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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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Visualization of phagosomal cysteine protease activity in vivo. (A) Schematic outline of the experimental design. (B–D) Proteins were separated by SDS-PAGE on a 12.5% gel and reactive proteases were visualized by streptavidin blotting. (B) Analysis of phagosomal proteases. J774 cells were incubated for 30 min at 37°C with latex beads previously coated with different concentrations of DCG-04 (pulse). After the removal of excess beads, cells were additionally incubated for 1 h at 37°C (chase). Cells were then directly lysed in 2× concentrated reducing sample buffer. Controls: on the left, cells were treated as in A, but free soluble DCG-04 was added during the pulse instead of DCG-04 coupled to beads, and on the right, cells were treated as in A, but DCG-04–coated beads were added just before lysis in the presence or absence of 100 μM JPM-565. (C) Analysis of phagosomal proteases comparing beads of 1- and 2-μm diameter. J774 cells were incubated for 30 min at 37°C with various concentrations of DCG-04 coupled to latex beads. Cells were then washed in PBS, incubated for an additional 60 min at 37°C, and lysed in sample buffer containing 100 μM JPM-565. (D) Labeling of phagosomal proteases requires phagocytosis. On the left, J774 cells were pretreated with the indicated inhibitors for 60 min (10μg/ml Cytochalasin D, 1mM Leupeptin, or 20 nM ConB), pulsed with beads coupled to 0.1 μM DCG-04 for 30 min at 37°C (lanes 1, 3, 4, and 5) or 4°C (lane 2), washed in PBS, and chased for 60 min at 37°C or 4°C before lysis in reducing sample buffer containing 100 μM JPM-565. On the right, cells were treated as shown on the left except that they were lysed at pH 5. Cell lysates were incubated with 10 μM DCG-04 for 60 min at 37°C. Note that the two differentially glycosylated forms of CatB appear as a doublet at low expression levels (left).
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fig2: Visualization of phagosomal cysteine protease activity in vivo. (A) Schematic outline of the experimental design. (B–D) Proteins were separated by SDS-PAGE on a 12.5% gel and reactive proteases were visualized by streptavidin blotting. (B) Analysis of phagosomal proteases. J774 cells were incubated for 30 min at 37°C with latex beads previously coated with different concentrations of DCG-04 (pulse). After the removal of excess beads, cells were additionally incubated for 1 h at 37°C (chase). Cells were then directly lysed in 2× concentrated reducing sample buffer. Controls: on the left, cells were treated as in A, but free soluble DCG-04 was added during the pulse instead of DCG-04 coupled to beads, and on the right, cells were treated as in A, but DCG-04–coated beads were added just before lysis in the presence or absence of 100 μM JPM-565. (C) Analysis of phagosomal proteases comparing beads of 1- and 2-μm diameter. J774 cells were incubated for 30 min at 37°C with various concentrations of DCG-04 coupled to latex beads. Cells were then washed in PBS, incubated for an additional 60 min at 37°C, and lysed in sample buffer containing 100 μM JPM-565. (D) Labeling of phagosomal proteases requires phagocytosis. On the left, J774 cells were pretreated with the indicated inhibitors for 60 min (10μg/ml Cytochalasin D, 1mM Leupeptin, or 20 nM ConB), pulsed with beads coupled to 0.1 μM DCG-04 for 30 min at 37°C (lanes 1, 3, 4, and 5) or 4°C (lane 2), washed in PBS, and chased for 60 min at 37°C or 4°C before lysis in reducing sample buffer containing 100 μM JPM-565. On the right, cells were treated as shown on the left except that they were lysed at pH 5. Cell lysates were incubated with 10 μM DCG-04 for 60 min at 37°C. Note that the two differentially glycosylated forms of CatB appear as a doublet at low expression levels (left).

Mentions: To check whether DCG-04–loaded streptavidin-coated beads can be acquired by phagocytes and target cysteine proteases in endosomal compartments, J774 cells were incubated with DCG-04–coated beads according to the procedure outlined in Fig. 2 A. To ensure that an adequate amount of beads was taken up, cells were pulsed with DCG-04–coated beads for 30 min. Excess beads were removed by washing and cells were chased for 60 min to allow the beads to reach the relevant endocytic compartments. At the end of the chase, cells were lysed in reducing SDS sample buffer immediately followed by heating to prevent postlysis modification of proteases by the probe. Samples were resolved by electrophoresis and analyzed by streptavidin blotting to visualize the DCG-04–modified polypeptides. DCG-04 coupled to beads labeled CatZ, CatB, CatS, and CatL, demonstrating that the activity of these four enzymes can indeed be visualized in phagosomes of live cells (Fig. 2 B). By loading the latex beads with increasing concentrations of DCG-04, the signal could be enhanced with maximal labeling being achieved at 0.1 μM DCG-04 (Fig. 2 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)

Visualization of phagosomal cysteine protease activity in vivo. (A) Schematic outline of the experimental design. (B–D) Proteins were separated by SDS-PAGE on a 12.5% gel and reactive proteases were visualized by streptavidin blotting. (B) Analysis of phagosomal proteases. J774 cells were incubated for 30 min at 37°C with latex beads previously coated with different concentrations of DCG-04 (pulse). After the removal of excess beads, cells were additionally incubated for 1 h at 37°C (chase). Cells were then directly lysed in 2× concentrated reducing sample buffer. Controls: on the left, cells were treated as in A, but free soluble DCG-04 was added during the pulse instead of DCG-04 coupled to beads, and on the right, cells were treated as in A, but DCG-04–coated beads were added just before lysis in the presence or absence of 100 μM JPM-565. (C) Analysis of phagosomal proteases comparing beads of 1- and 2-μm diameter. J774 cells were incubated for 30 min at 37°C with various concentrations of DCG-04 coupled to latex beads. Cells were then washed in PBS, incubated for an additional 60 min at 37°C, and lysed in sample buffer containing 100 μM JPM-565. (D) Labeling of phagosomal proteases requires phagocytosis. On the left, J774 cells were pretreated with the indicated inhibitors for 60 min (10μg/ml Cytochalasin D, 1mM Leupeptin, or 20 nM ConB), pulsed with beads coupled to 0.1 μM DCG-04 for 30 min at 37°C (lanes 1, 3, 4, and 5) or 4°C (lane 2), washed in PBS, and chased for 60 min at 37°C or 4°C before lysis in reducing sample buffer containing 100 μM JPM-565. On the right, cells were treated as shown on the left except that they were lysed at pH 5. Cell lysates were incubated with 10 μM DCG-04 for 60 min at 37°C. Note that the two differentially glycosylated forms of CatB appear as a doublet at low expression levels (left).
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Related In: Results  -  Collection

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fig2: Visualization of phagosomal cysteine protease activity in vivo. (A) Schematic outline of the experimental design. (B–D) Proteins were separated by SDS-PAGE on a 12.5% gel and reactive proteases were visualized by streptavidin blotting. (B) Analysis of phagosomal proteases. J774 cells were incubated for 30 min at 37°C with latex beads previously coated with different concentrations of DCG-04 (pulse). After the removal of excess beads, cells were additionally incubated for 1 h at 37°C (chase). Cells were then directly lysed in 2× concentrated reducing sample buffer. Controls: on the left, cells were treated as in A, but free soluble DCG-04 was added during the pulse instead of DCG-04 coupled to beads, and on the right, cells were treated as in A, but DCG-04–coated beads were added just before lysis in the presence or absence of 100 μM JPM-565. (C) Analysis of phagosomal proteases comparing beads of 1- and 2-μm diameter. J774 cells were incubated for 30 min at 37°C with various concentrations of DCG-04 coupled to latex beads. Cells were then washed in PBS, incubated for an additional 60 min at 37°C, and lysed in sample buffer containing 100 μM JPM-565. (D) Labeling of phagosomal proteases requires phagocytosis. On the left, J774 cells were pretreated with the indicated inhibitors for 60 min (10μg/ml Cytochalasin D, 1mM Leupeptin, or 20 nM ConB), pulsed with beads coupled to 0.1 μM DCG-04 for 30 min at 37°C (lanes 1, 3, 4, and 5) or 4°C (lane 2), washed in PBS, and chased for 60 min at 37°C or 4°C before lysis in reducing sample buffer containing 100 μM JPM-565. On the right, cells were treated as shown on the left except that they were lysed at pH 5. Cell lysates were incubated with 10 μM DCG-04 for 60 min at 37°C. Note that the two differentially glycosylated forms of CatB appear as a doublet at low expression levels (left).
Mentions: To check whether DCG-04–loaded streptavidin-coated beads can be acquired by phagocytes and target cysteine proteases in endosomal compartments, J774 cells were incubated with DCG-04–coated beads according to the procedure outlined in Fig. 2 A. To ensure that an adequate amount of beads was taken up, cells were pulsed with DCG-04–coated beads for 30 min. Excess beads were removed by washing and cells were chased for 60 min to allow the beads to reach the relevant endocytic compartments. At the end of the chase, cells were lysed in reducing SDS sample buffer immediately followed by heating to prevent postlysis modification of proteases by the probe. Samples were resolved by electrophoresis and analyzed by streptavidin blotting to visualize the DCG-04–modified polypeptides. DCG-04 coupled to beads labeled CatZ, CatB, CatS, and CatL, demonstrating that the activity of these four enzymes can indeed be visualized in phagosomes of live cells (Fig. 2 B). By loading the latex beads with increasing concentrations of DCG-04, the signal could be enhanced with maximal labeling being achieved at 0.1 μM DCG-04 (Fig. 2 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