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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 12, 2005 as doi:10.1096/fj.04-3525fje. |
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* National Institute of Sensory Organs, National Hospital Organization Tokyo Medical Center, Meguro-ku, Tokyo, Japan;
Department of Biomedical Science, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan;
The Corporation for Production and Research of Laboratory Primates, Tsukuba-shi, Ibaraki, Japan;
Department of Ophthalmology, Juntendo University Urayasu Hospital, Urayasu-shi, Chiba, Japan; and
|| Tsukuba Primate Research Center, National Institute of Biomedical Innovation, Tsukuba-shi, Ibaraki, Japan
1 Correspondence: Higashigaoka, Meguro-ku, Tokyo 152-8902, Japan. E-mail: iwatatakeshi{at}kankakuki.go.jp
SPECIFIC AIMS
A well-developed macula is found only in primates and birds, making a nonhuman primate model valuable in elucidating the etiology and the mechanism underlying macular degenerative diseases in humans. This study aimed to establish late- and early-onset macular degeneration in cynomolgus monkeys as animal models to study human age-related macular degeneration (AMD) by characterizing molecular composition of drusen and by evaluating involvement of anti-retinal autoimmunity in these diseases.
PRINCIPAL FINDINGS
1. Drusen in late-onset macular degeneration monkeys
The study was initiated from clinical observation of late-onset macular degeneration in monkeys. A total of 278 aged animals (mean age: 16.94 years) were funduscopically examined, and 32% of the population showed drusen-like spots in the macular region. Drusen are composed of debris-like material that accumulates with age below the retinal pigment epithelium (RPE) on Bruch’s membrane; its presence is generally accepted as a hallmark risk factor for developing AMD. The affected monkeys were further classified into two clinical entities by histological studies, one characterized by the formation of drusen in the foveal or parafoveal region and the other by degenerative changes of RPE cells such as hyperpigmentation, hypopigmentation, and vacuolation. We did not observe choroidal neovascularization, disciform scarring, geographic atrophy, or other advanced pathologic changes characteristic of later stage AMD. Photoreceptor inner and outer segments were histologically normal. Thus, for the late-onset macular degeneration monkeys, we consider drusen formation or RPE atrophy leading to abnormal fundus with pigmentary changes to be diagnostic of macular degeneration.
2. Immunohistochemical and direct proteome analysis of monkey drusen
The protein components of monkey drusen were analyzed by immunohistochemical methods and direct proteome analyses using mass spectrometry. In addition to late-onset macular degeneration monkeys, monkeys from a pedigree with an early-onset macular degeneration were examined. This early-onset macular degeneration has been shown to have an autosomal dominant inheritance and to develop macular drusen early in life, 
2 years of age.
Recent immunohistochemical studies in AMD have revealed that drusen contain protein molecules that mediate inflammatory and immune processes. Serial sections of the affected monkey retinas with drusen were incubated with antibodies directed against such molecules. All drusen in both late- and early-onset macular degeneration were heterogeneously bound by antibodies directed against apolipoprotein E (Fig. 1
a, b), amyloid P component (Fig. 1c, d
), complement component C5 (Fig. 1e, f
), the terminal C5b-9 complement complex (Fig. 1g, h
), and vitronectin (Fig. 1i, j
), a fluid phase inhibitor of complement cascade. The membrane-associated inhibitor of complement activation, membrane cofactor protein, was localized along the boundaries between drusen and RPE (Fig. 1k, l
).
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Drusen were isolated under a stereoscopic microscope from the eyes of late-onset macular degeneration monkeys. The isolated drusen (10 µg) were digested with trypsin and analyzed by liquid chromatography tandem mass spectroscopy (LC-MS/MS). We identified 60 proteins from three separate preparations and analyses, demonstrating that monkey drusen had some protein components in common with AMD drusen. These proteins included annexin V, clusterin, crystallins, and immunoglobulins in addition to the components identified by immunohistochemical studies such as apolipoprotein E, complement components, and vitronectin.
These results indicated that a common mechanism involving chronic inflammation mediated by complement activation is involved in drusen biogenesis in both late- and early-onset macular degeneration in monkeys and AMD.
3. Autoimmunity against retinal proteins in late-onset monkeys
The evidence of chronic complement activation at the site of drusen formation suggested that immune complex formation might be taking place via an immune response directed against retinal antigens. To evaluate the involvement of anti-retinal autoimmunity, sera from late-onset macular degeneration monkeys were immunoreacted with membrane blots of retinal proteins separated by SDS-PAGE. Sera collected from 20 affected animals and 10 age-matched control monkeys were used. Half of the sera from affected monkeys showed single or doublet reacting bands against 38, 40, 50, and 60 kDa proteins by Western blot. To identify these antigens, immunoblotting combined with 2-D electrophoresis was performed. The images of protein spots and chemiluminescent signals were merged, and the corresponding protein spots were excised. The excised protein spots were subjected to in-gel digestion with trypsin and analyzed by LC-MS/MS. As a result, annexin II was identified. The 40 kDa antigen was found to be µ-crystallin, but the 50 and 60 kDa proteins could not be identified.
Relative antibody titers against annexin II and µ-crystallin in sera were determined by ELISA in 42 affected and 41 age-matched control animals. The purified recombinant proteins were prepared. ELISA was performed with these recombinant proteins immobilized in 96-well plates. Relative antibody titers against annexin II in affected monkeys were significantly higher than those in control animals (P value <0.01) for the 38 kDa antigens. Antibody titers against µ-crystallin showed no significant difference between affected and unaffected monkeys, but several affected monkeys showed considerably elevated titers (360–610%).
CONCLUSIONS AND SIGNIFICANCE
AMD is the leading cause of blindness in individuals over the age of 60 in industrialized countries. Limited access to human retinal tissues and the lack of good animal models make this disease difficult to study. Earlier attempts to simulate AMD in experimental animals such as rodents have not fully replicated the clinical and histological features of the disease. On the other hand, Macaque monkeys have been known to develop macular degenerative changes with age, including pigment mottling, hyperpigmentation or hypopigmentation with drusen, consistent with the phenotype observed in the early stage of AMD. We recently reported a monkey pedigree with early-onset macular degeneration where drusen are observed <2 years after birth. Here, we compare the molecular composition of drusen from monkeys with late- and early-onset macular degeneration with human drusen. The investigation extended to hypothesize the involvement of anti-retinal autoimmunity in the etiology of AMD by identification of autoimmunoantigens expressed in RPE cells.
Proteome analyses and immunohistochemical studies of drusen composition demonstrated that monkey drusen had some protein components in common with AMD drusen including annexins, crystallins, immunoglobulins, apolipoprotein E, complement components, clusterin, and vitronectin (Fig. 1)
. Similarities in the molecular composition suggested that a common mechanism involving chronic inflammation mediated by complement activation is involved in drusen biogenesis in both late- and early-onset macular degeneration in monkeys and in AMD. We concluded that these monkey models could be an important bioresource to elucidate the mechanism of drusen formation and to test new diagnostic techniques and potential therapeutic strategies for prevention of AMD.
This study also described involvement of anti-retinal autoimmunity against annexin II and µ-crystallin for late-onset macular degeneration monkeys. The protein expression of annexin II was confirmed in the RPE cells both in vivo and in vitro. Although immunohistochemical analyses failed to detect annexin II in the retinal cross sections in this study, a previous report localized this protein to the basal plasma membrane of the RPE. Moreover, annexins were identified as drusen components by proteome analyses in this study, as is also reported in AMD. A possible pathological pathway whereby autoimmunity against annexin II could contribute to drusen formation is: 1) anti-annexin II immunoglobulins bind to the basal plasma membrane of the RPE; 2) the inactive C1 serum protein interacts with the Fc portion of the immunoglobulin; 3) this leads to formation of the C5b9 membrane attack complex; 4) causing damage to the RPE cells followed by shedding of the cell membranes in the sub-RPE space. Immune complex formation might continue in the resultant drusen cores leading to further development of drusen (Fig. 2
).
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Alternatively, anti-annexin II autoantibodies might contribute to the pathogenesis of the disease by impairing the normal activities of the protein, such as anti-inflammatory or phagocytic functions. High levels of anti-annexin autoantibodies have been reported in sera from patients with a common chronic inflammatory disease, rheumatoid arthritis (RA). Continuous production of annexin autoantibodies that inhibit anti-inflammatory activity of the protein is suspected of contributing for the pathogenesis of RA. On the other hand, antisera to RPE cell plasma membrane have been known to inhibit phagocytic activities of RPE cells. Disturbance of this function of RPE cells is likely to cause failure of ingestion of photoreceptor outer segments and accumulation of debris, leading to drusen formation. Autoantibodies against annexin II could inhibit anti-inflammatory or phagocytic functions of the protein and thereby contribute to the pathogenesis of the disease in late-onset monkeys.
Several affected monkeys showed considerably high antibody titers against µ-crystallin compared with the control group. Crystallins are proteins expressed in very high abundance in the lens that are critical to the refractivity and transparency of the organ, and are known to be synthesized by both the neurosensory retina and RPE, possibly functioning as stress proteins. Recently, crystallins were reported to be among the common proteins of drusen in AMD. We confirmed that crystallins are present in monkey drusen by proteome analyses. It can be hypothesized that injured RPE cells express µ-crystallin and shed their cell membranes with the proteins into sub-RPE space, exposing them as neo-autoantigens (Fig. 2)
.
It remains unclear whether autoantibodies against annexin II or µ-crystallin are the initial cause of the disease. It is possible that autoimmunity against these proteins might be the most critical event in the retina because annexin II is a ubiquitous protein and µ-crystallin is also expressed in brain, muscle, and kidney. Detailed clinical information on immunity in individual monkeys is essential to determine the primary event of this disease. Although further analyses are required to define the relationship between the autoantibodies and the pathogenesis of the disease, autoantigens identified in this study strongly suggest the involvement of anti-retinal autoimmunity in AMD. Defining the AMD-related autoantibodies may provide possible diagnostic tools for early detection and management of AMD.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3525fje; doi: 10.1096/fj.05-3525fje
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