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First published online 20 January 2009
doi: 10.1242/jcs.037200

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The scavenger receptor CD36 plays a role in cytokine-induced macrophage fusion

Laura Helming^*, Julia Winter and Siamon Gordon

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK

View larger version (44K): [in this window] [in a new window]   Fig. 1. Isolation of anti-CD36 antibodies blocking cytokine-induced macrophage fusion. (A) ThioM were labelled with CFSE and PKH26 and fusion induced by exposure to IL-4 in the presence of supernatants from three individual hybridoma lines MF2, MF3 and MF4. Macrophage fusion is represented by co-localization of the red and green fluorescent labels (yellow). (B) Quantitation of co-localization in the presence of the purified anti-CD36 antibodies MF2, MF3 and MF4 (20 µg/ml). Means±s.d. of 21 measurements combined from three independent experiments. (C) MF2, MF3, MF4 or isotype control (IgG2a, IgG2b) antibodies were covalently coupled to protein G beads and used for immunoprecipitation from ThioM lysates. Specific bands (100 kDa) could be detected on the silver-stained gel for MF2, MF3 and MF4 but not for isotype control antibodies. (D) Transient transfection of CHO cells with mCD36-YFP or YFP control vector, stained with MF2, 3 and 4. Positive staining was detected via anti-rat Alexa 555 (red). Positive staining with MF2, 3 and 4 was only detected in CHO cells expressing the mCd36-YFP fusion protein. (E) Western Blot analysis of wild-type (WT) and CD36-KO macrophage lysates using MF3, MF2, MF4 and anti-β-actin antibodies. ***P<0.0001, Mann Whitney Test, two-tailed.

View larger version (21K): [in this window] [in a new window]   Fig. 2. The involvement of CD36 in macrophage fusion. (A) BMM from wild-type (WT) and CD36-KO mice were induced to fuse by exposure to IL-4 and GM-CSF and stained with Hemacolor. (B) Macrophage fusion was quantified via the percentage of giant-cell nuclei relative to the total number of nuclei. (C) Fusion of wild-type and CD36-KO BMM in the presence of the anti-CD36 antibody (MF3, 20 µg/ml). (D,E) Shown are means±s.d. (n=8) *P=0.0404, ***P=0.0054 (Mann Whitney Test, two-tailed), representatives of three independent experiments are shown.

View larger version (62K): [in this window] [in a new window]   Fig. 3. The expression of CD36 during macrophage fusion. (A) FACS analysis of CD36 expression in ThioM after incubation with IL-4 using MF3 or isotype control IgG2a (grey shade) antibodies. Anti-rat Alexa 488 was used for detection (FL1-H). (B) Confocal microscopy of lamellipodia and cell-cell contacts (arrows) formed at the onset of macrophage fusion (ThioM, 6-8 hours). CD36 was labelled with MF3 and anti-rat Alexa 488 (green), actin with phalloidin-TRITC (red) and nuclei counterstained with DAPI (blue). (C) Lamellipodia and cell-cell contacts (arrows) in the presence of the anti-CD36 antibody MF3 (ThioM, 8 hours) and in (D) wild-type (WT) and CD36-KO BMM (incubated for 48 hours with IL-4/GM-CSF). Bar, 10 µm.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Expression of IL-4 induced markers. Real-time RT-PCR analysis of IL-4-induced markers in wild-type (WT) and CD36-KO BMM. Macrophages were stimulated for 24 hours. Means ±s.d. of duplicate reactions are shown. Arg1, Arginase 1; Chi3l3, Ym1; Tm7sf4, DC-STAMP; Retnla, Fizz1.

View larger version (35K): [in this window] [in a new window]   Fig. 5. The requirement of CD36 on one fusion partner. (A) Wild-type (WT) or CD36-KO BMM were labelled with PKH26 (red) and mixed with green CFSE-labelled wild-type ThioM and incubated with IL-4. Cross-fusion is represented by co-localization of the fluorescent labels (yellow). (B) Quantitation of macrophage fusion, shown are means ±s.d. (n=3). Quantitation was performed twice with similar results.

View larger version (35K): [in this window] [in a new window]   Fig. 6. A role for lipid recognition by CD36 and PS exposure and recognition during macrophage fusion. Quantitation of ThioM fusion in the presence of (A) oxLDL (50 µg/ml) or (B) PS liposomes (PS/PC, 50 µM). Means±s.d. combined from three independent experiments. (A) n=21, ***P<0.0001; (B) n=17, ***P=0.0003. Mann Whitney Test, two-tailed. (C) Binding of DiI-labelled PS liposomes (=FL2) to ThioM can be blocked by addition of anti-CD36 antibodies (MF3) but not control antibodies (IgG2a). This result was obtained in three independent experiments. (D) ThioM were plated with IL-4 on Permanox in the presence of annexinV-FITC for 18-24 hours. Nuclei were counterstained with DAPI. (E) Annexin V-FITC blocks IL-4 induced ThioM fusion. Means ±s.d., n=6, **P=0.0051, Mann Whitney Test, two-tailed. Shown are representatives of three independent experiments. Bar, 10 µm.

View larger version (15K): [in this window] [in a new window]   Fig. 7. CD36 is not involved in osteoclast formation. (A) TRAP staining and (B) quantitation of fusion in osteoclasts derived from wild-type (WT) and CD36-KO bone marrow progenitors by incubation with M-CSF/RANKL for 4 days. Shown are means±s.d. (n=6). Experiment was performed twice with similar results.

View larger version (24K): [in this window] [in a new window]   Fig. 8. Proposed mechanism of the involvement of CD36 during macrophage fusion. Localized areas of PS exposure on one fusion partner (macrophage 1) are recognized by CD36 on the other fusion partner (macrophage 2). Recognition of PS is required for efficient macrophage fusion.

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