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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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Cysteine protease activity in bone marrow–derived APCs. (A–C) Proteins were separated by SDS-PAGE on a reducing 12.5% SDS gel and reactive proteins were visualized by streptavidin blotting. (A) Analysis of the total contents in cysteine proteases of primary APCs. Total extracts (pH 5) from bone marrow cells cultured in GM-CSF for 6 d were incubated with 0.1 μM DCG-04 for 1 h at 37°C (left). Total extracts (pH 5) were incubated with 5 μM DCG-04 and subjected to immunoprecipitation using anti-CatB, anti-CatS, or anti-CatL antibodies (right). *, a labeled protease of unknown identity specific for bone marrow–derived APCs. (B) Analysis of the total contents in cysteine proteases of primary APCs from WT and Cat-deficient mice. Total extracts (pH 5) from bone marrow cells cultured in GM-CSF for 5 d were incubated with 0.1 μM DCG-04 for 1 h at 37°C. (C) Analysis of proteases incorporated into the phagosome of bone marrow–derived APCs during maturation. Cells cultured in GM-CSF for 6 d were incubated at 37°C with latex beads coupled to DCG-04 for 5 min. After the removal of excess beads, cells were additionally incubated at 37°C (chase). After each time point, cells were lysed in reducing sample buffer containing 100 μM JPM-565.
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fig4: Cysteine protease activity in bone marrow–derived APCs. (A–C) Proteins were separated by SDS-PAGE on a reducing 12.5% SDS gel and reactive proteins were visualized by streptavidin blotting. (A) Analysis of the total contents in cysteine proteases of primary APCs. Total extracts (pH 5) from bone marrow cells cultured in GM-CSF for 6 d were incubated with 0.1 μM DCG-04 for 1 h at 37°C (left). Total extracts (pH 5) were incubated with 5 μM DCG-04 and subjected to immunoprecipitation using anti-CatB, anti-CatS, or anti-CatL antibodies (right). *, a labeled protease of unknown identity specific for bone marrow–derived APCs. (B) Analysis of the total contents in cysteine proteases of primary APCs from WT and Cat-deficient mice. Total extracts (pH 5) from bone marrow cells cultured in GM-CSF for 5 d were incubated with 0.1 μM DCG-04 for 1 h at 37°C. (C) Analysis of proteases incorporated into the phagosome of bone marrow–derived APCs during maturation. Cells cultured in GM-CSF for 6 d were incubated at 37°C with latex beads coupled to DCG-04 for 5 min. After the removal of excess beads, cells were additionally incubated at 37°C (chase). After each time point, cells were lysed in reducing sample buffer containing 100 μM JPM-565.

Mentions: To establish our in vivo protease labeling assay, we used the J774 monocytic cell line, which has been extensively characterized in terms of phagosomal biogenesis using latex beads (13, 15). To identify the targets of DCG-04 in J774 cells, lysates were prepared at pH 5 and incubated with an increasing amount of the DCG-04, followed by SDS-PAGE and streptavidin blotting (Fig. 1 C). At least seven distinct polypeptides were detected in the 20–40-kD range, where most of the known active cysteine proteases are expected to migrate. To identify these proteases, DCG-04–labeled cell lysates were immunoprecipitated with antibodies directed against CatB, CatL, and CatS. This allowed the identification of three major DCG-04–labeled species as being CatB, CatL, and CatS (Fig. 1 D). The identity of these enzymes was additionally confirmed by comparing the labeling pattern of WT and CatB-, CatS-, or CatL-deficient cells (Fig. 4 B). The polypeptide strongly reactive with DCG-04 and of a mass slightly larger than that of CatB did not react with any of the antibodies tested (Fig. 1 C), including a CatH antiserum (unpublished data). DCG-04–labeled cell lysates were therefore incubated with streptavidin-coated agarose beads on a preparative scale and bound material was resolved by electrophoresis followed by Coomassie staining. The polypeptide of interest was excised, digested with trypsin, and analyzed by microbore and electron spray mass spectrometry, allowing its unambiguous identification as CatZ. The three top bands detected in J774 lysates (Fig. 1, C and D) were not considered in our analysis because they were never detected in in vivo labeling assays (see below).


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)

Cysteine protease activity in bone marrow–derived APCs. (A–C) Proteins were separated by SDS-PAGE on a reducing 12.5% SDS gel and reactive proteins were visualized by streptavidin blotting. (A) Analysis of the total contents in cysteine proteases of primary APCs. Total extracts (pH 5) from bone marrow cells cultured in GM-CSF for 6 d were incubated with 0.1 μM DCG-04 for 1 h at 37°C (left). Total extracts (pH 5) were incubated with 5 μM DCG-04 and subjected to immunoprecipitation using anti-CatB, anti-CatS, or anti-CatL antibodies (right). *, a labeled protease of unknown identity specific for bone marrow–derived APCs. (B) Analysis of the total contents in cysteine proteases of primary APCs from WT and Cat-deficient mice. Total extracts (pH 5) from bone marrow cells cultured in GM-CSF for 5 d were incubated with 0.1 μM DCG-04 for 1 h at 37°C. (C) Analysis of proteases incorporated into the phagosome of bone marrow–derived APCs during maturation. Cells cultured in GM-CSF for 6 d were incubated at 37°C with latex beads coupled to DCG-04 for 5 min. After the removal of excess beads, cells were additionally incubated at 37°C (chase). After each time point, cells were lysed in reducing sample buffer containing 100 μM JPM-565.
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Related In: Results  -  Collection

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fig4: Cysteine protease activity in bone marrow–derived APCs. (A–C) Proteins were separated by SDS-PAGE on a reducing 12.5% SDS gel and reactive proteins were visualized by streptavidin blotting. (A) Analysis of the total contents in cysteine proteases of primary APCs. Total extracts (pH 5) from bone marrow cells cultured in GM-CSF for 6 d were incubated with 0.1 μM DCG-04 for 1 h at 37°C (left). Total extracts (pH 5) were incubated with 5 μM DCG-04 and subjected to immunoprecipitation using anti-CatB, anti-CatS, or anti-CatL antibodies (right). *, a labeled protease of unknown identity specific for bone marrow–derived APCs. (B) Analysis of the total contents in cysteine proteases of primary APCs from WT and Cat-deficient mice. Total extracts (pH 5) from bone marrow cells cultured in GM-CSF for 5 d were incubated with 0.1 μM DCG-04 for 1 h at 37°C. (C) Analysis of proteases incorporated into the phagosome of bone marrow–derived APCs during maturation. Cells cultured in GM-CSF for 6 d were incubated at 37°C with latex beads coupled to DCG-04 for 5 min. After the removal of excess beads, cells were additionally incubated at 37°C (chase). After each time point, cells were lysed in reducing sample buffer containing 100 μM JPM-565.
Mentions: To establish our in vivo protease labeling assay, we used the J774 monocytic cell line, which has been extensively characterized in terms of phagosomal biogenesis using latex beads (13, 15). To identify the targets of DCG-04 in J774 cells, lysates were prepared at pH 5 and incubated with an increasing amount of the DCG-04, followed by SDS-PAGE and streptavidin blotting (Fig. 1 C). At least seven distinct polypeptides were detected in the 20–40-kD range, where most of the known active cysteine proteases are expected to migrate. To identify these proteases, DCG-04–labeled cell lysates were immunoprecipitated with antibodies directed against CatB, CatL, and CatS. This allowed the identification of three major DCG-04–labeled species as being CatB, CatL, and CatS (Fig. 1 D). The identity of these enzymes was additionally confirmed by comparing the labeling pattern of WT and CatB-, CatS-, or CatL-deficient cells (Fig. 4 B). The polypeptide strongly reactive with DCG-04 and of a mass slightly larger than that of CatB did not react with any of the antibodies tested (Fig. 1 C), including a CatH antiserum (unpublished data). DCG-04–labeled cell lysates were therefore incubated with streptavidin-coated agarose beads on a preparative scale and bound material was resolved by electrophoresis followed by Coomassie staining. The polypeptide of interest was excised, digested with trypsin, and analyzed by microbore and electron spray mass spectrometry, allowing its unambiguous identification as CatZ. The three top bands detected in J774 lysates (Fig. 1, C and D) were not considered in our analysis because they were never detected in in vivo labeling assays (see below).

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