Volume 15 Reports 1017 Number 12 Fig. 2. Trahecular meshwork from choroidal melanoma case 3, showing immunofluorescent staining for complement component C3. angle glaucoma. They observed cell-mediated im- of glaucomatous eyes, Arch. Ophthalmol. 68: munity, as indicated by leukocyte migration in- 643, 1962. hibition in only three of 10 patients with open- 2. Becker, B., Uneer, H-H., Coleman, S. L., et angle glaucoma and no other ocular diseases. al.: Plasma cells and gamma-globulin in tra- Evidence which may link open-angle glaucoma becular meshwork of eyes with primary open- to immunologic abnormalities include the obser- angle glaucoma, Arch. Ophthalmol. 70: 38, 1963. vation that such patients have a higher incidence 3. Myers, R. L.: Cell-mediated immunity: rele- of positive antinuclear antibody reaction (44 per vance to ocular diseases (Editorial on Recent cent), compared with nonglaucomatous controls Advances), INVEST. OPHTHALMOL. 14: 635, (7.5 per cent) and gg topical steroid responders 1975. (7 per cent).8 Also, certain HL-A antigens ap- 4. Leopold, I. H.: Clinical immunology in oph- pear in a higher percentage of open-angle glau- thalmology, Am. J. Ophthalmol. 81: 129, 1976. coma patients than in the normal population." 5. Allansmith, M. R., Whitney, C. R., McClellan, These studies differ, however, as to which of the B. H., et al.: Immunoglobulin in the human HL-A antigens are abnormally increased. Co- eye, Arch. Ophthalmol. 89: 36, 1973. operative studies are currently underway to more 6. Allansmith, M. R.: Personal communication. 7. Henley, W. L., Okas, S., and Leopold, I. H.: clearly define this question and to establish its Cellular immunity in chronic ophthalmic dis- significance in open-angle glaucoma, orders. 4. Leukocyte migration inhibition in diseases associated with glaucoma, Am. J. Appreciation is expressed to Ms. Linda Cleve- Ophthalmol. 76: 60, 1973. land for excellent technical assistance. 8. Waltman, S. R., and Yarian, D,: Antinuclear From the Duke University Eye Center and the antibodies in open-angle glaucoma, INVEST. Departments of Pathology, Duke University OPHTHALMOL. 13: 695, 1974. Medical Center and Veterans Administration Hos- 9. Henley, W. K., and Leopold, I. H.: The im- pital, Durham, N. C. This work was supported in portance of HL-A antigens in ophthalmology, part by the Electron Microscopy Laboratory, Am. J. Ophthalmol. 80: 774, 1975. Durham Veterans Administration Hospital, and a grant from the National Society for the Prevention of Blindness, New York, New York. Submitted for publication July 12, 1976. Reprint requests: Dr. Bruce Shields, Duke University Eye Center, Durham, N. C. 27710. Retinol- and retinoic acid-binding proteins: occurrence in human retina and absence Key words: immunofluorescence, immunoglobu- lins, antibodies, complement, trabecular mesh- from human cultured flbroblasts. DAVID work, open-angle glaucoma, choroidal melanoma. SWANSON, SIDNEY FUTTERMAN, AND JOHN REFERENCES C. SAARI. 1. Becker, B., Keates, E. U., and Coleman, S. L.: Low-molecular-weight retinol- and retinoic acid- Gamma-globulin in the trabecular meshwork binding proteins were shown to be present in the Downloaded from iovs.arvojournals.org on 09/26/2021 1018 Reports Investigative Ophthalmology December 1976 soluble fraction of human retinal tissue but ab- lium from 11 human eyes within 48 hours post- sent from human fbroblasts grown in tissue cul- mortem and stored frozen. Soluble protein was pre- ture. By the use of gel filtration and comparison pared by thawing, sonicating briefly to disperse the with bovine retinal tissue, the human intracellular tissue, centrifuging at 70,000 x g for 1 hour, and binding proteins were found to have molecular drawing off the supernatant. The protein concen- weights of approximately 17,000 daltons, which tration, roughly estimated by A2So measurement are comparable to the molecular weights of bo- using bovine albumin as standard, was adjusted to vine intracellular binding proteins. The quantity 12 mg. per milliliter by diluting the supernatant of retinoic acid bound exceeded that of retinol fivefold with buffer solution (0.05M Tris'Cl, pH by about eightfold. 7.5 and containing 0.2M NaCl). Human fibroblast supernatant. Fibroblasts ob- The presence and characterization of low-molec- tained by skin biopsy from a normal human volun- ular-weight retinol- and retinoic acid-binding teer were grown in Eagel's Minimal Essential proteins in the supernatant of bovine retina and Medium with 10 per cent fetal calf serum.7 Cells a variety of other human and animal tissues has were harvested between the seventh and thirteenth been previously reported.1"6 We undertook the passages by treatment with trypsin, rinsed three present study to determine whether human retinal times with normal saline, and stored at -67° C. tissue contained these intracellular binding pro- Approximately 0.5 ml. of packed cells suspended teins. In addition, because one would like to have in normal saline were thawed, sonicated, and available a noninvasive means to obtain human centrifuged at 70,000 x g for 1 hour. The fibro- intracellular binding proteins for vitamin A de- blast supernatant was concentrated with an Ami- rivatives, human fibroblasts from a skin biopsy and con UM-10 membrane to 2.0 ml. and a protein grown in tissue culture were analyzed for the concentration of 6.6 mg./ml. presence of these proteins. Presence of the bind- Bovine retinal supernatant. The method of ing proteins in cultured fibroblasts would permit preparation of supernatant from bovine retinal the use of a skin biopsy as an easily obtained tissue has been described.4 Protein concentration tissue source of these proteins for studies of their was adjusted by diluting with buffer to 12 mg./ml. possible role in retinal disease. to equal that of the human retinal supernatant Materials and methods. preparation. Human retinal tissue supernatant. Retinas were Incubation and analysis. One-milliliter samples grossly dissected from underlying pigment epithe- of supernatant were incubated for 20 minutes 15 20 25 30 35 Fraction number Fig. 1. Gel filtration of the soluble protein from human retina (—) bovine retina ( ) and human fibroblasts ( ) following incubation with 3H-retinol. The low-molecular-weighl binding protein for retinol appears in fractions 25 to 30. Mobilities of the human and bovine retinol-binding proteins are comparable with calculated values of the diffusion coefficient, Kd11—0.50 and 0.49, respectively. Downloaded from iovs.arvojournals.org on 09/26/2021 Volume 15 Reports 1019 Number 12 15 20 25 35 Fraction number Fig. 2. Gel filtration of the soluble protein from human retnia (—) bovine retina ( ) and human fibroblasts ( ) following incubation with 3H-retinoic acid. The low-molecular- weight binding protein for retinoic acid appears in fractions 26 to 33. High-molecular-weight protein capable (like albumin) of binding ligand and aggregated ligand appear in the vicinity of the void volume in fractions 15 to 19. with either 2 /iCi of ^H-retinol, specific activity weight binding proteins, it was estimated that 2.66 Ci per millimole (New England Nuclear, human retinas contain about eight times more Boston, Mass.); or 1.1 /tCi of HH-retinoic acid, binding protein for retinoic acid than for reti- specific activity 1.45 Ci. per millimole, generously nol. provided by Hoffmann-La Roche. After the ad- When the soluble protein of fibroblasts was dition of 100 mg. of sucrose to increase the den- analyzed following incubation, no peak of radio- sity, samples were analyzed by gel filtration with activity was present in the low-molecular-weight the above buffer through Sephadex G-75 columns protein region (Figs. 1 and 2). We conclude of 1.5 by 143 cm. Following gel filtration 0.2 ml. that the binding proteins are absent from human samples of each fraction (5.2 ml.) were counted in fibroblasts. This finding may represent the bio- 10 ml. of scintillation fluid. chemical correlate of the observation that vitamin Results and discussion. Analysis of supernatant A is not an essential component of fibroblast from human retinal tissue incubated with either tissue culture media.8 3H-retinol or 3H-retinoic acid revealed a peak of radioactivity corresponding to a protein of low molecular weight (Figs. 1 and 2). When com- We are indebted to Dr. John W. Chandler for advice and the use of his facilities for growing pared to similarly analyzed samples from bovine fibroblasts and to Ms. Patricia G. Skahen for retina, the binding proteins were calculated (see assistance with tissue culture techniques. Fig. 1 legend) to migrate at the same rate, indi- cating the molecular weights of the human intra- cellular binding proteins to be approximately equal From the Department of Ophthalmology, Univer- to those of the bovine, previously found to be sity of Washington School of Medicine, Seattle, 4 Wash. This study was supported by National In- about 17,000 daltons. This molecular weight is stitutes of Health Research Grants EY 00343 and lower than the molecular weight of 21,000 dal- EY 00529 from the National Eye Institute, and in tons obtained for human and bovine serum part by an unrestricted research grant from Re- retinol-binding protein.9-10 The intracellular re- search to Prevent Blindness, Inc. Submitted for tinol-binding protein is also distinguished from publication July 12, 1976. Reprint requests: David the serum retinol-binding protein by the observed Swanson, M.D., Department of Ophthalmology, failure of serum retinol-binding protein to ex- RJ-10, School of Medicine, University of Wash- change unlabeled retinol for 3H-retinol during ington, Seattle, Wash. 98195. incubation. On the basis of the specific activity of 3 3 the H-retinol and H-retinoic acid employed and Key words: retinol, retinoic acid, human retina, radioactivity recovered in the low-molecular- vitamin A, binding proteins, human fibroblasts. Downloaded from iovs.arvojournals.org on 09/26/2021 1020 Reports Investigative Ophthalmology December 1976 REFERENCES The hypothesis that an abnormality in the 1.