Macrophage Recognition and Phagocytosis of Apoptotic Fibroblasts Is Critically Dependent on Fibroblast-Derived Thrombospondin 1 and CD36

Reagents and Antibodies

Dulbecco’s modified Eagle’s medium (DMEM), RPMI 1640, containing penicillin, gentamicin and amphotericin, Ficoll-Paque, mannose, fucose, galactosamine, glucose, galactose, glucosamine, N-acetylglucosamine, propidium iodide, and Hoescht 33342 were purchased from Sigma Chemical Co. (St. Louis, MO). Annexin V-fluorescein isothiocyanate and terminal dUTP nick-end labeling (TUNEL) were purchased from Roche Diagnostics (Sydney, Australia). DiI(282) a long-chain dialkylaminostyryl dye, was purchased from Molecular Probes (Eugene, OR). FasL was purchased from Calbiochem (Sydney, Australia). Antibodies against TSP1 (clone A4.1) and CD36 (clone 1A7) and 185-1G20 (polyclonal) were purchased from Neomarkers (Freemont, CA). Antibodies against αvβ3, α5β1, and β1 were purchased from Chemicon (Melbourne, Australia). Disuccinimidyl suberate was purchased from Pierce (Rockford, IL). Coverslip chamber wells were obtained from Labtec (NUNC, Roskilder, Denmark). fMLP was purchased from Biomol (Plymouth, PA). Liposomes were prepared by sonication of pure phospholipids as described previously (Sigma). 11 Phospholipids were dissolved in chloroform/methanol and the solvent was evaporated under N₂ gas and the lipids were resuspended in phosphate-buffered saline (PBS) by vortexing. The mixtures were then sonicated for 5 minutes (Branson Sonifier 450). The liposomes contained phosphatidylcholine, phosphatidylcholine and phosphatidylserine (molar ratio, 70:30), or phosphatidylcholine and phosphatidylethanolamine (molar ratio, 70:30).

Macrophages

Heparinized peripheral blood was obtained from normal donors after informed written consent. Monocytes were isolated on Ficoll-Paque and macrophages were derived by culture of adherent monocytes were cultured for 5 to 7 days in RPMI 1640 supplemented with 10% fetal calf serum (FCS) and M-CSF as previously described. 12 Positive staining for CD68 staining was used to confirm the presence of macrophages. In this cell-culture system, 96 ± 3% of the cells were macrophages.

Apoptotic Fibroblasts

Primary cultures of fibroblasts were derived from normal human lung as previously described. 13 Fibroblasts were cultured in DMEM supplemented with 10% FCS, penicillin, gentamicin, and amphotericin. For experiments, fibroblasts were quiesced by the addition of serum-free DMEM for 16 hours, then rendered apoptotic by exposure to FasL (50 μg/ml) for 24 hours. Binding of FasL to Fas (CD95) on the cell-surface has been shown to induce apoptosis in multiple cell types. Fibroblasts were defined as apoptotic on the basis of morphological changes such as cell shrinkage and chromatin condensation as well as positive staining with TUNEL and annexin V-fluorescein isothiocyanate. Positive staining with propidium iodide identified necrotic cells. After exposure to FasL for 24 hours, 48 ± 3% of fibroblasts were found to be apoptotic and less than 1% were necrotic. In contrast 1.5 ± 0.3% of fibroblasts were apoptotic and less than 1% were necrotic when cultured in DMEM supplemented with 10% FCS.

Phagocytosis Assay

The phagocytic assay was performed as previously described. 14 Briefly, macrophages were plated on to coverslip chamber wells (10⁵ cells per well) in RPMI 1640. After adherence they were stained with DiI for 20 minutes. Macrophage viability was 98 ± 1% after this process. Apoptotic fibroblasts were suspended in RPMI and placed on the macrophage monolayer at a concentration of 10⁶ cells/well. This interaction was allowed to take place at 37°C for 1 hour. Loosely adherent fibroblasts were removed by washing with cold PBS five times. Fibroblasts were either untreated or incubated with antibodies to CD36, TSP1, and αvβ3 for 1 hour. In separate experiments, macrophages were incubated with antibodies to CD36, TSP1, αvβ3, or cyclic RGD peptides for 1 hour or alternatively incubated with liposomes and amino sugars. For all experiments, 200 cells/high-power field were counted and three high-power fields per section were analyzed (for a total of 600 cells). Fluorescent images were captured on a BioRad MRC 1000 confocal laser-scanning microscope. The phagocytic index was determined as the proportion of macrophages that had ingested TUNEL-positive fibroblasts expressed as a percentage of the total number of macrophages. Only one TUNEL-positive nucleus was observed in each macrophage.

Video Time-Lapse Microscopy

Fibroblasts (10⁴) were plated on a standard microscope slide and incubated with FasL for 24 hours. Two populations of macrophages were used to investigate the dynamics of chemotaxis and phagocytosis. One population was incubated with monoclonal antibodies to CD36 and the other population was stained with DiI. In a separate experiment one population of macrophages was incubated with monoclonal antibodies against TSP1 and the other population was stained with DiI. Macrophages (10⁵) were added to the fibroblasts after 24 hours of incubation with FasL. Phagocytosis of apoptotic cells was visualized by a Nikon Eclipse TE300 microscope (Lab Supply, Australia) (×40) throughout a 12-hour time period at a capture rate of 40 frames/minute. Fluorescent images were simultaneously captured at the beginning of the video time-lapse microscopy and at 6-hour intervals for 24 hours. The images were analyzed by Adobe Premier 5.1 software.

Flow Cytometry

Apoptotic fibroblasts and healthy fibroblasts grown in DMEM supplemented with 10% FCS were suspended in PBS and were dual stained with tetramethyl-rhodamine isothiocyanate-labeled antibodies to TSP1 or CD36 as well as with annexin V-fluorescein isothiocyanate according to the manufacturer’s specifications. For detection of intracellular CD36 staining, fibroblasts were permeabilized by treatment with 0.1% Triton X-100 for 3 minutes before antibody incubation. Labeled cells were analyzed on a FACScalibur (Becton Dickinson, San Jose, CA) and quantified by CellQuest software. Ten to thirty thousand events were collected for analysis.

Chemotaxis Assay

Chemotaxis assays were conducted in modified Boyden chambers (Neuroprobe, Cabin John, MD) as previously described. 15 Briefly, polycarbonate membrane filters (5 μm, Neuroprobe) were used to separate the upper wells from the lower wells. Apoptotic fibroblasts (10³/well) were seeded in the lower chambers and macrophages (10⁴/well) were added to the upper wells. Macrophages and apoptotic fibroblasts were incubated with antibodies and RGD peptides as described and the chambers were placed in a humidified incubator at 37°C for 90 minutes. Formyl-methionyl-leucyl-phenylalanine (fMLP) was used as positive control for chemotaxis. Filters were fixed, stained, and washed, and the number of macrophages that had migrated was evaluated by microscopy and direct cell counting.

Chemical Cross-Linking and Immunoprecipitation

Chemical cross-linking of surface proteins using disuccinimidyl suberate was performed on fibroblasts maintained in media containing 10% FCS (healthy) as well as fibroblasts serum-starved for 16 hours and exposed to FasL for a further 1 hour or for 24 hours. Immunoprecipitation of cell-surface proteins were performed as described previously. 16 In brief, cells were lysed in RIPA buffer (Roche Diagnostics) and triturated intermittently for 30 minutes. Insoluble material was removed by centrifugation at 14,000 rpm for 20 minutes at 4°C. The supernatant was precleared by incubation with protein A-agarose (Santa Cruz, San Diego, CA) for 1 hour at 4°C with end-over-end mixing. After centrifugation, supernatants were incubated with a monoclonal antibody to CD36 for 1 hour at 4°C. Immune complexes were precipitated by addition of protein A-agarose for 2 hours. The immune complexes that bound to agarose beads were then washed three times in RIPA buffer, boiled for 5 minutes, fractionated on a 4% stacking/12.5% resolving gel, and transferred to a polyvinylidene difluoride membrane. The membrane was blocked overnight in 5% skim milk powder in TTBS (Tris-buffered saline/0.25% Tween-20) and then incubated with anti-TSP1 antibody (1:500) for 1 hour. After washing, horseradish peroxidase-conjugated anti-mouse IgG was added (1:2000, 1 hour). After washing in TTBS, blots were developed by using enhanced chemiluminescence (ECL, Amersham) and exposed to ECL-Hyperfilm.

Thrombospondin Enzyme-Linked Immunosorbent Assay (ELISA)

Thrombospondin ELISA was performed using the sandwich method as previously described. 17 Each well of a 96-well ELISA plate (Corning, Cambridge, MA) was incubated with 100 μl of 1:1000 rabbit polyclonal anti-human TSP1 IgG overnight at room temperature and blocked with blocking solution (Block Ace; Dainihon, Osaka, Japan). The sample solution (100 μl) was added to each well and incubated for at 37°C for 1 hour, washed twice with washing solution (0.05% Nonidet P-40 in barbiturate-buffered saline). The wells were incubated with 1 μg/ml of mouse monoclonal antibody at 37°C for 1 hour, washed then incubated with 1 μg/ml of biotinylated horse polyclonal anti-mouse IgG (Vector, Burlingame, CA) and 1 μg/ml horseradish peroxidase-avidin complex (Vector). The bound antibody was detected by the addition of o-phenylene-diamine diluted in methanol containing 0.03% hydrogen peroxidase. The absorbance at 490 nm of each well was measured and the TSP1 concentration in each sample was determined from a standard curve prepared with purified human TSP1 at concentrations ranging from 0.39 to 3.5 ng/ml.

Confocal Microscopy

Cells were serum-starved for 16 hours and exposed to FasL (50 μg/ml) for a further 24 hours before being fixed in 1% paraformaldehyde for 30 minutes at room temperature and permeabilized for 3 minutes with 0.2% Triton X-100 in PBS containing 1% bovine serum albumin (BSA). Cells were rinsed in PBS and incubated with monoclonal antibody to TSP1 for 60 minutes at room temperature in PBS/1% BSA. From this stage onwards all incubations and washes were performed in the dark. After several washes in PBS, coverslips were incubated for 60 minutes in PBS containing rabbit anti-mouse IgGs conjugated to Alexa-488 and 1% BSA. Coverslips were rinsed in PBS and then incubated in with a polyclonal antibody to CD36 for 60 minutes at room temperature in PBS/1% BSA. After washes in PBS, cells were incubated with goat anti-rabbit IgGs conjugated to Alexa-546 and 1% BSA. Fibroblasts were then stained with Hoescht 333N2 nuclear stain (0.1 μg/ml) for 45 minutes at room temperature. After washing in PBS, coverslips were mounted with fade-resistant aqueous mounting medium. Co-localization of CD36 and TSP1 was examined on both healthy and apoptotic fibroblasts. Apoptotic fibroblasts were identified by their fragmented nuclei with Hoescht staining. Fluorescent images were obtained using a BioRad MRC1000 confocal laser-scanning microscope using COMOS software. Image processing was performed using Confocal Assistant software and Adobe Photoshop.

Statistical Analysis

Data are expressed as mean ± SE of at least four experiments. Statistical comparisons of mean data were performed using one-way analysis of variance and Student’s t-test with Bonferroni correction performed posthoc to correct for multiple comparisons. A P value <0.05 was regarded as statistically significant.

Apoptotic Fibroblasts Release TSP1

Figure 1 ▶ shows that the induction of apoptosis in fibroblasts induces the release of readily quantifiable levels of TSP1. Fibroblast cultures that were rendered quiescent by serum deprivation for 16 hours produced TSP1. However the induction of apoptosis by exposure to FasL (50 μg/ml) for an additional 24 hours produced a further significant increase of TSP1 from 1200 ± 15 pg/ml to 2100 ± 30 pg/ml (P < 0.01 compared to cells in serum-free media alone).

TSP1 Released from Apoptotic Cells Is a Chemotactic Agent for Macrophages

Having established that TSP1 was released by apoptotic fibroblasts, we sought to examine its role as a macrophage chemoattractant. Figure 2 ▶ shows that in a modified Boyden chamber assay, macrophage migration was significantly greater toward apoptotic fibroblasts than toward nonapoptotic cells (P < 0.05). Incubation of apoptotic fibroblasts with antibodies against TSP1 significantly inhibited macrophage chemotaxis (P < 0.01). Blockade of macrophage-derived CD36 also diminished their migration in response to signals released from the apoptotic cells, but not in response to fMLP. In contrast, blockade of αvβ3 integrins on the macrophage or CD36 on the apoptotic fibroblasts did not influence the chemotactic response. Similarly, incubation of either cell type with function-blocking antibodies recognizing αvβ5 and αvβ1 integrins was also without effect.

We confirmed the role of TSP1/CD36 in macrophage recognition and phagocytosis of apoptotic fibroblasts using video time-lapse microscopy. In these experiments, healthy macrophages appeared to migrate toward and phagocytose an apoptotic fibroblast, whereas macrophages treated with function-blocking antibodies against CD36 did not appear to actively phagocytose these cells (data not shown).

Surface Expression of CD36 and TSP1 Are Up-Regulated on Apoptotic Fibroblasts

Nonapoptotic fibroblasts appeared to contain an intracellular pool of CD36 as evidenced by the rightward shift in mean fluorescence intensity (Figure 3A) ▶ . Exposure of fibroblasts to FasL (50 μg/ml) for 24 hours induced a significant increase in TSP1 (Figure 3, B and C) ▶ and CD36 (Figure 3, D and E) ▶ mean fluorescence intensity compared to healthy untreated cells. Cell counts of 300 fibroblasts by confocal microscopy or 30,000 fibroblasts by flow cytometry demonstrated that the vast majority of apoptotic fibroblasts were also CD36^+ve and TSP1^+ve compared to healthy cells (Table 1) ▶ .

TSP1 Associates with CD36 on the Surface of Apoptotic Fibroblasts

Having demonstrated that expression of both TSP1 and CD36 were up-regulated on apoptotic fibroblasts, we sought to determine whether TSP1 was directly associated with CD36. Figure 4A ▶ shows a single band corresponding to the molecular weight of monomeric TSP1 (450 kd) in cell lysates immunoprecipitated with antibodies against CD36. After exposure of cells to FasL for 24 hours, an extra band corresponding to the expected size of TSP1 and CD36 (88 kd) is observed. In contrast, no extra band was detectable in cell lysates of healthy cells at 24 hours or in fibroblasts exposed to FasL for 1 hour. Visual confirmation of this association was provided by confocal microscopy (Figure 4; B, C, and D) ▶ . Apoptotic fibroblasts (nuclear fragmentation revealed by Hoechst 33342) that were double stained with antibodies to CD36 (green) and TSP1 (red), show distinct areas of co-localization (yellow). In contrast, healthy fibroblasts (intact nuclei with Hoechst staining) showed no co-localization.

Macrophages Use the αvβ3 Integrin, CD36, and TSP1 for the Recognition and Phagocytosis of Apoptotic Fibroblasts

Incubation of macrophages with function-blocking antibodies against αvβ3 reduced the percentage of macrophages that phagocytosed apoptotic fibroblasts from 90 ± 3% to 11 ± 1% (P < 0.01, Figure 5A ▶ ). This appeared specific for αvβ3 because the specific cyclic RGD peptide (GPenGRGDSPCA) also dose dependently inhibited phagocytosis, whereas the control peptide RGES (Figure 5A) ▶ and isotypically matched antibodies against αvβ5 and αvβ1 integrins did not influence phagocytosis. Incubation of macrophages with antibodies against CD36 and TSP1 also abrogated phagocytosis of apoptotic fibroblasts to the same level as inhibition by αvβ3 antibodies (Figure 5A) ▶ . Incubation of apoptotic fibroblasts with function-blocking antibodies to CD36 and TSP1 also inhibited phagocytosis (Figure 5B) ▶ .

Blockade of Phosphatidylserine or Amino Sugars Does Not Influence Phagocytosis of Apoptotic Fibroblasts

In contrast to inhibition of αvβ3, CD36, and TSP1, incubation of macrophages with liposomes containing phosphatidylserine or control liposomes containing phosphatidylcholine or a mixture of the two lipids had no effect on phagocytosis of apoptotic fibroblasts (Figure 6A) ▶ . Similarly, incubation of macrophages with a variety of specific amino sugars did not influence the ability of macrophages to phagocytose apoptotic fibroblasts (Figure 6B) ▶ .