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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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Addition of CD36Ab after OS binding to RPE is necessary and sufficient to increase the rate of OS internalization by RPE, independently of the OS binding receptor αvβ5. Differentiated monolayers of RPE-J cells were challenged for 2 h with FITC-OS in the presence of 50 μg/ml preimmune IgG or 50 μg/ml rat CD36 IgG. After 2 h, some samples were fixed to determine binding and internalization indices as shown in A. Striped bars, OS binding; filled bars, OS internalization; −, preimmune IgG control; +, CD36 IgG. (B) Identical samples as in A were washed with assay buffer 2 h after OS addition to remove unbound OS and further incubated in presence of either preimmune (−) or CD36 IgG (+). B shows the internalization indices of these samples determined 4 h after initial OS challenge (2 h after the removal of unbound OS). (C) We repeated the experiments shown in A and B but added αvβ5 inhibiting Ab P1F6 to all samples during the 2 h of OS challenge. P1F6 reduced OS binding at 2 h equally well in the presence of preimmune IgG, (−, dark striped bars) or of CD36 IgG (+, light striped bars). Internalization indices for both conditions were insignificant after 2 h (2 h, dark and light filled bars). Samples that were incubated with OS and P1F6 for 2 h were washed and incubated for another 2 h in the presence of preimmune (−) or CD36 IgG (+). Internalization indices that were determined after a total time of incubation of 4 h demonstrated that CD36 IgG accelerated internalization of OS prebound in the presence of P1F6 (4 h; −, preimmune IgG, +, CD36 IgG added after removal of unbound OS at 2 h). Note that the y axis scale in C is different from that in A and B. Values in A–C represent average OS indices ± SD (n = 3). (B and C). Statistical evaluation using Student's t test determined P < 0.001 for all changes compared with the appropriate control condition that were discussed as significant in the text. Note that all cells regardless of treatment completed internalization of prebound OS within 6 h after initial OS challenge (data not shown). Between the removal of unbound OS at 2 h and the termination of the experiment, the total amount of OS detected in each sample remained constant (data not shown). Thus, surface-bound OS did not dissociate from RPE cells and RPE cells did not degrade phagocytosed OS between 2 and 6 h after initial OS challenge.
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fig5: Addition of CD36Ab after OS binding to RPE is necessary and sufficient to increase the rate of OS internalization by RPE, independently of the OS binding receptor αvβ5. Differentiated monolayers of RPE-J cells were challenged for 2 h with FITC-OS in the presence of 50 μg/ml preimmune IgG or 50 μg/ml rat CD36 IgG. After 2 h, some samples were fixed to determine binding and internalization indices as shown in A. Striped bars, OS binding; filled bars, OS internalization; −, preimmune IgG control; +, CD36 IgG. (B) Identical samples as in A were washed with assay buffer 2 h after OS addition to remove unbound OS and further incubated in presence of either preimmune (−) or CD36 IgG (+). B shows the internalization indices of these samples determined 4 h after initial OS challenge (2 h after the removal of unbound OS). (C) We repeated the experiments shown in A and B but added αvβ5 inhibiting Ab P1F6 to all samples during the 2 h of OS challenge. P1F6 reduced OS binding at 2 h equally well in the presence of preimmune IgG, (−, dark striped bars) or of CD36 IgG (+, light striped bars). Internalization indices for both conditions were insignificant after 2 h (2 h, dark and light filled bars). Samples that were incubated with OS and P1F6 for 2 h were washed and incubated for another 2 h in the presence of preimmune (−) or CD36 IgG (+). Internalization indices that were determined after a total time of incubation of 4 h demonstrated that CD36 IgG accelerated internalization of OS prebound in the presence of P1F6 (4 h; −, preimmune IgG, +, CD36 IgG added after removal of unbound OS at 2 h). Note that the y axis scale in C is different from that in A and B. Values in A–C represent average OS indices ± SD (n = 3). (B and C). Statistical evaluation using Student's t test determined P < 0.001 for all changes compared with the appropriate control condition that were discussed as significant in the text. Note that all cells regardless of treatment completed internalization of prebound OS within 6 h after initial OS challenge (data not shown). Between the removal of unbound OS at 2 h and the termination of the experiment, the total amount of OS detected in each sample remained constant (data not shown). Thus, surface-bound OS did not dissociate from RPE cells and RPE cells did not degrade phagocytosed OS between 2 and 6 h after initial OS challenge.

Mentions: Interestingly, CD36 Abs affected OS internalization only at time points later than 2 h after OS challenge even though Abs were added to the cells with OS challenge. Therefore, we determined the window of time during which addition of stimulating concentrations of CD36 Ab was effective. To this end, we challenged RPE-J cells with FITC-OS for 2 h in the presence or absence of Ab. After 2 h we removed unbound OS and allowed phagocytosis to proceed in the presence or absence of Abs to determine their effect on the internalization rate of OS prebound to the RPE surface. As expected, RPE cells bound similar numbers of OS regardless of Ab treatment during the first 2 h of the experiment (Fig. 5 A, striped bars). Strikingly, addition of Ab after 2 h to cells previously unexposed to Ab was equally effective in increasing the internalization rate of surface-bound OS as the addition of Ab during both OS binding and internalization (Fig. 5 B, compare −/+ and +/+). Removal of Ab after the initial 2 h of OS binding resulted in the same internalization rate of bound OS as the absence of Ab throughout the experiment (Fig. 5 B, compare +/− and −/−). These data clearly demonstrated that the presence of stimulating concentrations of CD36 Ab during the internalization phase of OS phagocytosis was necessary and sufficient to accelerate internalization of bound OS by RPE.


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

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

Addition of CD36Ab after OS binding to RPE is necessary and sufficient to increase the rate of OS internalization by RPE, independently of the OS binding receptor αvβ5. Differentiated monolayers of RPE-J cells were challenged for 2 h with FITC-OS in the presence of 50 μg/ml preimmune IgG or 50 μg/ml rat CD36 IgG. After 2 h, some samples were fixed to determine binding and internalization indices as shown in A. Striped bars, OS binding; filled bars, OS internalization; −, preimmune IgG control; +, CD36 IgG. (B) Identical samples as in A were washed with assay buffer 2 h after OS addition to remove unbound OS and further incubated in presence of either preimmune (−) or CD36 IgG (+). B shows the internalization indices of these samples determined 4 h after initial OS challenge (2 h after the removal of unbound OS). (C) We repeated the experiments shown in A and B but added αvβ5 inhibiting Ab P1F6 to all samples during the 2 h of OS challenge. P1F6 reduced OS binding at 2 h equally well in the presence of preimmune IgG, (−, dark striped bars) or of CD36 IgG (+, light striped bars). Internalization indices for both conditions were insignificant after 2 h (2 h, dark and light filled bars). Samples that were incubated with OS and P1F6 for 2 h were washed and incubated for another 2 h in the presence of preimmune (−) or CD36 IgG (+). Internalization indices that were determined after a total time of incubation of 4 h demonstrated that CD36 IgG accelerated internalization of OS prebound in the presence of P1F6 (4 h; −, preimmune IgG, +, CD36 IgG added after removal of unbound OS at 2 h). Note that the y axis scale in C is different from that in A and B. Values in A–C represent average OS indices ± SD (n = 3). (B and C). Statistical evaluation using Student's t test determined P < 0.001 for all changes compared with the appropriate control condition that were discussed as significant in the text. Note that all cells regardless of treatment completed internalization of prebound OS within 6 h after initial OS challenge (data not shown). Between the removal of unbound OS at 2 h and the termination of the experiment, the total amount of OS detected in each sample remained constant (data not shown). Thus, surface-bound OS did not dissociate from RPE cells and RPE cells did not degrade phagocytosed OS between 2 and 6 h after initial OS challenge.
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fig5: Addition of CD36Ab after OS binding to RPE is necessary and sufficient to increase the rate of OS internalization by RPE, independently of the OS binding receptor αvβ5. Differentiated monolayers of RPE-J cells were challenged for 2 h with FITC-OS in the presence of 50 μg/ml preimmune IgG or 50 μg/ml rat CD36 IgG. After 2 h, some samples were fixed to determine binding and internalization indices as shown in A. Striped bars, OS binding; filled bars, OS internalization; −, preimmune IgG control; +, CD36 IgG. (B) Identical samples as in A were washed with assay buffer 2 h after OS addition to remove unbound OS and further incubated in presence of either preimmune (−) or CD36 IgG (+). B shows the internalization indices of these samples determined 4 h after initial OS challenge (2 h after the removal of unbound OS). (C) We repeated the experiments shown in A and B but added αvβ5 inhibiting Ab P1F6 to all samples during the 2 h of OS challenge. P1F6 reduced OS binding at 2 h equally well in the presence of preimmune IgG, (−, dark striped bars) or of CD36 IgG (+, light striped bars). Internalization indices for both conditions were insignificant after 2 h (2 h, dark and light filled bars). Samples that were incubated with OS and P1F6 for 2 h were washed and incubated for another 2 h in the presence of preimmune (−) or CD36 IgG (+). Internalization indices that were determined after a total time of incubation of 4 h demonstrated that CD36 IgG accelerated internalization of OS prebound in the presence of P1F6 (4 h; −, preimmune IgG, +, CD36 IgG added after removal of unbound OS at 2 h). Note that the y axis scale in C is different from that in A and B. Values in A–C represent average OS indices ± SD (n = 3). (B and C). Statistical evaluation using Student's t test determined P < 0.001 for all changes compared with the appropriate control condition that were discussed as significant in the text. Note that all cells regardless of treatment completed internalization of prebound OS within 6 h after initial OS challenge (data not shown). Between the removal of unbound OS at 2 h and the termination of the experiment, the total amount of OS detected in each sample remained constant (data not shown). Thus, surface-bound OS did not dissociate from RPE cells and RPE cells did not degrade phagocytosed OS between 2 and 6 h after initial OS challenge.
Mentions: Interestingly, CD36 Abs affected OS internalization only at time points later than 2 h after OS challenge even though Abs were added to the cells with OS challenge. Therefore, we determined the window of time during which addition of stimulating concentrations of CD36 Ab was effective. To this end, we challenged RPE-J cells with FITC-OS for 2 h in the presence or absence of Ab. After 2 h we removed unbound OS and allowed phagocytosis to proceed in the presence or absence of Abs to determine their effect on the internalization rate of OS prebound to the RPE surface. As expected, RPE cells bound similar numbers of OS regardless of Ab treatment during the first 2 h of the experiment (Fig. 5 A, striped bars). Strikingly, addition of Ab after 2 h to cells previously unexposed to Ab was equally effective in increasing the internalization rate of surface-bound OS as the addition of Ab during both OS binding and internalization (Fig. 5 B, compare −/+ and +/+). Removal of Ab after the initial 2 h of OS binding resulted in the same internalization rate of bound OS as the absence of Ab throughout the experiment (Fig. 5 B, compare +/− and −/−). These data clearly demonstrated that the presence of stimulating concentrations of CD36 Ab during the internalization phase of OS phagocytosis was necessary and sufficient to accelerate internalization of bound OS by RPE.

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