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Comparative Study
. 2009;214(7):495-505.
doi: 10.1016/j.imbio.2008.11.006. Epub 2009 Jan 20.

Differential role of the Ca(2+) sensor synaptotagmin VII in macrophages and dendritic cells

Steven M Becker  1 , Lélia Delamarre, Ira Mellman, Norma W Andrews
Affiliations
Comparative Study

Differential role of the Ca(2+) sensor synaptotagmin VII in macrophages and dendritic cells

Steven M Becker et al. Immunobiology. 2009.

Abstract

Synaptotagmin VII (Syt VII) is a Ca(2+) sensing molecule that regulates lysosomal exocytosis in several cell types. In macrophages (MØ), Syt VII is required for efficient uptake of large particle loads, by promoting the delivery of lysosomal membrane to phagocytic cups. Here we compare the phagocytic capacity of bone marrow-derived MØs and dendritic cells (DC), and show that the requirement for Syt VII correlates with the unique ability of MØs for continuous phagocytosis. In contrast to MØs, Syt VII(+/+) and Syt VII(-/-) immature DCs show similar levels of initial phagocytosis, followed by a marked decrease in particle uptake. [Ca(2+)](i) chelation and PI-3 kinase inhibition reduce particle uptake by MØs, but are markedly less inhibitory in DCs. Thus, immature DCs appear to lack the Syt VII, Ca(2+) and PI-3 kinase-dependent forms of phagocytosis that are present in MØs. Interestingly, expression of Syt VII is up-regulated during LPS-induced DC maturation, a stimulus that also induces Syt VII translocation from intracellular compartments to the plasma membrane. Syt VII(-/-) DCs show a delayed translocation of MHC class II to the cell surface during maturation, consistent with the possibility that Syt VII facilitates exocytosis and/or surface retention of molecules critical for antigen presentation.

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Figures

Fig. 1
Fig. 1
Early down-regulation of phagocytosis in immature DCs. (A, B) FACS analysis of zymosan uptake in MØs and DCs. Cells were incubated with Texas Red zymosan at 25 particles/cell for 0.5, 1, 2, 4, and 16 h. Extracellular zymosan was removed with extensive washes with ice cold PBS. Black line: background fluorescence of cells without exposure to zymosan. Samples were also gated by size to exclude any debris or residual particles. The images show that only very few externally attached particles remained after the washes, as detected with anti-zymosan antibodies (green). Bars = 10 μm. (C) Quantification of the geometric mean fluorescence of the FACS charts shown in A and B.
Fig. 2
Fig. 2
Phagocytosis of large particle loads is not Syt VII dependent in immature DCs. (A) Texas Red zymosan was added to DCs at 25 or 50 particles/cell, for 0.5, 1, 2, or 2.5 h, and the number of internalized particles was determined microscopically after washing, fixation and staining with anti-zymosan to detect extracellular particles. The data represents the mean +/− SD of internalized particles per 100 DCs. Over 300 cells per coverslip were counted in triplicate. (B) DCs from Syt VII+/+ or Syt VII−/− mice were stained live on ice for surface MHC-II, after incubation with (green) or without (blue) unlabeled zymosan at 50 particles/cell for 1 h at 37 °C. An isotype control antibody is shown in red. (C) Zymosan induces maturation in part of the DC population after 18 h. DCs from Syt VII+/+ mice were incubated with unlabeled zymosan at 50 particles/cell (green) or LPS (50 ng/ml) (blue) for 18 h, harvested and stained live with antibodies to MHC-II and CD86. Surface levels of MHC-II and CD86 from unstimulated (no zymosan or LPS) DCs cultured in parallel for 18 h are shown for comparison (red). The results shown are representative of several independent experiments.
Fig. 3
Fig. 3
Intracellular Ca2+ chelation inhibits zymosan uptake in a Syt VII-dependent manner by MØs, but not DCs. Fluorescent zymosan uptake was analyzed by FACS in Syt VII+/+ or Syt VII−/− MØs (A) and DCs (B) either with or without pretreatment of 20 μM BAPTA-AM. Zymosan was added at 25 particles/cell for 1 h. The results shown are representative of at least three independent experiments.
Fig. 4
Fig. 4
PI 3-kinase inhibition reduces zymosan uptake by MØs, but not by DCs. (A) FACS profiles of fluorescent zymosan uptake in MØs and DCs left untreated or pretreated with wortmannin (WM) at 5, 50, or 100 nM for 30 min. Zymosan was added at 25 particles/cell for 1 h. The “no zymosan” population reflects autofluorescence of cells without zymosan. (B) Quantification of percent phagocytosis in WM-treated versus untreated, determined from the geometric mean fluorescence of the FACS profiles shown in A. The data represents the mean +/− SD of triplicate experiments. The results shown are representative of at least three independent experiments.
Fig. 5
Fig. 5
Syt VII-YFP translocates from peripheral domains of lysosomal compartments to the plasma membrane during DC maturation. (A) Immature DCs were transduced with Syt VII-YFP (green) and loaded with Texas red (TR) dextran (red) prior to fixation. Images are 0.8 μm optical slices from the middle and top of a confocal Z-stack. Arrows point to peripheral domains of dextran loaded lysosomes containing Syt VII-YFP. (B) Live confocal image of a maturing (1 h LPS) DC loaded with TR-dextran (red) and transduced with MHC-II-GFP (green). (C) Live confocal images of DCs transduced with either MHC-II-GFP (green) [left] or Syt VII-YFP (green) [middle], loaded with TR-dextran (red), and induced to mature for 24 h with LPS/cluster disruption. Right panel: FACS profile comparing surface levels of endogenous Syt VII in immature DCs, or DCs induced to mature for 24 h with LPS/cluster disruption. Anti-Syt VII NH2-terminal antibodies were added at 4 °C to detect surface exposed Syt VII. The background control (black) represents secondary antibody alone. About 36% of LPS-treated cells had surface levels of Syt VII significantly above that of immature cells. The results shown are representative of several independent assays. Bars = 5 μm.
Fig. 6
Fig. 6
Syt VII is up-regulated in mature DCs. Syt VII mRNA transcript levels were measured using real-time qPCR on three independent preparations of CD11c+ purified immature and mature DCs. Values were normalized using β-actin transcript levels. The data shown represents +/− SD of triplicate determinations. Inset: 100 μg of whole cell lysates from CD11c+ purified immature (IM) and mature (M) DCs were run on SDS-PAGE and transferred to membrane. Western blotting using affinity purified polyclonal antibody to the amino-terminus of Syt VII detected a 66 kDa band which corresponds to the Syt VII α-isoform. Anti-β-actin antibodies (Abcam) were used as a loading control.
Fig. 7
Fig. 7
MHC-II surface transport is delayed in Syt VII-deficient cells. (A) Immature Syt VII+/+ or Syt VII−/− DCs were stimulated with LPS and cluster disruption and assessed for MHC-II and CD86 surface expression by FACS at indicated time points up to 12 h. Top profiles compare MHC-II levels, and bottom profiles compare CD86 levels. Syt VII+/+ DCs (blue), Syt VII−/− DCs (red). For clarity, the isotype control antibody pattern is only shown for the 12 h time point (right panels, grey). (B) Quantification of the geometric mean fluorescence of the charts shown in A. The results shown are representative of three independent experiments.
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