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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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Effects of stimulating and inhibiting concentrations of CD36 Ab on the kinetics of OS binding and internalization by human ARPE-19 and rat RPE-J cells. Differentiated ARPE-19 cells were challenged for different periods of time up to 5 h with FITC-OS in the presence of 50 μg/ml nonimmune mouse IgG (•), 50 μg/ml FA6–152 (▾), and 100 μg/ml FA6–152 (▪). For all time points, binding indices (A) and internalization indices (B) were determined as described for Fig. 2 and plotted against the time after OS challenge. Differentiated monolayers of rat RPE-J cells were challenged with FITC-OS as described for ARPE-19 cells in the presence of IgG fractions of preimmune serum (•) or rat CD36 antiserum (x), both at 50 μg/ml. Binding and internalization indices were determined as described for ARPE-19 cells and plotted against the time of OS challenge in (C) and (D), respectively. Values given are average OS index ± SD of three independent experiments for ARPE-19 cells and of five independent experiments for RPE-J cells. n.i., nonimmune.
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fig3: Effects of stimulating and inhibiting concentrations of CD36 Ab on the kinetics of OS binding and internalization by human ARPE-19 and rat RPE-J cells. Differentiated ARPE-19 cells were challenged for different periods of time up to 5 h with FITC-OS in the presence of 50 μg/ml nonimmune mouse IgG (•), 50 μg/ml FA6–152 (▾), and 100 μg/ml FA6–152 (▪). For all time points, binding indices (A) and internalization indices (B) were determined as described for Fig. 2 and plotted against the time after OS challenge. Differentiated monolayers of rat RPE-J cells were challenged with FITC-OS as described for ARPE-19 cells in the presence of IgG fractions of preimmune serum (•) or rat CD36 antiserum (x), both at 50 μg/ml. Binding and internalization indices were determined as described for ARPE-19 cells and plotted against the time of OS challenge in (C) and (D), respectively. Values given are average OS index ± SD of three independent experiments for ARPE-19 cells and of five independent experiments for RPE-J cells. n.i., nonimmune.

Mentions: We next investigated the effect of FA6–152 on the kinetics of OS binding and internalization by ARPE-19 cells. Fig. 3 A shows that, at early time points up to ∼2 h, during which RPE cells bind but do not internalize OS (6), all cells bound OS at the same rate regardless of Ab treatment. After 2 h, the time of onset of internalization by cultured RPE, cells treated with the inhibitory concentration of FA6–152 (100 μg/ml) had identical numbers of surface-bound OS as control cells. In contrast, cells treated with the stimulatory concentration of FA6–152 (50 μg/ml) had 15 to 35% fewer surface-bound OS than control cells. The decrease in cell surface-bound OS was probably due to an increased rate of internalization. Indeed, FA6–152 had opposite effects on the kinetics of OS internalization, confirming that the stimulating concentration of Ab accelerated engulfment beginning at 2 h while the higher concentration strongly inhibited engulfment (Fig. 3 B).


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

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

Effects of stimulating and inhibiting concentrations of CD36 Ab on the kinetics of OS binding and internalization by human ARPE-19 and rat RPE-J cells. Differentiated ARPE-19 cells were challenged for different periods of time up to 5 h with FITC-OS in the presence of 50 μg/ml nonimmune mouse IgG (•), 50 μg/ml FA6–152 (▾), and 100 μg/ml FA6–152 (▪). For all time points, binding indices (A) and internalization indices (B) were determined as described for Fig. 2 and plotted against the time after OS challenge. Differentiated monolayers of rat RPE-J cells were challenged with FITC-OS as described for ARPE-19 cells in the presence of IgG fractions of preimmune serum (•) or rat CD36 antiserum (x), both at 50 μg/ml. Binding and internalization indices were determined as described for ARPE-19 cells and plotted against the time of OS challenge in (C) and (D), respectively. Values given are average OS index ± SD of three independent experiments for ARPE-19 cells and of five independent experiments for RPE-J cells. n.i., nonimmune.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195987&req=5

fig3: Effects of stimulating and inhibiting concentrations of CD36 Ab on the kinetics of OS binding and internalization by human ARPE-19 and rat RPE-J cells. Differentiated ARPE-19 cells were challenged for different periods of time up to 5 h with FITC-OS in the presence of 50 μg/ml nonimmune mouse IgG (•), 50 μg/ml FA6–152 (▾), and 100 μg/ml FA6–152 (▪). For all time points, binding indices (A) and internalization indices (B) were determined as described for Fig. 2 and plotted against the time after OS challenge. Differentiated monolayers of rat RPE-J cells were challenged with FITC-OS as described for ARPE-19 cells in the presence of IgG fractions of preimmune serum (•) or rat CD36 antiserum (x), both at 50 μg/ml. Binding and internalization indices were determined as described for ARPE-19 cells and plotted against the time of OS challenge in (C) and (D), respectively. Values given are average OS index ± SD of three independent experiments for ARPE-19 cells and of five independent experiments for RPE-J cells. n.i., nonimmune.
Mentions: We next investigated the effect of FA6–152 on the kinetics of OS binding and internalization by ARPE-19 cells. Fig. 3 A shows that, at early time points up to ∼2 h, during which RPE cells bind but do not internalize OS (6), all cells bound OS at the same rate regardless of Ab treatment. After 2 h, the time of onset of internalization by cultured RPE, cells treated with the inhibitory concentration of FA6–152 (100 μg/ml) had identical numbers of surface-bound OS as control cells. In contrast, cells treated with the stimulatory concentration of FA6–152 (50 μg/ml) had 15 to 35% fewer surface-bound OS than control cells. The decrease in cell surface-bound OS was probably due to an increased rate of internalization. Indeed, FA6–152 had opposite effects on the kinetics of OS internalization, confirming that the stimulating concentration of Ab accelerated engulfment beginning at 2 h while the higher concentration strongly inhibited engulfment (Fig. 3 B).

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