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Differential roles of CD36 and alphavbeta5 integrin in photoreceptor phagocytosis by the retinal pigment epithelium.

Finnemann SC, Silverstein RL - J. Exp. Med. (2001)

Bottom Line: Early, CD36 antibodies had no effect on OS binding or internalization.Strikingly, antibodies were effective even if added to OS already bound by RPE. alphavbeta5 blocking antibody reduced OS binding equally well in the presence of CD36 antibodies but CD36 antibodies accelerated internalization of remaining bound OS.Our results demonstrate that CD36 ligation is necessary and sufficient to activate the OS internalization mechanism of RPE.

View Article: PubMed Central - PubMed

Affiliation: Margaret M. Dyson Vision Research Institute, Department of Ophthalmology and Department of Cell Biology, Weill Medical College of Cornell University, New York, NY 10021, USA. sfinne@med.cornell.edu

ABSTRACT
Retinal pigment epithelial (RPE) cells employ alphavbeta5 integrin and CD36 receptors to phagocytose photoreceptor outer segment fragments (OS). We explored special properties of RPE phagocytosis to identify the contribution of CD36 to RPE phagocytosis measuring effects of CD36 antibodies on OS binding and internalization kinetics. Early, CD36 antibodies had no effect on OS binding or internalization. Both control and CD36 antibody treated RPE initiated internalization approximately 2 hours after OS challenge. Later, bivalent CD36 IgG accelerated OS engulfment while monovalent Fab fragments inhibited engulfment. Cross-linking Fab fragments restored the accelerating activity of intact IgG. Strikingly, antibodies were effective even if added to OS already bound by RPE. alphavbeta5 blocking antibody reduced OS binding equally well in the presence of CD36 antibodies but CD36 antibodies accelerated internalization of remaining bound OS. Furthermore, CD36 ligation at either apical or basal RPE surface partially substituted for soluble factors that are required for internalization but not for binding of OS at the RPE apical surface. Our results demonstrate that CD36 ligation is necessary and sufficient to activate the OS internalization mechanism of RPE. They suggest that CD36 acts as a signaling molecule in postbinding steps of RPE phagocytosis independently of the OS binding receptor alphavbeta5 integrin.

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CD36 Ab binding of CD36 at the RPE basal surface stimulates internalization of OS bound to αvβ5 at the apical surface. RPE-J cells adherent on coverslips coated with nonimmune Ab (A) or CD36 Ab (B) for 6 h were stained with murine CD36 Ab. 2 x-y sections of each field are shown, corresponding to the basal surface plane (A-1, B-1) and to a plane 6 μm above the basal surface (A-2, B-2), representing a subapical-lateral plane of an RPE-J cell. Cover glass staining caused by coating Ab was subtracted from basal fields as background. Total height of cells seeded on coated overslips was ∼8–10 μm (data not shown). While most RPE cells were in contact with neighboring cells 6 h after seeding, cells exhibited membrane protrusions (A2, B1) and had not established a polarized phenotype. Green signals shows CD36 labeling while red nuclei stain serves as reference. Scale bars: 10 μm. In RPE-J cells on control Ab, CD36 localized to both basal and subapical plasma membrane (A-1, A-2). In contrast, in cells on CD36 Ab, CD36 was prominent at basal attachment sites (B-1) and mostly absent from the subapical plane (B-2) suggesting that the CD36 Ab trapped CD36 at the glass surface. (C) During 2 h of OS challenge, RPE-J cells on control or on CD36 Ab bound FITC-OS normally, independent of the presence (+) or absence (−) of FCS. αvβ5 inhibitory Ab P1F6 at 50 μg/ml interfered with OS binding (+, αvβ5 Ab) Shown are average OS indices ± SD (n = 3). (D) During 4 h of OS challenge, cells on nonimmune Ab internalized OS in the presence (+) but not the absence (−) of serum. In contrast, cells on CD36 Ab internalized increased numbers of OS in the absence of serum (−) (averages ± SD, n = 4, P < 0.005). Soluble CD36 Ab at 100 μg/ml that reduced internalization by control cells (averages ± SD, n = 3, P < 0.01) had no effect on internalization by cells whose CD36 was trapped at the basal surface.
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fig7: CD36 Ab binding of CD36 at the RPE basal surface stimulates internalization of OS bound to αvβ5 at the apical surface. RPE-J cells adherent on coverslips coated with nonimmune Ab (A) or CD36 Ab (B) for 6 h were stained with murine CD36 Ab. 2 x-y sections of each field are shown, corresponding to the basal surface plane (A-1, B-1) and to a plane 6 μm above the basal surface (A-2, B-2), representing a subapical-lateral plane of an RPE-J cell. Cover glass staining caused by coating Ab was subtracted from basal fields as background. Total height of cells seeded on coated overslips was ∼8–10 μm (data not shown). While most RPE cells were in contact with neighboring cells 6 h after seeding, cells exhibited membrane protrusions (A2, B1) and had not established a polarized phenotype. Green signals shows CD36 labeling while red nuclei stain serves as reference. Scale bars: 10 μm. In RPE-J cells on control Ab, CD36 localized to both basal and subapical plasma membrane (A-1, A-2). In contrast, in cells on CD36 Ab, CD36 was prominent at basal attachment sites (B-1) and mostly absent from the subapical plane (B-2) suggesting that the CD36 Ab trapped CD36 at the glass surface. (C) During 2 h of OS challenge, RPE-J cells on control or on CD36 Ab bound FITC-OS normally, independent of the presence (+) or absence (−) of FCS. αvβ5 inhibitory Ab P1F6 at 50 μg/ml interfered with OS binding (+, αvβ5 Ab) Shown are average OS indices ± SD (n = 3). (D) During 4 h of OS challenge, cells on nonimmune Ab internalized OS in the presence (+) but not the absence (−) of serum. In contrast, cells on CD36 Ab internalized increased numbers of OS in the absence of serum (−) (averages ± SD, n = 4, P < 0.005). Soluble CD36 Ab at 100 μg/ml that reduced internalization by control cells (averages ± SD, n = 3, P < 0.01) had no effect on internalization by cells whose CD36 was trapped at the basal surface.

Mentions: CD36 Ligation at the Basal Surface Induces Internalization of OS Bound to αvβ5 at the Apical Surface of RPE. Our results suggesting that CD36 ligation regulated the mechanism used by RPE to internalize OS fit two different scenarios: (i) ligated CD36 may interact directly with bound OS, with αvβ5 (possibly activating αvβ5's internalization function), or with other components of the internalization machinery at the apical surface of RPE, and (ii) ligated CD36 may initiate a cytoplasmic signaling mechanism that in turn activates components of the internalization machinery of RPE. To determine whether CD36 function required direct interaction of CD36 with the apical phagocytic machinery of RPE we tested the effects of CD36 ligation at the RPE basal surface on OS phagocytosis. Initial experiments determined that RPE-J cells adhered and spread equally well and appeared of similar morphology on CD36 and on nonimmune Ab-coated coverslips (data not shown). Regardless of their substrate, RPE-J cells required a minimum of 6 h to attach and spread. At this time, at which we used the cells for experiments, immunofluorescence labeling showed that the tight junction component ZO-1 was distributed diffusely in the cytoplasm differently from the circumferential localization characteristic for ZO-1 in polarized epithelia (data not shown). This demonstrated that cells did not possess functional tight junctions and, thus, lacked a plasma membrane protein permeability barrier. On nonimmune Ab, CD36 localized diffusely to the basal surface as well as to plasma membrane facing the medium (Fig. 7, A1 and A2). On CD36 Ab, CD36 localized predominantly to the basal surface in a punctate staining pattern confirming that immobilized CD36 Ab bound and redistributed CD36 (Fig. 7, B1 and B2). RPE-J cells on different Abs, via αvβ5, bound equal numbers of OS within 2 h (Fig. 7 C). However, attachment of RPE to CD36 Ab but not to nonimmune Ab promoted OS internalization in the absence of serum (Fig. 7 D, −). Cells seeded on either immobilized Ab internalized OS in the presence of serum (Fig. 7 D, +) but, importantly, inhibitory concentrations of soluble CD36 Ab added during OS challenge reduced OS internalization by cells on nonimmune Ab but had no effect on OS internalization by cells attached to CD36 Ab (Fig. 7 D, +, CD36 Ab). These data demonstrated that the effect of CD36 ligation was independent of interaction with OS or with the phagocytic machinery of RPE at the apical surface including αvβ5 integrin. This strongly supported hypothesis (2) that CD36 acted primarily as a signaling molecule in RPE phagocytosis whose ligation activated the RPE internalization machinery indirectly.


Differential roles of CD36 and alphavbeta5 integrin in photoreceptor phagocytosis by the retinal pigment epithelium.

Finnemann SC, Silverstein RL - J. Exp. Med. (2001)

CD36 Ab binding of CD36 at the RPE basal surface stimulates internalization of OS bound to αvβ5 at the apical surface. RPE-J cells adherent on coverslips coated with nonimmune Ab (A) or CD36 Ab (B) for 6 h were stained with murine CD36 Ab. 2 x-y sections of each field are shown, corresponding to the basal surface plane (A-1, B-1) and to a plane 6 μm above the basal surface (A-2, B-2), representing a subapical-lateral plane of an RPE-J cell. Cover glass staining caused by coating Ab was subtracted from basal fields as background. Total height of cells seeded on coated overslips was ∼8–10 μm (data not shown). While most RPE cells were in contact with neighboring cells 6 h after seeding, cells exhibited membrane protrusions (A2, B1) and had not established a polarized phenotype. Green signals shows CD36 labeling while red nuclei stain serves as reference. Scale bars: 10 μm. In RPE-J cells on control Ab, CD36 localized to both basal and subapical plasma membrane (A-1, A-2). In contrast, in cells on CD36 Ab, CD36 was prominent at basal attachment sites (B-1) and mostly absent from the subapical plane (B-2) suggesting that the CD36 Ab trapped CD36 at the glass surface. (C) During 2 h of OS challenge, RPE-J cells on control or on CD36 Ab bound FITC-OS normally, independent of the presence (+) or absence (−) of FCS. αvβ5 inhibitory Ab P1F6 at 50 μg/ml interfered with OS binding (+, αvβ5 Ab) Shown are average OS indices ± SD (n = 3). (D) During 4 h of OS challenge, cells on nonimmune Ab internalized OS in the presence (+) but not the absence (−) of serum. In contrast, cells on CD36 Ab internalized increased numbers of OS in the absence of serum (−) (averages ± SD, n = 4, P < 0.005). Soluble CD36 Ab at 100 μg/ml that reduced internalization by control cells (averages ± SD, n = 3, P < 0.01) had no effect on internalization by cells whose CD36 was trapped at the basal surface.
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fig7: CD36 Ab binding of CD36 at the RPE basal surface stimulates internalization of OS bound to αvβ5 at the apical surface. RPE-J cells adherent on coverslips coated with nonimmune Ab (A) or CD36 Ab (B) for 6 h were stained with murine CD36 Ab. 2 x-y sections of each field are shown, corresponding to the basal surface plane (A-1, B-1) and to a plane 6 μm above the basal surface (A-2, B-2), representing a subapical-lateral plane of an RPE-J cell. Cover glass staining caused by coating Ab was subtracted from basal fields as background. Total height of cells seeded on coated overslips was ∼8–10 μm (data not shown). While most RPE cells were in contact with neighboring cells 6 h after seeding, cells exhibited membrane protrusions (A2, B1) and had not established a polarized phenotype. Green signals shows CD36 labeling while red nuclei stain serves as reference. Scale bars: 10 μm. In RPE-J cells on control Ab, CD36 localized to both basal and subapical plasma membrane (A-1, A-2). In contrast, in cells on CD36 Ab, CD36 was prominent at basal attachment sites (B-1) and mostly absent from the subapical plane (B-2) suggesting that the CD36 Ab trapped CD36 at the glass surface. (C) During 2 h of OS challenge, RPE-J cells on control or on CD36 Ab bound FITC-OS normally, independent of the presence (+) or absence (−) of FCS. αvβ5 inhibitory Ab P1F6 at 50 μg/ml interfered with OS binding (+, αvβ5 Ab) Shown are average OS indices ± SD (n = 3). (D) During 4 h of OS challenge, cells on nonimmune Ab internalized OS in the presence (+) but not the absence (−) of serum. In contrast, cells on CD36 Ab internalized increased numbers of OS in the absence of serum (−) (averages ± SD, n = 4, P < 0.005). Soluble CD36 Ab at 100 μg/ml that reduced internalization by control cells (averages ± SD, n = 3, P < 0.01) had no effect on internalization by cells whose CD36 was trapped at the basal surface.
Mentions: CD36 Ligation at the Basal Surface Induces Internalization of OS Bound to αvβ5 at the Apical Surface of RPE. Our results suggesting that CD36 ligation regulated the mechanism used by RPE to internalize OS fit two different scenarios: (i) ligated CD36 may interact directly with bound OS, with αvβ5 (possibly activating αvβ5's internalization function), or with other components of the internalization machinery at the apical surface of RPE, and (ii) ligated CD36 may initiate a cytoplasmic signaling mechanism that in turn activates components of the internalization machinery of RPE. To determine whether CD36 function required direct interaction of CD36 with the apical phagocytic machinery of RPE we tested the effects of CD36 ligation at the RPE basal surface on OS phagocytosis. Initial experiments determined that RPE-J cells adhered and spread equally well and appeared of similar morphology on CD36 and on nonimmune Ab-coated coverslips (data not shown). Regardless of their substrate, RPE-J cells required a minimum of 6 h to attach and spread. At this time, at which we used the cells for experiments, immunofluorescence labeling showed that the tight junction component ZO-1 was distributed diffusely in the cytoplasm differently from the circumferential localization characteristic for ZO-1 in polarized epithelia (data not shown). This demonstrated that cells did not possess functional tight junctions and, thus, lacked a plasma membrane protein permeability barrier. On nonimmune Ab, CD36 localized diffusely to the basal surface as well as to plasma membrane facing the medium (Fig. 7, A1 and A2). On CD36 Ab, CD36 localized predominantly to the basal surface in a punctate staining pattern confirming that immobilized CD36 Ab bound and redistributed CD36 (Fig. 7, B1 and B2). RPE-J cells on different Abs, via αvβ5, bound equal numbers of OS within 2 h (Fig. 7 C). However, attachment of RPE to CD36 Ab but not to nonimmune Ab promoted OS internalization in the absence of serum (Fig. 7 D, −). Cells seeded on either immobilized Ab internalized OS in the presence of serum (Fig. 7 D, +) but, importantly, inhibitory concentrations of soluble CD36 Ab added during OS challenge reduced OS internalization by cells on nonimmune Ab but had no effect on OS internalization by cells attached to CD36 Ab (Fig. 7 D, +, CD36 Ab). These data demonstrated that the effect of CD36 ligation was independent of interaction with OS or with the phagocytic machinery of RPE at the apical surface including αvβ5 integrin. This strongly supported hypothesis (2) that CD36 acted primarily as a signaling molecule in RPE phagocytosis whose ligation activated the RPE internalization machinery indirectly.

Bottom Line: Early, CD36 antibodies had no effect on OS binding or internalization.Strikingly, antibodies were effective even if added to OS already bound by RPE. alphavbeta5 blocking antibody reduced OS binding equally well in the presence of CD36 antibodies but CD36 antibodies accelerated internalization of remaining bound OS.Our results demonstrate that CD36 ligation is necessary and sufficient to activate the OS internalization mechanism of RPE.

View Article: PubMed Central - PubMed

Affiliation: Margaret M. Dyson Vision Research Institute, Department of Ophthalmology and Department of Cell Biology, Weill Medical College of Cornell University, New York, NY 10021, USA. sfinne@med.cornell.edu

ABSTRACT
Retinal pigment epithelial (RPE) cells employ alphavbeta5 integrin and CD36 receptors to phagocytose photoreceptor outer segment fragments (OS). We explored special properties of RPE phagocytosis to identify the contribution of CD36 to RPE phagocytosis measuring effects of CD36 antibodies on OS binding and internalization kinetics. Early, CD36 antibodies had no effect on OS binding or internalization. Both control and CD36 antibody treated RPE initiated internalization approximately 2 hours after OS challenge. Later, bivalent CD36 IgG accelerated OS engulfment while monovalent Fab fragments inhibited engulfment. Cross-linking Fab fragments restored the accelerating activity of intact IgG. Strikingly, antibodies were effective even if added to OS already bound by RPE. alphavbeta5 blocking antibody reduced OS binding equally well in the presence of CD36 antibodies but CD36 antibodies accelerated internalization of remaining bound OS. Furthermore, CD36 ligation at either apical or basal RPE surface partially substituted for soluble factors that are required for internalization but not for binding of OS at the RPE apical surface. Our results demonstrate that CD36 ligation is necessary and sufficient to activate the OS internalization mechanism of RPE. They suggest that CD36 acts as a signaling molecule in postbinding steps of RPE phagocytosis independently of the OS binding receptor alphavbeta5 integrin.

Show MeSH
Related in: MedlinePlus