysis of the total contents in cysteine proteases of DCs treated or not with LPS during a 5-min pulse. Cells were treated as described in A. CD11c+ cells were lysed at pH 5 and incubated for 60 min with 5 μM DCG-04 at 37°C. (C) Uptake of fluorescent latex beads by bone marrow–derived DCs treated or not with LPS during a 5-min pulse. Cells were treated as described in A. The cells that did not internalize beads (∼50%) are not depicted in the histogram because FACS® settings were chosen to visualize the high fluorescence population only. (D) Analysis of proteases incorporated into phagosomes of peritoneal macrophages upon LPS activation. Experiments were conducted as described in A.
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fig6: LPS delays phagosome maturation in APCs. (A, B, and D) Labeled proteases were analyzed by SDS-PAGE on 12.5% reducing gel followed by streptavidin blotting. (A) Analysis of proteases incorporated into the phagosome of DCs (CD11c+) upon activation. Bone marrow cells cultured in GM-CSF for 6 d were incubated for 5 min at 37°C with fluorescent yellow beads coupled to DCG-04 in the presence or absence of 0.1 μg/ml LPS. Excess beads were removed and CD11c+ and CD11c− cells were separated. CD11c+ cells were additionally chased at 37°C. After chase, cells were lysed in reducing sample buffer containing 100 μM JPM-565. (B) Analysis of the total contents in cysteine proteases of DCs treated or not with LPS during a 5-min pulse. Cells were treated as described in A. CD11c+ cells were lysed at pH 5 and incubated for 60 min with 5 μM DCG-04 at 37°C. (C) Uptake of fluorescent latex beads by bone marrow–derived DCs treated or not with LPS during a 5-min pulse. Cells were treated as described in A. The cells that did not internalize beads (∼50%) are not depicted in the histogram because FACS® settings were chosen to visualize the high fluorescence population only. (D) Analysis of proteases incorporated into phagosomes of peritoneal macrophages upon LPS activation. Experiments were conducted as described in A.

Mentions: A remarkable trait of DCs is the phenotypic and functional change evoked by the exposure to inflammatory stimuli, such as bacterial products, e.g., LPS. Indeed, LPS increased B7.2 and MHC class II expression at the surface of our bone marrow–derived DCs (29 and unpublished data). To explore whether phagosome biogenesis in DCs is also modulated by exposure to LPS, day-6 bone marrow cultures were pulsed with DCG-04–coated beads in the presence or absence of LPS for 5 min. After removing excess beads, CD11c+ cells were isolated and chased at 37°C. The comparison of untreated and LPS-treated cells revealed drastic differences in the rates of phagosome maturation (Fig. 6 A). Indeed, the delivery of active proteases to the phagosome is considerably delayed in DCs pulsed in the presence of LPS because even after 60 and 120 min of chase, beads have not yet reached saturation (Fig. 6 A and unpublished data). This difference between LPS-treated and control cells does not result from reduced bead uptake by the cells pulsed in the presence of LPS (Fig. 6 C), nor from different amounts of active cysteine proteases (Fig. 6 B).


Analysis of protease activity in live antigen-presenting cells shows regulation of the phagosomal proteolytic contents during dendritic cell activation.

Lennon-Duménil AM, Bakker AH, Maehr R, Fiebiger E, Overkleeft HS, Rosemblatt M, Ploegh HL, Lagaudrière-Gesbert C - J. Exp. Med. (2002)

LPS delays phagosome maturation in APCs. (A, B, and D) Labeled proteases were analyzed by SDS-PAGE on 12.5% reducing gel followed by streptavidin blotting. (A) Analysis of proteases incorporated into the phagosome of DCs (CD11c+) upon activation. Bone marrow cells cultured in GM-CSF for 6 d were incubated for 5 min at 37°C with fluorescent yellow beads coupled to DCG-04 in the presence or absence of 0.1 μg/ml LPS. Excess beads were removed and CD11c+ and CD11c− cells were separated. CD11c+ cells were additionally chased at 37°C. After chase, cells were lysed in reducing sample buffer containing 100 μM JPM-565. (B) Analysis of the total contents in cysteine proteases of DCs treated or not with LPS during a 5-min pulse. Cells were treated as described in A. CD11c+ cells were lysed at pH 5 and incubated for 60 min with 5 μM DCG-04 at 37°C. (C) Uptake of fluorescent latex beads by bone marrow–derived DCs treated or not with LPS during a 5-min pulse. Cells were treated as described in A. The cells that did not internalize beads (∼50%) are not depicted in the histogram because FACS® settings were chosen to visualize the high fluorescence population only. (D) Analysis of proteases incorporated into phagosomes of peritoneal macrophages upon LPS activation. Experiments were conducted as described in A.
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fig6: LPS delays phagosome maturation in APCs. (A, B, and D) Labeled proteases were analyzed by SDS-PAGE on 12.5% reducing gel followed by streptavidin blotting. (A) Analysis of proteases incorporated into the phagosome of DCs (CD11c+) upon activation. Bone marrow cells cultured in GM-CSF for 6 d were incubated for 5 min at 37°C with fluorescent yellow beads coupled to DCG-04 in the presence or absence of 0.1 μg/ml LPS. Excess beads were removed and CD11c+ and CD11c− cells were separated. CD11c+ cells were additionally chased at 37°C. After chase, cells were lysed in reducing sample buffer containing 100 μM JPM-565. (B) Analysis of the total contents in cysteine proteases of DCs treated or not with LPS during a 5-min pulse. Cells were treated as described in A. CD11c+ cells were lysed at pH 5 and incubated for 60 min with 5 μM DCG-04 at 37°C. (C) Uptake of fluorescent latex beads by bone marrow–derived DCs treated or not with LPS during a 5-min pulse. Cells were treated as described in A. The cells that did not internalize beads (∼50%) are not depicted in the histogram because FACS® settings were chosen to visualize the high fluorescence population only. (D) Analysis of proteases incorporated into phagosomes of peritoneal macrophages upon LPS activation. Experiments were conducted as described in A.
Mentions: A remarkable trait of DCs is the phenotypic and functional change evoked by the exposure to inflammatory stimuli, such as bacterial products, e.g., LPS. Indeed, LPS increased B7.2 and MHC class II expression at the surface of our bone marrow–derived DCs (29 and unpublished data). To explore whether phagosome biogenesis in DCs is also modulated by exposure to LPS, day-6 bone marrow cultures were pulsed with DCG-04–coated beads in the presence or absence of LPS for 5 min. After removing excess beads, CD11c+ cells were isolated and chased at 37°C. The comparison of untreated and LPS-treated cells revealed drastic differences in the rates of phagosome maturation (Fig. 6 A). Indeed, the delivery of active proteases to the phagosome is considerably delayed in DCs pulsed in the presence of LPS because even after 60 and 120 min of chase, beads have not yet reached saturation (Fig. 6 A and unpublished data). This difference between LPS-treated and control cells does not result from reduced bead uptake by the cells pulsed in the presence of LPS (Fig. 6 C), nor from different amounts of active cysteine proteases (Fig. 6 B).

Bottom Line: Furthermore, the delivery of active proteases to the phagosome is significantly reduced after the activation of DCs with lipopolysaccharide.This observation is in agreement with the notion that DCs prevent the premature destruction of antigenic determinants to optimize T cell activation.Phagosomal maturation is therefore a tightly regulated process that varies according to the type and differentiation stage of the phagocyte.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA.

ABSTRACT
Here, we describe a new approach designed to monitor the proteolytic activity of maturing phagosomes in live antigen-presenting cells. We find that an ingested particle sequentially encounters distinct protease activities during phagosomal maturation. Incorporation of active proteases into the phagosome of the macrophage cell line J774 indicates that phagosome maturation involves progressive fusion with early and late endocytic compartments. In contrast, phagosome biogenesis in bone marrow-derived dendritic cells (DCs) and macrophages preferentially involves endocytic compartments enriched in cathepsin S. Kinetics of phagosomal maturation is faster in macrophages than in DCs. Furthermore, the delivery of active proteases to the phagosome is significantly reduced after the activation of DCs with lipopolysaccharide. This observation is in agreement with the notion that DCs prevent the premature destruction of antigenic determinants to optimize T cell activation. Phagosomal maturation is therefore a tightly regulated process that varies according to the type and differentiation stage of the phagocyte.

Show MeSH
Related in: MedlinePlus