Apoptotic retinal cell death induced by antirecoverin autoantibodies of cancer-associated retinopathy.
OBJECTIVE
Recoverin has been identified as a target autoantigen for antirecoverin antibodies found in the sera of some patients with cancer-associated retinopathy. The aim of this study was to investigate the role of antirecoverin antibodies in cancer-associated retinopathy.


METHODS
Human, rat, and rabbit antirecoverin antibodies were purified using a recoverin-affinity column. Purified biotinylated antibodies were cultured with recoverin-positive rat retinal cells E1A.NR3. Antibody uptake by retinal cells in vitro was analyzed by immunocytochemistry. Cytotoxic effect of antibodies on retinal cells was measured by the MTT colorimetric method. Apoptosis was shown by the ladder DNA fragmentation method and by fluorescent dye chromatin fragmentation analysis.


RESULTS
Antirecoverin antibodies obtained either from sera from five cancer-associated retinopathy patients or from sera of immunized animals were internalized by E1A.NR3 cells. Only specific, antirecoverin antibodies produced destruction of the cells in a dose- and time-dependent manner. Normal immunoglobulin G did not have such effects on retinal cells. No additional cell destruction was observed in the presence of complement as compared with cultures incubated with antirecoverin antibodies alone. Internucleosomal DNA fragmentation and presence of apoptotic cells was observed throughout the culture treated with recoverin specific antibodies but not with normal antibodies. Cells not expressing recoverin (Y79, PC12, and GH3) were not susceptible to cell destruction because of antirecoverin antibody action.


CONCLUSIONS
These studies showed that antibodies specific to recoverin are able to enter and cause death of cells expressing recoverin. In humans, autoantibodies originally elicited against recoverin expressed in tumor cells may damage retinal photoreceptors and play a role in the pathogenesis of cancer-associated retinopathy. Results suggest that autoantibody to recoverin, when given access to recoverin in the retina through the blood-retina barrier, could initiate photoreceptor degeneration leading to blindness. Such mechanism may be common for other paraneoplastic disorders or autoimmune diseases where antibodies interfere with the normal cell physiology.
Adamus
G
G
R.S. Dow Neurological Sciences Institute, Legacy-Good Samaritan Hospital and Medical Center, Portland, Oregon 97209, USA.
Machnicki
M
M
Seigel
G M
GM
D000818:Animals; D000951:Antigens, Neoplasm / Q000276:immunology*; D017209:Apoptosis; D001323:Autoantibodies / Q000502:physiology*; D002135:Calcium-Binding Proteins / Q000276:immunology* / Q000302:isolation & purification; D016923:Cell Death / Q000502:physiology; D002470:Cell Survival; D004396:Coloring Agents; D003165:Complement System Proteins / Q000502:physiology; D003602:Cytotoxicity, Immunologic / Q000502:physiology; D004247:DNA / Q000032:analysis; D053938:DNA Fragmentation / Q000502:physiology; D004306:Dose-Response Relationship, Immunologic; D005136:Eye Proteins; D051597:Hippocalcin; D006801:Humans; D007124:Immunoenzyme Techniques; D008074:Lipoproteins; D009419:Nerve Tissue Proteins; D011817:Rabbits; D051381:Rats; D011961:Receptors, Fc / Q000032:analysis; D051603:Recoverin; D012160:Retina / Q000166:cytology / Q000502:physiology*; D013778:Tetrazolium Salts; D013844:Thiazoles; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D001323:Autoantibodies; D002135:Calcium-Binding Proteins; D004396:Coloring Agents; D005136:Eye Proteins; D008074:Lipoproteins; D009419:Nerve Tissue Proteins; C496731:RCVRN protein, human; C496733:Rcvrn protein, rat; D011961:Receptors, Fc; D013778:Tetrazolium Salts; D013844:Thiazoles; D051603:Recoverin; D051597:Hippocalcin; D003165:Complement System Proteins; D004247:DNA; C022616:thiazolyl blue