Limits...
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.

Show MeSH

Related in: MedlinePlus

CD36 protein expression by stable human and rat-derived RPE derived cell lines. (A) Comparative immunoblotting of detergent extracts containing 20 μg total cellular protein followed by detection with rat CD36 antiserum shows that rat RPE-J cell lysates (RPE-J) contain ∼80% of the CD36 protein detected in lysates of RPE prepared from adult rat eyes (adult). Bands are specific for CD36 immunization, since they are absent in Western blot analysis probed with preimmune IgG. (B) Comparative immunoblotting of detergent extracts containing 100 μg total cellular protein followed by detection with human CD36 antiserum reveals the presence of CD36 protein of the expected molecular size of ∼88 kD in human ARPE-19 cells (ARPE-19) but not in human Bowes melanoma cells (Bowes), which are known not to express CD36 (reference 11). (C) Immunofluorescence labeling of paraformaldehyde fixed ARPE-19 cells with human CD36 mAb FA6–152 shows CD36 signals localizing to the RPE cell surface. (D) FA6–152 signals are specific since they are absent in parallel samples stained with nonimmune IgG.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2195987&req=5

fig1: CD36 protein expression by stable human and rat-derived RPE derived cell lines. (A) Comparative immunoblotting of detergent extracts containing 20 μg total cellular protein followed by detection with rat CD36 antiserum shows that rat RPE-J cell lysates (RPE-J) contain ∼80% of the CD36 protein detected in lysates of RPE prepared from adult rat eyes (adult). Bands are specific for CD36 immunization, since they are absent in Western blot analysis probed with preimmune IgG. (B) Comparative immunoblotting of detergent extracts containing 100 μg total cellular protein followed by detection with human CD36 antiserum reveals the presence of CD36 protein of the expected molecular size of ∼88 kD in human ARPE-19 cells (ARPE-19) but not in human Bowes melanoma cells (Bowes), which are known not to express CD36 (reference 11). (C) Immunofluorescence labeling of paraformaldehyde fixed ARPE-19 cells with human CD36 mAb FA6–152 shows CD36 signals localizing to the RPE cell surface. (D) FA6–152 signals are specific since they are absent in parallel samples stained with nonimmune IgG.

Mentions: We previously established that CD36 participates in the phagocytic mechanism of rat primary RPE, and that CD36 transfection renders human melanoma cells phagocytic for OS (9). As with primary RPE, stable RPE cell lines derived from rat (RPE-J) or human (ARPE-19) RPE phagocytose OS and use αvβ5 integrin to recognize OS (6). To determine whether these RPE cell lines also serve as a model system to study the role of CD36 in OS phagocytosis, we assessed CD36 expression by immunoblotting and immunofluorescence microscopy. As shown in Fig. 1 , both RPE-J and ARPE-19 cells expressed CD36; the amount of CD36 protein in RPE-J cells, as determined by immunoblot, was ∼80% of that in adult rat RPE (Fig. 1 A). Immunofluorescence staining showed that CD36 localized to the plasma membrane of confluent ARPE-19 cells (Fig. 1 C). Parallel samples stained with nonimmune mouse IgG did not fluoresce (Fig. 1 D).


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

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

CD36 protein expression by stable human and rat-derived RPE derived cell lines. (A) Comparative immunoblotting of detergent extracts containing 20 μg total cellular protein followed by detection with rat CD36 antiserum shows that rat RPE-J cell lysates (RPE-J) contain ∼80% of the CD36 protein detected in lysates of RPE prepared from adult rat eyes (adult). Bands are specific for CD36 immunization, since they are absent in Western blot analysis probed with preimmune IgG. (B) Comparative immunoblotting of detergent extracts containing 100 μg total cellular protein followed by detection with human CD36 antiserum reveals the presence of CD36 protein of the expected molecular size of ∼88 kD in human ARPE-19 cells (ARPE-19) but not in human Bowes melanoma cells (Bowes), which are known not to express CD36 (reference 11). (C) Immunofluorescence labeling of paraformaldehyde fixed ARPE-19 cells with human CD36 mAb FA6–152 shows CD36 signals localizing to the RPE cell surface. (D) FA6–152 signals are specific since they are absent in parallel samples stained with nonimmune IgG.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195987&req=5

fig1: CD36 protein expression by stable human and rat-derived RPE derived cell lines. (A) Comparative immunoblotting of detergent extracts containing 20 μg total cellular protein followed by detection with rat CD36 antiserum shows that rat RPE-J cell lysates (RPE-J) contain ∼80% of the CD36 protein detected in lysates of RPE prepared from adult rat eyes (adult). Bands are specific for CD36 immunization, since they are absent in Western blot analysis probed with preimmune IgG. (B) Comparative immunoblotting of detergent extracts containing 100 μg total cellular protein followed by detection with human CD36 antiserum reveals the presence of CD36 protein of the expected molecular size of ∼88 kD in human ARPE-19 cells (ARPE-19) but not in human Bowes melanoma cells (Bowes), which are known not to express CD36 (reference 11). (C) Immunofluorescence labeling of paraformaldehyde fixed ARPE-19 cells with human CD36 mAb FA6–152 shows CD36 signals localizing to the RPE cell surface. (D) FA6–152 signals are specific since they are absent in parallel samples stained with nonimmune IgG.
Mentions: We previously established that CD36 participates in the phagocytic mechanism of rat primary RPE, and that CD36 transfection renders human melanoma cells phagocytic for OS (9). As with primary RPE, stable RPE cell lines derived from rat (RPE-J) or human (ARPE-19) RPE phagocytose OS and use αvβ5 integrin to recognize OS (6). To determine whether these RPE cell lines also serve as a model system to study the role of CD36 in OS phagocytosis, we assessed CD36 expression by immunoblotting and immunofluorescence microscopy. As shown in Fig. 1 , both RPE-J and ARPE-19 cells expressed CD36; the amount of CD36 protein in RPE-J cells, as determined by immunoblot, was ∼80% of that in adult rat RPE (Fig. 1 A). Immunofluorescence staining showed that CD36 localized to the plasma membrane of confluent ARPE-19 cells (Fig. 1 C). Parallel samples stained with nonimmune mouse IgG did not fluoresce (Fig. 1 D).

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