Macrophage recognition and phagocytosis of apoptotic fibroblasts is critically dependent on fibroblast-derived thrombospondin 1 and CD36

Abstract

The induction of fibroblast apoptosis and their clearance by phagocytes is essential for normal wound healing and prevention of scarring. However, little is known about the clearance of apoptotic fibroblasts and whether apoptotic cells are active participants in the recruitment and activation of phagocytes. In this study, we provide the first evidence that apoptotic fibroblasts actively release increased amounts of thrombospondin (TSP1) to actively recruit macrophages. Expression of TSP1 and its receptor CD36 was increased on the surface of apoptotic fibroblasts. By chemical cross-linking and immunoprecipitation we show that TSP1 and CD36 were directly associated. This was confirmed by confocal microscopy. Blockade of either CD36 or TSP1 on apoptotic fibroblasts inhibited phagocytosis. Blockade of alpha v beta 3 integrins as well as CD36 and TSP1 on macrophages inhibited phagocytosis. In contrast, phosphatidylserine or lectins were not involved. These findings suggest that apoptotic fibroblasts release TSP1 as a signal to recruit macrophages while the up-regulated expression of the CD36/TSP1 complex on their cell surface may form a ligand bridging the fibroblast to a complex consisting of alpha v beta 3/CD36/TSP1 on macrophages. These results establish fundamental mechanisms for the clearance of apoptotic fibroblasts and may provide insights into the processes involved in normal wound repair.

Figure 1.

TSP1 is secreted by apoptotic fibroblasts. TSP1 was measured by ELISA in the supernatants obtained from fibroblasts cultured in serum-free DMEM and fibroblasts rendered apoptotic by exposure to serum-free media (sfm) for 16 hours and FasL for 24 hours. There was secretion of TSP1 from cells in serum-free media and cells exposed to FasL compared with the levels in serum-free media. There was a significant rise in TSP1 production by fibroblasts exposed to FasL compared to fibroblasts in serum-free media. Results represent mean ± SEM of five experiments. **, P < 0.01 compared to cells in sfm.

Figure 2.

TSP1 released by apoptotic fibroblasts is a chemoattractant for macrophages. Apoptotic fibroblasts were either untreated or incubated with antibodies against CD36 or TSP1. Macrophages were incubated with antibodies against CD36 or TSP1 or treated with various inhibitors. Incubation of apoptotic fibroblasts with antibodies against TSP1 and incubation of macrophages with antibodies against CD36 inhibited chemotaxis. Data represent mean ± SEM of five experiments. **, P < 0.01 compared to chemotaxis toward apoptotic fibroblasts.

Figure 3.

The expression of CD36 and TSP1 is up-regulated on the surface of apoptotic fibroblasts. Histograms of FACS analysis illustrating the presence of an intracellular pool of CD36 in healthy fibroblasts (A) and the up-regulation of TSP1 (B) and CD36 (D) on apoptotic fibroblasts as compared to healthy fibroblasts. Four-quadrant analysis demonstrates a significant number of annexin^+ve cells are also TSP1^+ve (top right) (C) and CD36^+ve (E). Nonapoptotic fibroblasts were cultured in DMEM supplemented with 10% FCS. Apoptotic fibroblasts were quiesced in serum-free DMEM for 12 hours and treated with FasL for 24 hours. Fibroblasts were suspended in 1% BSA/PBS and stained with tetramethyl-rhodamine isothiocyanate-labeled monoclonal antibodies to CD36 or TSP1. Similar results were obtained in each of four experiments.

Figure 4.

TSP1 associates with CD36 on apoptotic fibroblasts. A: Surface receptors on healthy fibroblasts and apoptotic fibroblasts were chemically cross-linked and protein was immunoprecipitated with antibodies against CD36, lysed, and the resultant protein complexes were then probed with antibodies against TSP1. CD36 associated with TSP1 on the apoptotic fibroblasts but not on healthy fibroblasts or on fibroblasts exposed to FasL for 1 hour. Similar results were obtained in each of three experiments. Double staining with CD36 (B; Alexa 546, red) or TSP1 (C; Alexa 488, green) reveals significant areas of co-localization (yellow staining in D) (↑). Fibroblasts that positively stained for CD36 and TSP1 were confirmed to be apoptotic by nuclear fragmentation on Hoescht 33342 (blue) staining. Healthy fibroblasts with intact nuclei do not demonstrate co-localization of CD36 and TSP1 ( ↑) Scale bar, 50 μm.

Figure 5.

Macrophages use the αvβ3/CD36/TSP1 complex to phagocytose apoptotic fibroblasts. A: Phagocytosis of apoptotic fibroblasts by human monocyte-derived macrophages was inhibited by incubation of macrophages with CD36, αvβ3, and TSP1 antibodies. Incubation of macrophages with the cyclic RGD peptide GPenGRGDSPCA also dose dependently inhibited phagocytosis. B: Incubation of apoptotic fibroblasts with antibodies against CD36 and TSP1 inhibited phagocytosis. Incubation with a function-blocking antibody against αvβ5 was without effect. *, P < 0.05; **, P < 0.01, compared to phagocytosis of apoptotic fibroblasts alone.

Figure 6.

A: Incubation of macrophages with liposomes containing phosphatidylserine (PS) (PC:PS, 70:30), phosphatidylcholine (PC), or phosphatidylethanolamine (PE) did not influence their ability to phagocytose apoptotic fibroblasts. B: Incubation with amino sugars also had no effect on the ability of macrophages to phagocytose apoptotic fibroblasts. Data represent mean ± SEM of five experiments. **, P < 0.01 compared to phagocytosis of apoptotic fibroblasts alone.