Interphotoreceptor Retinoid-Dinding Protein in Retinol Rod …iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/... · No. 5 Reports 845 tone for 10 min. Affinity-purified

844 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / Moy 1986 Vol. 27

position of anionic sites in Bruch's membrane of the rat. J His-tochem Cytochem 30:245, 1982.

3. Pino RM: Ultrastructural localization of lectin receptors on thesurface of the rat retinal pigment epithelium. Decreased sensitivityof the avidin-avidin-biotin method due to cell surface charge.J Histochem Cytochem 32:862, 1984.

4. Hynes RO and Yamada KM: Fibronectin: multifunctionalmodular glycoproteins. J Cell Biol 95:369, 1982.

5. Turksen K, Aubin JE, Sodek J, and Kalnins VI: Changes in thedistribution of laminin, fibronectin, type IV collagen and heparansulfate proteolgycan during colony formation by chick retinalpigment epithelial cells in vitro. Coll Relat Res 4:413, 1984.

6. Adler AJ and Klucznik KM: Proteins and glycoproteins of thebovine interphotoreceptor matrix: composition and fractionation.Exp Eye Res 34: 423, 1982.

7. Pino RM: Restriction to endogenous plasma proteins by a fe-nestrated capillary endothelium: an ultrastructural immunocy-tochemical study of the choriocapillary endothelium. Am J Anat172:279, 1985.

8. Novikoff AB, Leuenberger PM, NovikoffPM, and Quintana N:Retinal pigment epithelium. Interrelations of endoplasmic retic-ulum and melanosomes in the black mouse and its beige mutant.Lab Invest 40:155, 1979.

9. Mautner V and Hynes RO: Surface distribution of LETS proteinin relation to the cytoskeleton of normal and transformed cells.J Cell Biol 75:743, 1977.

10. Remold HG, Shaw JE, and David JR: A macrophage surfacecomponent related to fibronectin is involved in the response tomigration inhibitor factor. Cell Immunol 58:175, 1981.

Interphotoreceptor Retinoid-Dinding Protein

in Retinol Rod Cells ond Pineol Glond

Merlyn M. Rodrigues,* Joseph Hackerr,* Reginald Gaskins,* Barbara Wiggerr.t Ling Lee.fMichael Redmond, and Gerald J. Chaderf

Immunoelectron microscopic staining demonstrates Inter-photoreceptor Retinoid-Binding Protein (IRBP) in monkeyrod cell cytoplasm with virtually none in cone cells. The pinealalso contains significant amounts of IRBP demonstrating asimilarity of pinealocytes to rod but not cone photoreceptors.Invest Ophthalmol Vis Sci 27:844-850, 1986

Rods and cones are specialized photoreceptor cellsof the retina which mainly function in scotopic (low-light) and photopic (bright-light) vision respectively.Even though a rhodopsin-like photopigment is foundas low on the phylogenetic scale as Chlamydomonas,the origins and divergence of the two photoreceptorsare not well understood. Likewise, the developmentand function of the pineal gland in relationship to thevisual system is not clear, particularly with regard tothe similar nature or origins of pinealocytes and retinalrod or cone cells.

We and others have identified a large, soluble gly-coprotein, the Interphotoreceptor Retinoid-BindingProtein (IRBP) that may function as a vitamin Atransport vehicle between the neural retina and theretinal pigment epithelium (RPE). It is mainly an ex-tracellular protein of the retinal interphotoreceptormatrix (IPM).1 IRBP is the major soluble protein ofthe IPM, the only retinoid-binding protein in the sub-retinal space and binds endogenous or exogenouslyadded retinol in a light-dependent manner.2 The mainsite of cellular concentration and/or origin of this im-portant protein is yet unclear. We have reported pre-viously that the concentration of IRBP is greatly de-creased in inherited human retinal degenerations, cor-

responding to the primary loss of rod photoreceptorcells.3 The synthesis of IRBP by neural retina but notby RPE has been demonstrated in organ cultures ofmonkey and human retina.4'5 In toto, these studiesprovide only indirect evidence for the production ofIRBP by rod photoreceptor cells. Using immunocy-tochemical techniques at the light and electron micro-scopic level, we now report on the selective presenceof IRBP in rod photoreceptor cells of the primate retinaand that little if any IRBP is found in or associatedwith cone photoreceptor or Miiller (glial) cells. More-over, we also detect IRBP in the pineal gland, dem-onstrating a new biochemical link between this organand the retina.

Materials and Methods. Primary antisera to purifiedmonkey IRBP was raised in rabbits and affinity purifiedusing glutaraldehyde crosslinked IRBP-Sepharose im-munosorbent.6 The final protein concentration of thispreparation was 0.8 mg/ml. Absorption of the antibodywas accomplished by adding an excess of purifiedmonkey IRBP to the antibody, incubating at 4°C for24 hr and removing the antibody-antigen precipitateby centrifugation.

Rhesus monkey (Macacca mulatto) eyes were ob-tained fresh from 1-3-year-old animals. Immunocy-tochemical staining of their retinas was performed bylight microscopy using indirect immunofluorescentstaining and immunoperoxidase staining (avidin-bio-tin complex, ABC).7 Retinas were either fresh frozenor were fixed in 0.5-1.0% phosphate-buffered glutar-aldehyde for up to 2 hr, followed by freezing in liquidnitrogen. Frozen sections (6-/z thick) were fixed in ace-

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No. 5 Reports 845

tone for 10 min. Affinity-purified antibody was diluted1:100 or 1:200 in phosphate-buffered saline (PBS) pH7.4 prior to use. Following incubation in a moistchamber for 1 hr at room temperature, the antibodywas rinsed off and replaced with fluorescein-labeledgoat anti-rabbit IgG at a dilution of 1:20,applied for 30 min, then washed in buffer beforemounting.

Other eyes had an intravitreal injection of 0.5-1.0%phosphate-buffered glutaraldehyde 1-2 hr prior to sec-tioning and trimming the tissue for embedding inLowicryl. After fixation, tissues were washed in buffer,dehydrated in 50%, 75%, and 90% dimethyl formam-ide, and embedded in Lowicryl. Polymerization wasperformed by exposure to a UV light source at 4°C for24 hr, followed by polymerization at room temperaturefor 48-72 hr. The UV light source consisted of two 15-watt black light tubes producing 365-nm radiation.Immunoelectron microscopy of Lowicryl-embeddedtissue was performed using colloidal gold (15-20 nm)linked to goat anti-rabbit IgG or protein A.8 Controlsincluded nonimmune serum and antibodies absorbedwith purified IRBP antigen in both this case and in thelight microscopy experiments described above.

Fresh pineal glands from the same monkeys werefrozen for immunocytochemical staining for IRBP bythe ABC method. Other pineals, retinas and brainsamples were analyzed biochemically using an enzyme-linked immunoabsorbent assay (ELISA) developed inour laboratory that is specific for IRBP.9

Results. As expected, light microscopy revealed thepredominant staining for IRBP to be in the interpho-toreceptor space of peripheral, equatorial, and posteriorretina using both the immunofluorescence and im-munoperoxidase techniques. Immunofluorescent andimmunoperoxidase techniques demonstrated a similarpattern of distribution in the interphotoreceptor space(Fig. 1A, B). Staining was weak or not detectable inthe cone inner segments (Fig. 1A). There was a sharptransition zone at the ora serrata (Fig. 1C); stainingwas present in the peripheral retina but completely ab-sent in the ciliary epithelium. No staining was observedin controls using the antibody absorbed with purifiedantigen (Fig. 1D).

Immunoelectron microscopic staining with colloidalgold disclosed staining of rod inner segments (Fig. 2),as well as the cytoplasm of rod cells with antibodies toIRBP (up to 1:500 dilution). The rod outer segmentsand RPE showed much less staining although somestaining was observed along the RPE apical processes.Little if any staining was seen in any area of the conephotoreceptor cell. Miiller cells showed no IRBP im-munoreactivity. Immunoperoxidase staining of rodcells was also present. Similar immunoelectron micro-

scopic staining was obtained with the immunoperox-idase method (figure not shown); immunoreactivitywas more precise, however, with colloidal gold. In thefovea there was virtually a complete lack of staining ofcone inner and outer segments (Fig. 3).

The pineal gland showed IRBP-like immunoreac-tivity that appeared to have predominantly an intra-cellular localization by light microscopy (Fig. 1E). Im-munoelectron microscopy showed intracellular local-ization within cytoplasmic vesicles as well as someextracellular distribution (Fig. 4). ELISA of monkeypineal cytosol (ie, 100,000 X g supernatant) showedthe amount of IRBP in pineal to be approximately 7.0/zg/mg soluble protein in comparison to about 100 /xg/mg in whole retina cytosol and about 2 /ug/mg in ce-rebral cortex cytosol. No IRBP was detected in monkeyliver or testes.

Discussion. IRBP has now been well characterizedand localized mainly to the retinal interphotoreceptorspace (IPS).1 Since this protein could be of importancein intercellular retinoid transport, we felt it critical todetermine the cellular distribution and possible originof IRBP. Four cell types border the IPS: RPE cells,Miiller cells and rod and cone photoreceptor cells. Wehave previously excluded the RPE as the site of IRBPsynthesis in the primate retina.4 The retinal site of syn-thesis has been unclear however, although Hollyfieldet al5 have evidence that IRBP could be synthesizedmainly in rod cells of the human retina. In the presentpublication, we have now demonstrated that intracel-lular IRBP is localized primarily in rod photoreceptorcells of the primate retina. The outer segments of thesecells show much less reactivity compared with the innersegments and cytoplasm. Minimal reactivity was notedin cone cells, both in inner and outer segments. Thisdistribution is particularly evident in the specializedfoveal area of the primate retina in which the photo-receptor cells are virtually all of the cone type. Here,we found almost no staining in the cells and muchdiminished staining in the adjacent interphotoreceptorspace making it unlikely that cone cells synthesizemuch IRBP. Little or no staining was found in theMiiller cells, whereas the low level of staining in thephagocytic RPE cells could be ascribed to uptake fromthe IPS. As mentioned above, we have previously foundno evidence for IRBP synthesis by the RPE.4 Thus,IRBP might be considered to be a rod-specific markerprotein in the primate.

Although IRBP is clearly an "extracellular" proteinas demonstrated by both immunofluorescent and im-munoperoxidase light microscopy, less extracellularstaining is observed using the immunogold EM tech-nique than might have been expected. We assume thatthe bulk of extracellular IRBP is lost during EM fixation


846 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / May 1986 Vol. 27

„ * > * • •

rig. i . immunocytocnemistry in monkey retina. A, immunoiluorescent staining ot the lnterphotoreceptor matnx with anti-IKbP athmty-purity antibody. No reactivity of cone inner segments (arrow) is observed. Retinal pigment epithelium (•) is negative (X64). B, Immunoperoxidasestaining of interphotoreceptor space (X33O). C, Transition zone (arrow) at the ora serrata; staining of the interphotoreceptor space in peripheralretina and lack of reactivity in ciliary epithelium (X40). D, Absence of stain in peripheral retina treated with antibody previously absorbed withIRBP.13 E and F, Immunocytochemistry of monkey pineal gland. E, Staining of the monkey pineal gland with anti-IRBP antibodies, using theavidin-biotin-peroxidase method.14 F, Control of monkey pineal gland shows absence of stain with antibody previously absorbed with IRBP15

(E, F, X220).

and processing, due to its solubility in aqueous solu-tions. In a somewhat similar vein, we find no evidencefor preferential sequestering of the IRBP in rough en-doplasmic reticulum or Golgi apparatus, even thoughIRBP is a secreted protein. Although basically similar,our results do differ in some respects from data in recentabstracts concerning IRBP localization.10"12 Andersonet al12 for example, find IRBP in the cone-dominant

ground squirrel retina. Bunt-Milam and Saari10 de-scribed localization of IRBP in cytoplasmic granulesin the bovine retina, whereas we found a more gen-eralized distribution of IRBP in the monkey rod innersegment. These differences could be due to differencesin the primary antibody or to species variation; we havepreviously reported significant variability among sev-eral species in the biochemical detection of IRBP.13


No. 5 Reports 847

n g . L. upper, immunoeiectron microscopy ot Lowicryl-embedded peripheral monkey retina reacted with anti-IRBP antibodies linked tocolloidal gold-goat anti-rabbit IgG (20 nm; 1:100 dilution). Rod inner segments (R) display marked reactivity, whereas a cone cell (C) showsminimal or no reactivity. External limiting membrane is indicated (circle) (XI 5,840). Inset shows higher magnification of a portion of innersegment of a rod cell (R) stained with colloidal gold linked to goat anti-rabbit IgG (1:50 dilution) (X45,000). Lower, Control section treatedwith anti-IRBP antibody absorbed with IRBP followed by colloidal gold staining (XI 6,500).


848 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / May 1986 Vol. 27

Fig. 3. Immunoelectronmicroscopy of Lowicryl-embedded monkey maculareacted witn anti-IRBP an-tibodies linked to colloidalgold-goat anti-rabbit IgG (20nm; 1:100 dilution). Fovealcone inner segments (IS) intangential section show min-imal or no reactivity. Exter-nal limiting membrane is in-dicated (arrow). Residual in-terphotoreceptor matrixshows sparse granular stain-ing (XI 2,900). Inset showslight micrograph of the sametissue (Toluidine blue, X220).

Our antibody may also recognize determinants differ-ent from antibodies produced in other laboratories. Ifthe protein is processed prior to secretion, for example,our antibody may recognize an intracellular, nonpro-cessed form as well as an extracellular, processed form.

Technical factors could also play a role in these ap-parent differences. According to standard immuno-electron microscopic procedures,7 we have used shorterintervals of tissue fixation in weaker aldehyde solutionsfollowed by low temperature polymerization to retainantigenic sites that could otherwise be markedly di-minished or masked by more standard methods of fix-ation and processing used for electron microscopy. Wehave also considered that the intracellular localizationof IRBP in rod inner segments may reflect rod celldamage and imbibation of IRBP from the extracellularspace. We feel this is unlikely, however, since we wouldexpect other cell types to also be affected (eg, PE andcone cells) and see a more generalized distribution inseveral cell types. In fact, our data are consistent withthose of Hollyfield et al,5 indicating that the rod cellmay be the primary site of synthesis in human andmonkey retina.

Other important questions are raised by our sur-prising finding of IRBP-immunoreactivity in the pinealgland concerning similarities between this gland and

the retina. Zimmerman and Tso14 have described atransient photoreceptor-like differentiation of rat pi-nealocytes peaking in the early postnatal period thatmimics photoreceptor differentiation. Immunologicalevidence also links the mammalian eye and pinealgland; immunization with the highly antigenic retinalprotein "S-antigen" leads to inflammation of the pinealas well as the retina.15 Similarly, recent work from ourlaboratory indicates that purified IRBP causes selectiveinflammatory changes in both retina and pineal of ex-perimental animals.16 Our present results thus signif-icantly extend the evolutionary, developmental andpossibly functional analogy between pineal and retinalcells. The possibility of a similar function of IRBP inpineal and retina (eg, retinoid transport) is also raised,indicating that retinoids may play a more importantrole in pineal function than previously thought. It isalso interesting that IRBP-like immunoreactivity isfound within the pineal cells as well as extracellularly,perhaps reflecting the evolutionary transformation ofthe pineal from a true photoreceptor to a neurosecre-tory organ involved in secondary photic signal trans-duction.

In any event, IRBP has now been detected in rodphotoreceptor cells and pinealocytes in the primate butis found only in scant amounts in retinal cone pho-


No. 5 Reports 849

Fig. 4. Immunoelectronmicroscopy. Lowicryl-em-bedded monkey pineal re-acted with purified anti-IRBPantibody linked to colloidalgold-goat anti-rabbit IgG (20nm; 1:50 dilution). Reactivitywithin intracytoplasmic ves-icles (arrows) as well as scat-tered extracellular material(circles). Nucleus is indicated(N) (X21,000). Inset showslight micrograph of the typ-ical pineal lobules (Toluidineblue, X330).

toreceptors. This could reflect the evolutionary diver-gence of rod cells and pinealocytes on one hand andcones on the other.

Key words: interphotoreceptor retinoid-binding protein, ret-ina, rod photoreceptor, cone photoreceptor, pinealocyte,monkey fovea

From the Laboratory of Ophthalmic Pathology* and Laboratoryof Vision Research.t National Institutes of Health, Bethesda, Mary-

land. Submitted for publication: April 26, 1985. Reprint requests:Dr. Merlyn M. Rodrigues, Bldg. 10, Room ION 112, National EyeInstitute, National Institutes of Health, Bethesda, MD 20205.

References

1. Bunt-Milam AH and Saari JC: Immunocytochemical localiza-tion of two retinoid-binding proteins in vertebrate retina, J CellBiol 97:703, 1983.

2. Chader GJ and Wiggert B: Interphotoreceptor Retinoid-Binding


850 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / May 1986 Vol. 27

Protein. Characteristics in bovine and monkey retina. VisionRes 24:1605, 1984.

3. Bergsma DR, Wiggert B, Funahashi M, Kuwabara T, and ChaderGJ: Vitamin A receptors in normal and dystrophic human retina.Nature 265:66, 1977.

4. Wiggert B, Lee L, O'Brien PF, and Chader GJ: Synthesis of In-terphotoreceptor Retinoid-Binding Protein (IRBP) by monkeyretina in organ culture: effect of monensin. Biochem BiophysResCommun 118:789, 1984.

5. Hollyfield JG, Fliesler SJ, Rayborn ME, Fong S-L, Landers RA,and Bridges CD: Synthesis and secretion of interstitial retinol-binding protein by the human retina. Invest Ophthalmol Vis Sci26:58, 1985.

6. Kapoor CL and Cho-Chung US: Affinity purification of regu-latory subunits of cAMP-dependent protein kinase using cross-linked immunosorbent. J Immunol Methods 57:215, 1983.

7. Hsu SM, Raine L, and Fanger H: The use of avidin-biotin-per-oxidase complex (ABC) in immunoperoxidase techniques: acomparison between ABC and unlabeled antibody (PAP) pro-cedures. J Histochem Cytochem 29:577, 1981.

8. Roth J, Bendayan M, Carlemalm E, Villiger W, and GaravitoM: Enhancement of structural preservation and immunocyto-chemical staining in low temperature embedded pancreatic tissue.J Histochem Cytochem 29:663, 1981.

9. Wiggert B and Chader GJ: Monkey interphotoreceptor retinoid-binding protein (IRBP): isolation, characterization and synthesis.

In The Interphotoreceptor Matrix in Health and Disease, BridgesCD and Adler A, editors. New York, Alan R. Liss Inc., 1985,pp. 89-110.

10. Bunt-Milam AH and Saari JC: Immunocytochemical localiza-tion of interphotoreceptor retinol-binding protein in cytoplasmicvesicles of bovine photoreceptors. ARVO Abstracts. InvestOphthalmol Vis Sci 26(Suppl):17, 1985.

11. Schneider B, Papermaster DS, Liou G, and Bridges CD: EMimmunocytochemical localization of IRBP in vertebrate retinas.ARVO Abstracts. Invest Ophthalmol Vis Sci 26(Suppl):340,1985.

12. Anderson DH, Neitz J, Saari JC, Fenwick J, Kaska DD, JacobsGH, and Fisher SK: Identification and localization of retinoid-binding proteins in a cone-dominant retina. ARVO Abstracts.Invest Ophthalmol Vis Sci 26(Suppl):340, 1985.

13. Wiggert B, Bergsma DR, and Chader GJ: Retinol receptors ofthe retina and pigment epithelium: further characterization andspecies variation. Exp Eye Res 22:411, 1976.

14. Zimmerman BL and Tso MO: Morphologic evidence of pho-toreceptor differentiation of pinealocytes in the neonatal rat. JCell Biol 66:60, 1975.

15. Kaslow CM and Wacker WB: Pineal reactivity of antiretina sera.Invest Ophthalmol Vis Sci 16:181, 1977.

16. Gery I, Wiggert B, Redmond TM, Kuwabara T, Crawford M,Vistica B, and Chader GJ: IRBP-induced uveitis: a new exper-imental automimmune disease. ARVO Abstracts. InvestOphthalmol Vis Sci 26(Suppl):77, 1985.


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