Aminopeptidase N (CD13) is involved in phagocytic processes in human dendritic cells and macrophages

Abstract

Aminopeptidase N (APN or CD13) is a membrane ectopeptidase expressed by many cell types, including myelomonocytic lineage cells: monocytes, macrophages, and dendritic cells. CD13 is known to regulate the biological activity of various peptides by proteolysis, and it has been proposed that CD13 also participates in several functions such as angiogenesis, cell adhesion, metastasis, and tumor invasion. We had previously reported that, in human monocytes and macrophages, CD13 modulates the phagocytosis mediated by receptors for the Fc portion of IgG antibodies (FcγRs). In this work, we analyzed the possible interaction of CD13 with other phagocytic receptors. We found out that the cross-linking of CD13 positively modulates the phagocytosis mediated by receptors of the innate immune system, since a significant increase in the phagocytosis of zymosan particles or heat-killed E. coli was observed when CD13 was cross-linked using anti-CD13 antibodies, in both macrophages and dendritic cells. Also, we observed that, during the phagocytosis of zymosan, CD13 redistributes and is internalized into the phagosome. These findings suggest that, besides its known functions, CD13 participates in phagocytic processes in dendritic cells and macrophages.

Figure 1

Phenotypic differences in human monocyte-derived macrophages (MD-Ms) and monocyte-derived DCs (MD-DCs). Freshly isolated human monocytes were incubated in RPMI complete medium containing 1000 U/mL IL-4 and 800 U/mL GM-CSF (for MD-DCs) or complete RPMI medium without cytokines (for MD-Ms) for 7 days. The dot plots (a) show the populations of MD-Ms and MD-DCs obtained after 7 days in culture. Expression of CD14 (b) and CD1a (c) was evaluated using flow cytometry. Histograms correspond to MD-Ms (gray line), MD-DCs (black line), and isotype control (dashed line). MD-Ms were 95% CD14+ and 6% CD1a+. MD-DCs were 7% CD14+ and 84% CD1a+.

Figure 2

CD13 cross-linking enhances phagocytosis of zymosan by MD-DCs and MD-Ms. Cells were incubated with F(ab′)₂ fragments of mAb 452 (anti-CD13) at 4°C. After washing, the cells were incubated with 20 μg of zymosan-FITC in the presence (CD13xl) or absence of F(ab′)₂ fragments of goat anti-mouse antibodies for 60 min (MD-DCs) or 30 min (MD-Ms) at 37°C. The phagocytic index (a) (geometric mean of fluorescence intensity multiplied by the percentage of positive cells) and the percentage of fluorescence positive cells (b) (% of phagocytosis) were determined using flow cytometry. Data were normalized considering the value obtained in the absence of CD13 cross-linking as 100%; n = 8 for MD-DCs and n = 6 for MD-Ms. *P ≤ 0.05. Histograms of a representative experiment with MD-DCs (c) and MD-Ms (d): dashed lines: cells incubated with zymosan at 4°C; gray lines: cells incubated with zymosan at 37°C; black lines: cells incubated with zymosan at 37°C in the presence of CD13 cross-linking. In this experiment, the percentages of CD14+ cells were 96% in MD-Ms and 16% in MD-DCs, and the percentages of CD1a+ cells were 18% in MD-Ms and 93% in MD-DCs.

Figure 3

CD13 cross-linking enhances phagocytosis of E. coli by MD-Ms but not by MD-DCs. Cells were incubated with F(ab′)₂ fragments of anti-CD13 mAb at 4°C. After washing, the cells were incubated with 12 μg of E. coli-FITC, in the presence (CD13xl) or absence of F(ab′)₂ fragments of goat anti-mouse antibodies for 60 min (MD-DCs) or 30 min (MD-Ms) at 37°C. The phagocytic index (geometric mean of fluorescence intensity multiplied by the percentage of positive cells) (a) and the percentage of fluorescence positive cells (% of phagocytosis) (b) were determined using flow cytometry. Data from each donor were normalized considering the value obtained in the absence of CD13 cross-linking as 100%. (a) and (b) show the averages of 6 experiments with cells from different donors, *P ≤ 0.05. (c) and (d) show representative histograms of an experiment with MD-DCs (c) and MD-Ms (d) from a single donor, after trypan blue; dotted lines: cells incubated with E. coli at 4°C; gray lines: cells incubated with E. coli at 37°C; black lines: cells incubated with E. coli at 37°C in the presence of CD13 cross-linking. The percentages of FITC+ cells were 36 and 38% for MD-DCs and 54 and 69% for MD-Ms without and with CD13 cross-linking, respectively.

Figure 4

CD13 localizes at the phagosomal membrane after phagocytosis of zymosan particles in human monocyte-derived macrophages. Human MD-Ms were incubated with Texas Red-labeled zymosan particles for 30 min at 37°C. After washing away noningested particles, cells were fixed, permeabilized, and stained with anti-CD13 antibody and a secondary FITC-labeled goat anti-murine IgG. Samples were analyzed using confocal microscopy. From left to right: images of the same cell taken at 0.75 μm intervals in the z axis. Bar: 5.0 μm.

Figure 5

CD13 mediates internalization of particles in MD-DCs. MD-DCs were incubated with 2.5 μg F(ab′)₂ fragments of anti-CD13 mAb 452, with 2.5 μg anti-FcγRI mAb 32.2, or with no antibody (control) for 30 min at 4°C. After washing, cells were mixed with CFSC-SRBCs previously sensitized with F(ab′)₂ fragments of goat anti-mouse antibodies and incubated for 60 min at either 37°C or 4°C. Noningested SRBCs were lysed, and samples were analyzed using flow cytometry so as to determine the percentage and mean fluorescence intensity of positive cells. Phagocytic index was determined as indicated in Section 2. (a) and (b) show the average (±SD) phagocytic index and percentage of phagocytosis by MD-DCs obtained from monocytes of 8 different donors. *P ≤ 0.05. (c) shows representative dot plots of a single donor.

Figure 6

CD13 cross-linking does not modulate cytokine secretion induced by peptidoglycan from S. aureus. Human MD-DCs (gray bars) or MD-Ms (black bars) were stimulated with 10 μg of anti-CD13 mAb 452, or with 10 μg F(ab′)₂ fragments of anti-CD13 mAb plus secondary antibody, in the presence or absence of 10 μg PGN. After 18 h at 37°C, cells' supernatants were collected and assayed for the presence of IL-6, IL-10, IL-12, or TNFα using ELISA. Data shown are the average ± SD of three independent experiments with cells from three different donors.