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(Investigative Ophthalmology and Visual Science. 2006;47:968-976.)
© 2006 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.05-0685
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Serum Autoantibodies to {alpha}-Fodrin Are Present in 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. Wax3,4

1From the Departments of Ophthalmology and 2Clinical Chemistry and Laboratory Medicine, Johannes Gutenberg-University, Mainz, Germany; 3Alcon Laboratories Inc., Fort Worth, Texas; and the 4Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. Glaucoma is characterized by a progressive loss of retinal ganglion cells that results in a characteristic optic neuropathy associated with visual field loss. In previous studies, changes in the antibody profiles have been shown in the sera of patients with glaucoma, and these findings suggest a role for autoimmune involvement in the pathogenesis of glaucoma in some patients. The purpose of this study was to compare the antibody profiles 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 profiles 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 profiles in both study populations was demonstrated in the number and frequency of both up- and downregulation of antibody reactivities in patients with glaucoma of both national cohorts. The multivariate analysis of discriminance found a significant difference between 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). Furthermore, a newly described antibody biomarker in both study populations was identified as {alpha}-fodrin. Western blot results revealed that there was an increased frequency and enhanced immunoreactivity to {alpha}-fodrin (120 kDa) in the sera of patients with NPG. The presence of {alpha}-fodrin autoantibodies were confirmed by ELISA, in which a highly elevated anti-{alpha}-fodrin titer in patients with NPG was found to be significantly 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 identified 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 subjects. A newly described autoantibody to {alpha}-fodrin found in other neurodegenerative diseases such as Alzheimer’s, further implicate a role for autoimmunity and the neurodegenerative processes in glaucoma. The high correspondence of the autoantibody patterns found in the study populations from different continents provides further evidence that serum autoantibody patterns may be useful biomarkers for glaucoma detection or for determining prognosis in future studies by means of pattern-matching algorithms.

Glaucoma 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 field.

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 autoantibodies against ocular antigens are upregulated in serum of patients with glaucoma, such as heat shock proteins (HSPs),1 {gamma}-enolase,2 glutathione S-transferase,3 anti-phosphatidylserine,4 and glycosaminoglycans.5 These findings suggest that there may be changes in systemic humoral immunity underlying optic neuropathy in at least some patients with glaucoma. All these works have in common that they analyzed the immunoreactivity of only one or very few antibodies in the cohort populations that were studied.

Using an antibody profiling technique that is based on Western blot and digital image analysis combined with multivariate statistics and artificial neural networks, our group demonstrated significant and selective up- and downregulated autoantibody repertoires against ocular antigens in the sera of both primary open-angle (POAG) and normal-pressure glaucoma (NPG).6 7 The relationship of these autoantibodies to the pathogenesis of glaucoma is unclear. Some studies demonstrated 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 profiles found in patients with glaucoma,6 7 relatively few of the serum autoantibodies have been identified from previous studies. Nevertheless, it is often routine in clinical practice to use specific 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 profiling or specific autoantibodies have the potential to be used as diagnostic or therapeutic biomarkers in patients with glaucoma, it would first be important to investigate whether glaucoma patient autoantibody profiles are similar in different study populations. The purpose of this study was to analyze and compare the antibody profiles against optic nerve in patients with POAG or NPG and healthy subjects from Germany and the United States. In addition, we attempted to identify novel unidentified serum autoantibodies that were not reported in previous studies.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patients
One hundred twenty patients were included in the study: 60 from the United States and 60 from Germany. Each study subpopulation consisted of three groups: the first 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 Ophthalmology Department, University of Mainz, Germany, or at the Department of Ophthalmology and Visual Science, Washington University, St. Louis, Missouri. The patient classification 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 field, open iridocorneal angles, optic nerve cupping, and an absence of alternative causes of optic neuropathy (e.g., infection, inflammation, ischemic disease, and compressive lesions). The inclusion 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 field testing was performed with Goldmann perimetry. All patients 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 significant differences among the groups (P > 0.125). The patients of the U.S. cohorts have been described elsewhere in detail.1 12 The racial classification 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 –80°C.

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 bromphenol blue [pH 6.8]). The samples were first 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 –80°C for later analysis.

The bovine optic nerve extracts were used for 13.5% SDS-PAGE (MultiGel-Long; Biometra, Göttingen, 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 H+L, 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 flatbed scanner (Epson GT-9000; Epson Germany, Duüsseldorf, 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 profiles were compared by an analysis of discriminance.

Recently, this technique has been successfully used as standard protocol to analyze autoantibody repertoires in patients with myasthenia gravis,14 experimental uveitis,15 Tourette syndrome,16 and Sydenham chorea.17

Mass Spectrometry
Protein identification was performed by mass spectrometry (MS). The corresponding electrophoresed immunoreactive bands of the Western blot analysis were excised manually by a Pasteur pipette and transferred into a 1.5-mL reaction tube. Excised gel pieces were treated twice with 400 µL of 50% methanol containing 10% acetic acid and agitated for 45 minutes, followed by incubation in 400 µL of a buffer containing 100 mM ammonium bicarbonate (pH 8.0) with agitation for 30 minutes, followed by incubation in 400 µL of 50% acetonitrile containing 100 mM ammonium bicarbonate with agitation for 1 hour, and finally, incubation in 50 µL 100% acetonitrile with agitation for 15 minutes. Afterward, gel pieces were centrifuged (Concentrator; Eppendorf, Fremont, CA) to complete dryness. For rehydration and digestion, individual gel pieces were covered with 10 µL of a trypsin solution (0.04 µg/µL; Roche, Mannheim, Germany) for 10 minutes, followed by the addition of up to 20 µL 25 mM ammonium bicarbonate and incubation at 37°C 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 files and used for database searches with MASCOT (www.matrixscience.com/ Matrix Science, 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.

ELISA of {alpha}-Fodrin Antibodies
To assess the reactivity of autoantibodies in the sera of patients against the whole recombinant {alpha}-fodrin protein, we performed an ELISA (Aesculisa {alpha}-Fodrin A; Aesku Diagnostics, Wendelsheim, Germany). This ELISA uses human recombinant {alpha}-fodrin to test the antibody reactivity. The test was performed according to the guidelines of the manufacturer.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
All patients showed unique but similar complex staining patterns of IgG antibodies against optic nerve antigens. In Figure 1 representative randomly selected antibody profiles 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 antigen–antibody reactivity at this molecular weight. Several immunoreactive bands were present in each patient, but the figure 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
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  		FIGURE 1. Antibody profiles of 24 different patients: four control subjects (CTRL), four patients with POAG, and four patients with NPG from both countries. Right: patients from the German study; left: densitometric data from the U.S. patients. The intensity of antigen-antibody-reactivity (U) is plotted versus the molecular mass (kDa).

 

Figure 2
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  		FIGURE 2. This figure demonstrates some representative Western blot analysis from the U.S. study population (US) and the German population (GER). Furthermore, two negative controls (NC) are shown (secondary antibody only, no sera).

 
Figure 3 reveals the mean antibody reactivity in both control groups (from the U.S. and German substudies). The antibody profiles were analyzed for each patient in each group, and the average was calculated for each molecular weight. Both control groups revealed a complex autoantibody repertoire against optic nerve antigens, in agreement with our previous studies6 7 that revealed complex profiles of naturally occurring autoantibodies in healthy subjects.


Figure 3
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  		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).

 
Figures 4 and 5 demonstrate the differences in the mean antibody profiles of patients with POAG or NPG in both substudies (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 profiles of the POAG and NPG groups from the mean antibody profile of the control group (CTRL) was calculated.


Figure 4
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  		FIGURE 4. Comparison of the mean antigen-antibody reactivity of patients with POAG from the U.S. study population to the German. 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). All reactivities above zero represent upregulations in comparison with healthy subjects; all below zero are downregulations in comparison to the control group.

 

Figure 5
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  		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).

 
Figure 4 reveals the differences in mean antibody profiles between healthy subjects and patients with POAG from the 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 profiles in the NPG group.

In both the NPG and the POAG groups a very large similarity was found between the antibody profiles 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 furthermore, most of these regions corresponded to each other in the German and U.S. studies. Thus, reproducible up- and downregulation of autoantibodies were found, regardless of the country from which the patients were derived. There were surprisingly only a few regions that showed confounding effects 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 immunoreactivity in this region was negligible and similar in patients with POAG from each country.

The analysis of discriminance found a significant difference (P < 0.001) between glaucoma and control subjects in both countries, based on the complex antibody profiles, rather than on single peaks.

The analysis of discriminance is able to calculate a parameter, 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 understood as individual repertoire clustering. The closer the canonical 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 figure demonstrates that data for most of the patients with glaucoma (POAG or NPG) lay outside the accumulation of healthy subjects (CTRL).


Figure 6
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  		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.

 
In addition, the analysis of discriminance can calculate the Mahalanobis distances, which provide a measure of how different the clinical groups are from each other on average, based on the complex antibody profiles. 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 significant difference 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 discriminant function analysis, a model of discrimination is built step by step. Specifically, at each step, all immunoreactivities of each molecular weight are reviewed and evaluated to determine 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 demonstrates the 10 most significant molecular weights of immunoreactivities (i.e., the first 10 discriminatory steps), included in the analysis of discriminance. These antibodies, which have the most separation between glaucomatous or nonglaucomatous patterns, can also be considered antibody biomarkers for this disease.


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  		TABLE 1. The 10 Most Significant Biomarkers Derived from the Analysis of Discriminance

 
Although it is not necessary to know the identity of these antigens to consider them as useful in defining 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 identified as {alpha}-fodrin (spectrin). The analysis was repeated three times in other electrophoretic separations and yielded the same result. This was consistent with our findings of primary immunoreactive bands at 120 kDa and secondarily at ~150 kDa, which is a typical banding pattern for {alpha}-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).


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  		TABLE 2. Identification of {alpha}-Fodrin by Tandem Mass Spectrometry

 
To test whether the autoantibodies against the 120 kDa {alpha}-fodrin band recognize nondenatured fodrin, an ELISA with human recombinant {alpha}-fodrin as antigen was performed. The ELISA test confirmed the results from the Western blot analysis (Fig. 7) . The antibody titer against nondenatured {alpha}-fodrin was highly elevated in patients with NPG compared with the control 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). Furthermore, the {alpha}-fodrin titer in patients with NPG was significantly higher than in patients with POAG (NPG MW: 105.82; CTRL MW: 77.26; P = 0.04).


Figure 7
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  		FIGURE 7. Antibody titer ({alpha}-fodrin, mean ± SE) are plotted for healthy subjects (CTRL), patients with POAG, and those with NPG.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
There are several studies that propose an autoimmune mechanism may be involved prominently in patients with NPG.18

For example, an increased prevalence of monoclonal gammopathy,19 retinal immunoglobulin deposition,1 elevated serum titers of autoantibodies to many optic nerve5 and retinal antigens1 3 8 12 20 and abnormal T-cell subsets21 have been reported in many patients with glaucoma. Increased autoantibodies in the serum of patients with glaucoma include those to heat shock proteins (hsps), such as hsp60, hsp27, and {alpha}-crystallins,8 expression of which are upregulated in the glaucomatous 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 confirmed the results of preceding studies, in which it was found that the autoantibody profiles 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 profiles 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 findings 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 pathogenesis of glaucoma in some patients.

A key finding of the present study was the biochemical identification of a newly identified autoantibody in our patients that we identified as fodrin by MS. Fodrin (also called spectrin in erythrocytes) is a tetramer consisting of two distinct polypeptide chains, {alpha} and ß, 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 filaments to the plasma membrane in many different kinds of cells. Fodrin is also thought to be involved in the pathogenesis of other neurodegenerative diseases. In Alzheimer’s disease, an anti-brain spectrin immunoreactivity has been demonstrated.23 24 25 Furthermore, {alpha}-fodrin is a target of caspase-3 and is cleaved by caspases at very early stages of apoptosis leading to structural rearrangements, including membrane blebbing.26 The apoptotic cell death activates a proteolytic cascade, including caspase-8 and -3.27 Caspases are thought to play a major role in apoptotic processes in chronic neurodegenerative disorders such as Huntington’s, Parkinson’s, and Alzheimer’s diseases.28

In the literature, it is demonstrated that the proteolytic cleavage of {alpha}-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 specific for caspase-3 cleavage,26 whereas the 150 kDa results from cleavage by calpain, a protease 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 identified as {alpha}-fodrin. This result provides further hints of caspase activation in patients with glaucoma and is in accordance with other studies that demonstrated that {alpha}-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 evidence that there may be many shared mechanisms in the pathogenesis of glaucoma and other neurodegenerative diseases such as Alzheimer’s disease. However, the precise pathogenic significance of our findings 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 {alpha}-fodrin used as the antigen. Confirming the Western blot results, a highly elevated reactivity against {alpha}-fodrin was demonstrated in patients with NPG, whereas in patients with POAG, there was only a slight increase in activity compared with the control group.

The second major finding of our study was specific patterns of autoantibodies in patients with glaucoma that were characterized by both up- and downregulated areas of serum immunoreactivity in comparison to control subjects. A comparison of complex antibody profiles 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 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 quantitative data that can identify both up- and downregulated antibodies of specific molecular weight and thus reveal whether the antibody profiles are significantly different from the control group and specific to patients with glaucoma. Therefore, even without knowing the identity of those antibodies, these specific 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 patients 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 homogeneous and the population differences further minimized if more patients are included in future studies. Thus, our results suggest that it may be beneficial 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 develop 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-specific 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 associated with circulating autoantibodies, which bind self-antigens. 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 autoimmune 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 specificity of the body is reflected 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 influence their physiological functions, it becomes clear that the understanding of the role of up- and downregulation of autoantibodies is indeed complex. For example, although inhibition of the growth of neuronal processes by anti-NGF antibodies has been demonstrated,37 a combination of antibodies directed against different epitopes of NGF receptors results in a range of effects from mainly trophic influence 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 increasing 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 finding and relevance of decreased autoantibodies; however, associations and findings are now beginning to emerge in both the neurologic and diabetic disorders.43 44 Nevertheless, we demonstrated changes in the natural autoantibody profiles of patients with glaucoma as evidenced by altered serum immunoreactivities to both previously identified autoantigens and the newly identified autoantigen {alpha}-fodrin. We emphasize that it is premature to speculate that the changes we found in autoantibody profiles are pathogenic, epiphenomena, or a consequence of glaucomatous 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 laboratories,12 46 47 48 provide irrefutable evidence that many patients with glaucoma undergo a change in their natural autoimmunity profile, 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 patients with glaucoma.

We advocate that the reproducible and consistent findings 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 autoimmune diseases, such as myasthenia gravis and multiple sclerosis, that there is no or only a poor correlation between antibody titers and severity,50 51 future work should be conducted in this area, to investigate the correlation between the changes in antibody profiles and disease severity. To analyze this correlation, it might be more promising to screen for IgM antibody profiles than for IgG.

The identification of fodrin as one of the potential antibody biomarkers provides further evidence of common mechanisms in the pathogenesis of Alzheimer’s disease and glaucoma.

In summary, we found consistent autoantibody profiles in both the U.S. and German cohorts with both up- and downregulation of autoantibody activities compared with that in healthy subjects. The present study provides further evidence that changes in natural autoimmunity occur in some patients with glaucoma. The use of autoantibody profiles in patients with some forms of glaucoma could be a useful approach for glaucoma detection, monitoring, and possible guidance for using immunomodulating therapy.


   Footnotes
 
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 "advertisement" 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{at}eye-research.org.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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