Localization of cellular retinoic acid-binding protein to amacrine cells of rat retina

Exp. Eye Res. (1990)

50, 505-511

Localization VINOD

P. GAuP*,

of Cellular Amacrine A. MARGREET

Departments of “Ophthalmology

Retinoic Acid-binding Cells of Rat Retina DE LEEUW”,

Protein

ANN H. MILAM”AND

JOHN

to C. SAARPt

and bBiochemistry, School of Medicine, University of Washington, Seattle, WA 98195, U.S.A.

(Received 24 July 1989 and accepted 8 September 7989) Monoclonal antibodiesto performic acid-oxidizedcellular retinoic acid-bindingprotein (CRABP)from bovine retina were preparedby fusionof spleencellsfrom immunizedmicewith mousemyelomacells. Five antibodieswere studiedin detail. It wasestablishedby J%ISAthat the antibodiesreact with CRABP and oxidized CRABP.but not with other oxidized or unmodifiedretinoid-bindingproteins. Competitive ELISAdemonstratedthat the antibodiesreact with heat-denaturedantigenbut not with native protein. Western blotting and immunostaining,following sodiumdodecyl sulfategel electrophoresis,provided evidencefor recognitionof a singlecomponentin retinal supernatantswhosestaining is prevented by preabsorptionof the antibody with heat-denaturedCRABP. The insoluble fraction from a retinal homogenatecontains residual CRABPand two weakly-reacting components,whose staining is not affectedby preabsorptionof the antibody with antigen.Eachantibody producesthe samestainingpattern on cryostat sectionsof rat retina by indirect immunofluorescence. Amacrine somataon both sidesof the inner plexiform layer are labeled,as well asprocesses forming laminaewithin this layer, Theseresults suggestthat retinoic acid may play a functional role in the inner retina. Key words: amacrinecell; cellular retinoic acid-bindingprotein: immunocytochemistry: monoclonal antibody: performic acid-oxidizedantigens; retina : retinoic acid.

1. Introduction Different chemical forms of vitamin A mediate its diverse biologicai effects. Retinaldehyde is involved in vision (Wald, 1968), retinol is required for reproductive competence (Thompson, Howell and Pitt, 1964). and retinoic acid affects growth, morphogenesis,and epithelial differentiation (DeLuca, 19 79 ; Sporn and Roberts, 1984; Maden, 1985). Whereas the role of retinaldehyde in vision has been understood at the molecular level for years (Wald, 1968). mechanistic Information has been lacking for retinol and retinoic acid action. Recently retinoic acid was found to interact with soluble transcription activation factors (retinoic acid receptors), providing a plausible molecular mechanism for its effects on epithelial differentiation (Giguere et al.. 1987; Petkovich et al., 198 7). Evidence that retinoic acid acts asa morphogen (Maden, 1985) has been supported by the detection of a gradient of the retinoid in developing chick limb bud (Thaller and Eichele, 198 7 ; Brockes, 1989 : Giguere et al., 1989). Retinoic acid in tissuesis associatedwith CRABP, a specific carrier protein that solubilizes and probably controls local concentrations of free retinoid (Chytil and Ong, 1984; Maden et al., 1988). Several years ago, CRABP (Saari and Futterman, 19 76 ; Wiggert et al., 1978) and retinoic acid (Saari, Bredberg and * Current address: Department of Anatomy, Bowman School of Medicine, Winston-Salem, NC 2 7103. U.S.A. t For correspondence at: Department of Ophthalmology University of Washington, Seattle. WA 98195. U.S.A.

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s03.00/0

Gray RJ-10.

Garwin, 1982) were detected in bovine and rat retinas. Since retinoic acid has no known function in the visual cycle (Dowling and Wald, 1960), its presence suggeststhat it may be involved in other aspects of retinoid function in retina. Knowledge of the cellular localization of CRABP in retina would be useful in assessingthe function of retinoic acid in this tissue. In this communication, we describe the preparation and characterization of monoclonal antibodies (mabs) to CRABP purified from bovine retina, and localization of the binding protein by immunocytochemistry to a subset of amacrine cells in rat retina. More detailed anatomical and developmental studies are described elsewhere (Milam et al., in press: De Leeuw et af., in press),Accounts of this work have appeared in abstract form (Gaur et al., 1989; Bunt-Milam et al., 1989; De Leeuw et al., 1989).

2. Materials and Methods Materials

Materials were purchased from the following suppliers : bovine retinas (George A. Hormel Co., Austin, MN), secondary reagents for ELISA, immunostaining and immunocytochemistry (ICN Biomedicals, Inc., Lisle, IL, and Cooper Biomedical, Inc., Malvern, PA), Immulon microtiter plates (96-well) (DynaTech Inc., Chantilly, VA), nitrocellulose paper (BioRad Richmond, CA), HPLC solvents (Burdick and Jackson Laboratories, Inc., Muskegon, MI). Purified retinoidbinding proteins were prepared by methods previously described (Saari and Bredberg. 1988). 0 1990 AcademicPressLimited

V. P. GAUR

Preparation of Monoclonal Antibodies (mabs) Bovine retinal CRABP was purified to apparent homogeneity by published procedures (Saari and Bredberg, 1988) and oxidized with performic acid according to Moore (1963). Procedures used for fusion of spleen cells and mouse myeloma cells have been described previously (Gaur, Eldred and Sarthy, 1988). Five-week-old female BALB/c mice received an initial i.p. injection of 50 pg of performic acid-oxidized CRABP (ox-CRABP) emulsified in Freund’s complete adjuvant. A second i.p. injection of the same antigen, suspended in incomplete adjuvant, was administered 3 weeks later. After a further 2 l-day interval, the mice received three i.v. injections at 24-hr intervals, each containing 100 ,ug of ox-CRABP in 0.4 ml of physiological saline. Three days after the last i-v. injection, the mice were killed and spleen cells were used for fusion with NS-1 mouse myeloma cells. When cells in most wells were semi-confluent, culture supernatants were tested for the presence of antibodies to CRABP using an ELISA procedure described previously (BuntMilam and Saari, 1983). Hybridomas from cells producing antibodies were cloned at least three times by double dilution, using syngeneic red blood cells or thymocytes as feeder cells. Clones were retested and permanent lines were established. Cells ( 107) from selected clones were injected into BALB/c mice previously injected with 0.5 ml of pristane (2,5,10,14tetramethylpentadecane). Ascites was collected, tested for antibody activity and stored at - 70°C. IgG fractions were obtained using protein A-Sepharose (Bunt-Milam and Saari. 1983). SDS-PAGE and Western Blotting The gel system of Fairbanks, Steck and Wallach ( 19 71) was employed with 10 % polyacrylamide slabs. Gels were stained with Coomassie blue. Unstained portions of gels were transferred to nitrocellulose (Towbin, Staehelin and Gordon, 1979) for 1 hr and soaking of the nitrocellulose in bovine serum albumin (BSA, 0.1%) was limited to 1 hr to minimize loss of proteins. With these exceptions, the nitrocellulose sheets were stained with mabs using the procedure described in Gaur et al. (1988). For control experiments, the antibody was incubated for 90 min at room temperature with antigen that had been heated at 60°C for 10 min in phosphate buffered saline (PBS, 20 mM phosphate, pH 7.4, 0.15 mM NaCl) containing 0.3% Triton X-100 (see Fig. 6). ELlSA

Screening of hybridoma supernatants for antiCRABP employed the ELISA procedure described in Bunt-Milam and Saari (1983). Competitive IZLISA experiments involved preincubation of the mab with heat-denatured proteins at various concentrations for

ET AL

90 min at room temperature before use in the procedure. Antigens were denatured at 60°C for 10 min in PBS containing 0.3% Triton X-100 (Figs 2 and 4).

Rats were killed by an overdose of ether or sodium pentobarbital. Retinas were fixed in 4% paraformaldehyde in 0.13 M phosphate buffer, pH 7.4, for 6 hr at room temperature, and placed in 30% sucrose in the same buffer overnight. Cryosections were cut at 14 pm and mounted on gelatin-coated glass slides. The sections were processed for immunofluorescence as described previously (Bunt-Milam and Saari, 198 3). Controls included the use of non-immune mouse serum or mab C-l that had been incubated for 3 hr at 4°C with CRABP (40 ,ug ml-‘) previously heated at 60°C for 10 min in PBS containing 0.3% Triton X100. 3. Results and Discussion

CRABP and cellular retinol-binding protein CRBP are poor immunogens, perhaps because of their highly conserved amino acid sequences (Sundelin et al., 1985) and/or their acidic isoelectric points. Although antibodies to both proteins have been produced (Liou, Fong and Bridges, 1981: Ong, Crow and Chytil, 1982 ; Kato et al., 1985; Eriksson et al., 1987; Momoi et al., 1989), previous attempts in this laboratory to produce polyclonal antibodies to CRBP and CRABP were unsuccessful unless the proteins had been modified before use as an immunogen. Of the several modifications examined, performic acid oxidation (ox) of CRBP induced the highest anti-CRBP titers in rabbits (Saari et al., 1984). The results presented here document that ox-CRABP can be used to generate mabs to this protein. Culture supernatants from hybridomas were screened for the presence of anti-CRABP using an ELISA procedure. The wells of microtiter plates were coated with CRABP, whereas the animals had been immunized with ox-CRABP. From 576 wells containing hybridomas, eight colonies showed antiCRABP activity. Five strongly reactive clones were selected for ascites production and further characterization, The results obtained with mab C-l are representative of the other four mabs and are presented here. No anti-CRABP-producing hybridomas resulted from fusions utilizing spleen cells from animals immunized with unmodified CRABP. Several methods were employed to characterize the specificity of the mabs generated in this study. In initial tests, the specificity of the mabs was determined by screening for possible cross-reactivity with other purified retinoid-binding proteins. The amino acid sequence of rat liver CRBP is approximately 45 % identical to that of bovine adrenal and retinal CRABP

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FIG. 1. USA reactivity of mab C-l with retinoid-binding proteins. Wells of USA plates were coated with CRABP (m), CRBP(Cl), CRALBP(x). IRBP (A) or RBP (a). The amount of color generatedis expressedasa function of the dilution of mabC-l. The resultsindicate that the antibody is specificfor CRABP.

IO

20

30 CCRABPI

(pg

40 ml-‘)

50

60

FIG. 2. Effectof heating CRABPon recognitionby mabCl, assessed by competitive ELISA. Wells were coated with CRABP. Mab C-l was incubated for 90 min with several concentrationsof CRABPthat had beenheatedat 60°C for 0 min (0). 1 min (0). 5 min (m) or 10 min (0). The color generatedis displayedas a function of the concentration of competingantigen. The resultsdemonstratethat mab C-l reactswith heat-denaturedbut not native CRABP.

(Sundelin et al., 1985 : Crabb and Saari, 1986). CRALBP (cellular retinaldehyde-binding protein), IRBP (interphotoreceptor retinoid-binding protein) and RBP (retinol-binding protein) share the ability to bind retinoids, although their amino acid sequencesdo not appear to be related to one another or to CRABP (Pervaiz and Brew, 1987). Figure 1 illustrates that mab C-l does not recognize these other retinoidbinding proteins, indicating that the mab is highly specific for CRABP by this criterion. Further indications of specificity were obtained with competitive ELISA experiments in which mab C-l was preincubated with possible cross-reacting antigens before ELISA. Since CRABP usedas the antigen in mab production had been denatured by performic acid oxidation, the reactivity of mab C-l first was examined with native and heat-denatured CRABP. The results in Fig. 2 indicate that preincubation of the antibody with

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FIG.3. CompetitiveE&ISA with retinoid-bindingproteins. Wellswere coatedwith CRABP.Mab C-l waspreincubated for 15 min with heat-denaturedCRABP (m). CRBP (Cl), CRALBP (a), RBP (0). or IRBP (0). The results indicate that only heat-denaturedCRABPis able to competewith CRABPin the ELISA wells. native CRABP doesnot prevent color formation during the ELISA. However, use of CRABP heated at 60°C results in inhibition of color formation, indicating that the antibody recognizes denatured CRABP. Other heat-denatured, retinoid-binding proteins were then tested in similar competition experiments, illustrated in Fig. 3. None of the denatured retinoid-binding proteins other than CRABP is active in competition assays with mab C-l at concentrations up to 200 pg ml-‘. The observation that mab C-l reacts with CRABP coated on the surface of an ELISA plate (Fig. 1) but in competition experiments, only with CRABP that has been heated (Fig. 3) suggests that the protein is denatured by adsorption to the polystyrene of the ELISA plate. This latter conclusion is in accord with reports of surface denaturation of proteins adsorbed to plastics (Horbett and Brash, 1987). The competitive ELISA described may be of general utility in localizing epitopesto the interior or exterior of protein structures. Since CRABP had been oxidized with performic acid prior to immunization, it was important to demonstrate that the mabs did not recognize oxidized proteins nonspecifically. The results of Fig. 4 indicate that mab C-l recognizes CRABP and ox-CRABP but not ox-CRBP or ox-BSA. Performic acid oxidation modifies several amino acid residues, including cysteine, tyrosine, tryptophan and methionine (Moore, 1963 ; Hirs, 1967). Perhaps these modified residues and/or denaturation of the protein at low pH are responsible for the enhanced antigenicity resulting from the treatment. SDS-PAGE and Western blotting provided further indication of the specificity of the mabs. As shown in Fig. 5, mab C-l reacts with a single component that comigrates with authentic CRABP in supernatants derived from bovine or rat retinal homogenates. Analysis of the pellet reveals residual CRABP (Mr 16 kDa) and two other components (Mr 32 and

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5x104 Antibody

ET AL

5 x IO’ dilution

FIG. 4. ELISA reactivity of mab C-l with performic acidoxidized (ox) proteins. Wells of ELISA plates were coated with CRABP ( n ), ox-CRABP ( X ). ox-CRBP (0) and ox-BSA (4). The amount of color generated is expressed as a function of the dilution of mab C- 1. The results indicate that the antibody does not recognize oxidized proteins nonspecifically.

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FIG. 6. Effect of preabsorbtion of mab C-l with CRABP on Western blots. Samples of rat retinal supernatants (S) or pellets (P) were analyzed by SDS-PAGE and stained with mab C-l that had been preincubated with the following concentrations of heat-denatured CRABP : 0 ~1 mll’, lanes 1 and 2 : 25 ,ug mll’, lanes 3 and 4; 12.5 fig ml-.‘. lanes 5 and 6: 2.5 pugml-‘. lanes 7 and 8.

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+ 20 + 14

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4

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FIG. 5. Characterization of mab C-l by SDS-PAGE, Western blotting, and immunostaining. Samples were analyzed by SDS-PAGE and either stained with Coomassie Brilliant Blue (lanes l-3) or blotted to nitrocellulose and stained with mab C-l (lanes 4-7). Lane 1, bovine retinal supernatant : lane 2, purified bovine retinal CRABP; lane 3, molecular weight standards ; lane 4, rat retinal supernatant : lane 5, rat retinal pellet; lane 6. rat retinal homogenate: lane 7, bovine retinal CRABP. 34 kDa) that stain weakly. Preabsorption of the antigen with heat-denatured CRABP prevents reactivity with CRABP but does not affect the staining of the two other components (Fig. 6). We have not identified these components ; however, it is unlikely that their recognition could be responsible for the immunocytochemical localization described below, since that staining is abolished by preabsorption of the antibodies with heat-denatured antigen.

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5x104 Antibody

5x107 dllutlon

FIG. 7. Effect of treatment of the antigen with formaldehyde on recognition by the antibody. Wells were coated with CRABP and incubated with 4% paraformaldehyde for 0 min (M), 15 min (0). 30 min (A) and 60 min (0) before completion of ELISA. The results indicate that these simulated fixation conditions do not affect antibody recognition.

The effect of formaldehyde treatment on mab C-l reactivity was investigated, since this fixative is commonly employed in immunocytochemical studies. The wells of JZLISA plates were coated with CRABP and the adsorbed protein was incubated with 4% paraformaldehyde in PBS for 15. 30 or 60 min at room temperature. After rinsing the wells with PBS,

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F'IG. 8. lmmunofluorescence of rat retina. A and C. Sections were treated with mab C-l. B. Section was treated with nnab C-l preabsorbed with CRABP. The secondary antibody was goat anti-mouse IgG labeled with fluorescein isothiocyanate. hrote the localization to amacrine somata (arrows) and laminae in the inner plexiform layer (IPL). A, x 500: B and C. x 1251

mab C-l was used for ELISA. The results presented in Fig. 7 indicate that formaldehyde treatment does not affect recognition of the antigen. In rat retina, specific labeling with mab C-l is restricted to somata on both sides of the inner plexiform layer (Fig. 8). Four distinct laminae in the inner plexiform layer are formed by CRABP-positive processes. This labeling pattern corresponds to the distribution of amacrine cells in neural retina (Masland, 1988; Rodieck, 1988). More detailed evidence for an amacrine cell localization will be presented elsewhere (Milam et al., in press). No labeled processes are found in the outer plexiform or nerve fiber layers.

Specific labeling is absent in sections treated with nonimmune mouse serum or with anti-CRABP preabsorbed with heat-denatured CRABP [Fig. S(B)]. Localization of CRABP to amacrine cells of rat retina was unexpected. Two other retinoid-binding proteins,

CRALBP and CRBP, have been localized to Miiller cells of neural retina (Bunt-Milam and Saari, 1983; Bok, Ong and Chytil, 1984; Eisenfeld, Bunt-Milam and Saari, 1985). However, we have found an amacrine cell localization in all sub-primate species examined and the anticipated localization of CRABP solely to Miiller cells thus far has been found only in human and monkey retinas (Milam et al., in press). Amacrine cells show considerable biochemical and morphological heterogeneity (Masland, 1988). This study adds CRABP to the list of substances (neurotransmitters, associated enzymes and neuropeptides) that have been localized to subsets of amacrine cells. Although the functions of retinoic acid in other tissues have not been unequivocally determined, there is considerable evidence linking the retinoid to effects on differentiation and the generation of patterns during development (Strickland and Mahdavi. 19 78 ;

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DeLuca, 1979; Sporn and Roberts, 1984; Maden, 1985). Thus the presence of retinoic acid and its binding protein in retina (Ong et al., 1982 ; Saari et al., 1982 ; Kato et al., 1985; Blaner et al., 1986) suggests that it may be involved in similar processes in this tissue, since it can not be converted biologically to retinaldehyde or retinol and, therefore, can not participate in reactions of the visual cycle (Dowling and Wald, 1960). The precise patterns of inner plexiform laminations formed by subsets of amacrine cell processes (Masland, 1988) suggest that retinoic acid could be involved in their generation or maintenance. Experiments that examine the effect of retinoic acid depletion on retinal morphology and staining with anti-CRABP may elucidate on this hypothesis. Acknowledgments The authors acknowledge Lucille Bredberg for assistance in preparation of retinoid-binding proteins, Ingrid Klock and Faridah Dahlan for histologic help, and Brad Clifton, Ron Jones and Chuck Stephens for photographic help. This study was supported in part by NIH Grants EY02 3 17. EY01311, EY07031, and EY01730. the Lewis Berkowitz Family Foundation and National Retinitis Pigmentosa Foundation Fighting Blindness, and in part by an award from Research to Prevent Blindness, Inc. (R.P.B.). A. H. M. is a Senior Scholar of R.P.B. References Blaner. W. S.. Das, K., Mertz, J. R., Das, S. R. and Goodman, D. S. (1986). Effects of dietary retinoic acid on cellular retinol- and retinoic acid-binding protein levels in various rat tissues. 1. Lipid Res. 27, 1084-S. Bok. D., Ong, D. E. and Chytil, F. (1984). Immunocytochemical localization of cellular retinol binding protein in the rat retina. Invest. Ophthalmol. Vis. Sci. 25. 877-83, Brockes.J. P. (1989). Retinoids,homeoboxgenes.and limb morphogenesis. Neuron2, 1285-94. Bunt-Milam, A. H., De Leeuw,A. M.. Gaur, V. P. and Saari, J. C. (1989). Localization of cellular retinoic acidbinding protein in a subpopulation of GABAergic amacrinecells.Invest.Ophthalmol Vis. Sci. 30 (Suppl.). 344. Bunt-Milam, A. H. and Saari, J. C. (1983). Immunocytochemicallocalizationof two retinoid-bindingproteinsin vertebrate retina. J. CellBiol. 97, 703-12. Chytil, F. and Ong. D. E. (1984). Cellular retinoid-binding protein. In The Retinoids.Vol. 2 (Eds Sporn, M. B., Roberts, A. B. and Goodman, D. S.). Pp. 89-123. AcademicPress: Orlando. Crabb, J, W. and Saari, J. C. (1986). The completeaminoacid sequenceof the cellular retinoic acid-binding protein from bovine retina. Biochem.Int. 12, 391-5. De Leeuw,A. M., Gaur, V. P., Saari, J. C. and Bunt-Milam, A. H. (1989). Immunocytochemical localization of cellular retinol-, retinoic acid-, and retinaldehydebindingproteinsin rat retina during pre- and postnatal development.Invest. Ophthalmol.Vis. Sci. 30 (Suppl.), 345.

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