Glaucoma Patients from Germany and the United States
Franz H. Grus, 1 Stephanie C. Joachim, 1 Kai Bruns, 2 Karl J. Lackner, 2 Norbert Pfeiffer, 1 and Martin B. Wax 3,4 PURPOSE. Glaucoma is characterized by a progressive loss of retinal ganglion cells that results in a characteristic optic neu- ropathy associated with visual eld loss. In previous studies, changes in the antibody proles have been shown in the sera of patients with glaucoma, and these ndings suggest a role for autoimmune involvement in the pathogenesis of glaucoma in some patients. The purpose of this study was to compare the antibody proles against optic nerve antigens in patients with glaucoma in two different study populations from Germany and the United States. METHODS. One hundred twenty patients were included in the study, 60 from Germany and 60 from the United States: a control group (CTRL, n 20), a group of patients with primary open-angle glaucoma (POAG, n 20), and one group of patients with normal-pressure glaucoma (NPG, n 20) from each country. Western blot analyses against bovine optic nerve antigens were used to detect the IgG antibody patterns present in the patients sera. The complex antibody proles were analyzed by multivariate statistical techniques. RESULTS. Complex IgG autoantibody repertoires were present in all patients with glaucoma as well as healthy subjects from both the German and the United States study population. A large similarity between all antibody proles in both study populations was demonstrated in the number and frequency of both up- and downregulation of antibody reactivities in pa- tients with glaucoma of both national cohorts. The multivariate analysis of discriminance found a signicant difference be- tween the glaucoma groups and healthy subjects against optic nerve antigens. As in previous studies, the NPG group revealed the highest variance from the control group (P 0.01). Fur- thermore, a newly described antibody biomarker in both study populations was identied as -fodrin. Western blot results revealed that there was an increased frequency and enhanced immunoreactivity to -fodrin (120 kDa) in the sera of patients with NPG. The presence of -fodrin autoantibodies were con- rmed by ELISA, in which a highly elevated anti--fodrin titer in patients with NPG was found to be signicantly greater than in the control subjects (P 0.01) or age-matched patients with POAG (P 0.04). CONCLUSIONS. Complex IgG antibody patterns against optic nerve antigens can be reproducibly identied in the serum of study populations from the United States and Germany. In both cohorts, patients with glaucoma have characteristic differences in serum autoantibody repertoires from those in control sub- jects. A newly described autoantibody to -fodrin found in other neurodegenerative diseases such as Alzheimers, further implicate a role for autoimmunity and the neurodegenerative processes in glaucoma. The high correspondence of the auto- antibody patterns found in the study populations from differ- ent continents provides further evidence that serum autoanti- body patterns may be useful biomarkers for glaucoma detection or for determining prognosis in future studies by means of pattern-matching algorithms. (Invest Ophthalmol Vis Sci. 2006;47:968976) DOI:10.1167/iovs.05-0685 G laucoma represents a group of ocular disorders that are characterized by the loss of retinal ganglion cells and their axons, damage to the optic nerve, and gradual loss of visual eld. In past years, several studies provided evidence that there is a potential role for the immune system in the pathogenesis of glaucoma. These studies found that several serum autoantibod- ies against ocular antigens are upregulated in serum of patients with glaucoma, such as heat shock proteins (HSPs), 1 -eno- lase, 2 glutathione S-transferase, 3 anti-phosphatidylserine, 4 and glycosaminoglycans. 5 These ndings suggest that there may be changes in systemic humoral immunity underlying optic neu- ropathy in at least some patients with glaucoma. All these works have in common that they analyzed the immunoreactiv- ity of only one or very few antibodies in the cohort populations that were studied. Using an antibody proling technique that is based on Western blot and digital image analysis combined with multi- variate statistics and articial neural networks, our group dem- onstrated signicant and selective up- and downregulated au- toantibody repertoires against ocular antigens in the sera of both primary open-angle (POAG) and normal-pressure glau- coma (NPG). 6,7 The relationship of these autoantibodies to the pathogenesis of glaucoma is unclear. Some studies demon- strated that HSP-27 antibodies can trigger apoptotic cell death in retinal ganglion cells. 8,9 However, in general, studies that elucidate whether autoantibodies in patients with glaucoma represent epiphenomena, useful biomarkers, or genuine clues as to the pathogenesis to the disease have not been performed. Regarding the complex autoantibody proles found in pa- tients with glaucoma, 6,7 relatively few of the serum autoanti- bodies have been identied from previous studies. Neverthe- less, it is often routine in clinical practice to use specic autoantibodies or antibody patterns for neurologic disease screening or diagnosis. 10 Thus, it is not necessarily important to know the identity of all autoantibodies in these patterns to be able to use them as biomarkers for autoimmune diseases. We reasoned that if autoantibody proling or specic autoan- tibodies have the potential to be used as diagnostic or thera- peutic biomarkers in patients with glaucoma, it would rst be important to investigate whether glaucoma patient autoanti- From the Departments of 1 Ophthalmology and 2 Clinical Chemis- try and Laboratory Medicine, Johannes Gutenberg-University, Mainz, Germany; 3 Alcon Laboratories Inc., Fort Worth, Texas; and the 4 De- partment of Ophthalmology, University of Texas Southwestern Medi- cal Center, Dallas, Texas. Supported by Grant MAIFOR04 (University of Mainz) and DFG GR146314-1 (Deutsche Forschungs Gemeinschaft). Submitted for publication June 1, 2005; revised September 21, 2005; accepted January 26, 2006. Disclosure: F.H. Grus, None; S.C. Joachim, None; K. Bruns, None; K.J. Lackner, None; N. Pfeiffer, None; M.B. Wax, None The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked advertise- ment in accordance with 18 U.S.C. 1734 solely to indicate this fact. Corresponding author: Franz H. Grus, Universitaets-Augenklinik, Department of Ophthalmology, Johannes Gutenberg-University, Langenbeckstrasse 1, 55101 Mainz, Germany; grus@eye-research.org. Investigative Ophthalmology & Visual Science, March 2006, Vol. 47, No. 3 968 Copyright Association for Research in Vision and Ophthalmology body proles are similar in different study populations. The purpose of this study was to analyze and compare the antibody proles against optic nerve in patients with POAG or NPG and healthy subjects from Germany and the United States. In addi- tion, we attempted to identify novel unidentied serum auto- antibodies that were not reported in previous studies. MATERIALS AND METHODS Patients One hundred twenty patients were included in the study: 60 from the United States and 60 from Germany. Each study subpopulation con- sisted of three groups: the rst was a control group of 20 healthy volunteers without any ocular disorders (CTRL), the second consisted of patients with POAG (n 20), and the third was made up of patients with NPG (n 20). All patients had complete ophthalmic examinations at the Ophthal- mology Department, University of Mainz, Germany, or at the Depart- ment of Ophthalmology and Visual Science, Washington University, St. Louis, Missouri. The patient classication was in accordance with the guidelines of the European Glaucoma Society. 11 The inclusion criteria for NPG were intraocular pressure (IOP) without treatment of 21 mm Hg or lower on multiple measurements, glaucomatous optic disc damage with glaucomatous changes in the visual eld, open iridocorneal angles, optic nerve cupping, and an absence of alternative causes of optic neuropathy (e.g., infection, inammation, ischemic disease, and compressive lesions). The inclu- sion criteria for POAG were similar to those for NPG, except that the IOP had to be more than 21 mm Hg, in untreated or treated eyes, on at least one occasion, with no other reasons for elevated IOP, such as pseudoexfoliation or pigment dispersion. IOP was determined by Goldmann applanation tonometry, and visual eld testing was performed with Goldmann perimetry. All pa- tients with glaucoma had no history of other autoimmune diseases. Furthermore, 20 healthy subjects with no history of any ocular disorders or other autoimmune diseases were included as a control group in each substudy. The groups were matched for age and gender. The mean ages were 57 23.2 years (SD) in the control group, 64 9.8 years in the POAG group, and 70 9.5 years in the NPG group. There were no signicant differences among the groups (P 0.125). The patients of the U.S. cohorts have been described elsewhere in detail. 1,12 The racial classication of the U.S. group was 90% white and 10% black. The investigation was conducted in accordance with the tenets of the Declaration of Helsinki. Patients from the United States had blood samples drawn according to a protocol that was approved by the Barnes-Jewish Hospital Investigational Review Board and Washington University (St. Louis, Missouri). After they had given informed consent, blood samples were obtained from all patients. The samples were centrifuged and the serum stored for later examination at 80C. Western Blot Analysis The optic nerve was dissected from bovine eyes and homogenized in sample buffer (1 M Tris [pH 7.5], 10% SDS, dithiothreitol, and brom- phenol blue [pH 6.8]). The samples were rst boiled for 10 minutes and then centrifuged at 15000 rpm for 1 hour. They were then centrifuged several times afterward, and the pellet was discarded. The supernatant was centrifuged again and then stored at 80C for later analysis. The bovine optic nerve extracts were used for 13.5% SDS-PAGE (MultiGel-Long; Biometra, Gottingen, Germany). After electrophoresis, the gels were transferred onto nitrocellulose membranes (Protran BA 83; Schleicher and Schuell, Dassel, Germany) by using a semidry blotter (Biometra). The nitrocellulose membranes were cut into strips, and one strip was used per sample, as described previously. 6,13 The strips were incubated overnight with patients sera (1:40 dilution in wash buffer). After the strips were washed several times with Tris-buffered saline (TBS), they were incubated with 1:500 diluted secondary antibody: peroxidase-conjugated goat anti-human IgG (Immuno Pure HL, Pierce Biotechnology, IL) for 1 hour. After the strips were washed with TBS, the bands were developed by staining with 0.05% 4-chloro-1- naphthol (Sigma-Aldrich, Munich, Germany) with 0.015% hydrogen peroxide in 20% methanol in TBS for 20 minutes. Molecular weights were estimated for each band based on the distance migrated for 10 known molecular weight standards (BenchMark; Invitrogen, Karlsruhe, Germany). Data Analysis The data were acquired with a color atbed scanner (Epson GT-9000; Epson Germany, Duusseldorf, Germany). Digital image analysis and evaluation of Western blot analysis was performed (BioDocAnalyze; Biometra), which created densitometry data from the blots, showing the gray-scale values versus the molecular weight. Peak detection was performed (BioDocAnalyze; Biometra). From these detected peaks, a list of peak clusters was created. The software generates consistent peak sets across multiple densitometric data. When comparing a given protein peak across various samples, it is important to obtain an intensity value for each spectrum, even though they may not have been found during peak detection. In this study, a peak cluster was created if the given peak was found in 10% of all densitometric data. Furthermore, each densitograph of each Western blot is normalized according to the entire area under the curve. Thus, each variable of the data vector represents the percentage area of the peaks of the Western blot strip at the corresponding molecular weight. Based on the peak cluster list, multivariate statistical techniques were used to detect differences in the distribution of antibodies against optic nerve antigen in patients sera. The cluster list was exported to a statistical software program (Statistica; Statsoft, Tulsa, AZ). In the present study, the proles were compared by an analysis of discrimi- nance. Recently, this technique has been successfully used as standard protocol to analyze autoantibody repertoires in patients with myasthe- nia gravis, 14 experimental uveitis, 15 Tourette syndrome, 16 and Syden- ham chorea. 17 Mass Spectrometry Protein identication was performed by mass spectrometry (MS). The corresponding electrophoresed immunoreactive bands of the Western blot analysis were excised manually by a Pasteur pipette and trans- ferred into a 1.5-mL reaction tube. Excised gel pieces were treated twice with 400 L of 50% methanol containing 10% acetic acid and agitated for 45 minutes, followed by incubation in 400 L of a buffer containing 100 mM ammonium bicarbonate (pH 8.0) with agitation for 30 minutes, followed by incubation in 400 L of 50% acetonitrile containing 100 mM ammonium bicarbonate with agitation for 1 hour, and nally, incubation in 50 L 100% acetonitrile with agitation for 15 minutes. Afterward, gel pieces were centrifuged (Concentrator; Eppen- dorf, Fremont, CA) to complete dryness. For rehydration and digestion, individual gel pieces were covered with 10 L of a trypsin solution (0.04 g/L; Roche, Mannheim, Germany) for 10 minutes, followed by the addition of up to 20 L 25 mM ammonium bicarbonate and incubation at 37C for 7 hours. Digested samples were centrifuged for 1 minute at 13,000 rpm, and these tryptic digests were subsequently separated by HPLC (Thermo Electron, Waltham, MA) which is coupled online to an ion-trap electrospray MS-MS (LCQ, Deca Plus; Thermo Electron). MS/MS spectra were exported as Seaquest les and used for data- base searches with MASCOT (www.matrixscience.com/ Matrix Sci- ence, Boston, MA) using the NCBI (www.ncbi.nlm.nih.gov, National Institutes of Health, Bethesda, MD) and SwissProt (http://www.expasy. org/ Swiss Institute of Bioinformatics, Geneva, Switzerland) databases, both provided in the public domain. IOVS, March 2006, Vol. 47, No. 3 Serum Autoantibodies to -Fodrin in Glaucoma 969 ELISA of -Fodrin Antibodies To assess the reactivity of autoantibodies in the sera of patients against the whole recombinant -fodrin protein, we performed an ELISA (Aesculisa -Fodrin A; Aesku Diagnostics, Wendelsheim, Germany). This ELISA uses human recombinant -fodrin to test the antibody reactivity. The test was performed according to the guidelines of the manufacturer. RESULTS All patients showed unique but similar complex staining patterns of IgG antibodies against optic nerve antigens. In Figure 1 representative randomly selected antibody proles of 24 different patients from both study subpopulations (United States and Germany) are shown that include eight control subjects (CTRL), eight patients with POAG, and eight patients with NPG. In Figure 2, Western blots of patients are shown. As in the Western blots, the darker the bands, the higher the antigenantibody reactivity at this molecular weight. Several immunoreactive bands were present in each patient, but the gure demonstrates very clearly that the antibody patterns are so complex that it is not possible to differentiate between the clinical groups, just by inspecting them visually. FIGURE 1. Antibody proles of 24 different patients: four control sub- jects (CTRL), four patients with POAG, and four patients with NPG from both countries. Right: patients from the German study; left: densito- metric data from the U.S. patients. The intensity of antigen-antibody-re- activity (U) is plotted versus the mo- lecular mass (kDa). 970 Grus et al. IOVS, March 2006, Vol. 47, No. 3 Figure 3 reveals the mean antibody reactivity in both con- trol groups (from the U.S. and German substudies). The anti- body proles were analyzed for each patient in each group, and the average was calculated for each molecular weight. Both control groups revealed a complex autoantibody reper- toire against optic nerve antigens, in agreement with our pre- vious studies 6,7 that revealed complex proles of naturally occurring autoantibodies in healthy subjects. Figures 4 and 5 demonstrate the differences in the mean antibody proles of patients with POAG or NPG in both sub- studies (from the U.S. and Germany) from the control group. First, the mean antibody reactivities were calculated for the POAG and the NPG group, as described earlier. Furthermore, at each molecular weight, the difference in the mean antibody proles of the POAG and NPG groups from the mean antibody prole of the control group (CTRL) was calculated. Figure 4 reveals the differences in mean antibody proles between healthy subjects and patients with POAG from the FIGURE 2. This gure demonstrates some representative Western blot analysis from the U.S. study population (US) and the German popula- tion (GER). Furthermore, two negative controls (NC) are shown (sec- ondary antibody only, no sera). FIGURE 3. The mean antigen-antibody reactivity of control subjects (CTRL) from the U.S. and German study population. The reactivities were plotted against the corresponding molecular weight of the optic nerve antigen. The x-axis is the molecular mass (kDa) and the y-axis the density of the antigen-antibody reaction (U). FIGURE 4. Comparison of the mean antigen-antibody reactivity of pa- tients with POAG from the U.S. study population to the German. The differences from the control group in the mean antigenantibody reactivity were plotted against the corresponding molecular weight of the optic nerve antigen. The x-axis is the molecular mass (kDa) and the y-axis the difference in the density of the antigen-antibody reaction compared with the control group (U). All reactivities above zero represent upregulations in comparison with healthy subjects; all below zero are downregulations in comparison to the control group. FIGURE 5. The mean antigen-antibody reactivity of patients with NPG from the U.S. and German study population. The differences from the control group in the mean antigen-antibody reactivity were plotted against the corresponding molecular weight of the optic nerve antigen. The x-axis is the molecular mass (kDa) and the y-axis the difference in the density of the antigen-antibody reaction compared with the control group (U). IOVS, March 2006, Vol. 47, No. 3 Serum Autoantibodies to -Fodrin in Glaucoma 971 United States and Germany. Thus, if the values were higher than zero, we found an increase in antigen-antibody reactivity and if the values were less than zero, a decrease in antigen- antibody reactivity was found, compared with the control group. Figure 5 demonstrates these differential antibody pro- les in the NPG group. In both the NPG and the POAG groups a very large similar- ity was found between the antibody proles in the German and U.S. substudies. Furthermore, the graphs demonstrate for all groups that there are regions that are distinctly upregulated, but also regions that are clearly downregulated in comparison with healthy subjects (below zero in these graphs). And fur- thermore, most of these regions corresponded to each other in the German and U.S. studies. Thus, reproducible up- and down- regulation of autoantibodies were found, regardless of the country from which the patients were derived. There were surprisingly only a few regions that showed confounding ef- fects in the two national cohorts (e.g., at 35 kDa), where more immunoreactivity was present in the in patients with POAG from the United States. However, in comparison with control subjects in each country, the increase in immunoreac- tivity in this region was negligible and similar in patients with POAG from each country. The analysis of discriminance found a signicant difference (P 0.001) between glaucoma and control subjects in both countries, based on the complex antibody proles, rather than on single peaks. The analysis of discriminance is able to calculate a parame- ter, the so-called canonical roots, as a kind of similarity index of each Western blot. This can be used to illustrate the quality of discriminance between the samples and to demonstrate the sharpness of a diagnostic criterion. This can best be under- stood as individual repertoire clustering. The closer the canon- ical roots of the banding patterns of Western blot analysis, the more similar were the blots. Figure 6 shows the canonical roots of IgG antibodies of the three different groups for optic nerve antigens. The gure demonstrates that data for most of the patients with glaucoma (POAG or NPG) lay outside the accu- mulation of healthy subjects (CTRL). In addition, the analysis of discriminance can calculate the Mahalanobis distances, which provide a measure of how dif- ferent the clinical groups are from each other on average, based on the complex antibody proles. In terms of statistical distances, the NPG group was revealed to be the most different from all other groups (P 0.001; distance NPG-CTRL 2.91). However, the POAG group also revealed a signicant differ- ence from the control group (POAG-CTRL 2.13; P 0.01). Discriminant function analysis is used to determine which autoantibodies or immunoreactive bands discriminate between two or more naturally occurring groups. In stepwise discrimi- nant function analysis, a model of discrimination is built step by step. Specically, at each step, all immunoreactivities of each molecular weight are reviewed and evaluated to deter- mine which one will contribute most to the discrimination between groups. That immunoreactive band is then included in the model, and the process starts again. Table 1 demon- strates the 10 most signicant molecular weights of immuno- reactivities (i.e., the rst 10 discriminatory steps), included in the analysis of discriminance. These antibodies, which have FIGURE 6. Canonical roots of IgG serum autoantibodies of patients with POAG, patients with NPG, and healthy volunteers (CTRL). The analysis of discriminance is able to calculate a parameter, the so-called canonical roots, as a kind of similarity index of banding pattern on each Western blot. This method can be used to illustrate the quality of discriminance between the samples and to demonstrate the sharpness of a diagnostic criterion. The closer the canonical roots of samples were, the more similar the Western blot bands were. TABLE 1. The 10 Most Signicant Biomarkers Derived from the Analysis of Discriminance Variable Enter (E)/ Remove (R) Step F to Ent/Rem No. of Vars. In lambda F-Value df 1 df 2 P 115499 1 7.625 1 0.884 7.625 2 116 0.00077 9050 2 4.670 2 0.817 6.098 4 230 0.00011 31871 3 2.477 3 0.783 4.934 6 228 0.00009 63579 4 2.468 4 0.751 4.357 8 226 0.00007 24068 5 2.235 5 0.722 3.966 10 224 0.00005 10135 6 2.233 6 0.694 3.709 12 222 0.00004 15360 7 2.266 7 0.666 3.536 14 220 0.00003 61110 8 2.036 8 0.642 3.374 16 218 0.00003 104000 9 2.385 9 0.615 3.299 18 216 0.00002 75878 10 1.751 10 0.596 3.163 20 214 0.00002 A variable is dened as the immunoreactivity that occurs at a specic molecular weight. The table demonstrates the step number, the F to enter or remove, the number of variables in the model after the respective step, the overall value of Wilks lambda after the respective step, the F-value associated with the lambda, the degrees of freedom for that F-value and the corresponding probability. The stepwise procedure is guided by the respective F to enter and F to remove values. The F-value for a variable indicates its statistical signicance in the discrimination between groups, that is, it is a measure of the extent to which a variable makes a unique contribution to the prediction of group membership. Wilks lambda is calculated by the discriminant analysis, to assess the discriminatory power of the model and can have values in the range of 0 (perfect discrimination) to 1 (no discrimination). 972 Grus et al. IOVS, March 2006, Vol. 47, No. 3 the most separation between glaucomatous or nonglaucoma- tous patterns, can also be considered antibody biomarkers for this disease. Although it is not necessary to know the identity of these antigens to consider them as useful in dening patterns that are characteristic of patients with glaucoma, we attempted to identify the immunoreactive band that revealed the highest degree of difference between glaucoma and control subjects in this study. Therefore, the peak at approximately 120 kDa was tryptic digested and analyzed by tandem mass spectrometry. Table 2 demonstrates the tryptic peptides found in this mass spectrometry analysis: the band was identied as -fodrin (spectrin). The analysis was repeated three times in other electrophoretic separations and yielded the same result. This was consistent with our ndings of primary immunoreactive bands at 120 kDa and secondarily at 150 kDa, which is a TABLE 2. Identication of -Fodrin by Tandem Mass Spectrometry gi:17380501 Mass: 28,4462 Spectrin chain, brain: spectrin, non-erythroid chain; -II spectrin; fodrin chain. Peptides found: 1108.188073: ITALDEFATK 1154.923173: SSEEIESAFR 2146.975722: SADESGQALLAAGHYASDEVR Sequence: 1 MDPSGVKVLE TAEDIQERRQ QVLDRYHRFK ELSTLRRQKL EDSYRFQFFQ 51 RDAEELEKWI QEKLQVASDE NYKDPTNLQG KLQKHQAFEA EVQANSGAIV 101 KLDETGNLMI SEGHFASETI RTRLMELHRQ WELLLEKMRE KGIKLLQAQK 151 LVQYLRECED VMDWINDKEA IVTSEELGQD LEHVEVLQKK FEEFQTDLAA 201 HEERVNEVNQ FAAKLIQEQH PEEELIKTKQ EEVNAAWQRL KGLALQRQGK 251 LFGAAEVQRF NRDVDETIGW IKEKEQLMAS DDFGRDLASV QALLRKHEGL 301 ERDLAALEDK VKALCAEADR LQQSHPLSAN QIQVKREELI TNWEQIRTLA 351 AERHARLDDS YRLQRFLADF RDLTSWVTEM KALINADELA NDVAGAEALL 401 DRHQEHKGEI DAHEDSFKSA DESGQALLAA GHYASDEVRE KLSILSEERA 451 ALLELWELRR QQYEQCMDLQ LFYRDTEQVD NWMSKQEAFL LNEDLGDSLD 501 SVEALLKKHE DFEKSLSAQE EKITALDEFA TKLIQNNHYA MEDVATRRDA 551 LLSRRNALHE RAMHRRAQLA DSFHLQQFFR DSDELKSWVN EKMKTATDEA 601 YKDPSNLQGK VQKHQAFEAE LSANQSRIDA LEKAGQKLID VNHYAKEEVA 651 ARMNEVISLW KKLLEATELK GVKLREANQQ QQFNRNVEDI ELWLYEVEGH 701 LASDDYGKDL TNVQNLQKKH ALLEADVAAH QDRIDGTIQ ARQFQDAGHF 751 DAENIKKKQE ALVARYEALK EPMVARKQKL ADSLRLQQLF RDVEDEETWI 801 REKEPIAAST NRGKDLIGVQ NLLKKHQALQ AEIAGHEPRI KAVTQKGNAM 851 VEEGHFAAED VKAKLSELNQ KWEALKAKAS QRRQDLEDSL QAQQYFADAN 901 EAESWMREKE PIVGSTDYGK DEDSAEALLK KHEALMSDLS AYGSSIQALR 951 EQAQSCRQQV APMDDETGKE LVLALYDYQE KSPREVTMKK GDILTLLNST 1001 NKDWWKVEVN DRQGFVPAAY VKKLDPAQSA SRENLLEEQG SIALRQGQID 1051 NQTRITKEAG SVSLRMKQVE ELYQSLLELG EKRKGMLEKS CKKFMLFREA 1101 NELQQWINEK EAALTSEEVG ADLEQVEVLQ KKFDDFQKDL KANESRLKDI 1151 NKVAEDLESE GLMAEEVQAV QQQEVYGMMP RDEADSKTAS PWKSARLMVH 1201 TVATFNSIKE LNERWRSLQQ LAEERSQLLG SAHEVQRFHR DADETKEWIE 1251 EKNQALNTDN YGHDLASVQA LQRKHEGFER DLAALGDKVN SLGETAQRLI 1301 QSHPESAEDL KEKCTELNQA WTSLGKRADQ RKAKLGDSHD LQRFLSDFRD 1351 LMSWINGIRG LVSSDELAKD VTGAEALLER HQEHRTEIDA RAGTFQAFEQ 1401 FGQQLLAHGH YASPEIKEKL DILDQERTDL EKAWVQRRMM LDHCLELQLF 1451 HRDCEQAENW MAAREAFLNT EDKGDSLDSV EALIKKHEDF DKAINVQEEK 1501 IAALQAFADQ LIAVDHYAKG DIANRRNEVL DRWRRLKAQM IEKRSKLGES 1551 QTLQQFSRDV DEIEAWISEK LQTASDESYK DPTNIQSKHQ KHQAFEAELH 1601 ANADRIRGVI DMGNSLIERG ACAGSEDAVK ARLAALADQW QFLVQKSAEK 1651 SQKLKEANKQ QNFNTGIKDF DFWLSEVEAL LASEDYGKDL ASVNNLLKKH 1701 QLLEADISAH EDRLKDLNSQ ADSLMTSSAF DTSQVKEKRD TINGRFQKIK 1751 SMATSRRAKL SESHRLHQFF RDMDDEESWI KEKKLLVSSE DYGRDLTGVQ 1801 NLRKKHKRLE AELAAHEPAI QGVLDTGKKL SDDNTIGQEE IQQRLAQFVE 1851 HWKELKQLAA ARGQRLEESL EYQQFVANVE EEEAWINEKM TLVASEDYGD 1901 TLAAIQGLLK KHEAFETDFT VHKDRVNDVC TNGQDLIKKN NHHEENISSK 1951 MKGLNGKVSD LEKAAAQRKA KLDENSAFLQ FNWKADVVES WIGEKENSLK 2001 TDDYGRDLSS VQTLLTKQET FDAGLQAFQQ EGIANITALK DQLLAAKHIQ 2051 SKAIEARHAS LMKRWTQLLA NSATRKKKLL EAQSHFRKVE DLFLTFAKKA 2101 SAFNSWFENA EEDLTDPVRC NSLEEIKALR EAHDAFRSSL SSAQADFNQL 2151 AELDRQIKSF RVASNPYTWF TMEALEETWR NLQKIIKERE LELQKEQRRQ 2201 EENDKLRQEF AQHANAFHQW IQETRTYLLD GSCMVEESGT LESQLEATKE 2251 KHQEIRAMRS QLKKIEDLGA AMEEALILDN KYTEHSTVGL AQQWDQLDQL 2301 GMRMQHNLEQ QIQARNTTGV TEEALKEFSM MFKHFDKDKS GRLNHQEFKS 2351 CLRSLGYDLP MVEEGEPDPE FEAILDTVDP NRDGHVSLQE YMAFMISRET 2401 ENVKSSEEIE SAFRALSSEG KPYVTKEELY QNLTREQADY CVSHMKPYVD 2451 GKGRELPTAF DYVEFTRSLF VN In this analysis, three peptides at 1108, 1155, and 2147 Da were found, fragmented, and identied. According to database searches, the fragments belong to the fodrin chain. This analysis was repeated three times using different electrophoretic separations, and the result was conrmed. Peptides identied in bold. IOVS, March 2006, Vol. 47, No. 3 Serum Autoantibodies to -Fodrin in Glaucoma 973 typical banding pattern for -fodrin fragments. The 120-kDa band could be detected in approximately 78% of patients in POAG and CTRL, and in 95% of patients with NPG. The mean intensity of these bands was highly elevated in patients with NPG (0.51 0.09 [SE]) compared with healthy subjects (0.22 0.05) and patients with POAG (0.24 0.07; P 0.01). To test whether the autoantibodies against the 120 kDa -fodrin band recognize nondenatured fodrin, an ELISA with human recombinant -fodrin as antigen was performed. The ELISA test conrmed the results from the Western blot analysis (Fig. 7). The antibody titer against nondenatured -fodrin was highly elevated in patients with NPG compared with the con- trol group (NPG molecular weight [MW]: 105.82; CTRL MW: 77.26; P 0.01) and only slightly elevated in patients with POAG (POAG MW: 86.55; CTRL MW: 77.26; P 0.18). Fur- thermore, the -fodrin titer in patients with NPG was signi- cantly higher than in patients with POAG (NPG MW: 105.82; CTRL MW: 77.26; P 0.04). DISCUSSION There are several studies that propose an autoimmune mech- anism may be involved prominently in patients with NPG. 18 For example, an increased prevalence of monoclonal gam- mopathy, 19 retinal immunoglobulin deposition, 1 elevated se- rum titers of autoantibodies to many optic nerve 5 and retinal antigens 1,3,8,12,20 and abnormal T-cell subsets 21 have been re- ported in many patients with glaucoma. Increased autoantibod- ies in the serum of patients with glaucoma include those to heat shock proteins (hsps), such as hsp60, hsp27, and -crys- tallins, 8 expression of which are upregulated in the glaucoma- tous retina and optic nerve head. 22 Furthermore, these hsp antibodies are thought to induce neuronal apoptosis through the attenuation of the ability of native hsp27, to stabilize retinal actin cytoskeleton. 9 In this study, we demonstrated that complex natural IgG antibody repertoires exist, not only in both glaucoma groups (NPG and POAG), but also in healthy subjects. We conrmed the results of preceding studies, in which it was found that the autoantibody proles are more distinct from the control group in patients with NPG than in those with POAG, if optic nerve antigens were used as a substrate for blots against patient sera. 6 Other studies provided evidence that the antibody proles were more altered in patients with POAG than in those with NPG if retinal antigens were used as the substrate. 7 These studies by Grus et al. 6 and Joachim et al. 7 support the previous ndings of others, most notably Wax et al., 12 that characterized single antigen-antibody reactions and concluded that there might be an autoimmune mechanism involved in the patho- genesis of glaucoma in some patients. A key nding of the present study was the biochemical identication of a newly identied autoantibody in our patients that we identied as fodrin by MS. Fodrin (also called spectrin in erythrocytes) is a tetramer consisting of two distinct polypeptide chains, and , with molecular masses of 240 and 220 kDa. Fodrin is one of the major neuronal cytoskeleton proteins. Beside other functions, fodrin, together with ankyrin and protein 4.1, links actin laments to the plasma membrane in many different kinds of cells. Fodrin is also thought to be involved in the pathogenesis of other neurodegenerative dis- eases. In Alzheimers disease, an anti-brain spectrin immunore- activity has been demonstrated. 2325 Furthermore, -fodrin is a target of caspase-3 and is cleaved by caspases at very early stages of apoptosis leading to structural rearrangements, in- cluding membrane blebbing. 26 The apoptotic cell death acti- vates a proteolytic cascade, including caspase-8 and -3. 27 Caspases are thought to play a major role in apoptotic pro- cesses in chronic neurodegenerative disorders such as Hun- tingtons, Parkinsons, and Alzheimers diseases. 28 In the literature, it is demonstrated that the proteolytic cleavage of -fodrin typically leads to two characteristic bands: one at 150 kDa and one at 120 kDa. It has been demonstrated that the 120-kDa fragment is specic for caspase-3 cleavage, 26 whereas the 150 kDa results from cleavage by calpain, a pro- tease with features similar to caspases. In the present study, we found a high reactivity of antibodies to a 120-kDa fragment and a less expressed reactivity against a 150-kDa band. The 120-kDa band was identied as -fodrin. This result provides further hints of caspase activation in patients with glaucoma and is in accordance with other studies that demonstrated that -fodrin is cleaved by caspase-3 in a chronic ocular hypertensive rat model of glaucoma. 29,30 However, this effect cannot only be due to the caspase-3 cleavage, but can also occur under the chosen experimental conditions in reducing gels. The elevated immunoreactivity against fodrin provides evi- dence that there may be many shared mechanisms in the pathogenesis of glaucoma and other neurodegenerative dis- eases such as Alzheimers disease. However, the precise patho- genic signicance of our ndings remain the subject of further study. For example, we do not yet know if the autoantibodies trigger complex processes in the retinal ganglion cells such as destruction of part of the cytoskeleton. Such activity may lead to apoptosis and further cleavage of fodrin (e.g., by caspases) to yield a 120-kDa fragment of fodrin, which appears to be the predominant fodrin immunogen. In the present study, we used boiled antigen extracts to detect antibody reactivities in Western blot analysis. Therefore, we were able to detect only antibody reactions against linear epitopes representing the primary structure. This does not provide information about the reactivity against the native protein. Therefore, an ELISA test was performed with human recombinant -fodrin used as the antigen. Conrming the Western blot results, a highly elevated reactivity against -fo- drin was demonstrated in patients with NPG, whereas in pa- tients with POAG, there was only a slight increase in activity compared with the control group. The second major nding of our study was specic patterns of autoantibodies in patients with glaucoma that were charac- terized by both up- and downregulated areas of serum immu- noreactivity in comparison to control subjects. A comparison of complex antibody proles of glaucoma and control subjects, as done in the present study, rather than identifying individual autoantibodies in patient serum, may provide even greater information about the autoantibody repertoire in patients with FIGURE 7. Antibody titer (-fodrin, mean SE) are plotted for healthy subjects (CTRL), patients with POAG, and those with NPG. 974 Grus et al. IOVS, March 2006, Vol. 47, No. 3 glaucoma, particularly as it relates to antibodies against retinal or optic nerve antigens, and may provide further insight into the role of immune-mediated events. The characterization of a broad autoantibody repertoire in patients can provide quanti- tative data that can identify both up- and downregulated anti- bodies of specic molecular weight and thus reveal whether the antibody proles are signicantly different from the control group and specic to patients with glaucoma. Therefore, even without knowing the identity of those antibodies, these spe- cic patterns might be used as biomarkers for glaucoma. What is particularly notable is that there was a very large degree of consistency between the antibody patterns in pa- tients with glaucoma from the United States and Germany (Figs. 4, 5). There were only a very few antigen antibody reactions that were not unidirectionally up- or downregulated in both populations. These areas may become more homoge- neous and the population differences further minimized if more patients are included in future studies. Thus, our results suggest that it may be benecial to characterize autoantibody repertoires in patients with glaucoma and consider their use as either diagnostic or therapeutic biomarkers in both larger study populations and individual patients. Nevertheless, there are no detailed genealogies available of U.S. patients with glaucoma. Thus, the differences in cohorts cannot be ascribed to more than nationality, although we would imply that there are truly ethnic differences in the cohorts. The use of these antibody patterns may also lead to an earlier detection of the disease, because we know from other disease such as lupus erythematosus that antibody titers de- velop well before the clinical onset of the disease. 31,32 Many human diseases appear to be a result of autoimmune insult related to a loss of self-tolerance. Autoimmune diseases can be divided into organ-specic illnesses (e.g., thyroid disease, type- 1-diabetes, and myasthenia gravis) and systemic illnesses (e.g., rheumatoid arthritis and lupus erythematosus). Although, in most autoimmune diseases, the pathogenesis of autoimmune damage is unclear, virtually all autoimmune diseases are asso- ciated with circulating autoantibodies, which bind self-anti- gens. In contrast, the role of naturally occurring autoantibodies is still unclear. We hypothesize that the loss of attenuation of some endogenous autoantibodies may signify a loss of immune protection or signify an increased risk of development of au- toimmune disease. In addition, even though autoantibodies may not be directly responsible for the manifestation of the autoimmune disease, they are often markers of future diseases in presently healthy individuals. 33 Thus, antibody titers could be useful biomarkers in intervention trials that might prevent autoimmune disease manifestations before clinical signs appear. The molecular specicity of the body is reected in sets of anti-self receptors of autoreactive T-lymphocytes and natural autoantibodies, the totality of which forms the immunologic homunculus. 34,35 This natural autoantibody repertoire is very stable in healthy subjects. If we consider these natural autoantibodies to be regulatory factors, 36 potentially able to modulate the activity of target molecules and inuence their physiological functions, it be- comes clear that the understanding of the role of up- and downregulation of autoantibodies is indeed complex. For ex- ample, although inhibition of the growth of neuronal processes by anti-NGF antibodies has been demonstrated, 37 a combina- tion of antibodies directed against different epitopes of NGF receptors results in a range of effects from mainly trophic inuence up to the induction of neuronal differentiation. 38 Other regulatory effects of autoantibodies include their ability to activate an array of receptors in living cells, thereby increas- ing the production of the secondary messengers and inducing complex cascades. 36,39,40 The complex autoantibody repertoire in healthy subjects can be considered the gold standard. The effects of antibody levels that are outside these upper or lower limits, has not been well studied; however, in certain conditions, it is clear that pathologic sequelae will develop. Typically, this is the result of excessive antibody production as in the case of myasthenia gravis. 41,42 Less well understood is the nding and relevance of decreased autoantibodies; however, associations and ndings are now beginning to emerge in both the neurologic and diabetic disorders. 43,44 Nevertheless, we demonstrated changes in the natural autoantibody proles of patients with glaucoma as evidenced by altered serum immunoreactivities to both previously identied autoantigens and the newly identi- ed autoantigen -fodrin. We emphasize that it is premature to speculate that the changes we found in autoantibody proles are pathogenic, epiphenomena, or a consequence of glauco- matous optic neuropathy. What is clear, however, is that our current and previous work, 6 taken together with the published work of U.S. (for review, see Ref. 45) and Japanese laborato- ries, 12,4648 provide irrefutable evidence that many patients with glaucoma undergo a change in their natural autoimmunity prole, as evidenced by altered serum immunoreactivity in comparison to that in age-matched patients without glaucoma. Furthermore, this body of work is in accordance with the speculative hypothesis of Fisher et al., 49 who attempt to boost natural protective autoimmunity for therapeutic gain in pa- tients with glaucoma. We advocate that the reproducible and consistent ndings of autoantibodies present among patients with glaucoma in U.S., Japanese, 12 and German patients warrant further study to gain more insight of the role of autoantibodies in glaucomatous optic neuropathy. Beside further clinical studies, the role of autoantibodies should be investigated in autoimmune animal models to see whether the disease can be elicited by selective immunogens. Although it is well known from other autoim- mune diseases, such as myasthenia gravis and multiple sclero- sis, that there is no or only a poor correlation between anti- body titers and severity, 50,51 future work should be conducted in this area, to investigate the correlation between the changes in antibody proles and disease severity. To analyze this cor- relation, it might be more promising to screen for IgM antibody proles than for IgG. The identication of fodrin as one of the potential antibody biomarkers provides further evidence of common mechanisms in the pathogenesis of Alzheimers disease and glaucoma. In summary, we found consistent autoantibody proles in both the U.S. and German cohorts with both up- and down- regulation of autoantibody activities compared with that in healthy subjects. 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