Proc. Natl. Acad. Sci. USA Vol. 92, pp. 9176-9180, September 1995 Medical Sciences

Recoverin, a photoreceptor-specific calcium-binding , is expressed by the tumor of a patient with cancer- associated retinopathy ARTHUR S. POLANS*, DANUTA WITKOWSKA, TAMMIE L. HALEY, DRAKE AMUNDSON, LAWRENCE BAIZER, AND GRAZYNA ADAMUS R. S. Dow Neurological Sciences Institute, Legacy-Good Samaritan Hospital, Portland, OR 97209 Communicated by Lubert Stryer, Stanford University School of Medicine, Stanford, CA, June 13, 1995 (received for review February 6, 1995)

ABSTRACT Recoverin is a member ofthe EF-hand family in the gynecological tumors associated with these patients but of calcium-binding involved in the transduction of not in similar tumors obtained from individuals without neu- light by vertebrate photoreceptors. Recoverin also was iden- rological symptoms (5). Paraneoplastic syndromes ofthe visual tified as an autoantigen in the degenerative disease of the system also have been described (6-8). In patients with retina known as cancer-associated retinopathy (CAR), a para- cutaneous melanoma, diminished neurotransmission distal to neoplastic syndrome whereby immunological events lead to photoreceptors was associated with serum antibodies that the degeneration of photoreceptors in some individuals with stained a subset of bipolar cells. The retinal antigen has yet to cancer. In this study, we demonstrate that recoverin is ex- be identified (9-11), and retinal degeneration was not char- pressed in the tumor of a CAR patient but not in similar acteristic of the disorder. In contrast, cancer-associated reti- lung nopathy (CAR) is characterized by loss of photoreceptors, tumors obtained from individuals without the associated little change in the pigment epithelium or optic nerve head, retinopathy. Recoverin was identified initially by Western blot and limited signs of inflammation (12). Autoantibodies were analysis of the CAR patient's biopsy tissue by using anti- detected in the sera obtained from CAR patients and subse- recoverin antibodies generated against different regions ofthe quently used to identify the photoreceptor antigen as recov- recoverin molecule. In addition, cultured cells from the biopsy erin, a 23-kDa calcium-binding protein of the EF-hand family tissue expressed recoverin, as demonstrated by reverse tran- (13). Recoverin was detected in both rod and cone photore- scription-PCR using RNA extracted from the cells. The ceptor cells (14, 15), the primary sites of degeneration in the immunodominant region of recoverin also was determined in CAR syndrome, and significant biochemical information has this study by a solid-phase immunoassay employing overlap- since been obtained about the structure (16) and function ping heptapeptides encompassing the entire recoverin se- (17-20) of the protein. To determine whether recoverin could quence. Two linear stretches of amino acids (residues 64-70, induce immunological mechanisms that lead to the degener- Lys-Ala-Tyr-Ala-Gln-His-Val; and 48-52, Gln-Phe-Gln-Ser- ation of photoreceptors, rats were immunized with recoverin. Ile) made up the major determinants. One ofthe same regions High serum-antibody titers specific for recoverin, immuno- of the recoverin molecule (residues 64-70) also was uniquely competent T cells, and the degeneration ofphotoreceptor cells immunopathogenic, causing photoreceptor degeneration upon were observed in these animals (21, 22). immunization of Lewis rats with the corresponding peptide. The identity of the tumor antigen purportedly responsible These data demonstrate that the neural antigen recoverin for the CAR syndrome has not been ascertained. In this paper, more than likely is responsible for the immunological events we demonstrate that recoverin is expressed by the tumor of a associated with vision loss in some patients with cancer. These CAR patient. Identification was accomplished initially by data also establish CAR as one of the few autoimmune- Western blot analysis using lung tumor tissue from the patient, a series of CAR sera, and anti-peptide antibodies specific for mediated diseases for which the specific self-antigen is known. different regions of the recoverin molecule. Cultured cells derived from the patient's tumor tissue also expressed recov- Neurological disorders can be associated with cancer even erin, as demonstrated by reverse transcription-PCR (RT- though the tumor and its metastases have not entered the PCR) amplification of RNA extracted from the cells. The nervous system (1). These "remote effects" of cancer, or immunodominant region of recoverin also was determined in paraneoplastic syndromes, are thought to be autoimmune this study by ELISA using overlapping heptapeptides derived mediated. The expression of a tumor antigen supposedly leads from the recoverin sequence. The same region in the recoverin to an immunological response which then recognizes the same sequence proved to be immunopathogenic upon immunization antigen or a shared epitope in the nervous system. The of Lewis rats with the corresponding peptide. Other peptides autoimmune basis for a paraneoplastic disease was first dem- from the recoverin sequence were ineffective in eliciting onstrated for Lambert-Eaton myasthenic syndrome, a disor- photoreceptor degeneration. A simple model in which the der of the peripheral nervous system in which autoantibodies aberrant expression of recoverin in a subset of tumors leads to bind to presynaptic calcium channels at the neuromuscular specific autoantibodies, activated T cells, and concomitant loss junction (2, 3), thus interfering with the release of acetylcho- of photoreceptors forms the framework upon which further line and resulting in proximal muscle weakness and related experiments can be proposed. symptoms. In paraneoplastic cerebellar degeneration, a dis- ease of the central nervous system, autoantibodies to Purkinje MATERIALS AND METHODS cell antigens were detected in patients presenting with ataxia, nystagmus, and dysarthria (4). Upon autopsy, the afflicted Human eyes were obtained from the Lions Eye Bank of individuals displayed widespread loss of Purkinje cells in the Oregon, Legacy-Good Samaritan Hospital, Portland, OR. cerebellum. A Purkinje cell antigen subsequently was detected Abbreviations: CAR, cancer-associated retinopathy; SCCL, small cell carcinoma of the lung; RT-PCR, reverse transcription-PCR. The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed at: R. S. Dow payment. This article must therefore be hereby marked "advertisement" in Neurological Sciences Institute, Legacy-Good Samaritan Hospital accordance with 18 U.S.C. §1734 solely to indicate this fact. and Medical Center, 1120 NW 20th Avenue, Portland, OR 97209. 9176 Downloaded by guest on September 27, 2021 Medical Sciences: Polans et aL Proc. Natl. Acad. Sci. USA 92 (1995) 9177 Sera from CAR patients were obtained as reported previously (13) and according to approval by the Institutional Review OSL_ Board. Biopsy tissues were obtained from CAR patients, the Cooperative Human Tissue Network, Case Western Reserve ISL University, Cleveland, and the University of Florida, Gaines- ONL ville. Small cell carcinoma of the lung (SCCL) cell lines were obtained from the American Type Culture Collection. SCCL OPL cell line N417 was generously provided by Rhoda Manceckjee, Oregon Health Sciences University, Portland. Lewis rats were INL purchased from Harlan-Sprague-Dawley. IPL Recoverin Purification. Recoverin was isolated from human retinas as described (13, 23). Aliquots of the initial retinal homogenate were kept for SDS/PAGE and Western blot GCL analysis. Immunofluorescence Microscopy. Isolated human retinas FIG. 1. Fluorescence microscopy of human retinal cryosections stained with CAR serum (A) or CAR serum preadsorbed with 2.8 ,zg were fixed with 4% (wt/vol) paraformaldehyde in 0.1 M of purified human recoverin per ml (B). OSL, outer segment layer; sodium cacodylate buffer, pH 7.4, and embedded in acrylam- ISL, inner segment layer; ONL, outer nuclear layer; OPL, outer ide prior to freezing (13, 15). Cryosections (10 ,um) were plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; blocked to reduce nonspecific binding and incubated with sera GCL, ganglion cell layer. (1:350 dilution). Antibody binding was detected with biotin- ylated goat anti-human IgG (3 ,ug/ml) and streptavidin-Texas Animal Immunization. Female Lewis rats (150-180 g) were red conjugate (1 ,ug/ml). In some experiments, the primary inoculated in the hindfoot pad with a single dose of recoverin serum was preadsorbed with 1 ,ug of purified recoverin in a peptide (100 ,ug) as described (21). Peptides (20-mers) en- total volume of 350 t,l. compassing the entire recoverin sequence (residues 1-20, SDS/PAGE and Western Blot Analysis. Lung tumor tissues 21-40, and so on) were used as immunogens, and three animals and pelleted SCCL cells were disrupted in 125 mM Tris HCl, were immunized with each peptide. Eyes were removed 17 pH 6.8, containing 5 jig each of aprotinin and leupeptin per ml. days after immunization and fixed in 10% (vol/vol) buffered Proteins from the tumor homogenates and retinal homogenate formaldehyde prior to being embedded in paraffin. All animal and purified recoverin were resolved on an SDS/15% poly- procedures conformed to the Association for Research in acrylamide gel and electrotransferred to Immobilon (Milli- Vision and Ophthalmology resolution on the Use of Animals pore) (15). in Research. Lymphocyte-Proliferation Assay. Peripheral blood mono- nuclear cells were isolated by centrifugation by using His- RESULTS topaque 1077, and proliferative responses to recoverin were determined by measuring the incorporation of [3H]thymidine A 62-year-old male experienced sudden bilateral visual loss as described (21). Results are given in the form of a stimulation and complained of fuzzy central vision and a diminished sense index, calculated by dividing the mean cpm of cultures incu- of brightness in both eyes. Examination revealed both reduced bated with [3H]thymidine and recoverin by mean cpm of the color acuity and peripheral vision, although funduscopic and unstimulated control cultures incubated with only [3H]thymi- magnetic resonance imaging (MRI) studies were normal. dine. Severe dysfunction of both rods and cones characteristic of Epitope Mapping. Overlapping heptapeptides encompass- widespread photoreceptor degeneration was indicated by the ing the human recoverin sequence (24) were synthesized on patient's electroretinogram. There was no history or current polypropylene pins by following the manufacturer's recom- indication of cancer. mended procedures (Chiron Mimotopes, San Diego). 9-Flu- Detection of Anti-Recoverin Antibodies. Cryosections of orenylmethoxycarbonyl (FMOC)-protected amino acids and normal human retina were incubated with the patient's serum, 1-methyl-2-pyrrolidone were used during peptide synthesis. and antibody binding was detected by immunofluorescence Pins were blocked with 1% nonfat dry milk/0.5% bovine microscopy. As shown in Fig. IA, both rod and cone photo- serum albumin/0.1% Tween 20 in phosphate-buffered saline (PBS; 10 mM sodium phosphate, pH 7.4/150 mM NaCl). Sera, A a B b diluted in 1% bovine serum albumin in PBS, were incubated 94 .. _ _ with the pins for 2 h at room temperature. The binding of ,- antibodies was detected by incubation with anti-human IgG conjugated with horseradish peroxidase, followed by substrate (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) in 0.1 M citrate buffer, pH 5.0, containing 0.03% hydrogen peroxide for 15 min. 30 mo"W ELISA. Polyvinyl chloride microwell plates were coated with - recoverin and used to determine serum titers as described (25). Titers were determined by endpoint analysis compared with 20.1 normal human serum. Cell Culture. SCCL tumor biopsy tissue was obtained from the CAR patient and disrupted in RPMI medium 1640 con- taining 10% (vol/vol) fetal bovine serum and collagenase (200 14.4 units/ml) and then cultured for 18 hr. Cells were washed with fresh medium and cultured at a density of 5 x 105 cells per ml. and Western blot of a human retinal RT-PCR. RNA was isolated from pelleted SCCL cells FIG. 2. SDS/PAGE analysis (26) homogenate either stained with Coomassie blue (lane A) or incubated by using the Trizol reagent, and a cDNA corresponding to nt with CAR serum (lane a). SDS/PAGE and Western blot analysis of 100-216 of recoverin was cloned and sequenced as described purified human recoverin either stained with Coomassie blue (lane B) (27). or incubated with CAR serum (lane b). Downloaded by guest on September 27, 2021 9178 Medical Sciences: Polans et al. Proc. Natl. Acad. Sci. USA 92 (1995) receptors were stained in their entirety, while little labeling was 1 2 3 4 5 6 7 8 9 detected in more proximal portions of the retina. The immu- 94 nostaining demonstrated the presence of anti-retinal antibod- 67 -* -s ies characteristic of CAR (13, 28). Recoverin previously was identified as the photoreceptor autoantigen associated with 43 the CAR syndrome; therefore, immunohistochemistry was repeated with the patient's serum preincubated with purified 30 human recoverin. Fig. 1B shows that immunostaining was reduced significantly by preincubation with recoverin, thereby 20.1 - - demonstrating the specificity of the reaction shown in Fig. 1A. Western blot analysis was performed to confirm the pres- ence of anti-recoverin antibodies in the patient's serum. Pro- 14.4 teins from a human retinal homogenate were resolved by SDS/PAGE and either stained with Coomassie blue (Fig. 2, lane A) or transferred to Immobilon and incubated with the patient's serum (Fig. 2, lane a). A single immunoreactive band was detected at 23 kDa, which coincided with the molecular FIG. 3. Western blot analysis of CAR patient tumor tissue stained with CAR patient's own serum (lane 1), sera from two other CAR mass of human recoverin (24). Purified human recoverin also patients (lanes 2 and 3), normal human serum (lane 4), anti-N- was subjected to SDS/PAGE and stained with Coomassie blue terminal recoverin antibodies (lane 5), anti-C-terminal recoverin (Fig. 2, lane B) or electrotransferred and incubated with the antibodies (lane 6), or antibodies against residues 104-118 of the patient's serum (Fig. 2, lane b). The 23-kDa recoverin band recoverin sequence (lane 7). Western analysis also was performed with was immunostained. The presence of specific anti-recoverin tumor tissue isolated from a non-CAR patient diagnosed with SCCL antibodies in the patient's seruim helped confirm the diagnosis and stained with either anti-recoverin antibodies to the native protein of CAR. Western analysis also demonstrated the lack of (lane 8) or the same CAR serum used in lane 1. The positions of immunoreactivity associated with other potentially uveito- molecular mass standards (in kDa) are given on the left. genic proteins such as S-antigen (), interphotoreceptor retinoid-binding protein, or (Fig. 2, lane a). antibodies generated against peptides derived from different The level of anti-recoverin antibodies in the patient's serum regions of the recoverin sequence (15). Antibodies to the N was determined by ELISA over a period of six months; the and C termini of recoverin (Fig. 3, lanes 5 and 6, respectively) initial titer was 1:3000 and then increased to 1:6000. The recognized the same 23-kDa band, as did anti-peptide anti- response of the patient's peripheral blood lymphocytes to bodies to residues 104-118 (Fig. 3, lane 7). Proteins extracted recoverin was measured by a cell proliferation assay. The from an SCCL obtained from a patient without visual system results showed that the patient's lymphocytes responded mod- symptoms (25) were subjected to Western blot analysis with erately to recoverin stimulation (stimulation index = 2.8) when either anti-recoverin antibodies raised against the native pro- compared with the response of lymphocytes taken from nor- tein (Fig. 3, lane 8) or serum from a CAR patient (Fig. 3, lane mal individuals (stimulation index < 1.5), further suggesting an 9): no immunostaining was observed. The same result involvement of the immune system in the CAR syndrome. was Identification of Recoverin in the CAR Patient's Tumor. obtained when using other tumor tissues (Fig. 4A, lanes 1-3), Following serological testing, a chest x-ray revealed the pres- as well as when using cell lines (Fig. 4A4, lanes 4-10), from ence of a hilar mass in the patient's right lung which was later non-CAR patients with SSCL. Nonspecific bands were re- diagnosed by bronchoscopy as small cell carcinoma, interme- vealed by their reaction with normal rabbit serum (Fig. 44, diate cell variant. A protein extract from the tumor tissue then lane 11). was used for Western blot analysis. The patient's own serum, RNA was extracted from the CAR patient's SCCL cell line, containing anti-recoverin antibodies, stained a prominent as well as from SCCL tissues and cell lines from non-CAR protein band at 23 kDa (Fig. 3, lane 1). A higher molecular patients, to confirm the specific expression of recoverin. mass band reacted with the patient's serum but was labeled by Oligonucleotide primers flanking a 117-nt region ofthe human normal human serum as well (Fig. 3, lane 4). The 23-kDa band recoverin mRNA sequence were used for PCR. RNA extracted was immunoreactive with two additional CAR sera (Fig. 3, from the CAR patient's SCCL cell line produced a PCR lanes 2 and 3). Since human recoverin migrates at 23 kDa, lanes product of the expected size (Fig. 4B), while preparations from of the tumor biopsy material were stained with anti-recoverin non-CAR patients did not generate specific PCR products A B C 1 2 3 4 5 6 7 8 9 1011 1 9 A E r 7 P 01 0 94 - 67- 40- 3 0- 20.1- 14 .4-

FIG. 4. Western blot and RT-PCR analyses of SCCL tumor tissues and cell lines from non-CAR patients. (A) Proteins from tumor tissues (lanes 1-3) and cell lines (HTB171, N417, HTB119, HTB172, HTB175, HTB120, and HTB173; lanes 4-10, respectively) derived from non-CAR patients were stained with rabbit anti-recoverin antibodies. No recoverin was detected at 23 kDa. Nonspecific bands were detected when using normal rabbit serum (lane 11). (B) RNA extracted from the CAR patient's SCCL cell line was used for RT-PCR, and the products were resolved on an agarose gel and stained with ethidium bromide. A PCR product of the expected size was detected (left lane). Right lane consists of molecular size standards (123-bp ladder, BRL). (C) RNA extracted from the same non-CAR tumor tissues and cell lines as in A were processed for RT-PCR as in B. No PCR products corresponding to the expected mobility of recoverin were detected. The order of samples in C is the same as in A. Downloaded by guest on September 27, 2021 Medical Sciences: Polans et al. Proc. Natl. Acad. Sci. USA 92 (1995) 9179

2.0 100 TCCTTCCTGAAGGACTGTCCCACCGGCCGCATCACCCAG 138 TCCTTCCTGAAGGACTGTCCCACCGGCCGCATCACCCAG 34 S F L K D C P T G R I T Q 46 1.5

139 CAGCAGTTCCAGAGCATCTACGCCAAGTTCTTCCCCGAC 177 1.0 CAGCAGTTCCAGAGCATCTACGCCAAGTTCTTCCCCGAC 47 Q Q F Q S I Y A K FF P D 59 0.5 178 ACCGACCCCAAGGCCTACGCCCAGCATGTGTTCCGCAGC 216 11 kal. Im ACCGACCCCAAGGCCTACGCCCAGCATGTGTTCCGCAGC 4 60 T D P K A Y A Q H V F R S 72 0 20 40 60 80 100 120 140 160 180 200

FIG. 5. Expression of recoverin by cultured SCCL cells from a 3 CAR patient's tumor. RT-PCR amplification with RNA isolated from the CAR patient's SCCL cells demonstrated 100% identity between the known human recoverin sequence (top line) and the PCR product (middle line). Arrows indicate the PCR primers used for amplification. (Fig. 4C). Sequencing the PCR product derived from the CAR patient's cultured cells revealed 100% identity with the known recoverin sequence (Fig. 5). These results demonstrate that recoverin could be detected only in the tumor of the CAR patient. 0 20 40 60 80 100 120 140 160 180 200 Determining Immunodominant and Immunopathogenic Regions of Recoverin. Serum autoantibodies and T cells specific for recoverin were detected both in CAR patients and 0.8 in rodents immunized with recoverin (21). To characterize these responses further, the fine specificity for autoantibody binding to recoverin was determined. Peptides encompassing the entire human recoverin sequence were synthesized on pins 0.4 as overlapping heptapeptides (amino acids 1-7,2-8 and so on) and used in a solid-phase ELISAwith the CAR patient's serum to identify major determinants. Fig. 6 Top illustrates the ...... results. A major determinant was defined in the recoverin 0 20 40 60 80 100 120 140 160 180 200 sequence at amino acid residues 64-70 (Lys-Ala-Tyr-Ala-Gln- His-Val) and at a second site near residues 48-52 (Gln-Phe- Recoverin residue number Gln-Ser-Ile). These determinants did not change significantly FIG. 6. Epitope mapping of recoverin with sera from CAR patients between serum samples obtained over a 3-month period. In using a pin ELISA. Heptapeptides are numbered according to their addition, the same regions of the recoverin sequence were position in the human recoverin sequence and the residue at the N recognized by the sera of different CAR patients in this (Fig. terminus of the heptapeptide. (Top) CAR patient reported in this 6 Middle and Bottom) and a previous study (25). The similarity study. (Middle and Bottom) Two additional sera from different CAR between these CAR sera does not exclude the possibility that patients. Normal human sera resulted in absorbances of 0.15-0.20 for other patients may display additional or different binding sites, all peptide pins. depending upon their genetic predisposition. One of the immunodominant regions of the recoverin a-helix of EF-hand 2, is relatively sequestered near a hydro- molecule defined for these CAR patients also is pathogenic. phobic crevice (16), and is not predicted to be highly antigenic Lewis rats were immunized with peptides corresponding to based on hydrophilicity alone. different regions of the recoverin sequence. Immunization with a peptide comprising residues 61-80, containing the DISCUSSION major antibody determinant, resulted in signs of uveoretinitis and complete loss of photoreceptors (Fig. 7A). The features Although paraneoplastic syndromes are of unknown etiology, associated with this peptide-induced retinal degeneration were cumulative evidence supports an autoimmune component to the same as those reported for native recoverin (21). Immu- these diseases. Autoantibodies have been detected in the sera nization with other peptides, including one encompassing of patients afflicted with cancer and a wide variety of neuro- amino acid residues 48-52, did not lead to the degeneration of degenerative disorders, and at least one neural antigen has photoreceptors and resembled control conditions in which been identified in the tumors of the afflicted individuals (5). saline was substituted for recoverin (Fig. 7B). Interestingly, From previous studies, however, it is unclear whether the amino acid residues 64-70 promote both autoantibody binding tumor and the affected site in the nervous system share only and pathogenicity. This sequence is contained within the first a common sequence or the entire antigen. In the current study, FIG. 7. Histopathological changes A B in the retinas of Lewis rats immunized with a peptide comprising amino acids OSL 61-80 derived from the recoverin se- ONL quence (A) or saline control (B). The asterisk in A indicates the loss of pho- OPL toreceptor outer and inner segments, ^ * . while the arrow indicates remnant ~~ w"gNX^* z . INL bodies. Abbrevia- tions are the same as in the legend to GCL Fig. 1. Downloaded by guest on September 27, 2021 'Ft *4- -e~j>X;i*'tfflIPL 9180 Medical Sciences: Polans et al. Proc. Natl. Acad. Sci. USA 92 (1995)

recoverin was detected in the tumor of the CAR patient, thus autoimmunity, and neural degeneration and may provide supporting the hypothesis that the aberrant expression of the useful insights for the study of other ocular diseases. entire neural antigen underlies the immunological response leading to neural dysfunction. This research was supported by grants from the National Eye Both autoantibodies and T cells specific for recoverin have Institute (EY07089), the National Science Foundation (IBN-9409721), been detected in CAR patients, and photoreceptor degener- the Medical Research Foundation of Oregon, the Oregon Lions Sight ation ensues. The simplest model to account for these findings and Hearing Foundation, and the Legacy Research Services. consists of (i) the expression of recoverin in a subset of tumors 1. Brain, L. & Norris, F. H. (1965) The Remote Effects of Cancer on an response for recoverin, and (ii) immunological specific the Nervous System (Grune & Stratton, New York). which then leads to (iii) an immune-mediated degeneration of 2. Newsom-Davis, J. (1985) Seminars in Immunopathology (Spring- photoreceptors, the normal site of recoverin expression. Po- er, New York), Vol. 8, pp. 129-140. tential antigens like recoverin normally are unavailable for 3. Kim, Y. I. & Neher, E. (1988) Science 239, 405-408. immunological recognition at the inductive stage of the im- 4. Greenlee, J. E. & Lipton, H. I. (1986) Ann. Neurol. 19, 82-85. mune response due to sequestration within photoreceptors, an 5. Furneaux, H. M., Rosenblum, M. K., Dalmau, J., Wong, E., immune-privileged site. However, recoverin released by the Woodruff, P., Graus, F. & Posner, J. B. (1990) N. Engl. J. Med. tumor can trigger both cellular and humoral responses. 322, 1844-1851. The immunological events leading to photoreceptor degen- 6. Sawyer, R. A., Selhorst, J. B., Zimmerman, L. E. & Hoyt, W. F. eration can be partially reproduced in animals immunized with (1976) Am. J. Ophthalmol. 81, 606-613. S. E., Klein, R., Appen, R. & Choate, J. (1982) Cancer recoverin (21, 22), and an extensive evaluation of the animal 7. Kornguth, 50, 1289-1293. model is provided in the citations. After inoculation of Lewis 8. Keltner, J. L., Roth, A. M. & Chang, R. S. (1983) Arch. Ophthal- rats with recoverin, antibodies to recoverin but not to known mol. 101, 564-569. uveitogens, such as S-antigen (arrestin), interphotoreceptor 9. Alexander, K. R., Fishman, G. A., Peachey, N. S., Marchese, retinoid-binding protein, or rhodopsin, could be detected. A. L. & Tso, M. 0. M. (1992) Invest. Ophthalmol. Visual Sci. 33, Also, T cells specific for recoverin were detected. Therefore, 477-483. no secondary response occurred to other retinal antigens that 10. Milam, A. H., Saari, J. C., Jacobson, S. G., Lubinski, W. P., Feun, might be released during photoreceptor degeneration. Like- L. G. & Alexander, K. R. (1993) Invest. Ophthalmol. Visual Sci. wise, no responses to uveitogenic proteins other than recoverin 34, 91-100. were observed in the CAR patient (Fig. 2). An epiphenomenal 11. Weinstein, J. M., Kelman, S. E., Bresnick, G. H. & Kornguth, event involving induction by other retinal antigens, therefore, S. E. (1994) Ophthalmology 101, 1236-1243. 12. Buchanan, T. A. S., Gardiner, T. A. & Archer, D. B. (1984) Am. cannot explain the loss of photoreceptors; rather, a specific J. Ophthalmol. 97, 277-287. immunological response to recoverin more than likely under- 13. Polans, A. S., Buczylko, J., Crabb, J. & Palczewski, K. (1991) J. lies the degeneration. Cell Biol. 112, 981-989. In patients, the degeneration of photoreceptor cells is 14. Dizhoor, A., Ray, S., Kumar, S., Niemi, G., Spencer, M., Brolley, associated with the presence of anti-recoverin antibodies. How D., Walsh, K., Philipov, P., Hurley, J. & Stryer, L. (1991) Science the immune response leads to photoreceptor degeneration 251, 915-918. and, specifically, the role of autoantibodies in this have yet to 15. Polans, A. S., Burton, M. D., Haley, T. L., Crabb, J. W. & Palc- be determined. In animal studies (21), the retinopathy was zewski, K. (1993) Invest. Ophthalmol. Visual Sci. 34, 81-90. produced in Lewis rats by passive transfer of activated T cells 16. Flaherty, K. M., Zozulya, S., Stryer, L. & McKay, D. B. (1993) obtained from animals immunized with recoverin, but the Cell 75, 709-716. not stud- 17. Zozulya, S. & Stryer, L. (1992) Proc. Natl. Acad. Sci. USA 89, pathogenicity of anti-recoverin antibodies has been 11569-11573. ied. 18. Dizhoor, A. M., Chen, C.-K., Olshevskaya, E., Sinelnikova, V. V., An additional unresolved issue pertains to the expression of Phillipov, P. & Hurley, J. B. (1993) Science 259, 829-832. recoverin in a subset of tumors. The encoding recoverin 19. Kawamura, S. (1993) Nature (London) 362, 855-857. maps to human chromosome 17p13.1 (29, 30), which coincides 20. Gray-Keller, M. P., Polans, A. S., Palczewski, K. & Detwiler, with the position of tumor suppressor gene p53. A low- P. B. (1993) Neuron 10, 523-531. frequency mutation in p53 might explain both the develop- 21. Adamus, G., Ortega, H., Witkowska, D. & Polans, A. (1994) Exp. ment of the tumor and the expression of recoverin. This no Eye Res. 59, 447-456. doubt is a simplistic model, since the fine mapping between p53 22. Gery, I., Chanaud, N. P., III, & Anglade, E. (1994) Invest. and recoverin has not been performed. In addition, it is not Ophthalmol. Visual Sci. 35, 3342-3345. is the sole retinal expressed 23. Polans, A. S., Crabb, J. & Palczewski, K. (1993) in Methods in known whether recoverin antigen Neuroscience: Photoreceptor Cells, ed. Hargrave, P. (Academic, by the CAR patient's tumor. Recently, other retinal and neural New York), Vol. 15, pp. 248-260. have been mapped to the vicinity of recoverin and p53, 24. Wiechmann, A. F. & Hammarback, J. A. (1993) Exp. Eye Res. 56, thereby making it possible to determine whether other poten- 463-470. tial antigens are expressed in the tumors of CAR patients. 25. Adamus, G., Guy, J., Schmied, J. L., Arendt, A. & Hargrave, While autoimmune diseases are widespread, CAR is among P. A. (1993) Invest. Ophthalmol. Visual Sci. 34, 2626-2633. a select few for which the identity of the self-antigen is known 26. Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem. 162, 156- (31). Paraneoplastic syndromes like CAR, Lambert-Eaton 159. myasthenic syndrome, and paraneoplastic cerebellar degener- 27. Baizer, L., Ciment, G., Hendrickson, S. K., & Schafer, G. L. ation offer unique opportunities to identify self-antigens, since (1993) .F Neurochem. 61, 2054-2060. J. J. Jr. 99, can be detected in the as well as at the site 28. Rizzo, F., III, & Gittinger, W., (1992) Ophthalmology they patient's tumor, 1286-1295. of neural dysfunction. In contrast, while substantial evidence 29. Murakami, A., Yajima, T. & Inana, G. (1992) Biochem. Biophys. supports a role for S-antigen and interphotoreceptor retinoid- Res. Commun. 187, 234-244. binding protein as targets of the immune response in uveitis, 30. Wiechmann, A. F., Akots, G., Hammarback, J. A., Pettenati, an autoimmune disorder of the eye, the identity of the antigen M. J., Rao, P. N. & Bowden, D. W. (1994) Invest. Ophthalmol. at the inductive stage of the disease is less certain. Although Visual Sci. 35, 325-331. CAR is a rare disease, it is pertinent to studies of cancer, 31. Steinman, L. (1995) Cell 80, 7-10. Downloaded by guest on September 27, 2021