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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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αvβ5 integrin, but not αvβ3 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 αvβ5 inhibition. Binding indices are means ± SEM (n = 3) of results obtained with OS. Similar results were obtained challenging macrophages with apoptotic cells.
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Figure 10: αvβ5 integrin, but not αvβ3 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 αvβ5 inhibition. Binding indices are means ± SEM (n = 3) of results obtained with OS. Similar results were obtained challenging macrophages with apoptotic cells.

Mentions: To directly test the possibility that an intact actin cytoskeleton might be required for αvβ5-mediated particle binding, we challenged J774 macrophages with OS in the presence or absence of PMA and Cyt D. Fig. 10 shows particle binding determined at 18°C to minimize phagocytosis of bound particles. OS bound efficiently to control macrophages even in the presence of Cyt D (Fig. 10), in both cases in an β3 integrin–sensitive, αvβ5-independent manner (average β3 antibody inhibition 59% in control cells, 42% in Cyt D–treated cells). Increased OS binding in the presence of PMA exhibited the dual β3- and αvβ5-dependent mechanism (51% inhibited by β3 antibody, 39% inhibited by αvβ5 antibody), and blocking of both integrins with a combination of antibodies resulted in maximal inhibition of OS binding (75%). Strikingly, the addition of Cyt D decreased OS binding to PMA-treated cells by 40%, abolishing the increase induced by PMA. Addition of αvβ5-inhibiting antibody to these cells had no effect, whereas OS binding remained sensitive to β3-inhibiting antibodies (52% inhibition). Similar effects on PMA-induced particle binding only were observed when 1 μM latrunculin B, a different actin-disruptive drug, was substituted for Cyt D (data not shown). When we tested RPE-J particle binding in the presence of 5–20 μM Cyt D, we also found their binding activity reduced. However, the morphology of the epithelium was dramatically altered after 2 h of actin disruption. These experiments demonstrate that, in macrophages and RPE-J cells, binding of apoptotic cells or OS to αvβ5 integrin receptors but not to αvβ3 required intact actin microfilaments.


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)

αvβ5 integrin, but not αvβ3 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 αvβ5 inhibition. Binding indices are means ± SEM (n = 3) of results obtained with OS. Similar results were obtained challenging macrophages with apoptotic cells.
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Figure 10: αvβ5 integrin, but not αvβ3 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 αvβ5 inhibition. Binding indices are means ± SEM (n = 3) of results obtained with OS. Similar results were obtained challenging macrophages with apoptotic cells.
Mentions: To directly test the possibility that an intact actin cytoskeleton might be required for αvβ5-mediated particle binding, we challenged J774 macrophages with OS in the presence or absence of PMA and Cyt D. Fig. 10 shows particle binding determined at 18°C to minimize phagocytosis of bound particles. OS bound efficiently to control macrophages even in the presence of Cyt D (Fig. 10), in both cases in an β3 integrin–sensitive, αvβ5-independent manner (average β3 antibody inhibition 59% in control cells, 42% in Cyt D–treated cells). Increased OS binding in the presence of PMA exhibited the dual β3- and αvβ5-dependent mechanism (51% inhibited by β3 antibody, 39% inhibited by αvβ5 antibody), and blocking of both integrins with a combination of antibodies resulted in maximal inhibition of OS binding (75%). Strikingly, the addition of Cyt D decreased OS binding to PMA-treated cells by 40%, abolishing the increase induced by PMA. Addition of αvβ5-inhibiting antibody to these cells had no effect, whereas OS binding remained sensitive to β3-inhibiting antibodies (52% inhibition). Similar effects on PMA-induced particle binding only were observed when 1 μM latrunculin B, a different actin-disruptive drug, was substituted for Cyt D (data not shown). When we tested RPE-J particle binding in the presence of 5–20 μM Cyt D, we also found their binding activity reduced. However, the morphology of the epithelium was dramatically altered after 2 h of actin disruption. These experiments demonstrate that, in macrophages and RPE-J cells, binding of apoptotic cells or OS to αvβ5 integrin receptors but not to αvβ3 required intact actin microfilaments.

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
Related in: MedlinePlus