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Serum Autoantibodies to -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. 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. The present study provides further evidence
that changes in natural autoimmunity occur in some patients
with glaucoma. The use of autoantibody proles in patients
with some forms of glaucoma could be a useful approach for
glaucoma detection, monitoring, and possible guidance for
using immunomodulating therapy.
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