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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 Abs activate the OS internalization function of RPE in the absence of soluble factors. Differentiated monolayers of RPE-J cells were rinsed twice with warm serum free medium before challenge with OS resuspended in serum free medium in the presence of 50 μg/ml either of nonimmune IgG (n. i.), CD36 IgG (CD36), or oxLDL for 2 h or 5 h. Bars represent averages ±SD of three independent experiments. (A) Bars in A show binding (light bars) and internalization (dark bars) of OS in the absence of serum. Binding occurred in the absence of serum regardless of the stimulation of CD36. CD36 Abs increased internalization compared with nonimmune IgG (P < 0.02). (B) Cells received OS in the absence of serum for 2 h, at which unbound OS were removed. Cells were further incubated with or without 3% FCS (labeled: >2 h FCS, + or −) and in the presence of either preimmune or CD36 IgG (labeled: >2 h CD36 IgG, − or +). B shows the internalization indices of these samples determined 5 h after initial OS challenge (3 h after the removal of unbound OS). Remarkably, CD36 Abs were sufficient to accelerate internalization of prebound OS in the absence of serum (P < 0.001). (C) OxLDL (but not LDL, data not shown) had no effect on OS binding but increased OS internalization in the presence or absence of serum, as indicated in the Figure, during 5 h of coincubation with OS (P < 0.005). Thus, this multivalent CD36 ligand mimicked the effect observed with intact Ab.
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fig6: CD36 Abs activate the OS internalization function of RPE in the absence of soluble factors. Differentiated monolayers of RPE-J cells were rinsed twice with warm serum free medium before challenge with OS resuspended in serum free medium in the presence of 50 μg/ml either of nonimmune IgG (n. i.), CD36 IgG (CD36), or oxLDL for 2 h or 5 h. Bars represent averages ±SD of three independent experiments. (A) Bars in A show binding (light bars) and internalization (dark bars) of OS in the absence of serum. Binding occurred in the absence of serum regardless of the stimulation of CD36. CD36 Abs increased internalization compared with nonimmune IgG (P < 0.02). (B) Cells received OS in the absence of serum for 2 h, at which unbound OS were removed. Cells were further incubated with or without 3% FCS (labeled: >2 h FCS, + or −) and in the presence of either preimmune or CD36 IgG (labeled: >2 h CD36 IgG, − or +). B shows the internalization indices of these samples determined 5 h after initial OS challenge (3 h after the removal of unbound OS). Remarkably, CD36 Abs were sufficient to accelerate internalization of prebound OS in the absence of serum (P < 0.001). (C) OxLDL (but not LDL, data not shown) had no effect on OS binding but increased OS internalization in the presence or absence of serum, as indicated in the Figure, during 5 h of coincubation with OS (P < 0.005). Thus, this multivalent CD36 ligand mimicked the effect observed with intact Ab.

Mentions: In the absence of serum, OS remained externally bound to the surface of control RPE-J cells during 5 h of OS challenge. Addition of CD36 Ab at the time of OS challenge increased OS internalization 2.8-fold compared with cells receiving preimmune IgG (Fig. 6 A). To study whether CD36 Ab could activate internalization of OS prebound by RPE in the absence of serum and Ab, we challenged RPE-J cells for 2 h with OS in the absence of serum, removed unbound OS, and continued the incubation in the presence or absence of CD36 Abs and of heat-inactivated serum. RPE-J cells promptly internalized bound OS when serum was replenished but not when serum was omitted (Fig. 6 B, +/− and −/−). Strikingly, cells that received CD36 Abs after OS binding largely internalized prebound OS even in the absence of serum (Fig. 6 B, −/+). Furthermore, addition of oxidized LDL (oxLDL), a multivalent ligand for CD36 (21) had the same effect as addition of CD36 Ab, increasing OS internalization in the presence of serum and activating OS internalization in the absence of serum (Fig. 6 C). Lipoprotein-deficient serum fully retained the capacity of complete serum to initiate OS internalization (data not shown). Thus, low concentrations of oxLDL present in serum were not responsible for the serum effect on OS phagocytosis. However, cross-linking of CD36 using Abs or oxLDL partially substituted for unknown serum factor/s whose presence is necessary for the initiation of OS internalization by RPE in culture.


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

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

CD36 Abs activate the OS internalization function of RPE in the absence of soluble factors. Differentiated monolayers of RPE-J cells were rinsed twice with warm serum free medium before challenge with OS resuspended in serum free medium in the presence of 50 μg/ml either of nonimmune IgG (n. i.), CD36 IgG (CD36), or oxLDL for 2 h or 5 h. Bars represent averages ±SD of three independent experiments. (A) Bars in A show binding (light bars) and internalization (dark bars) of OS in the absence of serum. Binding occurred in the absence of serum regardless of the stimulation of CD36. CD36 Abs increased internalization compared with nonimmune IgG (P < 0.02). (B) Cells received OS in the absence of serum for 2 h, at which unbound OS were removed. Cells were further incubated with or without 3% FCS (labeled: >2 h FCS, + or −) and in the presence of either preimmune or CD36 IgG (labeled: >2 h CD36 IgG, − or +). B shows the internalization indices of these samples determined 5 h after initial OS challenge (3 h after the removal of unbound OS). Remarkably, CD36 Abs were sufficient to accelerate internalization of prebound OS in the absence of serum (P < 0.001). (C) OxLDL (but not LDL, data not shown) had no effect on OS binding but increased OS internalization in the presence or absence of serum, as indicated in the Figure, during 5 h of coincubation with OS (P < 0.005). Thus, this multivalent CD36 ligand mimicked the effect observed with intact Ab.
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fig6: CD36 Abs activate the OS internalization function of RPE in the absence of soluble factors. Differentiated monolayers of RPE-J cells were rinsed twice with warm serum free medium before challenge with OS resuspended in serum free medium in the presence of 50 μg/ml either of nonimmune IgG (n. i.), CD36 IgG (CD36), or oxLDL for 2 h or 5 h. Bars represent averages ±SD of three independent experiments. (A) Bars in A show binding (light bars) and internalization (dark bars) of OS in the absence of serum. Binding occurred in the absence of serum regardless of the stimulation of CD36. CD36 Abs increased internalization compared with nonimmune IgG (P < 0.02). (B) Cells received OS in the absence of serum for 2 h, at which unbound OS were removed. Cells were further incubated with or without 3% FCS (labeled: >2 h FCS, + or −) and in the presence of either preimmune or CD36 IgG (labeled: >2 h CD36 IgG, − or +). B shows the internalization indices of these samples determined 5 h after initial OS challenge (3 h after the removal of unbound OS). Remarkably, CD36 Abs were sufficient to accelerate internalization of prebound OS in the absence of serum (P < 0.001). (C) OxLDL (but not LDL, data not shown) had no effect on OS binding but increased OS internalization in the presence or absence of serum, as indicated in the Figure, during 5 h of coincubation with OS (P < 0.005). Thus, this multivalent CD36 ligand mimicked the effect observed with intact Ab.
Mentions: In the absence of serum, OS remained externally bound to the surface of control RPE-J cells during 5 h of OS challenge. Addition of CD36 Ab at the time of OS challenge increased OS internalization 2.8-fold compared with cells receiving preimmune IgG (Fig. 6 A). To study whether CD36 Ab could activate internalization of OS prebound by RPE in the absence of serum and Ab, we challenged RPE-J cells for 2 h with OS in the absence of serum, removed unbound OS, and continued the incubation in the presence or absence of CD36 Abs and of heat-inactivated serum. RPE-J cells promptly internalized bound OS when serum was replenished but not when serum was omitted (Fig. 6 B, +/− and −/−). Strikingly, cells that received CD36 Abs after OS binding largely internalized prebound OS even in the absence of serum (Fig. 6 B, −/+). Furthermore, addition of oxidized LDL (oxLDL), a multivalent ligand for CD36 (21) had the same effect as addition of CD36 Ab, increasing OS internalization in the presence of serum and activating OS internalization in the absence of serum (Fig. 6 C). Lipoprotein-deficient serum fully retained the capacity of complete serum to initiate OS internalization (data not shown). Thus, low concentrations of oxLDL present in serum were not responsible for the serum effect on OS phagocytosis. However, cross-linking of CD36 using Abs or oxLDL partially substituted for unknown serum factor/s whose presence is necessary for the initiation of OS internalization by RPE in culture.

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