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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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Different rates of protease acquisition by the phagosome of macrophages and DCs. (A) Surface expression of CD11c (top) and MHC class II (bottom) surface in sorted bone marrow–derived APCs. Cells were treated as described in C (see below), incubated with the appropriate antibody, and analyzed by cytofluorometry. Dotted line in top shows isotype control antibody. (B) Uptake of fluorescent latex beads by sorted bone marrow–derived APCs. Cells were treated as described in C. 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. (C–D) Proteins were separated by SDS-PAGE on a 12.5% gel and reactive proteases were visualized by streptavidin blotting. (C) Analysis of proteases incorporated into the phagosome of macro-phages (CD11c−) and DCs (CD11c+). Cells cultured in GM-CSF for 6 d were incubated for 5 min at 37°C with fluorescent yellow beads coupled to DCG-04. Excess beads were removed and cells were separated into CD11c+ and CD11c− cells by MACS at 4°C. Equal cell numbers were additionally incubated at 37°C (chase). After the chase, cells were lysed in reducing sample buffer containing 100 μM JPM-565. (D) Analysis of the total content of cysteine proteases of macrophages (CD11c−) and DCs (CD11c+). Cells were treated as described in C. CD11c− and CD11c+ cells were lysed at pH 5 and incubated for 60 min with 5 μM DCG-04 at 37°C.
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fig5: Different rates of protease acquisition by the phagosome of macrophages and DCs. (A) Surface expression of CD11c (top) and MHC class II (bottom) surface in sorted bone marrow–derived APCs. Cells were treated as described in C (see below), incubated with the appropriate antibody, and analyzed by cytofluorometry. Dotted line in top shows isotype control antibody. (B) Uptake of fluorescent latex beads by sorted bone marrow–derived APCs. Cells were treated as described in C. 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. (C–D) Proteins were separated by SDS-PAGE on a 12.5% gel and reactive proteases were visualized by streptavidin blotting. (C) Analysis of proteases incorporated into the phagosome of macro-phages (CD11c−) and DCs (CD11c+). Cells cultured in GM-CSF for 6 d were incubated for 5 min at 37°C with fluorescent yellow beads coupled to DCG-04. Excess beads were removed and cells were separated into CD11c+ and CD11c− cells by MACS at 4°C. Equal cell numbers were additionally incubated at 37°C (chase). After the chase, cells were lysed in reducing sample buffer containing 100 μM JPM-565. (D) Analysis of the total content of cysteine proteases of macrophages (CD11c−) and DCs (CD11c+). Cells were treated as described in C. CD11c− and CD11c+ cells were lysed at pH 5 and incubated for 60 min with 5 μM DCG-04 at 37°C.

Mentions: Mouse bone marrow cells were cultured in GM-CSF, allowing the isolation of two distinct types of professional APCs: DCs (CD11c+) and macrophages (CD11c−; see Fig. 5) . The CD11c− cell population may also contain neutrophils from the granulocyte lineage whose development is equally promoted by GM-CSF. Like macrophages, neutrophils are highly phagocytic cells that are part of the innate immune system but also have the ability to mediate antigen processing and presentation (28). To first analyze the cysteine protease content of bone marrow–derived APCs, day-6 cultures were directly lysed at pH 5 and labeled with soluble DCG-04. Although the activity of CatZ, CatB, CatS, and CatL was detected in these primary APCs, they displayed a more complex pattern of active cysteine proteases than the J774 and RAW264.7 cell lines. In particular, we observed an additional labeled protein migrating at a mass slightly larger than CatS, which was not seen in J774 and RAW264.7 cell lysates (Fig. 4). DCG-04 modification followed by streptavidin-mediated retrieval of the polypeptide and mass spectrometry allowed us to identify it as CatH. The labeling of cells from CatB, CatS, and CatL knockout mice showed that the labeled polypeptides identified as corresponding to these enzymes by immunoprecipitation do not include any other protease (Fig. 4 B). In addition, the presence in bone marrow APC lysates of a labeled polypeptide that comigrates with CatL could be inferred from labeling bone marrow APCs from CatL knockout mice (longer exposed blots; unpublished data). Additional analysis will be needed to establish the identity of this protease.


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)

Different rates of protease acquisition by the phagosome of macrophages and DCs. (A) Surface expression of CD11c (top) and MHC class II (bottom) surface in sorted bone marrow–derived APCs. Cells were treated as described in C (see below), incubated with the appropriate antibody, and analyzed by cytofluorometry. Dotted line in top shows isotype control antibody. (B) Uptake of fluorescent latex beads by sorted bone marrow–derived APCs. Cells were treated as described in C. 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. (C–D) Proteins were separated by SDS-PAGE on a 12.5% gel and reactive proteases were visualized by streptavidin blotting. (C) Analysis of proteases incorporated into the phagosome of macro-phages (CD11c−) and DCs (CD11c+). Cells cultured in GM-CSF for 6 d were incubated for 5 min at 37°C with fluorescent yellow beads coupled to DCG-04. Excess beads were removed and cells were separated into CD11c+ and CD11c− cells by MACS at 4°C. Equal cell numbers were additionally incubated at 37°C (chase). After the chase, cells were lysed in reducing sample buffer containing 100 μM JPM-565. (D) Analysis of the total content of cysteine proteases of macrophages (CD11c−) and DCs (CD11c+). Cells were treated as described in C. CD11c− and CD11c+ cells were lysed at pH 5 and incubated for 60 min with 5 μM DCG-04 at 37°C.
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Related In: Results  -  Collection

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fig5: Different rates of protease acquisition by the phagosome of macrophages and DCs. (A) Surface expression of CD11c (top) and MHC class II (bottom) surface in sorted bone marrow–derived APCs. Cells were treated as described in C (see below), incubated with the appropriate antibody, and analyzed by cytofluorometry. Dotted line in top shows isotype control antibody. (B) Uptake of fluorescent latex beads by sorted bone marrow–derived APCs. Cells were treated as described in C. 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. (C–D) Proteins were separated by SDS-PAGE on a 12.5% gel and reactive proteases were visualized by streptavidin blotting. (C) Analysis of proteases incorporated into the phagosome of macro-phages (CD11c−) and DCs (CD11c+). Cells cultured in GM-CSF for 6 d were incubated for 5 min at 37°C with fluorescent yellow beads coupled to DCG-04. Excess beads were removed and cells were separated into CD11c+ and CD11c− cells by MACS at 4°C. Equal cell numbers were additionally incubated at 37°C (chase). After the chase, cells were lysed in reducing sample buffer containing 100 μM JPM-565. (D) Analysis of the total content of cysteine proteases of macrophages (CD11c−) and DCs (CD11c+). Cells were treated as described in C. CD11c− and CD11c+ cells were lysed at pH 5 and incubated for 60 min with 5 μM DCG-04 at 37°C.
Mentions: Mouse bone marrow cells were cultured in GM-CSF, allowing the isolation of two distinct types of professional APCs: DCs (CD11c+) and macrophages (CD11c−; see Fig. 5) . The CD11c− cell population may also contain neutrophils from the granulocyte lineage whose development is equally promoted by GM-CSF. Like macrophages, neutrophils are highly phagocytic cells that are part of the innate immune system but also have the ability to mediate antigen processing and presentation (28). To first analyze the cysteine protease content of bone marrow–derived APCs, day-6 cultures were directly lysed at pH 5 and labeled with soluble DCG-04. Although the activity of CatZ, CatB, CatS, and CatL was detected in these primary APCs, they displayed a more complex pattern of active cysteine proteases than the J774 and RAW264.7 cell lines. In particular, we observed an additional labeled protein migrating at a mass slightly larger than CatS, which was not seen in J774 and RAW264.7 cell lysates (Fig. 4). DCG-04 modification followed by streptavidin-mediated retrieval of the polypeptide and mass spectrometry allowed us to identify it as CatH. The labeling of cells from CatB, CatS, and CatL knockout mice showed that the labeled polypeptides identified as corresponding to these enzymes by immunoprecipitation do not include any other protease (Fig. 4 B). In addition, the presence in bone marrow APC lysates of a labeled polypeptide that comigrates with CatL could be inferred from labeling bone marrow APCs from CatL knockout mice (longer exposed blots; unpublished data). Additional analysis will be needed to establish the identity of this protease.

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