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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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Inhibition but not stimulation of OS internalization by monovalent Fab fractions of CD36 Abs. 5 h of challenge with FITC-OS of human (A) and rat (B) RPE was followed by fluorescence scanning to calculate binding and internalization indices as described in Fig. 2. Light bars represent the binding index, dark bars the internalization index for each condition. Values in A and B represent averages ± SD of three independent experiments. (A) The presence of Fab fractions of FA6–152 IgG at 20 and 50 μg/ml, marked 20 and 50, respectively, did not significantly alter the amount of OS bound or internalized by ARPE-19 cells. Control cells received Fab fragments of nonimmune IgG at 50 μg/ml. (B) Fab fractions of rat CD36 IgG at 50 μg/ml, marked 50, decreased the OS internalization index of rat RPE-J cells to 54% of the index of cell receiving preimmune Fab fractions (Student's t test P < 0.005). Changes in bound OS were not significant. Cross-linking of CD36 Fab at 20 and 50 μg/ml, marked 20 and 50, using goat anti–rabbit Fab IgG at 10 μg/ml reversed the effect of CD36 Fab alone. Using Student's t test, these changes were significant with P < 0.005. n.i., nonimmune.
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fig4: Inhibition but not stimulation of OS internalization by monovalent Fab fractions of CD36 Abs. 5 h of challenge with FITC-OS of human (A) and rat (B) RPE was followed by fluorescence scanning to calculate binding and internalization indices as described in Fig. 2. Light bars represent the binding index, dark bars the internalization index for each condition. Values in A and B represent averages ± SD of three independent experiments. (A) The presence of Fab fractions of FA6–152 IgG at 20 and 50 μg/ml, marked 20 and 50, respectively, did not significantly alter the amount of OS bound or internalized by ARPE-19 cells. Control cells received Fab fragments of nonimmune IgG at 50 μg/ml. (B) Fab fractions of rat CD36 IgG at 50 μg/ml, marked 50, decreased the OS internalization index of rat RPE-J cells to 54% of the index of cell receiving preimmune Fab fractions (Student's t test P < 0.005). Changes in bound OS were not significant. Cross-linking of CD36 Fab at 20 and 50 μg/ml, marked 20 and 50, using goat anti–rabbit Fab IgG at 10 μg/ml reversed the effect of CD36 Fab alone. Using Student's t test, these changes were significant with P < 0.005. n.i., nonimmune.

Mentions: The concentration dependence of their effects suggested that the Abs may act by cross-linking CD36 receptors at the RPE surface. Therefore, we tested the effects of monovalent Fab fragments of FA6–152 or of anti–rat CD36 IgG on OS internalization. Unlike the intact Ab, Fab fragments did not accelerate OS internalization (Fig. 4) . However, addition of secondary Abs to cross-link rat CD36 Fab fragments partially restored the stimulatory effect of CD36 Abs increasing the amount of internalized OS by 2.6-fold as compared with cells treated with CD36 Fab fragments alone or by 34% compared with control cells treated with preimmune Fab fragments plus cross-linker (Fig. 4 B). In the absence of cross-linker, anti–rat CD36 Fab fragments reduced OS internalization by 46% (Fig. 4 B). Addition of cross-linking secondary Abs to FA6–152 Fab fragments did not rescue the Ab's effect on OS uptake suggesting that the generation of Fab fragments may have altered antigen recognition by the Ab. Taken together, these results suggested that stimulation but not inhibition of the RPE's internalization rate required bivalent CD36 Abs.


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

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

Inhibition but not stimulation of OS internalization by monovalent Fab fractions of CD36 Abs. 5 h of challenge with FITC-OS of human (A) and rat (B) RPE was followed by fluorescence scanning to calculate binding and internalization indices as described in Fig. 2. Light bars represent the binding index, dark bars the internalization index for each condition. Values in A and B represent averages ± SD of three independent experiments. (A) The presence of Fab fractions of FA6–152 IgG at 20 and 50 μg/ml, marked 20 and 50, respectively, did not significantly alter the amount of OS bound or internalized by ARPE-19 cells. Control cells received Fab fragments of nonimmune IgG at 50 μg/ml. (B) Fab fractions of rat CD36 IgG at 50 μg/ml, marked 50, decreased the OS internalization index of rat RPE-J cells to 54% of the index of cell receiving preimmune Fab fractions (Student's t test P < 0.005). Changes in bound OS were not significant. Cross-linking of CD36 Fab at 20 and 50 μg/ml, marked 20 and 50, using goat anti–rabbit Fab IgG at 10 μg/ml reversed the effect of CD36 Fab alone. Using Student's t test, these changes were significant with P < 0.005. n.i., nonimmune.
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fig4: Inhibition but not stimulation of OS internalization by monovalent Fab fractions of CD36 Abs. 5 h of challenge with FITC-OS of human (A) and rat (B) RPE was followed by fluorescence scanning to calculate binding and internalization indices as described in Fig. 2. Light bars represent the binding index, dark bars the internalization index for each condition. Values in A and B represent averages ± SD of three independent experiments. (A) The presence of Fab fractions of FA6–152 IgG at 20 and 50 μg/ml, marked 20 and 50, respectively, did not significantly alter the amount of OS bound or internalized by ARPE-19 cells. Control cells received Fab fragments of nonimmune IgG at 50 μg/ml. (B) Fab fractions of rat CD36 IgG at 50 μg/ml, marked 50, decreased the OS internalization index of rat RPE-J cells to 54% of the index of cell receiving preimmune Fab fractions (Student's t test P < 0.005). Changes in bound OS were not significant. Cross-linking of CD36 Fab at 20 and 50 μg/ml, marked 20 and 50, using goat anti–rabbit Fab IgG at 10 μg/ml reversed the effect of CD36 Fab alone. Using Student's t test, these changes were significant with P < 0.005. n.i., nonimmune.
Mentions: The concentration dependence of their effects suggested that the Abs may act by cross-linking CD36 receptors at the RPE surface. Therefore, we tested the effects of monovalent Fab fragments of FA6–152 or of anti–rat CD36 IgG on OS internalization. Unlike the intact Ab, Fab fragments did not accelerate OS internalization (Fig. 4) . However, addition of secondary Abs to cross-link rat CD36 Fab fragments partially restored the stimulatory effect of CD36 Abs increasing the amount of internalized OS by 2.6-fold as compared with cells treated with CD36 Fab fragments alone or by 34% compared with control cells treated with preimmune Fab fragments plus cross-linker (Fig. 4 B). In the absence of cross-linker, anti–rat CD36 Fab fragments reduced OS internalization by 46% (Fig. 4 B). Addition of cross-linking secondary Abs to FA6–152 Fab fragments did not rescue the Ab's effect on OS uptake suggesting that the generation of Fab fragments may have altered antigen recognition by the Ab. Taken together, these results suggested that stimulation but not inhibition of the RPE's internalization rate required bivalent CD36 Abs.

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