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Monocyte-derived dendritic cells exhibit increased levels of lysosomal proteolysis as compared to other human dendritic cell populations.

McCurley N, Mellman I - PLoS ONE (2010)

Bottom Line: Unlike other DCs, MDDCs also fail to reduce their intralysosomal pH in response to maturation stimuli.Human DCs are comparable to murine DCs in exhibiting a markedly reduced level of lysosomal proteolysis.Thus, caution should be exercised when using human MDDCs as a model for DC function and cell biology.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Ludwig Institute for Cancer Research, Yale University School of Medicine, New Haven, Connecticut, United States of America.

ABSTRACT

Background: Fine control of lysosomal degradation for limited processing of internalized antigens is a hallmark of professional antigen presenting cells. Previous work in mice has shown that dendritic cells (DCs) contain lysosomes with remarkably low protease content. Combined with the ability to modulate lysosomal pH during phagocytosis and maturation, murine DCs enhance their production of class II MHC-peptide complexes for presentation to T cells.

Methodology/principal findings: In this study we extend these findings to human DCs and distinguish between different subsets of DCs based on their ability to preserve internalized antigen. Whereas DCs derived in vitro from CD34+ hematopoietic progenitor cells or isolated from peripheral blood of healthy donors are protease poor, DCs derived in vitro from monocytes (MDDCs) are more similar to macrophages (M Phis) in protease content. Unlike other DCs, MDDCs also fail to reduce their intralysosomal pH in response to maturation stimuli. Indeed, functional characterization of lysosomal proteolysis indicates that MDDCs are comparable to M Phis in the rapid degradation of antigen while other human DC subtypes are attenuated in this capacity.

Conclusions/significance: Human DCs are comparable to murine DCs in exhibiting a markedly reduced level of lysosomal proteolysis. However, as an important exception to this, human MDDCs stand apart from all other DCs by a heightened capacity for proteolysis that resembles that of M Phis. Thus, caution should be exercised when using human MDDCs as a model for DC function and cell biology.

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MDDCs and CD34DCs are similar in activity of other lysosomal hydrolases.(A) Cell lysates made from cultures of MΦs, MDDCs, and CD34DCs were incubated together with fluorescent substrates specific for acid phosphatase, α-mannosidase, and β-glucuronidase in either reaction buffer (pH 4.5) or control buffer (pH 7.4). After a 60-minute reaction, detection of the reaction product was measured with a fluorescence spectrophotometer. (B) Compendium of lysosomal hydrolase activity measurements relative to MΦs. While the non-protease acid hydrolases show a similar magnitude of activity in MDDCs and CD34DCs, the differences in proteolysis between the two subsets are accentuated. Immature (i-) and mature (m-) DCs; relative fluorescence units (RFU).
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pone-0011949-g003: MDDCs and CD34DCs are similar in activity of other lysosomal hydrolases.(A) Cell lysates made from cultures of MΦs, MDDCs, and CD34DCs were incubated together with fluorescent substrates specific for acid phosphatase, α-mannosidase, and β-glucuronidase in either reaction buffer (pH 4.5) or control buffer (pH 7.4). After a 60-minute reaction, detection of the reaction product was measured with a fluorescence spectrophotometer. (B) Compendium of lysosomal hydrolase activity measurements relative to MΦs. While the non-protease acid hydrolases show a similar magnitude of activity in MDDCs and CD34DCs, the differences in proteolysis between the two subsets are accentuated. Immature (i-) and mature (m-) DCs; relative fluorescence units (RFU).

Mentions: The initial investigation of lysosomal degradation in DCs of mice demonstrated that, in contrast to proteolysis, DCs were comparable to MΦs in lysosomal degradation of non-protein substrates [5]. Indeed, this finding is consistent with the observation that post-translational modifications of proteins only rarely contribute to the cognate T cell epitopes bound to class II MHC [15], [16], [17], perhaps because these modifications are removed in lysosomes. We therefore investigated whether the attenuated proteolytic capacity of human CD34DCs was due to an overall decrease in lysosomal hydrolytic activity or whether it was protease-specific. Cell-free extracts were prepared from MΦs, MDDCs, and CD34DCs and were tested against substrates specific for the activity of lysosomal acid phosphatase, β-glucuronidase, and α-mannosidase. In contrast to the marked difference in protease activity between MDDCs and CD34DCs, these other lysosomal hydrolases were comparable in activity between the two DC subsets (Fig. 3A). Though the greatest difference in hydrolytic activity was seen between the mature CD34DCs and the immature MDDCs when assaying for β-glucuronidase activity, this difference was at most 5-fold, substantially less then the 28-fold difference in protease activity between these two DC types (Fig. 3B). The difference in lysosomal hydrolytic capacity between MDDCs and CD34DCs was therefore predominantly limited to proteolysis, analogous to our previous findings using bone marrow-derived mouse DCs vs. mouse macrophages [5].


Monocyte-derived dendritic cells exhibit increased levels of lysosomal proteolysis as compared to other human dendritic cell populations.

McCurley N, Mellman I - PLoS ONE (2010)

MDDCs and CD34DCs are similar in activity of other lysosomal hydrolases.(A) Cell lysates made from cultures of MΦs, MDDCs, and CD34DCs were incubated together with fluorescent substrates specific for acid phosphatase, α-mannosidase, and β-glucuronidase in either reaction buffer (pH 4.5) or control buffer (pH 7.4). After a 60-minute reaction, detection of the reaction product was measured with a fluorescence spectrophotometer. (B) Compendium of lysosomal hydrolase activity measurements relative to MΦs. While the non-protease acid hydrolases show a similar magnitude of activity in MDDCs and CD34DCs, the differences in proteolysis between the two subsets are accentuated. Immature (i-) and mature (m-) DCs; relative fluorescence units (RFU).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2914042&req=5

pone-0011949-g003: MDDCs and CD34DCs are similar in activity of other lysosomal hydrolases.(A) Cell lysates made from cultures of MΦs, MDDCs, and CD34DCs were incubated together with fluorescent substrates specific for acid phosphatase, α-mannosidase, and β-glucuronidase in either reaction buffer (pH 4.5) or control buffer (pH 7.4). After a 60-minute reaction, detection of the reaction product was measured with a fluorescence spectrophotometer. (B) Compendium of lysosomal hydrolase activity measurements relative to MΦs. While the non-protease acid hydrolases show a similar magnitude of activity in MDDCs and CD34DCs, the differences in proteolysis between the two subsets are accentuated. Immature (i-) and mature (m-) DCs; relative fluorescence units (RFU).
Mentions: The initial investigation of lysosomal degradation in DCs of mice demonstrated that, in contrast to proteolysis, DCs were comparable to MΦs in lysosomal degradation of non-protein substrates [5]. Indeed, this finding is consistent with the observation that post-translational modifications of proteins only rarely contribute to the cognate T cell epitopes bound to class II MHC [15], [16], [17], perhaps because these modifications are removed in lysosomes. We therefore investigated whether the attenuated proteolytic capacity of human CD34DCs was due to an overall decrease in lysosomal hydrolytic activity or whether it was protease-specific. Cell-free extracts were prepared from MΦs, MDDCs, and CD34DCs and were tested against substrates specific for the activity of lysosomal acid phosphatase, β-glucuronidase, and α-mannosidase. In contrast to the marked difference in protease activity between MDDCs and CD34DCs, these other lysosomal hydrolases were comparable in activity between the two DC subsets (Fig. 3A). Though the greatest difference in hydrolytic activity was seen between the mature CD34DCs and the immature MDDCs when assaying for β-glucuronidase activity, this difference was at most 5-fold, substantially less then the 28-fold difference in protease activity between these two DC types (Fig. 3B). The difference in lysosomal hydrolytic capacity between MDDCs and CD34DCs was therefore predominantly limited to proteolysis, analogous to our previous findings using bone marrow-derived mouse DCs vs. mouse macrophages [5].

Bottom Line: Unlike other DCs, MDDCs also fail to reduce their intralysosomal pH in response to maturation stimuli.Human DCs are comparable to murine DCs in exhibiting a markedly reduced level of lysosomal proteolysis.Thus, caution should be exercised when using human MDDCs as a model for DC function and cell biology.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Ludwig Institute for Cancer Research, Yale University School of Medicine, New Haven, Connecticut, United States of America.

ABSTRACT

Background: Fine control of lysosomal degradation for limited processing of internalized antigens is a hallmark of professional antigen presenting cells. Previous work in mice has shown that dendritic cells (DCs) contain lysosomes with remarkably low protease content. Combined with the ability to modulate lysosomal pH during phagocytosis and maturation, murine DCs enhance their production of class II MHC-peptide complexes for presentation to T cells.

Methodology/principal findings: In this study we extend these findings to human DCs and distinguish between different subsets of DCs based on their ability to preserve internalized antigen. Whereas DCs derived in vitro from CD34+ hematopoietic progenitor cells or isolated from peripheral blood of healthy donors are protease poor, DCs derived in vitro from monocytes (MDDCs) are more similar to macrophages (M Phis) in protease content. Unlike other DCs, MDDCs also fail to reduce their intralysosomal pH in response to maturation stimuli. Indeed, functional characterization of lysosomal proteolysis indicates that MDDCs are comparable to M Phis in the rapid degradation of antigen while other human DC subtypes are attenuated in this capacity.

Conclusions/significance: Human DCs are comparable to murine DCs in exhibiting a markedly reduced level of lysosomal proteolysis. However, as an important exception to this, human MDDCs stand apart from all other DCs by a heightened capacity for proteolysis that resembles that of M Phis. Thus, caution should be exercised when using human MDDCs as a model for DC function and cell biology.

Show MeSH