In vivo, apoptotic cells are efficiently removed by professional or nonprofessional phagocytes, a process thought to be essential for tissue remodeling and resolution of inflammation. Macrophages recognize apoptotic cells by several mechanisms, including recognition of exposed phosphatidylserine (PS); however, PS recognition on apoptotic cells has not been identified as a feature of human macrophages. The purpose of this study was to determine whether human monocyte-derived macrophages could be stimulated to recognize PS, defined as inhibition of phagocytosis by PS-containing liposomes. We also assessed the potential roles for scavenger receptors, CD14, and lectins. Uptake of apoptotic neutrophils into unstimulated macrophages was blocked about 50% by Arg-Gly-Asp-Ser and anti-αv, and up to 20% by oxidized low density lipoprotein and N-acetylglucosamine, implying a major role for integrin and minor roles for scavenger and lectin receptors. Uptake into macrophages stimulated with β-1,3-glucan was blocked 50% by PS liposomes and 40% by oxidized low density lipoprotein, suggesting that the macrophages had switched from using integrin to recognition of PS. MEM-18 and 61D3 (anti-CD14 mAbs) were poor inhibitors of apoptotic neutrophil uptake, but good inhibitors of apoptotic lymphocyte uptake. The switch to PS recognition was accompanied by down-regulation of αvβ3 expression and function. Anti-CD36 blocked uptake into unstimulated or stimulated macrophages, suggesting CD36 involvement not only with the αvβ3 integrin mechanism (as previously reported) but also with PS recognition. A maximum of 70% inhibition was achieved by combining anti-CD36 with either anti-av or PS liposomes.

The process of apoptosis promotes rapid removal of cells in situ, whether it occurs during tissue remodeling, negative immune selection, or the resolution of inflammation. Of the numerous cells reported to recognize and remove apoptotic bodies, the macrophage is the most prominent. By phagocytosing dying cells before they undergo secondary necrosis, macrophages remove cellular debris and prevent spillage of toxic materials that might amplify tissue injury (1). Ingesting apoptotic cells also actively suppresses macrophage production of proinflammatory cytokines and some icosanoids (2, 3). In these ways, macrophages remove effete cells in a controlled, nonreactive manner.

Macrophages appear to recognize apoptotic cells via different mechanisms, including integrin, phosphatidylserine (PS)3 recognition, scavenger receptors, and lectins. Human monocyte-derived macrophages (HMDM) (4, 5, 6), human alveolar macrophages (7), and murine bone marrow-derived macrophages (8) seem to phagocytose apoptotic cells via the αvβ3 integrin system, which on human macrophages associates with CD36. This complex then binds to secreted thrombospondin (TSP), which binds to an undefined ligand on the apoptotic cell (6). Elicited murine peritoneal macrophages, phorbol ester-treated THP-1 cells, and glucan-stimulated mouse bone marrow-derived macrophages have been shown to recognize exposed PS on the surface of apoptotic cells (9, 10). Although the recognition is stereospecific, the putative PS receptor (PSR) has not yet been characterized. Third, macrophages may use one or more members of the scavenger receptor family (SR) to engulf apoptotic cells. SRA-I/II on murine thymic macrophages and elicited peritoneal macrophages (11), SRB-1 on transfected CHO cells (12), CLA-1 on transfected HEK293 cells (13), CD36 (6), and macrosialin/CD68 on resident mouse peritoneal macrophages (14, 15, 16) all reportedly mediate binding and/or uptake of apoptotic cells. Of these, SR-B1, CLA-1, CD36, and CD68 have been reported to bind to PS- and phosphatidylinositol (PI)-containing liposomes (13, 14, 15, 16, 17); however, it remains unclear whether they recognize PS on apoptotic cells. Fourth, HMDM have been reported to phagocytose apoptotic leukocytes using CD14, inhibited specifically by the mAbs 61D3 and MEM-18 (18, 19). How CD14 functions is not known. Some reports differentiate it from the αvβ3/CD36/TSP system (18) and the putative PSR (20), whereas Pradhan and co-workers suggest that these are all part of a single, large receptor complex (21). Lastly, mouse resident peritoneal macrophages have been shown to use a lectin that is inhibited by N-acetylglucosamine (NAG) (22).

Of these recognition systems, only the αvβ3/CD36/TSP system and CD14 have been demonstrated in HMDM uptake of apoptotic cells. Human macrophages have been shown to recognize PS on sickled red cells or symmetric red cell ghosts (23, 24, 25, 26); however, it has been suggested that they are unable to recognize PS on apoptotic cells (4, 27, 28). We have shown that recognition of PS on apoptotic cells by murine bone marrow-derived macrophages can by induced after treatment with a digestible particulate stimulus such as β-glucan (10, 29); however, the lack of an Ab to mouse CD36 prevented our assessment of its involvement. Our objectives for this study were to determine whether PS recognition could be induced in HMDM, what role, if any, CD36 played in PS recognition, and what other receptors might be involved in phagocytosis by either stimulated or unstimulated macrophages. For the purposes of this study, we have defined PS recognition strictly as inhibition of phagocytosis by PS-containing liposomes, although others have suggested that inhibition by symmetric red cell ghosts in the absence of inhibition by PS liposomes is also suggestive of PS recognition (21).

Sterile plastic ware was purchased from Becton Dickinson (Franklin Lakes, NJ) and Corning Costar (Cambridge, MA). DMEM, RPMI 1640, HBSS, and PBS were obtained from Life Technologies (Grand Island, NY). X-Vivo 10 medium was obtained from BioWhittaker (Walkersville, MD). FCS was purchased from Gemini Bioproducts (Calabasas, CA). Acetylated low density lipoprotein (AcLDL), low density lipoprotein (LDL), and oxidized low density lipoprotein (OxLDL) were obtained from BTI (Stoughton, MA). Phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), PI, and PS were purchased from Avanti Polar Lipids (Alabaster, AL). Sterile β-1,3-glucan was purchased from Accurate Chemical Co. (Westbury, NY). BSA, human serum albumin, antibiotics, sterile 1.1-mm latex beads, dimethoxybenzidine (o-dianisidine HCl), NAG, Arg-Gly-Glu-Ser (RGES), Arg-Gly-Asp-Ser (RGDS), l-α-glycerophosphorylserine (GPS), l-α-glycerophosphorylcholine, phospho-l-serine, phospho-d-serine (PDS), EDTA, and all remaining chemicals were purchased from Sigma (St. Louis, MO).

Monoclonal IgM mouse anti-human CD36 (clone CB38/NL07), monoclonal IgM mouse anti-CD15 (clone H198), monoclonal IgM mouse anti-CD15s (clone 2F3), monoclonal IgG1 mouse anti-human CD45, CD61 (β3), CD51/61(αvβ3) CD64, CD16, and monoclonal IgG2b mouse anti-human Cd32 were purchased from PharMingen (San Diego, CA); IgG1 anti-human αv derived from mouse ascites was obtained from Life Technologies; and two monoclonal mouse anti-human CD68 Abs were purchased from Cortex (IgG1; San Leandro, CA) and Monosan (IgM; Amuden, Netherlands). The mAb 61D3 (IgG1 mouse anti-human) was a gift from J. D. Capra and Drs. Andrew Devitt and Christopher Gregory. MEM-18 (mouse IgG1) was purchased from Monosan/Sanbio and TUK4 was purchased from Caltag Laboratories (Burlingame, CA). Tetramethyl rhodamine isothiocyanate- or Cy3-conjugated AffiniPure F(ab′)2 of goat anti-mouse IgG and IgM Abs were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).

Healthy unmedicated adult human subjects donated 400 ml of whole blood under a protocol approved by National Jewish Center’s institutional review board. Mononuclear cells were separated by dextran sedimentation and centrifugation through a discontinuous plasma-Percoll gradient as described previously (30). After three washes with HBSS, the cells were suspended at 4 × 106/ml in DMEM, and plated in 48-well tissue-culture plates at 0.5 ml/well. Following 1-h incubation at 37°C in 5% CO2, the cell layer was washed twice to remove nonadherent leukocytes, and the adherent monocytes were given X-Vivo 10 medium containing 10% (v/v) pooled human serum (heated at 65°C for 30 min) plus 100 U/ml penicillin and 100 mg/ml streptomycin (HMDM medium). The monocytes were cultured in 37°C in 5% CO2, with medium exchanged on days 3 and 7. Day 7–10 HMDM were used, according to preliminary studies for maximal macrophage uptake of apoptotic cells (data not shown).

Neutrophils were isolated from the modified dextran/plasma Percoll preparation of human blood as described above (30), washed twice with HBSS, and suspended at 5 × 106/ml in RPMI 1640 containing 10% (v/v) FCS. With this preparation, neutrophils were >98% viable and pure, as measured by trypan blue exclusion and histologic analysis.

Day 5–8 HMDM were stimulated with 25 μg/ml β-1,3-glucan or 5:1 latex beads, suspended in HMDM medium. After 48 h the monolayer was washed twice with HBSS to remove noningested particles before examination for recognition of apoptotic cells.

Neutrophils were placed in 75-cm2 tissue culture flasks, irradiated with a 302-nm UV transilluminator for 10 min, transferred to 50-ml plastic tubes, and rotated end-over-end in a 5% CO2 incubator at 37°C for 3 h. The cells were then washed twice and resuspended in RPMI plus 10% FCS at 1 × 108/ml for use in the phagocytosis assay. This treatment induced 77 ± 1.7% apoptosis by analysis of morphologic changes with <3% necrosis, as assessed microscopically for nuclear condensation and trypan blue exclusion (n = 24). These apoptotic neutrophils have been shown to express PS externally using annexin-V-FITC labeling and flow cytometry (31). For some experiments, apoptotic neutrophils were opsonized as previously described with mouse anti-CD45 and rabbit anti-mouse IgG (2).

For some experiments, Jurkat T cells were used. They were routinely cultured in RPMI and 20% FCS containing l-glutamine, penicillin, and streptomycin. To induce apoptosis, Jurkat cells were irradiated as described for neutrophils.

Uptake was determined as previously described (1, 32). Apoptotic neutrophils were added to day 7–10 HMDM at a 5:1 ratio in HMDM medium and incubated for 2 h at 37°C in 5% CO2 to achieve maximal neutrophil uptake, based on preliminary time-course and dose-response studies (data not shown). The monolayer was then washed with PBS to remove attached noningested neutrophils, fixed with 1% formalin, and treated with dimethoxybenzidine to stain for myeloperoxidase (MPO) as a marker of the ingested neutrophils (1, 32). The macrophages themselves were routinely negative for peroxidase staining. Using ×40 light microscopy, one investigator (M.L.W.) examined the macrophages for uptake of apoptotic neutrophils, counting two replicate wells each of 200 HMDM/well, and a second investigator (V.A.F.) made blinded confirmation of representative plates. The phagocytic index (φI) was calculated as the percentage of phagocytosing macrophages × the average number of neutrophils ingested per macrophage. Macrophages that showed discrete, round, MPO-positive inclusion(s) were scored as having ingested one (or more) apoptotic neutrophils, while HMDM that showed diffusely MPO-stained cytoplasm, presumably due to earlier digestion of one or more neutrophils, were scored as having ingested only one neutrophil. Those neutrophils whose margins extended more than 50% beyond the edge of the macrophage cell membrane were scored as noningested. (This convention may have underestimated the number of phagocytosed neutrophils, because ultrastuctural analyses have shown macrophages to engulf such neutrophils with a thin wall of cytoplasm (32).) To verify that the results were not specific for neutrophils, apoptotic Jurkat cells were offered; phagocytosis was evaluated in the same way, except that the MPO stain was not used.

Although the absolute value for the control phagocytic index varied daily between HMDM preparations, within each experiment this value was highly reproducible. To facilitate comparison of the results, we report the phagocytic index in several experiments as the percentage of the mean of the control phagocytic index replicates for that experiment (normalized φI).

To study HMDM use of the candidate receptors, the following ligands, Abs, and controls were used: RGDS, RGES, anti-human αv, anti-human CD36, GPS, GPC, PLS, PDS, AcLDL, OxLDL, LDL, anti-human CD68, and NAG. Unilamellar phospholipid vesicles were prepared as previously described (9). PC liposomes were made at 100 mol % PC, and PA, PE, PI, and PS liposomes were mixed at 70 mol % PC/30 mol % phospholipid. Macrophages were preincubated with the receptor ligands, Abs, or their negative controls for 30 min before adding apoptotic neutrophils. All inhibitors were examined for their ability to inhibit phagocytosis nonspecifically by examining the uptake of zymosan and apoptotic neutrophils opsonized with CD45 as previously described (2). In no case were any of the inhibitors effective in decreasing phagocytosis of either of these particles (data not shown). As an additional control, opsonized neutrophils were fed to macrophages in the presence or the absence of anti-CD64, anti-CD32, and anti-CD16 (each at 50 μg/ml).

HMDM were cultured in 100-mm bacteriologic plates to facilitate harvesting. They were still adherent, and preliminary experiments showed that they phagocytose apoptotic cells using mechanisms identical with those cultured on tissue culture plastic (not shown). Day 5 HMDM were stimulated with 25 μg/ml glucan or were left unstimulated. After 48 h macrophages were harvested by replacing the medium with ice-cold HBSS plus 10 mM EDTA and gently detaching cells with a cell scraper. HMDM were then washed twice with HBSS to remove EDTA and were resuspended in HBSS to 5 × 105/ml. Ninety-six-well, flat-bottom tissue culture plates were precoated for 2 h at 4°C with 50 μl of HBSS containing 100 μg/ml anti-CD36, 100 μg/ml human serum albumin, or 100 μg/ml anti-CD45. After rewarming the coated plates to 37°C, 50 μl of the HMDM suspension/well was adhered over 30 min at 37°C in 5% CO2 before adding apoptotic neutrophils for the phagocytosis assay. In select plates, HMDM were treated with 0.1 mmol of PS liposomes or 150 μg/ml OxLDL for 30 min before adding apoptotic cells.

HMDM were cultured in bacteriologic plates. Day 5 HMDM were stimulated with 25 μg/ml glucan or were left unstimulated. After 48 h, select plates were pretreated for 30 min with 0.1 mmol of PS liposomes or 150 μg/ml OxLDL. Macrophages were harvested as described above, added at 1 × 106/100 μl in HBSS to 96-well round-bottom tissue culture plates, and washed twice with chilled Krebs-Ringers phosphate buffer with dextrose plus 0.01% BSA. Primary Ab was added for a 30-min incubation on ice (anti-CD36, 100 μg/ml; 61D3, 12.5 μg/ml; anti-CD45, 100 μg/ml; anti-VnR: anti-CD61 or anti-CD51/61, and anti-CD68, all at 1/20 dilution). After three washes, the cells were incubated for 30 min on ice in the dark with 100 μg/ml of the relevant secondary Ab (tetramethyl rhodamine isothiocyanate- or Cy3-conjugated F(ab′)2 goat anti-mouse IgG or IgM). HMDM were then washed a final three times and suspended at 1 × 106/ml in Krebs-Ringers phosphate buffer with dextrose plus 0.01% BSA. Samples were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, Sunnyvale, CA).

For phagocytosis studies, a mixed effects model was used to analyze data for each inhibitor (33), with the response variable being the absolute value of φI. If the distribution of φI indicated a high degree of positive skewness, it was transformed using a natural logarithm. The covariance structure of the repeated measurements was evaluated by comparing Akaike’s information criterion between models, specifying several commonly occurring covariance structures (34). In every case, we found the data to conform to either a compound symmetric or an unstructured covariance pattern. Pairwise multiple comparisons between conditions were made using the Tukey-Kramer multiple comparisons procedure at the 0.05 level of significance. All analyses were performed in SAS using the Mixed procedure (35). Although statistical analysis was conducted on the absolute or log-transformed value of the φI, we reported the results using normalized φI to facilitate comparison.

To confirm use of the αvβ3(VnR)/CD36/TSP mechanism in phagocytosing apoptotic neutrophils and to test for a potential switch to the PS recognition mechanism, we examined the function of these receptors in control, glucan-stimulated, and latex-stimulated macrophages. The tetrapeptide RGDS was used to block integrin (αvβ3) binding, and PS liposomes were used to bind the putative PSR, with RGES and PC liposomes as negative controls. In unstimulated HMDM, pretreatment with RGDS, but not RGES (not shown), blocked phagocytosis of apoptotic neutrophils by approximately 50% (Fig. 1). Increasing the concentration of RGDS to 10 mM reduced phagocytosis no further (data not shown). Neither PS nor PC liposomes were active in inhibiting apoptotic cell uptake. Latex treatment did not change this inhibition pattern (Fig. 1). In contrast, glucan treatment was associated with loss of inhibition by RGDS and acquisition of inhibition by PS liposomes (Fig. 1). Testing concentrations from 5–75 μg/ml, we found 25 μg/ml glucan to maximize both the macrophage’s capacity for phagocytosis and the generation of PS-inhibitable activity (data not shown). This concentration was therefore used in subsequent experiments, although it should be noted that this level is not achievable in mycotic infections in vivo. After testing this concentration for 24, 48, and 72 h, we found that PS-inhibitable uptake was maximal following 48 h of treatment, so this time point was chosen for all additional experiments. Inhibition of apoptotic cell uptake by PS liposomes was likewise dependent on the concentration of total phospholipid. Concentrations of PS liposomes from 0.001–0.1 mM caused progressive inhibition of apoptotic cell uptake into stimulated HMDM, while higher concentrations had no further effect (not shown). In contrast, we found that opsonized apoptotic cells were taken up by nearly all the macrophages; the mean percent positive macrophages for unstimulated cells was 86.7 ± 7.8%, and that for stimulated cells was 91.2 ± 6.3%. For both populations of cells, 90% inhibition of uptake was achieved using a combination of mAbs against Fc receptors (anti-CD64, anti-CD32, and anti-CD16; data not shown).

FIGURE 1.

Human macrophages can be stimulated to recognize PS on apoptotic cells. Day 5 HMDM were stimulated with 25 μg/ml glucan or 5:1 latex beads. Forty-eight hours later, unstimulated or stimulated HMDM were pretreated with 0.1 mM PS-containing liposomes or 1.0 mM RGDS before phagocytosing apoptotic neutrophils. ∗, p = < 0.010 for all indicated comparisons (n = 12).

FIGURE 1.

Human macrophages can be stimulated to recognize PS on apoptotic cells. Day 5 HMDM were stimulated with 25 μg/ml glucan or 5:1 latex beads. Forty-eight hours later, unstimulated or stimulated HMDM were pretreated with 0.1 mM PS-containing liposomes or 1.0 mM RGDS before phagocytosing apoptotic neutrophils. ∗, p = < 0.010 for all indicated comparisons (n = 12).

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The specificity of PS inhibition was probed by comparing PS liposomes with those containing other neutral and anionic phospholipids, including PC, PA, PI, and PE. Only pretreatment with PS liposomes significantly reduced phagocytosis of apoptotic cells by stimulated HMDM (Fig. 2). To confirm recognition of the polar head group of PS, we examined the inhibitory action of water-soluble phosphoester derivatives of PS using GPS and PLS, with GPC and PDS as negative controls (Fig. 3). Again, in glucan-stimulated HMDM, PS liposomes markedly reduced macrophage ingestion of apoptotic neutrophils, while PC liposomes did not. Similarly, GPS and PLS blocked uptake, while GPC and PDS were inactive. These results imply that stimulated HMDM did not simply interact with apoptotic neutrophils through their negative charge or hydrophobicity, but that they specifically recognized PS on the surface of the apoptotic cell. None of the inhibitors had any effect on the uptake of zymosan or opsonized apoptotic cells.

FIGURE 2.

Inhibition of uptake by liposomes is restricted to those containing PS. To determine the specificity of PS inhibition, glucan-stimulated HMDM were pretreated for 30 min with liposomes containing PS, PC, PA, PI, or PE before phagocytosing apoptotic neutrophils. ∗, p < 0.0001 (n = 6).

FIGURE 2.

Inhibition of uptake by liposomes is restricted to those containing PS. To determine the specificity of PS inhibition, glucan-stimulated HMDM were pretreated for 30 min with liposomes containing PS, PC, PA, PI, or PE before phagocytosing apoptotic neutrophils. ∗, p < 0.0001 (n = 6).

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FIGURE 3.

Structural analogues of PS also inhibit phagocytosis by glucan-stimulated macrophages. Glucan-stimulated HMDM were pretreated for 30 min with 0.1 mmol of PS or PC liposomes or with structural analogues of PS, including 1.0 mM GPS and PLS, or GPC and PDS as negative controls, before phagocytosing apoptotic neutrophils. ∗, p < 0.0001 (n = 4).

FIGURE 3.

Structural analogues of PS also inhibit phagocytosis by glucan-stimulated macrophages. Glucan-stimulated HMDM were pretreated for 30 min with 0.1 mmol of PS or PC liposomes or with structural analogues of PS, including 1.0 mM GPS and PLS, or GPC and PDS as negative controls, before phagocytosing apoptotic neutrophils. ∗, p < 0.0001 (n = 4).

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These findings confirmed that unstimulated macrophages phagocytosed apoptotic neutrophils using the αvβ3/CD36/TSP mechanism as previously reported by Savill and co-workers (6), but glucan stimulation promoted a switch to use of the proposed PSR. Further, the PSR accounted for a similar portion of apoptotic cell uptake into glucan-stimulated macrophages (∼50%), as did the αvβ3/CD36/TSP mechanism into unstimulated HMDM.

The next step was to use Abs to αvβ3 and CD36 to determine their ability to inhibit phagocytosis of apoptotic cells by unstimulated and glucan-stimulated HMDM. RGDS and PS liposomes were used to demonstrate the ability of the macrophages to switch to the PSR. As expected, in unstimulated HMDM, anti-CD36, anti-αv, and RGDS reduced phagocytosis by 30–40%, while PS liposomes had no inhibitory effect (Fig. 4). Following glucan stimulation, there was loss of RGDS and anti-αv inhibition, but gain of PS inhibition. Anti-CD36 blocked phagocytosis by both. Other IgM Abs against surface Ags (CD15, expressed at low levels, and CD15s, expressed at high levels, determined by flow cytometry) were also used at the same concentrations; these had no effect on the uptake of apoptotic cells (data not shown). These findings argued that CD36 was required for phagocytosis of apoptotic cells by both resting and stimulated HMDM.

FIGURE 4.

Abs against CD36 inhibit uptake of apoptotic cells by macrophages that recognize PS. To determine the involvement of CD36 and the VnR, unstimulated or glucan-stimulated HMDM were pretreated for 30 min with 100 μg/ml anti-CD36 or a 1/20 dilution of anti-αv before phagocytosing apoptotic neutrophils. RGDS (1.0 mM) and PS liposomes (0.1 mmol) were used in parallel to indicate the receptor switch (n = 3).

FIGURE 4.

Abs against CD36 inhibit uptake of apoptotic cells by macrophages that recognize PS. To determine the involvement of CD36 and the VnR, unstimulated or glucan-stimulated HMDM were pretreated for 30 min with 100 μg/ml anti-CD36 or a 1/20 dilution of anti-αv before phagocytosing apoptotic neutrophils. RGDS (1.0 mM) and PS liposomes (0.1 mmol) were used in parallel to indicate the receptor switch (n = 3).

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Expression of αvβ3 and CD36 on unstimulated and stimulated macrophages was determined by flow cytometry. We found that 58.6 ± 11.9% (range, 37.2–70.0%) of unstimulated macrophages were CD36 positive. After glucan stimulation, the percentage of CD36-expressing macrophages was unchanged at 58.9 ± 10.6% (range, 43.6–76.2%; n = 7). We also observed no change in CD45 expression. Using anti-CD51/61 and anti-CD61 Abs to detect αvβ3, we found a shift in fluorescence in all the cells that was not seen when the isotype control was used, suggesting that all the macrophages expressed this VnR at low levels. Despite low expression on unstimulated cells, we observed a statistically significant decrease in expression on glucan-stimulated macrophages (Fig. 5). This decrease may account in part for why this Ab no longer inhibits phagocytosis by stimulated cells; however, it may be accompanied by decreased function of the αvβ3 receptor, as we suggested for mouse macrophages (10)

FIGURE 5.

Flow cytometric analysis of unstimulated and stimulated macrophages for expression of VnR. For comparison, the average mean fluorescence intensity for CD36+ cells was >400 for both populations, and the average mean fluorescence intensity for CD45+ cells was >250 (n = 12). Stimulated macrophages showed a statistically significant decrease in binding to anti-CD51/61 and anti-CD61 (p < 0.0001). There was no change in the expression of CD36 or CD45.

FIGURE 5.

Flow cytometric analysis of unstimulated and stimulated macrophages for expression of VnR. For comparison, the average mean fluorescence intensity for CD36+ cells was >400 for both populations, and the average mean fluorescence intensity for CD45+ cells was >250 (n = 12). Stimulated macrophages showed a statistically significant decrease in binding to anti-CD51/61 and anti-CD61 (p < 0.0001). There was no change in the expression of CD36 or CD45.

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It was striking to observe that despite the mechanism used, only 50% inhibition was achieved, suggesting that other mechanisms were contributing to uptake of apoptotic cells. We sought to clarify this issue by examining the inhibition of other receptors reported to mediate phagocytosis of apoptotic cells, including SRs other than CD36, CD14 (recognized by the mAb 61D3), and lectins (11, 12, 13, 14, 15, 16, 17, 18, 19, 22).

SRs are a rapidly enlarging receptor class recognizing different lipoprotein derivatives. SRA-I/II bind AcLDL and OxLDL, but not LDL or PS (35, 36); the structurally related MARCO receptor (which has expression restricted to splenic and lymph node macrophages) likewise binds AcLDL (37, 38). Members of the B class of SRs, SR-B1 and the human homologue CLA-1, CD36, and macrosialin (CD68), an OxLDL receptor, exhibit different binding affinities for these lipoproteins, but all appear capable of binding PS and PI at least when offered in a liposome (13, 15, 16). SR-B1 and CLA-1 reportedly bind all three lipoproteins (3, 37, 39). CD36 binds OxLDL and not LDL (40); its binding of AcLDL is controversial (37, 40). CD68 binds OxLDL alone (41, 42, 43). To test the roles of these various SR in apoptotic cell uptake, we pretreated HMDM with 150 μg/ml of AcLDL, OxLDL, or LDL. In our system, these concentrations did not appear to be toxic, as the macrophages could still phagocytose zymosan and opsonized apoptotic cells (not shown). In unstimulated HMDM, OxLDL, but not AcLDL or LDL, inhibited phagocytosis by approximately 20%. In glucan-stimulated HMDM, the inhibitory effect of OxLDL was more pronounced, reducing phagocytosis by almost 40%, while AcLDL and LDL were again inactive (Fig. 6). Subsequent dose-response studies, using up to 250 μg/ml OxLDL did not further inhibit phagocytosis in either unstimulated or stimulated macrophages (data not shown).

FIGURE 6.

To assay for use of SR, unstimulated and glucan-stimulated HMDM were pretreated for 30 min with 150 μg/ml AcLDL, LDL, or OxLDL before phagocytosing apoptotic neutrophils (n = 12). ∗, p = 0.032; #, p < 0.0001.

FIGURE 6.

To assay for use of SR, unstimulated and glucan-stimulated HMDM were pretreated for 30 min with 150 μg/ml AcLDL, LDL, or OxLDL before phagocytosing apoptotic neutrophils (n = 12). ∗, p = 0.032; #, p < 0.0001.

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The unique inhibitory effect of OxLDL suggested that both groups of macrophages might recognize apoptotic neutrophils through either CD36 or CD68. SR-AI/II, MARCO, and SR-B1 were unlikely to be involved, because AcLDL never significantly inhibited apoptotic cell uptake in either group of macrophages and because the first two do not appear capable of binding PS. CD36 and CD68 were reasonable candidates to mediate HMDM phagocytosis of apoptotic cells, because macrophages express these receptors. CD36 is expressed on the surface of human macrophages (6), and CD36 transfection confers the capacity to recognize apoptotic cells on nonprofessional phagocytes (27). Macrosialin/CD68 is localized to lysosomal granules of murine and human macrophages (44); however, Steinberg and co-workers have shown surface expression as well and have implicated this receptor in the uptake of apoptotic cells by mouse peritoneal macrophages (14, 15). Since CD36 was expressed on both unstimulated and glucan-stimulated macrophages; it was important to determine whether CD68 was also expressed, and if Abs to CD68 could inhibit phagocytosis. Using two different anti-CD 68 Abs in flow cytometry experiments, we were unable to demonstrate CD68 expression on the surface of HMDM, stimulated or not (not shown). These Abs also did not inhibit phagocytosis. It was likely, then, that inhibition by OxLDL was not caused by its binding to CD68.

Another reported mechanism for HMDM phagocytosis of apoptotic cells is the 61D3-inhibitable receptor CD14 (18, 19, 45), which might function separately (18, 20) or jointly with the integrin and phospholipid receptors (21). Using the 61D3 mAb, we achieved inconsistent results; in some experiments we observed 20% inhibition of apoptotic neutrophil uptake, and in some experiments we observed no inhibition. This may have been due to donor variation or differences in the preparations of 61D3 that we used, although we observed no differences in the ability of these preparations to bind to macrophages as detected by flow cytometry. We therefore purchased a second anti-CD14 Ab (MEM-18), which was shown by Devitt and colleagues to inhibit the binding of apoptotic lymphocytes to CD14 (19). Levels of CD14 expression detected by MEM-18 were similar to those detected by 61D3; 38% of cells from either unstimulated or stimulated macrophage populations cultured for 7 days were positive (data not shown). We then compared the effects of MEM-18 and TUK-4 (an anti-CD14 Ab that does not inhibit the interaction of apoptotic cells with macrophages (19)) on the phagocytosis of apoptotic neutrophils and lymphocytes (irradiated Jurkat T cells). The results of these experiments are shown in Fig. 7. Although MEM-18 was effective in reducing the uptake of apoptotic Jurkat T cells by both unstimulated and stimulated macrophages (p < 0.003), it was not effective in inhibiting neutrophil uptake. Taken together with the data we collected using 61D3, these results suggest that CD14 mediates the binding and uptake of apoptotic lymphocytes, but that it is of minor importance in mediating the binding or phagocytosis of apoptotic neutrophils.

FIGURE 7.

CD14 is involved in the uptake of apoptotic lymphocytes, but not apoptotic neutrophils, by human macrophages. Unstimulated or stimulated macrophages were pretreated with a 1/20 dilution of either MEM-18 or TUK-4 for 30 min before addition of apoptotic Jurkat T cells or apoptotic neutrophils. PS liposomes (0.1 mM) and RGDS (1 mM) were used as controls to verify that the glucan stimulation was effective (n = 4). ∗, p ≤ 0.003.

FIGURE 7.

CD14 is involved in the uptake of apoptotic lymphocytes, but not apoptotic neutrophils, by human macrophages. Unstimulated or stimulated macrophages were pretreated with a 1/20 dilution of either MEM-18 or TUK-4 for 30 min before addition of apoptotic Jurkat T cells or apoptotic neutrophils. PS liposomes (0.1 mM) and RGDS (1 mM) were used as controls to verify that the glucan stimulation was effective (n = 4). ∗, p ≤ 0.003.

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Lastly, to determine whether HMDM used a lectin-like receptor to phagocytose apoptotic neutrophils, we examined their ingestion of apoptotic cells in the presence of NAG. In unstimulated macrophages, NAG pretreatment reduced the phagocytosis of apoptotic cells by approximately 20% (not shown); this difference was statistically significant (p < 0.04; n = 12) and differs from the previous findings of Savill et al. (46). Increasing the NAG dose to as high as 40 mM inhibited phagocytosis no further (data not shown). In glucan-stimulated HMDM, NAG had no significant effect. Despite the statistical significance, it seems unlikely that lectins contribute in a major way to phagocytosis by human macrophages, and, in fact, the effect of NAG may be nonspecific.

After observing the inhibitors’ individual effects on HMDM phagocytosis of apoptotic cells, it became important to determine whether combining inhibitors would give additive effects. Using RGDS to block the αvβ3/CD36/TSP mechanism, PS liposomes to inhibit the PSR, OxLDL to block SR (presumably CD36), and NAG to inhibit a possible lectin-like receptor, we pretreated unstimulated and stimulated HMDM with the following combinations before addition of apoptotic neutrophils: NAG and OxLDL; NAG and RGDS; RGDS and OxLDL; NAG, OxLDL, and RGDS; and PS and OxLDL. No inhibitor or combination of inhibitors reduced phagocytosis much below 50% of control levels, and we observed no additive effects (Fig. 8). Using the combination of anti-αv and anti-CD36 in unstimulated macrophages, we achieved a maximal inhibition of 68.3 ± 3.2% (n = 6); the addition of 61D3 to this combination showed a slight, but not statistically significant, additive effect (74.3 ± 5.5%). Using the combination of anti-CD36 and PS liposomes in stimulated cells, we achieved a maximal inhibition of 70.8 ± 4.5% (n = 6); adding 61D3 to this combination gave 75.3 ± 3.7% inhibition. The effects of MEM-18 were the same as those of 61D3 (not shown).

FIGURE 8.

To determine whether inhibitors could act synergistically, both control macrophages (A) and glucan-stimulated macrophages (B) were pretreated for 30 min with the combinations shown before addition of neutrophils (n = 4).

FIGURE 8.

To determine whether inhibitors could act synergistically, both control macrophages (A) and glucan-stimulated macrophages (B) were pretreated for 30 min with the combinations shown before addition of neutrophils (n = 4).

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Because CD36 appeared to be required for the uptake of apoptotic cells into both unstimulated and glucan-stimulated HMDM and because PS liposomes and OxLDL failed to inhibit uptake additively into stimulated HMDM, we questioned whether the putative PS receptor we have proposed might be CD36. In fact, CD36 has been reported to recognize PS because binding of AcLDL to CD36-transfected COS cells can be competed with PS liposomes (17). In addition, Ryeom et al. have shown that CD36 is expressed on retinal pigment epithelium that phagocytoses photoreceptor outer segments in a PS-inhibitable fashion (47). However, PI liposomes are equally or more effective in both these systems, and we did not observe inhibition of phagocytosis by PI liposomes (see Fig. 2). Nevertheless, we hypothesized that if CD36 was able to recognize PS on apoptotic cells, binding of its Ab would be inhibited by PS liposomes. This is not an unreasonable hypothesis, as Ramprasad et al. have shown that binding of Abs to CD68 (OxLDLR) can inhibit OxLDL binding to THP-1 cells and vice versa (15). After harvesting, macrophages were pretreated with either PS liposomes or OxLDL before addition of anti-CD36 Ab and were examined by flow cytometry. Following PS liposome pretreatment, the percentage of CD36-positive HMDM remained unchanged in both unstimulated and stimulated macrophages (CD36-positive macrophages (unstimulated HMDM, 57.4 ± 15.7%; unstimulated HMDM and PS liposomes, 59.4 ± 15.7%; glucan-stimulated HMDM, 61.5 ± 13.5%; glucan-stimulated HMDM and PS liposomes, 62.7 ± 8.38%; n = 4). Similarly, following OxLDL pretreatment, unstimulated and stimulated HMDM did not change their expression of CD36 (CD36-positive macrophages: control HMDM, 60.2%; control HMDM and OxLDL, 61.7%; glucan-stimulated HMDM, 62.23%; glucan-stimulated HMDM and OxLDL, 65.8%; n = 2). We next attempted to down-regulate CD36 expression by adding PS liposomes or OxLDL for 30 min before harvesting the macrophages for flow cytometry. CD36 expression was not effected by either pretreatment (data not shown). These data implied that CD36 was not the PS receptor; however, it was possible that the epitope to which the Ab bound was not the epitope that bound to the apoptotic cell. This interpretation seems less likely given that these Abs did inhibit phagocytosis of PS-expressing apoptotic cells. We therefore hoped to clarify this issue by down-regulating the expression of CD36 with anti-CD36 before phagocytosis.

We plated macrophages on anti-CD36-coated dishes before assaying the phagocytosis of apoptotic neutrophils in the presence or the absence of PS liposomes or OxLDL. Anti-CD45 was used as the control. Unstimulated macrophages plated on anti-CD36 showed the expected reduction in uptake of apoptotic cells (55.2 ± 3%; n = 6); neither PS liposomes nor OxLDL decreased phagocytosis further (data not shown). Stimulated macrophages plated on anti-CD36 also had reduced apoptotic cell uptake (39.5 ± 10%; n = 6); OxLDL failed to inhibit phagocytosis further, and PS inhibited uptake by an additional 10%, which was not statistically significant (data not shown). Macrophages adhered to plates coated with human serum albumin or anti-CD45 Ab, as nonspecific controls, showed no change in phagocytosis, and the patterns of inhibition by RGDS and PS were consistent with whether the cells were stimulated. Our interpretation of these data is that CD36 is a necessary cofactor for phagocytosis by PS-recognizing macrophages, but that it may not necessarily function as a PSR.

Our data show that human macrophages can be induced to recognize PS on apoptotic cells and that the phagocytosis associated with this recognition requires CD36. Mature, unstimulated HMDM cultured for 7–10 days phagocytosed apoptotic neutrophils primarily through the αvβ3/CD36/thrombospondin system described by Savill and co-workers (6). In contrast, HMDM that had been stimulated with glucan lost the ability to use this recognition process but acquired the ability to recognize PS. Previous investigators have examined human macrophages for use of the proposed PS receptor and were unable to identify it (4, 27, 28). However, those studies were with unstimulated macrophages, which, as confirmed here, do not use this recognition system. CD36 appeared to function in both groups of macrophages (25–40 and 40–55% uptake, respectively), suggesting a role in both recognition mechanisms. CD14 played a minor role, if any, in the uptake of apoptotic neutrophils by either unstimulated or stimulated macrophages, although selected Abs against this Ag inhibited the uptake of apoptotic lymphocytes. Our findings agree with those of Flora and colleagues, who have shown that the CD14 mechanism appears to be most efficient for some lymphocytes and that uptake of apoptotic neutrophils or peripheral blood neutrophils is inhibited poorly by 61D3 (18). At this point, it is unclear whether the CD14 mechanism shows any connection or cooperativity with the αvβ3/CD36/TSP or the PSR mechanisms, but the fact that macrophages that fail to use CD14 still recognize PS on apoptotic neutrophils suggests that CD14 is not the PSR we have proposed. We could not find evidence for the use of SRs other than CD36; however, identification of new SRs remains an active area of investigation. In both stimulated and unstimulated macrophages, a large proportion of the uptake (30–50%) was not blocked by any of the inhibitors we used, suggesting that additional mechanisms remain uncharacterized.

The nature of the receptor(s) that mediate PS recognition remains unclear. The binding of AcLDL to CD36 can be competed by PS liposomes, suggesting that CD36 can bind PS directly (17) and may itself be a PSR capable of both recognition and initiation of apoptotic cell uptake. In fact, Ryeom et al. have shown that CD36 on retinal pigment epithelium can bind and internalize PS-containing liposomes, and that anti-CD36 and PS liposomes can inhibit the uptake of PS-expressing photoreceptor outer segments (47). However, PI liposomes function equally well or better in both these systems, and we have been unable to demonstrate inhibition of phagocytosis using this anionic phospholipid or AcLDL on either human (see Fig. 2) or mouse macrophages (9). Although we found that down-regulation of CD36 was associated with a loss of PS inhibition of stimulated macrophages, the binding of anti-CD36 was not inhibited by PS liposomes, and CD36 was not down-modulated by PS liposomes. These data suggest that CD36 may act as a necessary cofactor for either the VnR system (for which the ligand has not been defined) or PS recognition (for which the receptor has not been defined). It is highly likely that there may be multiple PSRs, some of which remain to be identified. In our hands, PS recognition is specific, in that phagocytosis is inhibited stereospecifically by PS and its analogues, GPS and phosphoserine. Other anionic phospholipids have no effect, arguing against any of the currently identified class B SRs mediating this function.

It is also highly likely that the ligands on apoptotic cells are complex. We have used an operational definition for the PSR we have proposed, which is inhibition of phagocytosis by PS liposomes. Pradhan et al. have shown that inhibition of phagocytosis by unstimulated macrophages can be inhibited by symmetric red cell ghosts and have suggested that this implies PS recognition. They therefore hypothesize that murine macrophages use multiple, partly overlapping systems to recognize apoptotic cells (21). In our hands, however, symmetric red cells and sickled red cells, like PS liposomes, only inhibited phagocytosis of stimulated macrophages and had no effect on unstimulated macrophages (unpublished data). If human macrophages recognize PS on symmetric ghosts and sickled red cells (23, 24, 25, 26), why do they only appear to see it on apoptotic cells after they are stimulated? It is possible that PS on a symmetric red cell is not equivalent to that on an apoptotic cell. Data supporting this have been provided by Terpstra et al., who showed that binding of macrophages to oxidized red cells was calcium dependent, whereas binding to apoptotic cells was not (48). We also found that binding or phagocytosis of apoptotic cells by PS-recognizing macrophages is calcium independent (data not shown). These data lend support to the idea proposed by Pradhan et al. that apoptotic ligands are complex structures.

In summary, then, PS recognition of apoptotic cells appears to be an inducible function in both human and mouse macrophages. CD36 seems to be an important cofactor for at least two different recognition mechanisms: that mediated by the VnR and that mediated by PSR. Why are there so many different receptor systems for apoptotic cells? One could speculate a number of reasons. First, removal of apoptotic cells may be so critical to normal tissue structure and function that multiple mechanisms have been developed. Second, one of the critical functions of the apoptotic cell when phagocytosed is to actively suppress macrophage proinflammatory functions, which may be mediated by one or more of these receptor mechanisms (2, 3). Third, there may be variations in ligand expression among different types of apoptotic cells. Support for this is provided by the work of Flora et al. (18) and Hart et al. (49). The former showed that the CD14 recognition mechanism appeared to be more effective for removal of lymphocytes than neutrophils, which our data confirm; the latter showed that CD44 treatment of human macrophages enhanced phagocytosis of apoptotic neutrophils but not lymphocytes. However, in most studies as well as those described herein there is a significant proportion of the recognition and uptake that is not blocked by any of the inhibitors identified to date, either alone or in combination. The clear implication is that a major mechanism for recognition and uptake of apoptotic cells remains to be identified.

We thank Drs. J. D. Capra, Andrew Devitt, and Christopher Gregory for the kind provision of 61D3 mAb, and Drs. John Savill, Christopher Gregory, and Daniel Steinberg for their helpful discussions on this fascinating topic. We also acknowledge Sally Kreiss for human blood cell preparation, Becky Bucher-Bartelson and David McCormick for guidance with statistical analysis, and William Townend for assistance with flow cytometry.

1

This work was supported in part by the Will Rogers Foundation, National Institutes of Health Grant R01GM48211, and the Cancer Center at University of Colorado Health Sciences Center.

3

Abbreviations used in this paper: PS, phosphatidylserine; HMDM, human monocyte-derived macrophages; TSP, thrombospondin; PSR, phosphatidylserine receptor; SR, scavenger receptor; PI, phosphatidylinositol; NAG, N-acetylglucosamine; AcLDL, acetylated low density lipoprotein; LDL, low density lipoprotein; OxLDL, oxidized low density lipoprotein; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; RGES, Arg-Gly-Glu-Ser; RGDS, Arg-Gly-Asp-Ser; GPS, l-α-glycerophosphorylserine; GPC, l-α-glycerophosphorylcholine; PLS, phospho-l-serine; PDS, phospho-d-serine; MPO, myeloperoxidase; φI, phagocytic index; VnR, vitronectin receptor.

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