Cell Culture.
J774.A1 murine macrophages, NRK-F49 rat fibroblasts, and rat RPE-J cells were obtained from American Type Culture Collection and routinely maintained in DMEM supplemented with 10 or 4% (RPE) FCS. For binding and phagocytosis experiments, macrophages and fibroblasts were seeded at confluence on glass coverslips for 30 min or overnight before use. RPE-J cells were differentiated on semipermeable filters as described previously 36. Rat primary cells were isolated from adult Long Evans rats (Richard Harlan, Inc.) immediately after CO₂ asphyxiation. Rat bone marrow–derived macrophages and rat blood–derived monocyte macrophages were prepared and maintained according to published procedures 1337. In brief, blood-derived monocytes were isolated by plastic adherence after Ficoll gradient purification of leukocytes from anticoagulant-treated rat blood. Bone marrow cells were flushed aseptically from femur bones of Long Evans rats, washed, seeded, and used without repassaging. All primary macrophages were cultured for 6–7 d before use in IMDM, supplemented with 10% autologous serum; bone marrow–derived cell medium was supplemented with 5% L cell–conditioned medium (Sigma Chemical Co.) as a source of M-CSF 13. Primary rat neutrophils, which were isolated from fresh rat blood according to established protocols 38, underwent spontaneous apoptosis over 24 h in culture. HL-60 cells, a gift from Dr. F. Maxfield (Weill Medical College of Cornell University) were maintained in 10% FCS in RPMI. To induce differentiation and apoptosis, HL-60 cells were cultured in the dark at 2 x 10⁵ cells/ml in growth medium containing 1 µM all-trans retinoic acid for 6 d. Before binding or phagocytosis assays, apoptotic cells were routinely tested for viability by trypan blue exclusion and phosphatidylserine surface exposure staining with FITC–annexin V. All populations used were >95% viable and >60% annexin V positive.

Preparation of Photoreceptor OS.
OS were isolated according to established protocols from bovine eyes obtained fresh from the slaughterhouse 39. OS were stored suspended in 10 mM sodium phosphate, pH 7.2, 0.1 M sodium chloride, 2.5% sucrose at –80°C. Before use, OS were thawed and labeled by addition of 20% vol of 1 mg/ml FITC or 0.2 mg/ml Texas red (both from Molecular Probes) in 0.1 M sodium bicarbonate, pH 9.0, for 1 h at room temperature in the dark, before being washed and resuspended in cell culture medium.

Opsonization of Zymosan.
Zymosan particles were opsonized with serum complement components as described previously 40. In brief, 1% zymosan particles in PBS were boiled for 30 min and opsonized in 50% bovine serum in HBSS for 1 h at 37°C. Washed particles were labeled with FITC as described for OS, and stored at 4°C.

Particle Binding and Phagocytosis Experiments.
To study particle binding, phagocytes at confluence were challenged with 10 particles per cell in growth medium (RPE) or serum-free growth medium (other cells) for the duration of the experiment, washed three times with PBS containing 1 mM MgCl₂ and 0.2 mM CaCl₂ (PBS-CM), and fixed in ice-cold methanol. OS were covalently labeled with FITC or Texas red, and thus could be observed directly. Apoptotic cells were visualized by TUNEL staining or by granulocyte-specific myeloperoxidase immunofluorescence staining using FITC- or Texas red–conjugated secondary antibodies. Nuclei were counterstained with DAPI or propidium iodide at 1 ng/ml in PBS-CM. For competition experiments, OS and apoptotic cells were labeled with different fluorochromes. Particle labeling with either fluorochrome yielded identical results. Unlabeled particles competed with fluorescence-labeled particles for binding and phagocytosis by all cell types. For inhibition experiments, GRGDSP or GRADSP peptides (Calbiochem) were used at 1 mg/ml. Effects of peptides and antibodies were concentration dependent as established previously 26, and maximal effective concentrations of antibodies, 20–50 µg/ml, were used throughout this study. Concentrations of pharmacological reagents were as follows: calphostin C at 100 nM (light activated), cytochalasin D (Cyt D) at 5–20 µM, Gö6976 at 10 nM, hypericin at 5 µM, and latrunculin B at 1 µM. PMA was routinely used at 50 nM; 16–160 nM gave similar results. Cells were pretreated for times indicated in the figure legends with activators or inhibitors before challenge with OS or apoptotic granulocytes in the continuous presence of reagent. Cell viability and morphology remained unchanged, and none of the cell types initiated detectable apoptosis over the course of the experiments. Since experiments were performed on confluent cells, the spreading effect of PMA on macrophages was negligible.

Phagocytosis or binding of OS was quantified by fluorescence scanning of fixed samples as described 26. Samples were scanned with a STORM 860 Imager, at 950 V (blue or red fluorescence setup; Molecular Dynamics). Areas representing the binding by 1–2 x 10⁵ phagocytes were selected, and the fluorescent signals were quantified with ImageQuant 1.2 (Molecular Dynamics). Within one experiment, particle counts directly correlated with particle binding. To compare particle binding of different cell types, as RPE cells and macrophages, the fluorescence of propidium iodide (nuclei, red) and the particle-derived FITC fluorescence were both measured in each field. The binding plus internalization index or the binding index (bound particles at early times of phagocytic challenge) were calculated dividing particle counts by nuclei counts, thereby normalizing for phagocyte numbers. Quenching of fluorescence derived from externally bound particles using trypan blue 2641 allowed determination of the internalization index. Phagocytosis or binding of apoptotic cells was quantified following the same procedure based on TUNEL or myeloperoxidase immunostaining fluorescence emissions; binding indices were determined dividing by nuclei counts of control fields, so as not to count apoptotic nuclei. Microscopic observation revealed that 75% of RPE-J cells and >90% of J774 cells had bound or phagocytosed an average of 5 ± 1 OS after 2 h. Using the double fluorescence scanning method on the same samples, this translated into a mean index combining binding plus internalization of 7.6 ± 0.9 for macrophages and 6.3 ± 0.9 for RPE. After 30 min, 75% of macrophages had bound multiple OS; this translated to a mean binding index of 3.8 ± 0.4. At this time point, <20% of particles had been internalized by macrophages, similar to the 2-h time point of RPE 2641.

Immunofluorescence Microscopy.
Samples were fixed in ice-cold methanol or 4% paraformaldehyde in PBS-CM and processed as described previously 42. Samples were observed with a Nikon fluorescence microscope E600. Digital images were acquired with a back-illuminated cooled CCD camera (CCD1000 PB; Princeton Instruments), translated using MetaMorph (Universal Imaging), and recompiled in Adobe Photoshop^® 4.0. Horizontal (x–y) sections were acquired at 0.5-µm steps using a z motor (Prior), and out of focus light was removed using MetaMorph.

Cell Fractionation.
Integrin cytoskeletal association was assayed by cell fractionation into detergent-soluble and insoluble fractions according to previously described protocols 43. Confluent cells in 6-cm dishes, preincubated with pharmacological reagents or vehicle as before binding or phagocytosis assays, were extracted in 800 µl 50 mM MES, 5 mM MgCl₂, 3 mM EGTA, 0.5% Triton X-100, pH 6.4, for 40 s at room temperature (soluble fraction). The remaining cellular material was completely solubilized in an equal volume of RIPA (50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 2 mM CaCl₂, 1 mM MgCl₂, 0.1% SDS, 1% sodium desoxicholate, 1% Triton X-100) (insoluble fraction). Equal volumes of soluble and insoluble fractions were compared by 7.5% SDS-PAGE and immunoblotting. For immunoprecipitation of integrins from the different fractions, the soluble fraction was harvested as described above, whereas the insoluble fraction was obtained by scraping the remaining cellular material in an equal volume of 50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 2 mM CaCl₂, 1 mM MgCl₂, 1% Triton X-100, 1% NP-40 supplemented with 2 mM each of aprotinin, leupeptin, pepstatin, iodoacetamide, and PMSF, and 1 mM N-ethylmaleimide (IP lysis buffer), vortexing for 30 min at 4°C. This procedure completely solubilized integrin proteins. Immunoprecipitations were performed from this insoluble and the soluble fraction as follows.

Surface Protein and Immunoprecipitations, SDS-PAGE, and Immunoblotting.
For metabolic labeling, confluent cells on plastic dishes were starved for 90 min in cysteine, methionine-free DMEM, and labeled with 0.1 mCi/ml ³⁵S Express (NEN) for 12–14 h. Preincubation with pharmacological reagents or vehicle as control was performed as before binding/phagocytosis assays. To biotinylate surface proteins, cells were incubated with Sulfo-NHS-LC biotin (Pierce Chemical Co.) at 0.5 mg/ml in PBS-CM twice for 20 min on ice. Excess reactive biotin was quenched in 50 mM NH₄Cl in PBS-CM. Cells were lysed in IP lysis buffer for 30 min. Protein concentration of supernatant lysates was determined according to Bradford 44. Crude lysates were analyzed by nonreducing 7.5% SDS-PAGE followed by Western blot detection of integrins as published previously 45. Streptavidin-agarose precipitation has been described in detail elsewhere 46. Immunoprecipitates were formed incubating precleared lysate with 5 µg P1F6 IgG or nonimmune mouse IgG, with 5 µg rabbit anti–mouse IgG, and with 5 mg protein A–sepharose, each for 1 h at 4°C. Samples were washed four times in 50 mM Tris-HCl, pH 8.5, 0.5 M NaCl, 2 mM CaCl₂, 1 mM MgCl₂, 1 mg/ml egg albumin, 0.1% Triton X-100 eluted in nonreducing or reducing SDS sample buffer, and analyzed on 10% SDS-PAGE followed by fluorography or blotting. Blots were incubated with integrin antibodies, horseradish peroxidase–conjugated streptavidin, or secondary antibodies, followed by ECL (NEN) detection. X-ray films were scanned, and signals were quantified using NIH Image 1.61.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Macrophages and RPE Cells Bind, Nonpreferentially, OS and Apoptotic Cells but Use Different Integrins for This Purpose.
Blood monocyte or bone marrow–derived macrophages use vβ3 integrin receptors to bind apoptotic cells before phagocytosis 7, whereas RPE cells bind OS via vβ5 integrins 2526. We initially tested the hypothesis that the characteristic choice of different integrin receptors by macrophages and RPE cells was determined by differences in the bound particles. To this end, we exposed the phagocytes to the physiologic ligands of the other cell, i.e., macrophages to OS and RPE cells to apoptotic cells (Fig. 1). Rat bone marrow macrophages and the murine macrophage cell line J774, which retains the vβ3 integrin–dependent apoptotic cell clearance mechanism of noninflammatory monocyte macrophages 13, efficiently bound and phagocytosed OS (Fig. 1, a and b). Primary cultures of rat RPE, and rat (RPE-J) and human (ARPE-19) RPE cell lines identified and took up apoptotic granulocytes (and other apoptotic cells; data not shown), but not their nonapoptotic counterparts (Fig. 1c–f). Most subsequent studies were performed on J774 macrophages and RPE-J cells, but were confirmed on rat bone marrow–derived macrophages and primary rat RPE cultures.

View larger version (72K): [in this window] [in a new window] [Download PPT slide]   Figure 1 Macrophages take up OS, and RPE cells recognize and phagocytose apoptotic cells. (a) Phase-contrast and (b) merged fluorescence image of rat bone marrow–derived macrophage phagocytosing FITC-labeled OS (green) 40 min after particle challenge. Macrophage nuclei were stained with propidium iodide (red). (c) Phase-contrast and (d) merged fluorescence image of RPE-J cell after internalization of an apoptotic granulocyte, 3 h after phagocytic challenge. Apoptotic DNA was stained with TUNEL (green), nuclei with propidium iodide. (e and f) RPE-J monolayer challenged for 2 h with equal numbers of HL-60 cells, which had been induced to undergo apoptosis (f) or not (e). Samples were stained for myeloperoxidase to detect HL-60 cells (green) and counterstained with propidium iodide. RPE-J cells take up exclusively cells from the apoptotic population. Bound or phagocytosed HL-60 cells were also positive in TUNEL staining (data not shown). Nonapoptotic and apoptotic HL-60 cells expressed identical amounts of myeloperoxidase (data not shown). Scale bars, 10 µm.

View larger version (35K): [in this window] [in a new window] [Download PPT slide]   Figure 2 Apoptotic cells and OS compete for binding to vβ3 of macrophages and for binding to vβ5 of RPE cells. (a) Time course of combined OS binding plus internalization by rat bone marrow–derived macrophages (x) and NRK-F49 fibroblasts (•) compared with RPE-J cell combined binding plus internalization of OS () and apoptotic cells (). J774 binding and internalization were indistinguishable from those of rat primary macrophages. (b) Integrin dependence and RGD peptide inhibition of binding of OS (black bars) and apoptotic cells (white bars) by RPE-J cells and by J774 macrophages. Mean OS binding indices in the absence of apoptotic cells were 6.3 ± 0.9 for RPE-J and 3.8 ± 0.4 for J774, after 2 h or 30 min of particle challenge, respectively. At these time points, particle phagocytosis accounted for <20% of total particle count. Mean apoptotic cell binding indices in the absence of OS were 5.5 ± 0.4 for RPE-J and 3.7 ± 0.5 for J774, at the same time points. (c) Quantitative competition of OS with apoptotic cells for phagocyte binding. RPE-J and J774 macrophages were challenged with different ratios of OS and apoptotic cells, for 2 h or 30 min, respectively. Particles were labeled with different fluorophores (FITC and Texas red) and quantified by fluorescence scanning. 100% represents binding of each particle in the absence of the other; for binding indices, see b. OS, black bars; apoptotic cells, white bars. Results represent means ± SEM (n = 5). When challenged with equal numbers of OS and apoptotic cells, both phagocytes seem to prefer apoptotic cells. However, this may be attributed to the larger size of apoptotic cells compared with OS (see Fig. 1).

Binding of Apoptotic Cells and OS by vβ5 Is Dormant in Macrophages but May Be Activated by PKC.
We tested three hypotheses that might account for particle binding by different integrin binding receptors in macrophages and RPE cells. Hypothesis 1 was that cell type–specific integrin protein expression determined receptor availability for particle binding. However, Fig. 3 shows that selective integrin expression was not involved, as both β3 and β5 were expressed at similar levels by J774 cells, rat bone marrow–derived macrophages, and RPE-J cells. Immunoprecipitation of vβ5 from RPE and macrophage lysate using the antibody P1F6, which recognizes only intact heterodimers, and coimmunoprecipitation of β3 integrin with v integrin confirmed the formation of vβ3 (data not shown) and vβ5 receptors (see Fig. 8). We have shown previously that the steady state distribution of β3 integrins is basolateral in the RPE 26. Although this does not exclude a temporary presence of vβ3 at the apical phagocytic surface, this spatial segregation may render it less available for efficient apoptotic cell or OS binding by the RPE than vβ5, which localizes apically and cytoplasmically. In contrast, double immunofluorescence staining with antibodies recognizing the β3 extracellular domain and with P1F6 antibodies specific for the extracellular face of the vβ5 receptor complex showed that in nonpermeabilized macrophages, both antigens were localized in the same optical sections of the plasma membrane of a given cell, even if their distribution within the plane of the membrane differed. Like β3 integrins, vβ5 receptors localized partially to basal attachment sites of macrophages but were also available at their free surface for binding to apoptotic cells or OS.

View larger version (49K): [in this window] [in a new window] [Download PPT slide]   Figure 3 J774 and rat macrophages express similar levels of v, β3, and β5 integrin subunits as rat RPE-J cells and rat NRK-F49 fibroblasts. Proteins were detected by comparative immunoblotting after SDS-PAGE of 50 µg each of detergent lysates of RPE-J cells, J774 macrophages, primary rat monocytes (Mono), and rat NRK-F49 fibroblasts. Tested rodent primary macrophages and cell lines expressed levels of β5 integrin protein comparable to RPE cells, while an earlier study did not detect β5 in human monocyte–derived macrophages (reference 29). This may be attributed to species differences, or to differences in in vitro monocyte maturation conditions.

View larger version (84K): [in this window] [in a new window] [Download PPT slide]   Figure 4 β3 integrin and vβ5 integrin localize to attached and free macrophage plasma membrane. β3 integrins but not vβ5 integrin redistribute to attachment sites in macrophages seeded on fibrinogen substrate. Immunodetection of surface integrins was performed on unpermeabilized cells with β3-specific or vβ5 receptor–specific antibodies. Top and bottom panels shown are en face views of the apical and the attached cell surface, respectively. (a) β3 and vβ5 integrin distribution in J774 macrophages attached to laminin-coated coverslips for 30 min. β3 and vβ5 are present on both surfaces, as demonstrated by the immunofluorescence signals in horizontal optical sections at the levels of attached (a1 and a3) and free (a2 and a4) cell surfaces. (b) Same staining as in a, of J774 macrophages seeded on fibrinogen for 30 min, reveals specific relocalization of β3 integrins to attachment sites. Compare a1 with b1, a2 with b2. Scale bars, 10 µm.

View larger version (36K): [in this window] [in a new window] [Download PPT slide]   Figure 5 Effects of attachment on different extracellular substrates or β3-specific antibodies on J774 macrophage apoptotic cell or OS binding and internalization. J774 macrophages were seeded on coated coverslips in serum-free medium for 30 min. Particles were added for 30 min (a) or 90 min (b) in the presence or absence of inhibitory antibodies or peptides. White (a) and gray (b) bars, hatched or not, represent mean binding plus internalization indices ± SEM (n = 6). Black bars represent mean internalization indices ± SEM (n = 3). Note that cells seeded for 30 min before particle challenge, as studied in this experiment, bound fewer particles than cells which had attached overnight (see Fig. 2). Results represent data from OS challenges. Identical results were obtained challenging with apoptotic cells. Cell adhesion or morphology did not notably change due to attachment to the β3 antibody or to different substrates (LN, laminin; Fb, fibrinogen; VN, vitronectin). Attachment to vβ5 or β1 antibody had no effect on particle binding (data not shown).

View larger version (32K): [in this window] [in a new window] [Download PPT slide]   Figure 6 vβ5 apoptotic cell or OS binding activity requires PKC stimulation. Endogenous PKC signaling preactivates vβ5 binding by RPE cells, and stimulation of PKC induces a novel vβ5-dependent binding mechanism in macrophages and fibroblasts. Cal C, calphostin C. 100% binding represents binding of DMSO controls for each phagocyte. Results are expressed as means ± SEM, of six independent experiments performed in duplicate. Using the Student's t test, P < 0.001 for all conditions discussed as significant in the text. (a) Effects of PKC activators and inhibitors on RPE-J particle binding. Longer preincubation time increased the effects of all PKC inhibitors used, suggesting that immediately before particle addition, RPE vβ5 is already activated. Mean binding index of controls (100%) after 2 h of particle challenge was 5.39 ± 0.9 regardless of preincubation time. (b) Effect of PKC activators and inhibitors on macrophage particle binding. J774 or rat primary macrophages were preincubated for 30 min or 3 h in growth medium with pharmacological reagents or solvent, before OS challenge for 30 min. Similar results were obtained incubating with apoptotic cells, which competed with OS for binding to PMA-activated macrophages (data not shown). Binding index of controls (100%) was 4.2 ± 0.7. (c) Activation of vβ5-mediated particle binding in NRK-F49 fibroblasts induced by PMA treatment. Cells were preincubated with reagents for 30 min before challenge with OS for 2 h, at which time point numbers of internalized apoptotic cells or OS were insignificant. Mean binding index of control fibroblasts was 1.36 ± 0.2, and of PMA-treated cells was 3.4 ± 0.5.

To determine if the stimulated vβ5 integrin–mediated particle binding in macrophages exhibited typical features of OS binding by RPE cells, we compared the temperature sensitivity and kinetics of particle binding and internalization by J774 cells, in the presence or absence of PMA. Total (bound plus internalized) and internalized particles were measured to calculate binding and internalization indices plotted in Fig. 7. Onset of binding of apoptotic cells or OS to control and PMA-stimulated cells occurred with the same rapid kinetics typical of macrophages (Fig. 7 a, solid lines). When challenged with particles for 30 min or longer, both control and PMA-stimulated cells increasingly internalized bound material (Fig. 7 a, dotted lines), resulting in a decrease in particles detected bound to the cell surface. J774 cells, which were equilibrated to 18°C before particle challenge, also bound both particles with the same fast kinetics regardless of whether they had been preincubated with PMA or with solvent alone, albeit slightly delayed regarding the 37°C time course (Fig. 7 b, solid lines). Under these conditions, <10% of total particles were internalized regardless of PMA treatment and time of incubation (Fig. 7 b, dotted lines). Finally, incubation at 14°C or on ice during particle challenge abolished particle binding with or without PMA treatment (Fig. 7c and Fig. d), while allowing normal binding of complement-opsonized zymosan via macrophage Mβ2, sensitive to M-blocking antibody 5C6 (data not shown). These experiments demonstrate that apoptotic cell or OS binding by macrophages via vβ3 or vβ3 plus vβ5 integrin shows the same temperature requirement as particle binding via vβ5 to RPE cells. On the other hand, PMA-stimulated macrophages, which possess an activated vβ5 integrin, bind apoptotic cells or OS with the same rapid kinetics as control macrophages, which do not use vβ5, indicating that RPE particle binding does not occur slowly due to an intrinsic property of vβ5.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Figure 7 Temperature sensitivity of apoptotic cell or OS binding and internalization by macrophages. Particle binding by macrophages, which use both vβ3 and vβ5, retains the fast macrophage binding kinetics and onset of binding. J774 cells were challenged with apoptotic cells or OS after 20 min preincubation with PMA () or solvent alone (•) and preequilibration to different temperatures for 20 min. To establish binding indices for all temperatures, internalized particle counts were subtracted from counts representing bound plus internalized particles. Binding indices are plotted in solid lines for four temperatures: (a) incubation at 37°C, (b) 18°C, (c) 14°C, and on ice (at 0°C) (d). At both 37°C and 18°C, binding by PMA-treated cells exceeded that by control cells by approximately the same margin at all times. Thus, the onset of vβ5-mediated binding by macrophages was fast and there was no lag phase as characteristic for vβ5 binding by RPE. Reequilibration of cells to permissive temperatures after 60 min on ice restored their binding activity, and led to increased binding in the presence of PMA (data not shown), excluding nonspecific cell damage. Internalization indices are plotted in dotted lines. Internalization occurred regardless of treatment at 37°C (a). Cells at 18°C bound but did not internalize particles (b), but internalized prebound particles within 45 min after being shifted to 37°C (data not shown). Complement-opsonized zymosan binding occurred efficiently at all temperatures (data not shown). Values shown represent means ± SEM (n = 3) of results obtained with OS. Identical results were obtained challenging macrophages with apoptotic cells.

PKC-activated vβ5 Is Linked to the Actin Cytoskeleton.

View larger version (56K): [in this window] [in a new window] [Download PPT slide]   Figure 9 PKC activation or inhibition affects the cytoskeletal stabilization of vβ5 integrin receptors in RPE and in macrophages. (a and b) β5 but not β3 resistance to nonionic detergent extraction correlated with PKC activation in RPE and macrophages. Pretreatment conditions were as in Fig. 8. G+P represents treatment with Gö6976 for 15 min, followed by the addition of PMA to this inhibitor medium for 30 min. After pretreatment, cells were separated in detergent-soluble and insoluble fractions as described in Materials and Methods. Protein concentrations in the fractions revealed that 35% total cellular protein was present in the soluble fraction, and 65% in the insoluble fraction. This distribution was the same comparing RPE and macrophages and did not change after treatment with PKC activators or inhibitors. Integrin distribution in subcellular fractions were compared by Western blotting with β5 polyclonal antibody (upper lanes) or β3 antibody 26 (lower lanes) after nonreducing SDS-PAGE. Lanes with odd numbers are soluble fractions (labeled s), and lanes with even numbers are the corresponding insoluble fractions (labeled i), of cell types and treatment as indicated (see legend to Fig. 8). Actin microfilament disruption with 10 µM Cyt D completely solubilized β3 and β5 (a and b, lanes 7 and 8) and prevented PMA-induced recruitment of β5 into insoluble fractions (a and b, lanes 9 and 10). Shown here is one representative experiment out of a total of five experiments. Corresponding soluble and insoluble fractions were separated in duplicate on the same gels and blotted for β5 or β3. The appearance of unidentified secondary bands in some β5 blots was variable and did not correlate with pharmacological treatment. (c) PKC activation or inhibition regulated extractability of vβ5 heterodimeric receptors. Metabolically labeled cells were pretreated and separated into soluble (lanes 1, 2, 5, 7, 8, and 11, labeled s) and insoluble fractions (lanes 3, 4, 6, 9, 10, and 12, labeled i). From each fraction, immunoprecipitates with nonimmune mouse IgG (lanes 1, 3, 7, and 9) or P1F6 antibody (all other lanes) were analyzed by nonreducing SDS-PAGE and fluorography. Note that J774 immunoprecipitates with P1F6 after separation of cells into soluble and insoluble fractions exhibited lower backgrounds than immunoprecipitates from total cell lysates (see Fig. 8 a, lanes 5 and 6), possibly due to the dilution of proteins that bound nonspecifically to P1F6 immune complexes. Quantification values given in the text represent means of four experiments, of which one representative experiment is shown.

View larger version (42K): [in this window] [in a new window] [Download PPT slide]   Figure 10 vβ5 integrin, but not vβ3 integrin, requires actin microfilaments for apoptotic cell or OS binding. J774 macrophages were preincubated with or without 10 µM Cyt D for 5 min, then PMA or solvent was added for an additional 25 min. Cells were equilibrated to 18°C for 20 min and challenged with FITC-OS or apoptotic cells for 45 min. At this time point, 18°C binding was similar in amount to binding at 37°C (see Fig. 7). Binding by cells treated with DMSO or PMA alone reproduced the integrin inhibition pattern established for 30-min challenge at 37°C (see Fig. 6 b). Both integrins bound particles independently, as dual inhibition had an additive effect on particle binding by PMA-treated macrophages (cross-hatched bars). Cyt D reduced the particle binding index of PMA-activated samples to that of vehicle controls and abolished its sensitivity to vβ5 inhibition. Binding indices are means ± SEM (n = 3) of results obtained with OS. Similar results were obtained challenging macrophages with apoptotic cells.

	Discussion

Top Abstract Materials and Methods Results Discussion References

Recognition Mechanisms of RPE Cells and Monocyte Macrophages Do Not Distinguish between Surface Signals of Photoreceptor Outer Segments and Signals Exposed during Apoptosis.
Although RPE cells lack inflammatory phagocytic mechanisms mediated in immune cells by Fc receptors, complement receptors, and glycan receptors, they possess a highly efficient clearance pathway for OS. The RPE phagocytic mechanism for OS and apoptotic cells has a capacity similar to the monocyte macrophage phagocytic mechanism. Our results confirm and extend earlier reports that described the extraordinary specificity of the RPE recognition machinery towards OS 20 and of the machinery used by noninflammatory macrophages for apoptotic over normal cells 4.OS and apoptotic cells quantitatively compete for both monocyte macrophage and RPE binding, demonstrating that the binding receptors used by either cell type to initiate phagocytosis of these types of particles do not discriminate between them. They also suggest that at least some of the local surface changes elicited by the daily photoreceptor outer segment renewal program are functionally equivalent to the recognition signals exposed during apoptosis.

RPE Cells Use vβ5 Integrin, Whereas Macrophages Use vβ3 Integrin to Bind Both OS and Apoptotic Cells.
Surprisingly, rat primary and murine J774 macrophages used vβ3 integrin to bind OS, even though they expressed abundant vβ5 integrin at the cell surface. The partial reduction in particle binding observed when using vβ3 inhibitory antibodies and RGD peptides on macrophages confirms earlier reports that they also use nonintegrin binding receptors for apoptotic cells or OS, presumably in parallel mechanisms (for a review, see reference 11). In contrast, RPE cells likely use vβ5 integrin as primary binding receptor for OS, as vβ5-inhibiting antibodies abolish 85% of particle binding. RPE cells bound apoptotic cells as efficiently as OS via an vβ5-dependent pathway, even though they express equal levels of β3 integrins as macrophages. Since β3 integrins in RPE cells are polarized at the basolateral surface of the RPE, their availability for particle binding may be limited. Although we expect this hypothesis to be difficult to test experimentally in epithelial cells, macrophages do not share similar permeability barriers between plasma membrane domains. Indeed, on appropriate immobilized substrates or specific antibodies, β3 integrins were efficiently trapped at macrophage attachment sites, leaving vβ5 receptors exposed at the particle contact surface of the phagocytes. In spite of their favorable exposure, vβ5 receptors failed to mediate apoptotic cell or OS binding. These experiments clearly demonstrate that neither the ligand (OS versus apoptotic cells), the exclusive expression of a given integrin receptor (vβ3 versus vβ5), nor its localization at the site of particle contact is sufficient to generate the characteristic receptor selectivity of RPE and macrophage particle recognition.

The Selection of a Particular Integrin Receptor by a Given Phagocyte Is Determined by the Activation of Specific Signaling Pathways.
Our results show that specific cellular context determines which integrin receptor, vβ3 or vβ5, will be activated for recognition and binding of OS and apoptotic cells. Pharmacological studies revealed that the PKC signaling pathway plays a key role in the activation of vβ5 for particle binding. Interestingly, RPE cells possess a maximally activated vβ5 integrin–gated binding mechanism that can be blocked by PKC inhibitors but cannot be enhanced by PKC stimulators. In contrast, monocyte macrophages constitutively express a functionally equivalent binding mechanism insensitive to PKC, in which vβ3 plays an important role, but activate a dormant vβ5 binding mechanism upon PKC stimulation that acts in parallel with their vβ3 pathway. Although there is no evidence to date supporting a functional role for the novel vβ5-mediated particle recognition mechanism in phagocytosis by monocyte macrophages, it is likely that this route may be important in vivo. Indeed, an active vβ5-dependent phagocytic recognition mechanism for apoptotic cells was recently described in immature dendritic cells 29, and our results predict that PKC signaling may also be involved in the regulation of vβ5 binding activity in these cells. Future studies should address the molecular mechanisms underlying vβ5 integrin activation by cellular inside-out signaling mechanisms involving PKC (for a recent review, see reference 52). It is equally important to understand the unavailability of the abundant vβ3 integrin receptors in the RPE, which, as mentioned above, may be related to their subcellular distribution, and the vβ3 activation in macrophages.

Temperature Sensitivity (18°C) May Be an Intrinsic Property of Recognition Receptors for Apoptotic Cells and OS, Whereas the Kinetics and the Restoration of v Integrin–mediated Particle Binding Are Determined by Cellular Context.
Particle binding involving both vβ3 and vβ5 integrins share a requirement of temperatures of at least 18°C, which may be a characteristic property of receptor mechanisms recognizing apoptotic cells and OS. Importantly, our results show that the activation of vβ5-mediated particle recognition in macrophages by PKC did not result in slower binding or a lag phase after particle challenge, as would have been expected from the very slow kinetics of this process in RPE cells. Furthermore, RPE cells retain their vβ5 integrin–dependent activity even after successive phagocytic challenges in vivo, and in vitro (Finnemann, S.C., unpublished data). In contrast, macrophages lose their vβ3 integrin–dependent phagocytic recognition mechanism upon an initial particle challenge 2. Thus, RPE cells are permanent noninflammatory phagocytes whose clearance mechanism exhibits unique binding characteristics that cannot solely be attributed to intrinsic properties of their primary recognition receptor, vβ5 integrin.

PKC Activation or Inhibition Controls vβ5 Integrin Function as Binding Receptor for Apoptotic Cells or OS by Regulating Its Interaction with Cytoskeletal Elements: vβ5 but Not vβ3 Integrin–mediated Particle Binding Requires Intact Actin Microfilaments.
Our findings agree well with earlier investigations which demonstrated that vβ5 but not vβ3 integrin function is sensitive to modulation by PKC signaling 33495053. However, this is the first report to demonstrate a specific effect of PKC on the association of vβ5 itself with the actin cytoskeleton. Our biochemical assays did not determine whether PKC induced a direct or indirect association of vβ5 with actin. Lewis et al. 50 have shown a correlation between PMA activation of vβ5 and a redistribution or activation of several cytoskeleton-associated molecules, e.g., -actinin, tensin, and vinculin. These actin-associated proteins may mediate both mechanical as well as signaling roles of integrin receptors. Cytoskeleton-associated proteins and their roles in apoptotic cell or OS binding and subsequent noninflammatory phagocytosis have yet to be studied in detail. Before inflammatory phagocytosis, particle binding to surface Fc receptors or Mβ2 integrins appears to be independent of the integrity of the actin cytoskeleton 5455. The hypothesis that binding of the same particle to a different integrin binding receptor, in response to phagocyte- rather than ligand-specified signals, induces different outside-in signaling effects, which ultimately determines properties of later phases of phagocytic clearance, is intriguing and will be the subject of further studies. The unique susceptibility of RPE phagocytes to genetic manipulation by viral or plasmid expression vectors in vitro and in vivo 4256 will certainly be helpful in future studies designed to characterize the roles of specific actin binding and regulatory proteins in apoptotic cell or OS binding and phagocytosis.