Autoantibodies Against Retinal Bipolar Cells in Cutaneous Melanoma-Associated Retinopathy

Transcription

1 Autoantibodies Against Retinal Bipolar Cells in Cutaneous Melanoma-Associated Retinopathy Ann H. Milam,* John C. Saari*-\ Samuel G. Jacobson,% Wojciech P. Lubinski,% Lynn G. Feun,% and Kenneth R. Alexander Purpose. This study's goal was to determine the pathophysiology of the retinopathy that occurs in patients with metastatic cutaneous melanoma and sudden onset of night blindness, the so-called melanoma-associated retinopathy (MAR) syndrome. We tested the hypothesis that sera from two MAR patients contained autoantibodies that reacted with "on" bipolar cells of the human retina. Methods. Immunofluorescence was performed on cryostat sections of unfixed normal human retinas. Sera and IgG fractions were tested from the two MAR patients and 38 control subjects (28 patients with metastatic melanoma, but no visual symptoms; two patients with non-mar retinopathy; and eight normal subjects). Results. The sera and IgG fractions from both MAR patients but from none of the control subjects produced heavy immunostaining of bipolar cells, which were identified as rod bipolars by a double labeling procedure using anti-protein kinase C. Conclusions. We hypothesize that MAR patients generate autoantibodies against a melanoma antigen that cross react with bipolar cells of the retina. These antibodies, by an unknown mechanism, may cause abnormalities of the rod and cone systems that are characteristic of MAR. Invest Ophthalmol Vis Sci. 1993;34: JL wo different types of paraneoplastic retinopathy have been described: cancer-associated retinopathy (CAR), 1 " 10 ' and cutaneous malignant melanomaassociated retinopathy (MAR). 11 " 14 In CAR, which is From the Departments of *Ophthalmology and f Biochemistry, University of Washington, Seattle; Department of%ophthahnology, Bascom Palmer Eye Institute, and ^Department of Medicine, Sylvester Comprehensive Cancer Center, University of Miami, Miami, Florida; and ^Department of Ophthalmology, University of Illinois at Chicago, Chicago, Illinois. Supported by the National Retinitis Pigmentosa Foundation, Inc., Baltimore, Maryland; by NIH grants EY01311 (AHM), EY02317 (JCS), EY0I730, EY05627 (SCJ), and EY08301 (KRA); by a departmental award from Research to Prevent Blindness, Inc., New York, New York; and by the Chatlos Foundation, Inc., Longwood, Florida. Ann H. Milam is a Senior Scholar and Samuel G. Jacobson iv a Dolly Green Scholar of Research to Prevent Blindness, Inc. Submitted for publication: June 16, 1992; accepted August 8, Proprietary interest Category: N. Reprint requests to: Ann H. Milam, Department of Ophthalmology, RJ- 10, University of Washington, Seattle, WA associated with small-cell carcinoma of the lung and other tumors, patients may have visual symptoms before the identification of the tumor, and progressive visual loss typically evolves over weeks to months. There is clinical, electroretinographic and histopathologic evidence of dysfunction and death of rod and cone photoreceptors. 1 " 10 The sera of some CAR patients contain antibodies that react with photoreceptors and ganglion cells of the retina. 3 " 9 Recent reports indicate that at least one of the retinal antigens is recoverin, a calcium-binding protein isolated initially from bovine rod outer segments. 15 " 18 A working hypothesis for the pathophysiology of CAR is that antibodies generated against a cancer cell antigen cross react with certain cells of the retina and cause retinal pathology. In MAR, patients have a relatively acute onset of night blindness months to years after diagnosis of metastatic cutaneous melanoma. They typically have a sen- Invesiigative Ophthalmology & Visual Science, January 1993, Vol. 34, No. 1 Copyright Association for Research in Vision and Ophthalmology 91

2 92 Investigative Ophthalmology & Visual Science, January 1993, Vol. 34, No. 1 sation of shimmering lights, elevated dark-adapted thresholds, and an electroretinogram (ERG) resembling that found in some forms of congenital stationary night blindness (CSNB). 11 " 14 Results of retinal function tests on MAR patients suggest that, unlike CAR, photoreceptor function is intact but that signal transmission between photoreceptors and secondorder interneurons of the retina is defective. 11 " 14 Specifically, involvement of "on" bipolar cells was suggested. 13 A recent report indicated that serum from one MAR patient showed evidence of antibodies reacting against retina (components not specified) but not against the retinal CAR antigen. 13 To our knowledge, no detailed immunocytochemical or biochemical studies of MAR patients have been published. We had the opportunity to examine a patient with metastatic cutaneous melanoma who had visual function test results like those described previously for MAR patients. In this report, we demonstrate that sera from this patient and another well-documented MAR patient 13 cross react with human retinal bipolar cells, and we correlate this new observation with the retinal function abnormalities in MAR. We also document clinical, retinal function and serologic findings in both MAR patients before and after treatment with oral corticosteroids, a trial prompted by the recent report that steroid treatment was valuable in a patient with CAR. 10 MATERIALS AND METHODS Case History In June 1987, a 36-year-old man (MAR patient no. 1) had a cutaneous malignant melanoma removed from his left lower leg. In December 1990, left inguinal lymph nodes were found to be positive for melanoma and were excised. Clinical and radiologic examinations since that time have not revealed further evidence of metastatic disease. No chemotherapy has been administered. In January 1991, the patient experienced an abrupt onset in his left eye of "moving lights" that appeared to encircle fixation. A week later, he noted that night vision was darker in the left eye than in the right eye. There was further loss of night vision in the left eye over several days, more evident centrally than peripherally. He then noted an interocular asymmetry in visual acuity, with the left eye having a localized central blur. Vision in the left eye in daylight also seemed brighter and colors were more vivid. In March 1991, night vision in the his right eye diminished over several days until the level of night vision disturbance became equal in both eyes. Lights also became bright in the right eye under daylight conditions. According to clinical records, the ERGs performed at that time showed "a decreased scotopic b-wave and normal photopic responses, as seen in congenital stationary night blindness." By September 1991, the abnormal brightness in both eyes under daylight conditions was less pronounced, but there was a sensation of "looking through water." He also described prolonged "after images." In December 1991, the patient was evaluated at the Bascom Palmer Eye Institute. Best corrected visual acuity was 20/20 (+0.50) OD and 20/40 (+0.50) OS. With an Amsler grid, the patient described a small central "gray zone" in the left eye but no distortion; the right eye had no abnormalities. Color vision tested with the Farnsworth Panel D-15 was normal in the right eye but abnormal in the left eye without a specific axis of confusion. Goldmann kinetic perimetry with the V-4e target showed full visualfields;the I-4e target was detectable only within the isopter. No afferent pupillary defects were present and intraocular pressures were normal. Slit-lamp examination revealed normal anterior segments, but there were fine cells in each vitreous. On funduscopic examination, the retinal pigment epithelium appeared normal (except for slight mottling in the mid periphery), and the foveolar reflex appeared broader and flatter in the left eye than in the right. Retinal arteries were slightly attenuated. A serum sample was taken. A trial of oral corticosteroids was started in the third week of January 1992 at 60 mg/day of prednisone. The dosage was reduced by 5 mg/wk until March 13, 1992, when the dose was 20 mg/day. This level of prednisone was maintained. Serum samples were taken on January 17, 1992, before prednisone was initiated and then at 2 wk intervals until February 28, On March 31, 1992, the patient was re-evaluated at the Bascom Palmer Eye Institute. While on steroids, he noted a diminution of the sensation of "looking through water" and a decrease in the annoying brightness of lights, but no noticeable difference in night vision. Visual acuities, Farnsworth Panel D-15 results, and the clinical examination were unchanged. Kinetic visualfieldswere unchanged with the V-4e target, but the I-4e target was now detectable to the isopter. A fluorescein angiogram showed subtle RPE transmission in the periphery of both eyes and widening of the foveal avascular zone in the left eye. A serum sample was repeated. Subjects Two patients with MAR (patients no. 1 and no. 2) were included in this study. Details of the clinical course and retinal function abnormalities of patient no. 2 were published. 13 Sera from the two MAR patients and 38 other subjects were evaluated by immunocytochemical and biochemical techniques. The other subjects in-

3 Autoantibodies Against Retinal Bipolar Cells in MAR 93 eluded 28 patients with cutaneous malignant melanoma (ages yr) who had no visual symptoms (four patients at Stage I; one at Stage II; six at Stage III; and 17 at Stage IV); one patient with autosomal recessively inherited CSNB (age 73); one patient with birdshot chorioretinopathy (age 38); and eight normal subjects (ages 21-59). Visual function tests were performed on MAR patient no. 1 and on five patients with CSNB. The CSNB patients were as follows: One patient (age 73) had autosomal recessive CSNB; three patients (ages 9, 21, and 25) had X-linked CSNB; and one patient had multiplex CSNB (age 42). Informed consent was obtained from all participating subjects. Visual Function Tests Two-color (500 nm and 650 nm) dark-adapted static threshold perimetry was performed using a full field test strategy of 75 loci on a 12 grid. S (blue) cone perimetry was performed with a 440 nm stimulus on a yellow adapting field using a central test of 48 loci on a 4 grid. Spectral sensitivity was measured with 15 monochromatic targets in the dark-adapted state and in the presence of a white background (10 cd/m 2 ). Details of these methods have been published. 19 " 22 Full field rod, mid spectral cone, and S cone ERGs were performed according to methods previously described. 23 " 25 Waveforms were measured conventionally. 23 Light-adapted ERGs to 1 Hz flashes of white light also were analyzed after filtering with a bandpass of Hz. Immunocytochemistry Normal human retinas 3 hr or less post mortem were obtained from the University of Washington Lions' Eye Bank. Samples from retinas either unfixed or fixed (4% paraformaldehyde in 0.1 mol/1 phosphate buffer (ph 7.4) at room temperature; 15 min to 6 hr) were infiltrated with 30% sucrose in the same buffer at 4 C, cryosectioned at 12 ixm thickness, mounted on subbed slides, air dried, and stored at 20 C. Plastic rings were mounted around the sections to form incubation wells, and the sections were pretreated for 30 min at room temperature in a blocking solution that contained 1% horse serum, 1% bovine serum albumin and 0.05% Triton X-100 in phosphate buffered saline (PBS), followed by overnight incubation at 4 C in the serum or immunoglobulin (IgG) fraction from each patient at a dilution of 1:100 in PBS with 0.3% Triton X-100. We chose this dilution after determining that less dilute control sera or IgG fractions produced higher background or nonspecific staining of the retinas. The sections were rinsed in PBS for 30 min (two times) at room temperature, incubated for 1 hr at room temperature in secondary antibody (goat antihuman IgG labeled with fluorescein isothiocyanate [FITC] at 1:50 in PBS with 0.3% Triton X-100), rinsed with PBS for 30 min (twice) at room temperature, and coverslipped in 90% glycerol in PBS containing 2% l,4-diazabicyclo(2,2,2) octane. For double labeling experiments, cryostat sections were incubated overnight at 4 C with a mixture of patient IgG (1:100) and an antibody made in mouse against protein kinase C, a specific marker for rod bipolar cells 26 (1:50; Seikagaku Kogyo Co., Letc. Chuo-ku, Tokyo, Japan). After a rinse in PBS, the sections were treated with a mixture of secondary antibodies (goat anti-human and antimouse IgG) labeled with FITC and rhodamine, respectively. The sections were processed as just described and photographed as consecutive exposures of the same field with alternate filters for FITC and rhodamine. 27 Double labeling experiments also were performed using MAR IgG and an antibody (R6B6, 1:1000) prepared in rabbit that recognizes glycine-extended cholecystokinin precursors, a marker for blue cone bipolar cells, 28 or with MAR IgG and an antibody preparation made in rabbit against recoverin (1:250), a marker for certain cone bipolar cells. 29 Biochemistry IgG was isolated from 1 ml of serum with 0.5 ml Hi- Trap columns of protein A-Sepharose (Pharmacia Fine Chemicals, Inc., Piscataway, NJ) using the manufacturer's recommended procedure. IgG fractions and sera were stored frozen at 80 C. Human retinas were homogenized in 0.25 mol/1 sucrose, 10 mmol/1 3-[N-morpholino]propane sulfonic acid, ph 7, 0.1 jumol/1 leupeptin, 1 mmol/1 phenylmethane sulfonyl fluoride, 1 mmol/1 dithiothreitol (all from Sigma Chemical Corp.; St. Louis, MO) with five strokes of a glass/glass Tenbroeck homogenizer. Supernatant and pellet fractions were generated by centrifugation at 100,000 X g for 1 hr and by resuspending the pellet to the original volume in the above buffer without sucrose. Retinal fractions were stored frozen at 80 C in small portions. Samples of retinal homogenate, supernatant, or pellet were analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) systems run at alkaline ph 30 or neutral ph. 31 A Hoefer (Hoefer Scientific Instruments, San Francisco, CA) Mini-Gel apparatus was employed with 0.75 mm-thick gels. Samples were dissolved in the appropriate sample application buffer and immediately immersed in boiling water for 2 min. Approximately 75 /ig of protein was applied to each lane. Conditions for neutral PAGE included 7.5% gels, a ratio of acrylamide to bis-acrylamideof 27:1, a running ph of 7.2, and no stacking gel. Conditions for alkaline ph PAGE included 7.5% gels, a ratio of acrylamide to bis-acrylamide of 38:1, a running ph of 8.8, and 10% stacking gel. Gels or polyvi-

4 94 Investigative Ophthalmology 8c Visual Science, January 1993, Vol. 34, No. 1 nylidene difluoride (PVDF) blots were stained with Coomassie blue. 31 Molecular weight standards were obtained from Diversified Biotech (Newton Centre, MA; pre-stained, mid-range kit). For Western blotting, separated proteins were transferred to PVDF membranes using conditions previously described. 32 PVDF transfers were probed with sera or IgG fractions at 1:100 dilution, according to the instructions obtained with an alkaline phosphatase-based system (ProtoBlot; Promega Corp., Madison, WI) or a horseradish peroxidase-based system (VectaStain ABC; Vector Laboratories, Inc., Burlingame, CA). For enzyme-linked immunosorbent assay (ELISA), microtiter plates were coated with human retinal supernatant (homogenized in buffer or buffered detergent), diluted to a concentration that produced a color response about 10% above background with control antisera (about 38 fxg/m\). Serial dilutions of control and patient sera and IgG fractions then were analyzed by ELISA using a previously described procedure. 31 RESULTS Visual Function Tests Figure 1 (upper) compares the ERG results from MAR patient no. 1 with those from a normal subject and a patient with CSNB. The dark-adapted ERGs of the CSNB and MAR patients both showed an abnormally decreased b-wave amplitude at lower stimulus intensities, and the b- to a-wave ratio was reduced at higher intensities. The light-adapted ERGs of the CSNB patient also appeared different from normal, a finding that has been attributed to the absence of an "on" component 13 and to abnormal oscillatory potentials (OPs). 33 " 35 The light-adapted MAR waveforms resembled those of CSNB but were not exactly the same. Figure 1 (lower left) shows that the difference in appearance of light-adapted waveforms in CSNB and MAR may be partly the result of a difference in OPs. The filtered CSNB waveform had a less complex pattern of OPs than normal, 33 " 35 but the same analysis of the MAR waveform indicated that OPs were very reduced, if not unmeasurable. A similar finding was obtained in MAR patient no. 2. The cone flicker ERG of the MAR patient (Fig. 1, lower right) also differed from those of normal subjects and CSNB patients. Although normal in amplitude, the MAR flicker ERG had very slow time to peak (42 ms, OD; 41 ms, OS) compared to normal (mean 27 ms; SD = 1.0; n = 37) 24 and to the range of results in CSNB patients (30-35 ms; n = 5). Figure 2 (upper) shows dark-adapted perimetry results in the MAR patient. There was no measurable rod sensitivity in the central visual field and very reduced rod sensitivity elsewhere in the field. Mean rod NORMAL NORMAL CSNB MAR OSCILLATORY POTENTIALS CONE FLICKER FIGURE l. Electroretinograms in a normal subject, a 21-yearold man with X-linked CSNB, and MAR patient no. 1. (Top) ERGs to different intensities of white light stimuli in the dark- and light-adapted states. The five relative stimulus intensities in log units are, from bottom to top: 1.1, 0.7, -0.4, -0.2, and 0. (Bottom left) Oscillatory potentials from filtering ( Hz) the light-adapted waveform to stimulus intensity (Bottom right) Cone flicker ERGs (29 Hz) to white light stimuli at intensity 0.7. Stimulus onset is at trace onset. sensitivity loss across the visualfieldwas 36 db (SD, 3.0 db), falling within the range found in our CSNB patients (28-41 db; n = 4). A dark-adapted spectral sensitivity function from a superior field locus (Fig. 2, lower left) illustrates that rod-mediated function was measurable, albeit severely reduced. Cone sensitivity to the 650 nm stimulus, dark-adapted, was reduced at most test loci (compared to normal results to 650 nm at the cone plateau). 21 Mean loss was 11 db (SD, 4.0 db), which also is within the range for CSNB patients (6-12 db; n = 4). S cone function in the MAR patient was assessed with psychophysics and the ERG. With S cone perimetry, there was no detectable function, except at fixation, where sensitivity was reduced by about 10 db. A light-adapted spectral sensitivity function from an infe-

5 Autoantibodies Against Retinal Bipolar Cells in MAR il ROD SENSITIVITY LOSS S 36 - CONE SENSITIVITY LOSS 12 - o CE 1- LU O o LU N T N T ECCENTRICITY (deg) DARK-ADAPTED LIGHT-ADAPTED " 400 WAVELENGTH (nm) FIGURE 2. (Top) Gray scale displays of rod (measured at 500 nm) and cone (measured at 650 nm) sensitivity losses across the visual field of the right eye of MAR patient no. 1. Rod sensitivity was measured at 500 nm and cone sensitivity was measured at 650 nm. There are 16 levels of gray (0-54 db for rods; 0-25 db for cones). Black indicates no detection of the stimulus. White indicates test result was within 2 standard deviations of the mean normal sensitivity for that locus. (Bottom) Spectral sensitivity measurements in the dark-adapted state (at a locus 24 superior and 12 temporal from fixation) and light-adapted state (12 inferior) in the MAR patient (triangles) compared to normal results (area between solid lines represents range of normal data; n = ] 0, ages yr). The dark-adapted data can be fit by a combination of the CIE scoiopic sensitivity curve 42 (shifted vertically by about 3 log units relative to the patient data at 500 nm) and a peripheral cone function' 13 (equated at 600 nm). rior field locus (Fig. 2, lower right) illustrates the abnormality at shorter wavelengths. In contrast, two CSNB patients had normal S cone sensitivity at fixation and at a few paracentral loci, and there was detectable (although reduced) function at about 60% of the test loci. Therefore, the MAR patient showed a greater degree of S cone sensitivity loss by pe rime try than these CSNB patients. S cone ERGs in the MAR patient were not detectable. Visual function tests were repeated after the patient underwent a trial of corticosteroids and while he still was on 20 mg/day of prednisone. Rod and cone ERG results did not show a trend between visits that would suggest change in response threshold or suprathreshold amplitude or timing. There was a small but statistically significant increase between visits in mean sensitivity from dark-adapted perimetry results, as analyzed by the paired t-test (P < 0.001). The mean increase of 2 db for rod sensitivity and 2.5 db for cone sensitivity, however, may not represent true sensitivity change if long-term fluctuation complicates interpretation of changes less than 3-5 db in MAR, as it can in glaucoma. 36 Immunocytochemistry Indirect immunofluorescence was performed on sections of fresh, unfixed human retina using serum from MAR patients no. 1 and no. 2 and the following control patients: 28 patients with melanoma but no retinopathy, two patients with other retinal diseases, and eight normal subjects. Sera from most control patients produced no specific immunolabeling of retina sections (Fig. 3, left). Cells of the retinal pigment epithelium were filled with bright yellow-orange autofluore-

6 96 Investigative Ophthalmology & Visual Science, January 1993, Vol. 34, No. 1 FIGURE 3. Cryostat sections of normal unfixed human retina processed for indirect immunofluorescence. (Left) Section processed with IgG (1:100) from a normal subject. Note autolluorescent lipofuscin granules (*) in the retinal pigment epithelium (RPE) and scattered throughout the photoreceptor layer (p). The remainder of the neural retina shows a dull green autolluorescence, including a group of ganglion cells (g). (X68.) (Right) Section of same retina processed using IgG (1:100) from MAR patient no. 1. In addition to autolluorescent structures found in Figure 3 (left), note brightly stained cells (arrows) in the inner nuclear layer and the row of bright dots (arrowheads) in the outer plexiform layer. The nerve fiber layer (n) also is immunoreactive with the MAR serum, as found in some controls. (X68.) scent lipofuscin granules, and the retina showed a dim green autofluorescence throughout (Fig, 3, left). A similar result was obtained using no primary antibody before treatment with secondary antibody. Sera from some control subjects (n = 3) and melanoma patients (n = 7) produced higher nonspecific background labeling of all parts of the retina and specific staining of filamentous structures in the nerve fiber layer (Fig. 4, left). Similar immunostaining of retinal neurofilaments by sera from a few normal human subjects was reported previously. 37 Sera from the two MAR patients produced very weak labeling of rod outer segments and strong labeling of filaments in the nerve fiber layer (Figs. 3, right, and 4, right). In addition, both MAR sera produced strong labeling of some but not all cells lying midway in the inner nuclear layer plus afinelybeaded band in the outer plexiform layer (Figs. 3, right, and 4, right). These cells tentatively were identified as bipolar cells. The inner plexiform layer of the unfixed retinas was poorly preserved, and only hints of axons could be traced from the labeled cells. Immunocytochemistry was repeated using serum samples from MAR patient no. 1 taken throughout his course of steroid treatment and from MAR patient no. 2 before and after similar steroid treatment. All serum samples from both MAR patients produced equivalent specific labeling of the population of bipolar cells. None of the sera or IgG fractions from the normal subjects, patients with other retinal diseases, or non-mar melanoma patients produced this labeling of bipolar cells. Because the labeled cells appeared to be bipolar cells, double labeling experiments were performed using a mixture of each MAR patient's serum and antiprotein kinase C, a specific marker for rod bipolar cells. 26 The labeled cells were photographed alternately with filters for FITC and rhodamine. Although labeling of bipolar cells with anti-protein kinase C was patchy in unfixed sections, it was found that many of the cells labeled with each of the two MAR sera also were labeled with the antibody against protein kinase C (Fig. 5). These results indicate that the retinal neurons labeled with sera from MAR patients are bipolar cells and that many of them are rod bipolar cells. The double-labeling experiments were repeated using MAR sera and the antibody preparation against protein kinase C on sections of human retinas that had been fixed with paraformaldehyde. Rod bipolar cells were labeled with anti-protein kinase C in retinas that had been fixed from zero to two hr, but no longer. However, no specific labeling was found in any of the fixed retinas with MAR sera, even after fixation for only 15 min. This result, indicates that the human bipolar cell antigen that is recognized by MAR sera is ex- FIGURE 4. Cryostat sections of another human retina processed as in Figure 3. (Left) Section processed with IgG (1:100) from another normal subject. Note autoi'luorescent lipofuscin in the RPE (*), higher nonspecific background autofluorescence, labeled filamentous structures (n) in the nerve fiber layer, and autottuorescent ganglion cells (g). Sera and IgGs from a few other normal subjects and non- MAR melanoma patients produced this pattern of irnmunolabeling. (X68.) (Right) Section processed with IgG (1:100) from MAR patient no. 2, illustrating brightly stained cells in the inner nuclear layer (arrows) and a beaded band in the ouler plexiform layer (arrowheads). The nerve fiber layer (n) also is labeled, as found in some normal subjects and other melanoma patients. (X68.)

7 Autoantibodies Against Retinal Bipolar Cells in MAR * FIGURE 5. Normal retina processed as in Figure 3 using IgG (1:100) from MAR patient no. 2 mixed with anti-protein kinase C (1:50), followed by a mixture of anti-human IgG labeled with FITC and anti-mouse IgG labeled with rhodamine. (Left) Photograph taken usingfiltersfor FITC fluorescence. Note specific labeling by MAR IgG of bipolar cells (arrowheads) in the inner nuclear layer. Lipofuscin in the RPE (*) is autofluorescent, and the nerve fiber layer (n) is inmiunostained. (X250.) (Right) The same section photographed with filters for rhodamine fluorescence to reveal labeling by anti-protein kinase C, a specific marker for rod bipolar cells. Note that some of the cells labeled with the IgG from MAR patient no. 2 also are labeled with anti-protein kinase C. Axonal processes of the rod bipolar cells in the inner plexiform layer are indicated by arrows. (X250.) PAGE and Western blot analysis to detect the antigen responsible for bipolar cell staining. Gel systems were run at neutral or alkaline phs, and two detection methods were employed during the study. Comparable results were found with both detection methods, at both phs. Representative results are shown for retinal homogenates at alkaline ph (Fig. 6) using Protoblot reagents. Control and patient sera or IgG appeared to recognize numerous retinal components. However, several proteins were labeled in the absence of primary antibody (designated by brackets in Fig. 6). Sera and IgG from MAR patient no. 1 stained a component or components of approximately 75 kd (doublet) in retinal homogenates (arrow; Fig. 6, lane 3). However, a comigrating component also was stained by IgG from a normal subject (Fig. 6, lane 1), by IgG from a melanoma patient without MAR (Fig. 6, lane 2), and not by IgG from MAR patient no. 2 (Fig. 6, lane 4). Thus, staining of this prominent component did not correlate with the staining of bipolar cells found with the two MAR sera. Further analysis of homogenate, pellet, and supernatant retinal fractions did not reveal a corn if tremely sensitive to fixation and probably would not be detected in sections processed for immunocytochemistry by conventional fixation techniques. The double labeling experiment also was performed on unfixed cryostat sections using MAR sera and anti-cholecystokinin precursor, a marker for blue cone bipolar cells, 28 or using anti-recoverin (CAR antigen), a marker for rods, cones, and certain cone bipolar cells On unfixed sections, neither anticholecystokinin precursor or anti-recoverin produced immunolabeling of the specific bipolar and photoreceptor cell types previously shown to be labeled by these antibodies in fixed retinas. 17 ' 28 ' 29 We suggest that the lack of labeling with these antibodies results from the fact that cholecystokinin precursor and recoverin are soluble cytoplasmic proteins, not membrane-associated, and they probably are removed from the unfixed sections in the first rinse after overnight incubation in primary antibody. Biochemistry Samples of homogenates or supernatants and pellets derived from human retinas were analyzed by SDS FIGURE 6. Screening of MAR patients' and control IgGs by Western analysis alkaline ph gel system. Proteins of a human retinal homogenate were separated by SDS-PAGE, electroblotted to PVDF membranes and probed with IgG (1:100), followed by Protoblot reagents. Lane 1, normal subject no. 3; lane 2, non-mar melanoma patient; lane 3, MAR patient no. 1; lane 4, MAR patient no. 2. Brackets at right designate proteins that stained in the absence of primary antibody. The arrow designates the reactive band observed initially with IgG from MAR patient no. 1 and subsequently with IgGs from several of the controls. Comparable results were obtained at neutral ph and using VectaStain reagents. Numbers on the left are the molecular weights of standard proteins, in thousands.

8 98 Investigative Ophthalmology & Visual Science, January 1993, Vol. 34, No. 1 ponent that was recognized uniquely by the two MAR sera. ELISA plates were coated with supernatants from human retinas solubilized with buffer or buffered detergent and were tested for reactivity with IgG from MAR patients and controls. No differences were noted. DISCUSSION Two mechanisms have been hypothesized as the pathophysiologic basis of paraneoplastic retinopathy in CAR and MAR: direct toxic damage to the retina by a circulating product of the tumor cells, or damage by an autoantibody initially directed against a tumorspecific epitope that cross reacts with a retinal antigen 3,5-10,12-16,38 Specific immunostaining of a population of retinal bipolar cells by sera from two MAR patients provides support for the latter hypothesis, although cross reactivity of our patients' sera with melanoma cells has not yet been tested because their tumor cells are not available. Although the mechanism by which circulating antibodies cause dysfunction of retinal neurons is unknown, it has been shown experimentally that vascular perfusion of rat eyes with homologous antibodies against arrestin, a photoreceptor-specific protein, produces ERG abnormalities. 39 Our results support a previous prediction 13 that specific damage by autoantibodies to rod bipolar cells in MAR patients may be related to their visual function alterations. Our immunocytochemical studies revealed that many of the bipolar cells labeled with sera from two MAR patients also were labeled with antiprotein kinase C, a marker for rod bipolar cells. 26 A defect at the level of the rod bipolar cell leading to impaired signal transmission from rod photoreceptors would be consistent with the reduced b-wave amplitude but preserved a-wave in the dark-adapted ERG, the normal rhodopsin levels by fundus reflectometry," and the marked rod-mediated sensitivity losses across the visual field by dark-adapted perimetry. The pathophysiology that leads to abnormal conemediated function in MAR, however, needs further clarification. MAR patients have been found to have a cone pathway ERG "on" response defect, 13 oscillatory potential abnormalities in the photopic ERG, middle/ long wavelength cone sensitivity losses across the visual field, and short wavelength (blue) cone function abnormalities Although functional retinal tests and immunologic staining implicate components of the same pathway, there is no direct evidence in CAR or MAR that would link the presence of circulating autoantibodies to retinal components with retinal dysfunction. The evidence for blue cone dysfunction in MAR patient no. 1 and two other MAR patients 1314 prompted us to determine whether the bipolar cells labeled with the two MAR sera also were labeled with anti-cholecystokinin precursor, a marker for primate blue cone bipolar cells. 28 Although this antibody preparation labels blue cone bipolar cells in fixed human retinas, 2829 it did not label bipolar cells in the unfixed human retinas used in the present study to demonstrate labeling of bipolar cells by MAR sera. Cholecystokinin precursor is stored as a soluble protein within secretory granules, and we believe this protein probably was removed from the unfixed sections during the first rinse after overnight incubation in the primary antibody. Immunocytochemistry using anti-recoverin (CAR antigen) 1516 on unfixed retina sections also failed to label photoreceptors 1517 and certain cone bipolar cells, 29 probably for the same reason, because recoverin also is a soluble protein. We were unable to detect the antigen responsible for the immunostaining of rod bipolar cells by MAR sera using biochemical techniques. This is in contrast to a recent study on CAR patients, where specific staining of a 26 kd retinal component, subsequently identified as recoverin, was clearly evident on Western blots. 15 We noted sensitivity of bipolar cell immunostaining to even brief fixation by paraformaldehyde, suggesting that antigenicity is abolished by amino group modification and perhaps by conformational pertubation. Denaturation by sodium dodecyl sulfate may have disrupted a conformational epitope, accounting for the absence of specific staining by the two MAR sera on Western blots. It also is possible that a component stained specifically by MAR sera comigrates with a nonspecifically stained component and thus is hidden. Among the many melanoma-associated antigens that have been described are cell surface gangliosides and proteoglycans. 40 ' 41 Thus, the antigen responsible for bipolar staining may not be a protein. Further work is needed to document the MAR retinal antigen. The clinical and retinal function findings of MAR patient no. 1 in this study provided further observations on this disease. First, the dysfunction seemed to manifest in one eye before the other. The patient was an excellent observer, and the history of symptoms occurring in one eye weeks before the other eye was convincing. MAR patient no and two other MAR patients recently described by Pollack et al 38 related a similar sequence of events. Second, although previous work has emphasized the similarity of dysfunction in MAR to that in CSNB, we found functional differences between the MAR and CSNB patients, specifically in cone system function, including reduced oscillatory potentials, altered cone flicker timing, an abnormal S cone ERG, and elevated S cone thresholds. Third, unlike in CAR, 10 we found no definite beneficial effects of steroid therapy on the vision of MAR

9 Autoantibodies Against Retinal Bipolar Cells in MAR 99 patient no. 1. Serial monitoring by immunocytochemistry and measurements of retinal function in this MAR patient provided no strong evidence to warrant such therapy in other MAR patients. MAR patient no. 2 also showed no change in serum reactivity with bipolar cells or in the ERG 13 after steroid treatment. Many important questions remain to be answered, including whether a retinal bipolar cell component shares an epitope with malignant melanoma cells, and whether MAR patients' antibodies penetrate the blood-retinal barrier and cause the observed bipolar cell dysfunction, perhaps by interfering with a natural receptor/ligand interaction. To our knowledge, histopathology has not yet been performed on an eye from an MAR patient, but is needed to determine if bipolar cells are abnormal and if immune complexes are present. Improved understanding of the pathophysiology of this syndrome is needed to develop rational therapy for MAR. Acknowledgments We are grateful to the following colleagues for providing information or sera from their patients: Drs. G. A. Fishman, A. S. Polans, S. C. Pollock, M. O. M. Tso, and R. Weleber; Drs. ]. Del Valle and A. M. Dizhoor for providing antibodies; Dr. J. Davis for referring the patient with birdshot chorioretinopathy; Drs. E. L. Chuang, J. L. Stone, and A. V. Cideciyan for critical comments; and B. Koernig and D. Slaughter for clinical coordination of this study. We thank J. Chang, G. Garwin, and I. Klock for excellent technical assistance, C. Stephens, R. Jones, and B. Clifton for photographic help, and K. Allen and S. Groves of the University of Washington Lions' Eye Bank for providing retinas used in this study. Key Words autoantibodies, cancer-associated retinopathy, congenital stationary night blindness, melanoma-associated retinopathy, retinal bipolar cell. References 1. Sawyer RA, Selhorst JB, Zimmerman LE, Hoyt WF. 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