CD36 modulates migration of mouse and human macrophages in response to oxidized LDL and may contribute to macrophage trapping in the arterial intima - PubMed (original) (raw)

. 2009 Jan;119(1):136-45.

doi: 10.1172/JCI35535. Epub 2008 Dec 8.

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CD36 modulates migration of mouse and human macrophages in response to oxidized LDL and may contribute to macrophage trapping in the arterial intima

Young Mi Park et al. J Clin Invest. 2009 Jan.

Abstract

The trapping of lipid-laden macrophages in the arterial intima is a critical but reversible step in atherogenesis. However, the mechanism by which this occurs is not clearly defined. Here, we tested in mice the hypothesis that CD36, a class B scavenger receptor expressed on macrophages, has a role in this process. Using both in vivo and in vitro migration assays, we found that oxidized LDL (oxLDL), but not native LDL, inhibited migration of WT mouse macrophages but not CD36-deficient cells. We further observed a crucial role for CD36 in modulating the in vitro migratory response of human peripheral blood monocyte-derived macrophages to oxLDL. oxLDL also induced rapid spreading and actin polymerization in CD36-sufficient but not CD36-deficient mouse macrophages in vitro. The underlying mechanism was dependent on oxLDL-mediated CD36 signaling, which resulted in sustained activation of focal adhesion kinase (FAK) and inactivation of Src homology 2-containing phosphotyrosine phosphatase (SHP-2). The latter was due to NADPH oxidase-mediated ROS generation, resulting in oxidative inactivation of critical cysteine residues in the SHP-2-active site. Macrophage migration in the presence of oxLDL was restored by both antioxidants and NADPH oxidase inhibitors, which restored the dynamic activation of FAK. We conclude therefore that CD36 signaling in response to oxLDL alters cytoskeletal dynamics to enhance macrophage spreading, inhibiting migration. This may induce trapping of macrophages in the arterial intima and promote atherosclerosis.

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Figures

Figure 1

Figure 1. Macrophage migration in vivo is inhibited by NO2LDL in a CD36-dependent manner.

(A) Thioglycollate-elicited peritoneal macrophages were isolated by lavage and counted 4 hours after mice were injected intraperitoneally with LPS. In some cases, mice were pretreated with NO2LDL or control LDL (NO2–LDL; 50 μg) prior to LPS injection. Data are plotted as the migration index, defined as [1 – (peritoneal macrophage count from each animal/average number of peritoneal macrophages of the thioglycollate-only control group mice)] × 100 (%). n = 10–15 per group; significance was determined by ANOVA and Bonferroni’s multiple comparison test. (B) Migration was evaluated as in A, but in WT and _Cd36_-null mice. (C) Peritoneal macrophages from LPS- (upper panel) or LPS- and NO2LDL-injected (lower panel) WT mice were stained with annexin V and 7-amino-actinomycin D (7-AAD) and subjected to analysis by flow cytometry.

Figure 2

Figure 2. Murine and human macrophage migration in vitro is inhibited by NO2LDL in a CD36-dependent manner.

(A) Murine peritoneal macrophages were added to the upper chamber of the Transwell with or without NO2LDL (50 μg/ml) and allowed to migrate through the porous membrane into the lower chamber containing medium alone or medium with monocyte chemotactic protein–1 (MCP-1). Migrated cells on the lower side of the membrane were stained with DAPI and counted under a fluorescence microscope using a ×10 objective. (B) NO2–LDL, Cu2+oxLDL, Ac-LDL, HDL (50 μg/ml each), or TSP-1 (20 nM) was added to the migration chamber and migration quantified as above. (C) Peritoneal macrophages from WT and _Cd36_-null mice were exposed to 0, 25, and 50 μg/ml NO2LDL and migrated cells quantified as in A. (D) Human peripheral blood monocyte–derived macrophages were treated with isotype-matched control IgG or anti-CD36 monoclonal antibody (5 μg/ml) and were added to the migration chamber with or without NO2LDL (50 μg/ml). Macrophage migration was quantified as in A.

Figure 3

Figure 3. NO2LDL induces rapid macrophage spreading and actin polymerization in a CD36-dependent manner.

(A) Macrophages from WT and _Cd36_-null mice were plated on serum-coated glass coverslips, incubated with NO2–LDL or NO2LDL (50 μg/ml) at 37°C, and then photographed after 5 minutes (original magnification, ×94.5). (B) Quantitative comparison of cell spreading was obtained by calculating the percentage of spread cells at each time point. (C) Mean cell surface areas were measured by confocal microscopy and quantitative comparisons obtained between macrophages from WT and _Cd36_-null mice. (D) Peritoneal macrophages from WT and _Cd36_-null mice were exposed to 50 μg/ml NO2LDL and then stained with fluorescein-phalloidin to detect polymerized actin. Fluorescence intensity was assayed by flow cytometry. The left histogram shows fluorescence intensity in WT cells with (blue) or without (red) exposure to NO2LDL, while the right histogram shows fluorescence intensity in _Cd36_-null cells.

Figure 4

Figure 4. NO2LDL induces phosphorylation of macrophage FAK in a CD36- and Src kinase–dependent manner, and FAK mediates macrophage spreading in response to NO2LDL.

(A) WT and _Cd36_-null mouse peritoneal macrophages were exposed to 50 μg/ml NO2LDL for the indicated times at 37°C, and then lysates were subjected to Western blot analysis to detect levels of FAK Tyr576/577 phosphorylation using an antibody specific for the phosphorylated form. Immunoblotting with anti-FAK antibody was used for loading control, and fold changes were calculated from scanned images. (B) WT mouse peritoneal macrophages were exposed to NO2LDL or NO2–LDL (50 μg/ml) at 0°C or 37°C and examined as in A. (C) Peritoneal macrophages were preincubated with Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(_t_-butyl)pyrazolo[3,4-d]pyrimidine (PP2; 10 μM) or vehicle control along with 50 μg/ml NO2LDL for the indicated times. Cells were then lysed and subjected to immunoblotting to detect FAK Tyr576/577 phosphorylation and Src kinase Tyr416 phosphorylation. (D) Peritoneal macrophages were immunoprecipitated with anti-CD36 antibody or nonimmune IgA, and the precipitates were then analyzed by immunoblot using anti-FAK (top), anti-CD36 (middle), and anti–SHP-2 antibodies (bottom). (E) Comparison of FAK Tyr576/577 phosphorylation kinetics in NO2LDL- (solid line) and TSP-1–treated cells (dashed line).

Figure 5

Figure 5. FAK mediates macrophage spreading in response to NO2LDL.

(A) Mouse peritoneal macrophages were preincubated with FAK inhibitors (PF-573,228 and PF-562,271; 10 μM for each) for 1 hour and incubated with or without NO2LDL at 37°C. Cells were photographed after 5 minutes (original magnification, ×94.5). (B) Mean cellular area was measured by confocal microscopy, and quantitative comparisons between FAK inhibitor–treated and untreated cells were obtained.

Figure 6

Figure 6. Inactivation of SHP-2 by NO2LDL.

(A) Peritoneal macrophages from WT and _Cd36_-null mice were exposed to 50 μg/ml NO2LDL for the indicated times. Western blot analysis of lysates was performed with an antibody against phospho–SHP-2 (Tyr580) (upper blots) and total SHP-2 (t–SHP-2; lower blots). Dephosphorylation of SHP-2 was rapid and sustained in WT but not _Cd36_-null cells. (B) Lysates from macrophages incubated with NO2LDL for the indicated time periods were incubated with 5-F-IAA to acetylate available cysteine residues. Cell lysates were then immunoprecipitated with anti–SHP-2 antibody and analyzed by SDS-PAGE. The fluoresceinated SHP-2 band was detected by in situ fluorescence scanning of the gel. Decreased band intensity of fluoresceinated SHP-2 indicates time-dependent oxidation of the active site cysteine and inactivation of SHP-2.

Figure 7

Figure 7. Macrophage exposure to NO2LDL induces generation of ROS in a CD36-dependent manner.

(A) Peritoneal macrophages were exposed to 50 μg/ml NO2LDL or NO2–LDL for 30 and 60 minutes. ROS were then detected with the fluorescent probe 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) by fluorescence microscopy (original magnification, ×40). Quantification by counting fluorescent cells is shown in B. (C) Fluorimetric quantification of ROS generation in a separate study comparing macrophages from WT with those from _Cd36_-null mice. (D) Peritoneal macrophages were pretreated with antioxidants (20 mM NAC and 100 μM resveratrol) or NADPH oxidase inhibitors (10 μM apocynin and 4 μM DPI) and then exposed to 50 μg/ml NO2LDL. ROS were detected as in A and B. Res, resveratrol; Apo, apocynin.

Figure 8

Figure 8. Antioxidants and NADPH oxidase inhibitors restore dynamic activation of FAK and macrophage migration.

(A) Peritoneal macrophages were pretreated with antioxidant (20 mM NAC) or NADPH oxidase inhibitor (10 μM apocynin) and incubated with 50 μg/ml NO2LDL for the indicated times. Cells were then lysed and subjected to immunoblotting to detect FAK Tyr576/577 phosphorylation. (B) Peritoneal macrophages were pretreated with antioxidants or NADPH oxidase inhibitors and then loaded into a Boyden chamber with NO2LDL. Migrated macrophages were counted after 16 hours as described in Figure 2. †P < 0.05, *_P_ > 0.05 compared with no treatment.

Figure 9

Figure 9. Model depicting CD36-dependent mechanism of macrophage trapping in the neointima.

oxLDL interacts with CD36 to induce a signaling cascade that leads to activation of specific Src kinases (e.g., Lyn), which in turn phosphorylate and activate FAK and lead to actin polymerization. oxLDL interactions with CD36 also lead to NADPH oxidase–mediated generation of intracellular ROS, which in turn induce oxidative inactivation of SHP-2, resulting in sustained FAK activation that perturbs cytoskeletal disassembly. The net effects are enhancement of cell spreading with concomitant inhibition of migration and therefore trapping of cells in the neointima. PTP, protein tyrosine phosphatase.

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