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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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Changes in cysteine protease activity during phagosome biogenesis. (A–D) Proteins were separated by SDS-PAGE on a reducing 12.5% SDS gel and reactive proteins were visualized by streptavidin blotting. (A) Analysis of proteolytic activities incorporated into the phagosome of J774 cells during maturation. Cells were incubated at 37°C with latex beads coupled to DCG-04 as indicated for different periods of time (pulse). After removing 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. (B) Purification of phagosomes and analysis of their proteolytic contents. J774 cells were incubated for 7.5 min at 37°C with DCG-04–coated beads (pulse). After removal of excess beads, cells were incubated for the times indicated at 37°C (chase). Subcellular fractionation was then performed using a sucrose gradient. Both phagosomal (+beads) and nonphagosomal fractions (−beads) were recovered and directly lysed in reducing SDS sample buffer. (C) Comparison of the total contents in cysteine proteases of J774 and RAW264.7 (Raw) cells. Lysates (pH 5) were incubated with various concentrations of DCG-04 for 1 h at 37°C. (D) Analysis of proteolytic activities incorporated into the phagosome of RAW264.7 cells during maturation. Cells were treated as in A.
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fig3: Changes in cysteine protease activity during phagosome biogenesis. (A–D) Proteins were separated by SDS-PAGE on a reducing 12.5% SDS gel and reactive proteins were visualized by streptavidin blotting. (A) Analysis of proteolytic activities incorporated into the phagosome of J774 cells during maturation. Cells were incubated at 37°C with latex beads coupled to DCG-04 as indicated for different periods of time (pulse). After removing 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. (B) Purification of phagosomes and analysis of their proteolytic contents. J774 cells were incubated for 7.5 min at 37°C with DCG-04–coated beads (pulse). After removal of excess beads, cells were incubated for the times indicated at 37°C (chase). Subcellular fractionation was then performed using a sucrose gradient. Both phagosomal (+beads) and nonphagosomal fractions (−beads) were recovered and directly lysed in reducing SDS sample buffer. (C) Comparison of the total contents in cysteine proteases of J774 and RAW264.7 (Raw) cells. Lysates (pH 5) were incubated with various concentrations of DCG-04 for 1 h at 37°C. (D) Analysis of proteolytic activities incorporated into the phagosome of RAW264.7 cells during maturation. Cells were treated as in A.

Mentions: To monitor the activity of individual cysteine proteases at different stages of phagosome maturation, J774 cells were pulsed and chased with DCG-04–coated beads for different times. We observed a time-dependent increase in active protease labeling (Fig. 3 A). As previously shown by Garin et al. (15), this suggests that active cysteine proteases are not delivered into the phagosome synchronously, but rather incorporated gradually. CatZ and CatB activities were the first detected by DCG-04 labeling (Fig. 3 A), which is consistent with the previous observation that both enzymes are present in early endosomes (15). When cells were chased for longer periods, an increase in total labeling was observed, indicating that the more mature phagosomes were also more proteolytically active. Compared with CatZ, the increase in activity of CatB was more substantial at the later time points, consistent with a continuous delivery of active CatB into the maturing phagosome as previously observed (Fig. 3 A; reference 15). We detected active CatS and CatL only after a 15-min pulse and a 30-min chase, with an additional increase at later chase times (Fig. 3 A). After 2 h of chase, CatB, CatS, CatL, and CatZ activities were all established components of phagosomal proteolytic content (Fig. 3 A). No additional increase in signal was observed between 60 and 120 min of chase (Fig. 3 A). This suggests that DCG-04–coated beads may have reached saturation after these longer chase periods (Fig. 3 A), as it was previously shown that CatS is still delivered 12 h after phagosome formation (15).


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)

Changes in cysteine protease activity during phagosome biogenesis. (A–D) Proteins were separated by SDS-PAGE on a reducing 12.5% SDS gel and reactive proteins were visualized by streptavidin blotting. (A) Analysis of proteolytic activities incorporated into the phagosome of J774 cells during maturation. Cells were incubated at 37°C with latex beads coupled to DCG-04 as indicated for different periods of time (pulse). After removing 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. (B) Purification of phagosomes and analysis of their proteolytic contents. J774 cells were incubated for 7.5 min at 37°C with DCG-04–coated beads (pulse). After removal of excess beads, cells were incubated for the times indicated at 37°C (chase). Subcellular fractionation was then performed using a sucrose gradient. Both phagosomal (+beads) and nonphagosomal fractions (−beads) were recovered and directly lysed in reducing SDS sample buffer. (C) Comparison of the total contents in cysteine proteases of J774 and RAW264.7 (Raw) cells. Lysates (pH 5) were incubated with various concentrations of DCG-04 for 1 h at 37°C. (D) Analysis of proteolytic activities incorporated into the phagosome of RAW264.7 cells during maturation. Cells were treated as in A.
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fig3: Changes in cysteine protease activity during phagosome biogenesis. (A–D) Proteins were separated by SDS-PAGE on a reducing 12.5% SDS gel and reactive proteins were visualized by streptavidin blotting. (A) Analysis of proteolytic activities incorporated into the phagosome of J774 cells during maturation. Cells were incubated at 37°C with latex beads coupled to DCG-04 as indicated for different periods of time (pulse). After removing 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. (B) Purification of phagosomes and analysis of their proteolytic contents. J774 cells were incubated for 7.5 min at 37°C with DCG-04–coated beads (pulse). After removal of excess beads, cells were incubated for the times indicated at 37°C (chase). Subcellular fractionation was then performed using a sucrose gradient. Both phagosomal (+beads) and nonphagosomal fractions (−beads) were recovered and directly lysed in reducing SDS sample buffer. (C) Comparison of the total contents in cysteine proteases of J774 and RAW264.7 (Raw) cells. Lysates (pH 5) were incubated with various concentrations of DCG-04 for 1 h at 37°C. (D) Analysis of proteolytic activities incorporated into the phagosome of RAW264.7 cells during maturation. Cells were treated as in A.
Mentions: To monitor the activity of individual cysteine proteases at different stages of phagosome maturation, J774 cells were pulsed and chased with DCG-04–coated beads for different times. We observed a time-dependent increase in active protease labeling (Fig. 3 A). As previously shown by Garin et al. (15), this suggests that active cysteine proteases are not delivered into the phagosome synchronously, but rather incorporated gradually. CatZ and CatB activities were the first detected by DCG-04 labeling (Fig. 3 A), which is consistent with the previous observation that both enzymes are present in early endosomes (15). When cells were chased for longer periods, an increase in total labeling was observed, indicating that the more mature phagosomes were also more proteolytically active. Compared with CatZ, the increase in activity of CatB was more substantial at the later time points, consistent with a continuous delivery of active CatB into the maturing phagosome as previously observed (Fig. 3 A; reference 15). We detected active CatS and CatL only after a 15-min pulse and a 30-min chase, with an additional increase at later chase times (Fig. 3 A). After 2 h of chase, CatB, CatS, CatL, and CatZ activities were all established components of phagosomal proteolytic content (Fig. 3 A). No additional increase in signal was observed between 60 and 120 min of chase (Fig. 3 A). This suggests that DCG-04–coated beads may have reached saturation after these longer chase periods (Fig. 3 A), as it was previously shown that CatS is still delivered 12 h after phagosome formation (15).

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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