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Macrophage and retinal pigment epithelium phagocytosis: apoptotic cells and photoreceptors compete for alphavbeta3 and alphavbeta5 integrins, and protein kinase C regulates alphavbeta5 binding and cytoskeletal linkage.

Finnemann SC, Rodriguez-Boulan E - J. Exp. Med. (1999)

Bottom Line: In RPE, alphavbeta5 binding was maximally activated even before any phagocytic challenge and was reduced by PKC inhibitors.Furthermore, alphavbeta5 but not alphavbeta3 particle binding required actin microfilaments.These data constitute the first evidence that noninflammatory phagocytes actively regulate the earliest phase of phagocytic clearance, particle binding, by controlling receptor activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Margaret M. Dyson Vision Institute, New York, New York 10021, USA. sfinne@mail.med.cornell.edu

ABSTRACT
Noninflammatory monocyte macrophages use alphavbeta3 integrin to selectively bind apoptotic cells, initiating their phagocytic removal. In a related process, the retinal pigment epithelium (RPE) employs alphavbeta5 integrin to recognize spent photoreceptor outer segment particles (OS). Here, we show that apoptotic cells and OS compete for binding to these receptors, indicating that OS and apoptotic cells expose surface signals recognizable by alphavbeta3 and alphavbeta5. Particle binding to alphavbeta5 required protein kinase C (PKC) activation. In RPE, alphavbeta5 binding was maximally activated even before any phagocytic challenge and was reduced by PKC inhibitors. In macrophages, it was dormant but became activated upon PKC stimulation. PKC-activated alphavbeta5-mediated binding in macrophages differed from constitutive binding to the same integrin receptor in RPE cells in that the former followed much faster kinetics, similar to particle binding mediated by alphavbeta3. Activation of alphavbeta5 for particle binding correlated with its recruitment into a detergent-insoluble fraction, a process sensitive to pharmacological modulation of PKC in both types of phagocytes. Furthermore, alphavbeta5 but not alphavbeta3 particle binding required actin microfilaments. These data constitute the first evidence that noninflammatory phagocytes actively regulate the earliest phase of phagocytic clearance, particle binding, by controlling receptor activity.

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β3 integrin and αvβ5 integrin localize to attached and free macrophage plasma membrane. β3 integrins but not αvβ5 integrin redistribute to attachment sites in macrophages seeded on fibrinogen substrate. Immunodetection of surface integrins was performed on unpermeabilized cells with β3-specific or αvβ5 receptor–specific antibodies. Top and bottom panels shown are en face views of the apical and the attached cell surface, respectively. (a) β3 and αvβ5 integrin distribution in J774 macrophages attached to laminin-coated coverslips for 30 min. β3 and αvβ5 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.
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Figure 4: β3 integrin and αvβ5 integrin localize to attached and free macrophage plasma membrane. β3 integrins but not αvβ5 integrin redistribute to attachment sites in macrophages seeded on fibrinogen substrate. Immunodetection of surface integrins was performed on unpermeabilized cells with β3-specific or αvβ5 receptor–specific antibodies. Top and bottom panels shown are en face views of the apical and the attached cell surface, respectively. (a) β3 and αvβ5 integrin distribution in J774 macrophages attached to laminin-coated coverslips for 30 min. β3 and αvβ5 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.

Mentions: Hypothesis 2, applicable to macrophages, was that their preferred use of αvβ3 for apoptotic cell or OS binding might be based on the faster early kinetics of this pathway over αvβ5-mediated uptake (Fig. 2 a). To facilitate detection of a hidden αvβ5 particle binding activity, αvβ3 receptors were depleted from the free macrophage surface. When compared with control cells plated on laminin (which is not a substrate for either αvβ3 or αvβ5), a large fraction of αvβ3 receptors was recruited to the attached surface in macrophages plated on the specific αvβ3 substrate fibrinogen (47; compare Fig. 4, a1 and a2, with Fig. 4, b1 and b2), resulting in a reduction in particle binding of 40% after 30 min of particle challenge (Fig. 5 a), and a 44% decrease of combined binding and internalization even after 90 min (Fig. 5 b). αvβ5 receptor distribution remained unchanged on fibrinogen, as judged from the similar appearance of αvβ5 immunofluorescence signals on both attached and open cell surfaces on laminin and fibrinogen (compare Fig. 4, a3 and a4, with Fig. 4, b3 and b4). However, the remaining binding at 30 min and the combined binding plus internalization at 90 min were still reduced by β3 but not by αvβ5 function-blocking antibodies (Fig. 5). Incubation with β3 antibody in the presence of the peptide inhibitor GRGDSP did not have an additive effect (but the peptide alone reduced binding by ∼30%; data not shown, and see Fig. 2 b), indicating that αvβ3 was the only RGD-sensitive receptor, which mediates apoptotic cell or OS recognition in this system. However, ligand binding by leukocyte β2 integrins may not be inhibitable by RGD peptides 48. At the 90-min time point, at least 65% of the particles had been internalized (Fig. 5 b, black bars). Comparing total and internal particles of laminin- and fibrinogen-seeded macrophages, differences in internalized particles were less pronounced than differences in surface-bound particles (as deduced by subtracting internal from total particles), confirming that attachment to fibrinogen inhibited particle binding but not internalization (Fig. 5 b, gray and black bars). Similar results were obtained when cells were plated on β3 integrin antibodies or on vitronectin, a substrate for both αvβ3 and αvβ5 (Fig. 5). These experiments indicate that αvβ5 integrin receptors are present in macrophages but are not active in apoptotic cell or OS binding.


Macrophage and retinal pigment epithelium phagocytosis: apoptotic cells and photoreceptors compete for alphavbeta3 and alphavbeta5 integrins, and protein kinase C regulates alphavbeta5 binding and cytoskeletal linkage.

Finnemann SC, Rodriguez-Boulan E - J. Exp. Med. (1999)

β3 integrin and αvβ5 integrin localize to attached and free macrophage plasma membrane. β3 integrins but not αvβ5 integrin redistribute to attachment sites in macrophages seeded on fibrinogen substrate. Immunodetection of surface integrins was performed on unpermeabilized cells with β3-specific or αvβ5 receptor–specific antibodies. Top and bottom panels shown are en face views of the apical and the attached cell surface, respectively. (a) β3 and αvβ5 integrin distribution in J774 macrophages attached to laminin-coated coverslips for 30 min. β3 and αvβ5 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.
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Related In: Results  -  Collection

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Figure 4: β3 integrin and αvβ5 integrin localize to attached and free macrophage plasma membrane. β3 integrins but not αvβ5 integrin redistribute to attachment sites in macrophages seeded on fibrinogen substrate. Immunodetection of surface integrins was performed on unpermeabilized cells with β3-specific or αvβ5 receptor–specific antibodies. Top and bottom panels shown are en face views of the apical and the attached cell surface, respectively. (a) β3 and αvβ5 integrin distribution in J774 macrophages attached to laminin-coated coverslips for 30 min. β3 and αvβ5 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.
Mentions: Hypothesis 2, applicable to macrophages, was that their preferred use of αvβ3 for apoptotic cell or OS binding might be based on the faster early kinetics of this pathway over αvβ5-mediated uptake (Fig. 2 a). To facilitate detection of a hidden αvβ5 particle binding activity, αvβ3 receptors were depleted from the free macrophage surface. When compared with control cells plated on laminin (which is not a substrate for either αvβ3 or αvβ5), a large fraction of αvβ3 receptors was recruited to the attached surface in macrophages plated on the specific αvβ3 substrate fibrinogen (47; compare Fig. 4, a1 and a2, with Fig. 4, b1 and b2), resulting in a reduction in particle binding of 40% after 30 min of particle challenge (Fig. 5 a), and a 44% decrease of combined binding and internalization even after 90 min (Fig. 5 b). αvβ5 receptor distribution remained unchanged on fibrinogen, as judged from the similar appearance of αvβ5 immunofluorescence signals on both attached and open cell surfaces on laminin and fibrinogen (compare Fig. 4, a3 and a4, with Fig. 4, b3 and b4). However, the remaining binding at 30 min and the combined binding plus internalization at 90 min were still reduced by β3 but not by αvβ5 function-blocking antibodies (Fig. 5). Incubation with β3 antibody in the presence of the peptide inhibitor GRGDSP did not have an additive effect (but the peptide alone reduced binding by ∼30%; data not shown, and see Fig. 2 b), indicating that αvβ3 was the only RGD-sensitive receptor, which mediates apoptotic cell or OS recognition in this system. However, ligand binding by leukocyte β2 integrins may not be inhibitable by RGD peptides 48. At the 90-min time point, at least 65% of the particles had been internalized (Fig. 5 b, black bars). Comparing total and internal particles of laminin- and fibrinogen-seeded macrophages, differences in internalized particles were less pronounced than differences in surface-bound particles (as deduced by subtracting internal from total particles), confirming that attachment to fibrinogen inhibited particle binding but not internalization (Fig. 5 b, gray and black bars). Similar results were obtained when cells were plated on β3 integrin antibodies or on vitronectin, a substrate for both αvβ3 and αvβ5 (Fig. 5). These experiments indicate that αvβ5 integrin receptors are present in macrophages but are not active in apoptotic cell or OS binding.

Bottom Line: In RPE, alphavbeta5 binding was maximally activated even before any phagocytic challenge and was reduced by PKC inhibitors.Furthermore, alphavbeta5 but not alphavbeta3 particle binding required actin microfilaments.These data constitute the first evidence that noninflammatory phagocytes actively regulate the earliest phase of phagocytic clearance, particle binding, by controlling receptor activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Margaret M. Dyson Vision Institute, New York, New York 10021, USA. sfinne@mail.med.cornell.edu

ABSTRACT
Noninflammatory monocyte macrophages use alphavbeta3 integrin to selectively bind apoptotic cells, initiating their phagocytic removal. In a related process, the retinal pigment epithelium (RPE) employs alphavbeta5 integrin to recognize spent photoreceptor outer segment particles (OS). Here, we show that apoptotic cells and OS compete for binding to these receptors, indicating that OS and apoptotic cells expose surface signals recognizable by alphavbeta3 and alphavbeta5. Particle binding to alphavbeta5 required protein kinase C (PKC) activation. In RPE, alphavbeta5 binding was maximally activated even before any phagocytic challenge and was reduced by PKC inhibitors. In macrophages, it was dormant but became activated upon PKC stimulation. PKC-activated alphavbeta5-mediated binding in macrophages differed from constitutive binding to the same integrin receptor in RPE cells in that the former followed much faster kinetics, similar to particle binding mediated by alphavbeta3. Activation of alphavbeta5 for particle binding correlated with its recruitment into a detergent-insoluble fraction, a process sensitive to pharmacological modulation of PKC in both types of phagocytes. Furthermore, alphavbeta5 but not alphavbeta3 particle binding required actin microfilaments. These data constitute the first evidence that noninflammatory phagocytes actively regulate the earliest phase of phagocytic clearance, particle binding, by controlling receptor activity.

Show MeSH