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. Author manuscript; available in PMC: 2011 May 4.
Published in final edited form as: J Neurochem. 2009 Mar 19;109(4):1144–1156. doi: 10.1111/j.1471-4159.2009.06042.x

Figure 3. TREM2/TREM2-L interactions are important for phagocytosis, and TREM2 is required for efficient phagocytosis of apoptotic neuronal cells but not of beads.

Figure 3

(A) Phagocytosis of apoptotic Neuro2A cells (>40% Annexin Vhi) by BV2 microglial cells cocultured at an E:T of 1:10 as assessed by flow cytometry (left column) or fluorescence confocal microscopy (right panels). Histograms indicate the percent of BV2 cells that have internalized CM-DiI-labeled staurosporine-treated Neuro2A cells during 1h assays. Images of single optical sections (<1 μm) were obtained by confocal microscopy with a 60x magnification lens of BV2 cells labeled with an APC-conjugated anti-CD11b mAb (blue) and cocultured with CM-DiI+ apoptotic Neuro2A cells (red). Images indicate uptake of neuronal debris by BV2 cells. Phagocytosis is inhibited by cytochalasin D in both assays. Scale bars represent 10 μm. (B) Phagocytosis of apoptotic Neuro2A cells is reduced in BV2 cells following lentiviral-mediated RNAi against TREM2. RNAi reduced the surface expression of TREM2 up to 84% as detected by flow cytometry (left). Quantification of 6 experiments shows that phagocytosis by BV2 cells deficient in TREM2 is reduced to 15±5% (mean ± SEM) from 36±5% for untreated BV2 cells and from 34±5% for BV2 cells transduced with empty virus (*p-value < 0.05) (right). (C) A mAb to TREM2 partially but significantly inhibits phagocytosis of apoptotic Neuro2A cells by BV2 microglia. Representative flow cytometric histograms assessing phagocytosis in the presence of a blocking TREM2 mAb (Clone 78.18) or an isotype control mAb (rat IgG1) is shown (left). Summary of 6 experiments, showing a reduction to 23.7 ± 0.9% of effector cells engulfing targets compared to untreated (34.8 ± 2.9%) and control mAb (36.7 ± 2.1%) treated BV2 cells (right). The TREM2 mAb partially decreases phagocytosis by 32-35%. (**p-value ≤ 0.005, ***p-value ≤ 0.0005). (D) Reduction of microglial TREM2 by RNAi does not reduce BV2 phagocytosis of microspheres (here at 1:50 E:T ratio, top row), although the same cells again show a loss of phagocytosis of apoptotic Neuro2A cells at 1:10 E:T (sts N2A, bottom row) during 1h assays. BV2 cells were left untreated or subjected to cytochalasin D or infected with TREM2 shRNA or empty virus. Representative flow cytometric histograms are shown (n=2).