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Characteristic epitope recognition pattern of autoantibodies against eukaryotic ribosomal protein L7 in systemic autoimmune diseases.

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ARTHRITIS & RHEUMATISM
Vol. 40, No. 4, April 1997, pp 661-671
0 1997, American College of Rheumatology
661
CHARACTERISTIC EPITOPE RECOGNITION PATTERN OF
AUTOANTIBODIES AGAINST EUKARYOTIC RIBOSOMAL, PROTEIN L7
IN SYSTEMIC AUTOIMMUNE DISEASES
ELL1 NEU, PETER H. HEMMERICH, HANS-HARTMUT PETER, ULRICH KRAWINKEL,
and ANNA H. VON MIKECZ
Objective. To define the epitope-recognition pattern and the fine specificity of the autoantibody response to protein L7 in patients with rheumatic
diseases.
Methods. The epitope-recognition pattern was
studied by enzyme-linked immunosorbent assay utilizing overlapping fragments of L7. The fine specificity was
examined by binding inhibition and isoelectric focusing.
Results. We observed a disease-specific epitoperecognition pattern of anti-17 autoantibodies. There
was one immunodominant epitope that was recognized
by all anti-L7-positive sera from patients with systemic
lupus erythematosus (SLE), rheumatoid arthritis (RA),
and systemic sclerosis (SSc). Additional recognition of
minor epitopes was observed; it arises by intramolecular
epitope spreading and was correlated with disease activity in SLE patients. SSc patients differed from SLE
and RA patients in that their sera did not recognize
certain minor epitopes. The major epitope was recognized by high-affinity autoantibodies of limited heterogeneity. Minor epitopes were recognized by heterogeneous low-affinity autoantibodies.
Conclusion. The anti-17 autoantibody response is
oligoclonal. Additional B cell clones are activated by
antigen during active phases of disease.
Supported by the Deutsche Forschungsgemeinschaft through
SFB 156. Dr. Neu received “Graduiertenforderung” from the Land
Baden-Wurttemberg.
Elli Neu, PhD, Peter H. Hemmerich, PhD (current address:
Institut fur Molekulare Biotechnologie e.V., Jena, Germany), Ulrich
Krawinkel, PhD, Anna H. von Mikecz, PhD (current address: Umwelthygiene Institut, Universitat Dusseldorf, Dusseldorf, Germany):
Universitat Konstanz, Konstanz, Germany; Hans-Hartmut Peter, MD:
Medizinische Universitatsklinik Freiburg, Freiburg, Germany.
Address reprint requests to Elli Neu, PhD, Kennedy Institute
of Rheumatology, 1 Aspenlea Road, Hammersmith, London W6 8LH,
U.K.
Submitted for publication May 7, 1996; accepted in revised
form October 15, 1996.
Systemic autoimmune diseases are characterized
by the occurrence of multiple autoantibody specificities
targeting nuclear and cellular particles (for review, see
refs. 1 and 2), yet a pathologic role is discussed only for
a few of them. Anti-DNA antibodies, for example, are
thought to cause tissue injury by deposition of antigenantibody complexes in the kidneys, skin, and vessel walls
(3,4). However, autoantibodies serve as important diagnostic tools for the clinician, since distinct autoantibody
profiles are characteristic of a particular rheumatic
disease. Well-known examples are the systemic lupus
erythematosus (SLE)-specific antibodies directed
against double-stranded DNA (dsDNA) or the Sm antigen (1). Moreover, defining certain autoantigenic
epitopes can refine the clinical diagnosis (5,6). This has
been demonstrated with the anti-RoISS-A response,
which is found in patients who have SLE and Sjogren’s
syndrome (SS) (1). Bozic et a1 (7) showed that anti-Ro
52 antibodies from SLE patients mostly recognize 1
major epitope between amino acids 216 and 292, while
antibodies from SS patients generally recognize multiple
B cell epitopes between amino acids 55 and 292.
Autoantibodies often target multiple epitopes on
their respective autoantigens, as has been shown for La
(S), Ro (9), lamin B (lo), fibrillarin ( l l ) , and others. The
recognition of multiple epitopes can arise by an event
referred to as “epitope spreading.” For example, in 3 SS
patients, it was observed that in the very early stage of
the anti-La autoantibody response, only 1 immunodominant epitope was recognized. With time, the response
broadened to include other epitopes (12). Epitope
spreading was also observed in the anti-topoisomerase I
autoantibody response of a patient who had systemic
sclerosis (SSc) (13). Further evidence of epitope spreading is based on results obtained from animal models.
James et a1 (14) described epitope spreading in rabbits
after immunization with an Sm B/B’-derived octapeptide. The animals developed antibodies that bound not
NEU ET AL
662
only these octapeptides, but also many other octapeptides of Sm B/B’, and finally, they developed antibodies
that targeted other spliceosomal proteins. Intra- and
intermolecular epitope spreading was also reported by
Topfer et a1 (15), who immunized mice with either La or
Ro and obtained antibodies that targeted epitopes located on both La and Ro.
A common feature of self and non-self B cell
epitopes is that they reside in hydrophilic and highly
conserved domains (16). Accordingly, autoantigenic B
cell epitopes often seem to represent functional domains
of the autoantigen (17). Autoantigenic epitopes recognized in systemic connective tissue diseases are generally
either basic or contain extended, multivalent, chargerich segments such as a-helices (18).
We recently reported that patients with SLE,
mixed connective tissue disease (MCTD), rheumatoid
arthritis (RA), SSc, and SS frequently develop an autoimmune response to ribosomal protein 1 7 (19). Depending on the method of detection, frequencies of anti-17
positivity in patients with SLE range from 34% to 75%,
and the frequency is 50% in MCTD and SSc patients
(19,20). Three linear epitopes which fulfill the structural
criteria described above have been defined on this
molecule (21). In the present study, we define the
epitope-recognition pattern and the fine specificity of
the anti-17 autoantibody response in patients with rheumatic diseases.
PATIENTS AND METHODS
Patient sera. Patient sera were collected at the Division of Rheumatology and Clinical Immunology, Freiburg
University Hospital, at the Rheumaforschungsinstitut Aachen,
and at the Division of Immunology and Transfusion Medicine
of the Medizinische Hochschule Hannover. Patients fulfilled
the American College of Rheumatology (ACR; formerly, the
American Rheumatism Association) criteria for SLE (22), RA
(23), and SSc (24).
A 3-letter code or numbers were used to identify sera.
Sera obtained from healthy blood donors served as controls.
One patient, MLH, was a 17-year-old female who
was first diagnosed as having SLE in December 1990. The
diagnosis was based on 7 of 11 ACR criteria: malar rash,
arthritis, pleuritis and interstitial lung disease, leukopenia- and
thrombocytopenia, glomerulonephritis, antinuclear antibody
(ANA) titer of maximally 1:6,400, and anti-dsDNA antibodies.
She also showed intermittent signs of complement consumption. During a phase of strong disease activity (July 1991 to
September 1992), she experienced several infections (Salmonella enteritidis in August 1991, Listeria monocytogenes in
August 1992, and herpes zoster in November 1991). Attempts
to control the SLE with azathioprine (until July 1991), cyclosporin A (August 1991 to April l992), and methotrexate and
chloroquine (September 1991 to November 1991) failed. Only
when cyclophosphamide was introduced into the therapeutic
1
248
L7
---
PEPTIDE I
(1 - 56)
PEPTIDEII
(12-64)
PEPTIDEIII
(27-72)
PEPTIDE IV
(41 - 80)
PEPTIDE V
(54 - 88)
PEPTIDE VI
(78 - 127)
PERIDEVII
(118- 167)
PEPTIDE VIII (158 - 207)
PEPTIDE 1X
(198 - 248)
Figure 1. Schematic presentation of full-length L7 and 9 overlapping
fragments, designated peptides I-IX. Numbers indicate amino acid
positions.
regimen (April 1992 to April 1993) did the disease activity
gradually improve. Throughout the entire observation period,
the patient received prednisone at a daily dosage of 0.1-0.5
m a g of body weight, depending on disease activity.
Purification of fusion proteins. Full-length protein L7
fused to Schistosoma juponicum glutathione-S-transferase
(GST-L7) (25), 9 nested fragments of L7 fused to GST (the
designation “peptide I to peptide IX” corresponds to the
previous designation “epitope I to epitope IX’) (21), and GST
were expressed in Escherichia coli and purified by affinity
chromatography as described elsewhere (26). The purity of the
fusion proteins was assessed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Protein concentrations were determined by the Bradford method.
Enzyme-linkedimmunosorbentassay (ELISA) for antiL7 autoantibodies.ELISAs for the detection of autoantibodies
against full-length protein L7 were performed as described
(20). ELISAs for the detection of autoantibodies against
peptides I-IX were performed correspondingly. Screenings
were carried out with sera diluted 1:400.
Immunoblotting. The immunoblotting analysis was
performed as previously described (21).
Competitive binding inhibition of the ELISA with
GST-fused peptides I1 and IX. Microtiter plates were coated
with recombinant fusion proteins. Sera diluted 1:200 in PBS
containing 0.1% Tween 20, 2% milk powder, and different
concentrations (lO-‘M to 10 -9M) of the respective competitor
were added. As a control the same reaction was performed
with GST as a mock competitor. Inhibition was expressed as
(OD492
nm with competitor - background)
x 100%
(OD492
nm without competitor - background)
where OD,,, nm = optical density at 492 nm. The average
relative affinity of anti-17 autoantibodies for a given peptide is
expressed as the competitor concentration at which 50%
inhibition of binding (ICso) is achieved.
Biotinylation of GST-L7 and GST. GST-L7 and GST
were dialyzed against sodium borate buffer (O.ZM, pH 8.8).
N-hydroxysuccinimide biotin (10 mg/ml in DMSO; Sigma, St.
Louis, MO) was added to the protein solution at a ratio of 250
mg/mg of protein, and was incubated at room temperature for
HUMORAL AUTOIMMUNE RESPONSE TO PROTEIN L7
Patient Dx
MSR
RJR
m
WFR
HGN
BBR
vwl
EKS
MGR
DDZ
EMT
ADE
HSN
ETE
EVK
EBN
CGS
BK
MR
BE
SR
1
2
3
5
13
14
18
21
25
JKB
ECA
SDR
AHN
MLH
RMD
GRF
HWR
WWG
I
II
111
IV
v
VI
VII
Vlll
0.130
0.005
0.089
0.096
0.030
0.041
0.028
0.029
0.079
0.036
0.050
0.072
0.096
0.009
0.092
0.092
0.011
0.087
0.026
0.007
0.097
0.016
0.085
0.017
0.039
0.082
0.022
0.140
0.057
0.015
0.100
0.049
0.008
0.135
0.125
0.132
0.065
0.082
0.119
0.057
0.129
0.092
0.074
0.091
0.092
0.030
0.028
0.003
0.063
0.160
0.074
0.110
0.053
0.107
0.138
0.036
0.079
0.085
0.066
0.105
0.006
0.019
0.049
0.028
0.040
0.054
0.028
0.067
0.000
0.010
0.036
0.101
0.047
0.091
0.067
0.066
0.000
0.115
0.078
0.000
0.055
0.024
0.023
0.057
0.026
0.049
0.008
0.079
0.009
0.038
0.023
0.067
0.038
0.079
0.055
0.109 0.001
0.111 0.029
0.024 0.129
0.052 0.032
0.086 0.105
0.067 0.056
0.079
0.035
0.108
QLm
0.081 0.039
0.121 0.044
0.098 0.032
0.012 0.002
0.081 0.066
0.042 0.026
0.065 0.034
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
0.001
0.073
SLE
SLE
SLE
SLE
SLE
SLE
SLE
SLE
SLE
SLE
SLE
SLE
SLE
SLE
SLE
SLE
SLE
SLE
SLE
SLE
ssc
ssc
ssc
0.001
0.062
0.099
0.010
0.100 a;Zee 0.117
0.000
0.028
0.000
GEL2
0.039
0.051
0. I00
0.009
0.018
0.000
0.003
0.060
0.018
K
0.002 0.000 0.092 0.048
0.107 0.099 0.167 0.111
0.028 0.059 0.001 0.167
IX
asu
E
E
0.139
0.091
0.002
0.078
Qa-2
0.155
0.142
0.014
0.090
0.000
0.121
0.032
0.076
0.069
0.159
E
e,zLz
0.019
0.064
0.120
0.054
0.086
0.146
0.045
0.178
Patient Dx
HKY
MKG
RJE
MGK
IBR
GWR
MSR
MKZ
HDH
EGS
M
m
FSR
BDL
HSL
CKH
SPR
AST
2
3
4
5
6
7
8
9
10
13
14
16
17
18
19
20
22
23
24
25
26
27
28
29
30
31
ssc
ssc
ssc
ssc
ssc
ssc
ssc
ssc
ssc
ssc
ssc
ssc
ssc
ssc
ssc
ssc
ssc
ssc
ssc
ssc
ssc
ssc
ssc
ssc
ssc
ssc
ssc
SSC
ssc
ssc
ssc
ssc
ssc
ssc
ssc
ssc
ssc
ssc
ssc
ssc
ssc
ssc
ssc
I
0.003
0.000
0.124
0.010
0.096
0.029
0.069
0.000
0.123
0.091
0.001
0.098
0.003
0.059
0.007
0.085
0.106
0.022
0.000
0.034
0.039
0.025
0.037
0.01 1
0.033
0.035
0.037
0.046
0.052
0.034
0.0.59
0.021
0.038
0.024
0.059
0.042
0.054
0.036
0.029
0.040
0.029
0.036
0.000
I1
663
111
IV
v
VI
VII
0.018
0.1 16 0.010 0.000 0.123
0.091 0.002 0.063 0.099
0.000 0.097 0.107 0.022
0.039 0.059 0.111 0.069
0.108 0.007 0.092 0.073
0.096 0.086 0.029 0.108 0.007
0.019 0.089
0.149
0. 102 0.098 0.139 0.038
0.128 0.059 0.029 0.093 0.100
0.001 0.091 0.000 0.029 0.100
0.099 0.002
0.029 0.019
0.039 0.158 0.049 0.019 0.078
0.167
0.028 0.079 0.067
0.092
0.006 0.071 0.100
0.067 0.073 0.025 0.000 0.061
0.093 0.105 0.001 0.098
0.003 0.011 0.032 0.117
0.044 0.000 0.053 0.000 0.000
0.116 0.016 0.024 0.069 0.054
0.117 0.013 0.035 0.068 0.173
0.064 0.010 0.013 0.024 0.092
0.105 0.066 0.058 0.093 0.075
0.050 0.007 0.010 0.076 0.155
0.111 0.020 0.013 0.003 0.080
0.111 0.028 0.032 0.032 0.113
0.137 0.022 0.041 0.027 0.093
0.037 0.060 0.064 0.164
0.032 0.041 0.111
0.030 0.051 0.045
0.044 0.055 0.046 0.159
0.013 0.016 0.01 1 0.038
0.102 0.019 0.030 0.051 0.124
0.104 0.012 0.025 0.01 1 0.066
0.123 0.049 0.072 0.050 0.125
0.121 0.018 0.032 0.113 0.156
0.043 0.059 0.103 0.136
0.019 0.037 0.059 0.168
0.105 0.015 0.036 0.01 1 0.073
0.140 0.033 0.053 0.069 0.170
0.115 0.016 0.030 0.054 0.139
0.113 0.028 0.038 0.020 0.067
0.032 0.000 0.000 0.000 0.037
Eg
Lul
u
ti??
%? K
E
ti$??
w
i2i-Y
%?
I
ti!?
Vlll
0.189
0.018
0.029
0.047
0.068
0.091
0.107
0.017
0.000
0.019
0.048
0.025
0.098
0.100
0.029
0.007
0.038
0.069
0.000
0.098
0.075
0.059
0.099
0.080
0.039
0.053
0.048
0.086
0.111
0.067
0.083
0.022
0.057
0.022
0.072
0.114
0.114
0.091
0.046
0.095
0.068
0.028
0.008
IX
0.018
0.001
0.179
0.006
0.000
0.169
0.100
0.099
0.019
0.092
0.017
0.028
0.096
0.091
0.019
0.029
0.075
0.044
0.027
0.076
0.076
0.049
0.089
0.020
0.074
0.083
0.100
0.130
0.066
0.111
0.118
0.032
0.074
0.078
0.094
0.070
0.108
0.080
0.061
0.097
0.081
0.074
0.008
Figure 2. Reactivity of anti-17-positive sera with 9 overlapping glutathione-S-transferase-fusedfragments of L7, designated peptide I to peptide
IX, as shown by optical density (OD) values on enzyme-linked immunosorbent assay (1:400 dilution). Cut-off for positivity was OD,,, nm 0.200 units,
as described elsewhere (20). Positive values are shown in bold and are underlined. Dx = diagnosis; RA = rheumatoid arthritis; SLE = systemic lupus
erythemdtosus; ssc = systemic sclerosis.
4 hours. Afterwards, 20 ml of 1M NH,CI was added per 250 ml
of ester, and this was incubated for 10 minutes at room
temperature. Subsequently, the protein solution was dialyzed
against phosphate buffered saline (PBS).
Isoelectric focusing (IEF) and ligand blotting. IEF was
performed with the PhastSystem (Pharmacia, Uppsala, Sweden). PhastGel IEF 3-9, covering pH range 3-9, and PhastGel
IEF 5-8, covering pH range 5-8, were used to focus patient
sera in homogeneous 5% polyacrylamide gels. IEF was performed according to the recommendations of the supplier.
After completion of IEF, proteins were blotted onto nitrocellulose membranes (Hybond-C Super; Amersham,
Braunschweig, Germany), using the PhastTransfer Semi-dry
Transfer Kit (Pharmacia).
The nitrocellulose membranes were preincubated with
25 mM Tris, 192 mM glycine for 15 minutes. After transfer,
they were blocked with 4% nonfat dry milk powder and 0.1%
Tween 20 in PBS (PBS-T), pH 8, for 1 hour at room temperature. Biotinylated GST-L7 and GST were diluted 1:1,000 in
PBS-T containing 2% dried milk powder and incubated with
the membranes for 1 hour at room temperature. After 4
washes with PBS-T, the membranes were incubated with
peroxidase-conjugated streptavidin (Dianova, Hamburg, Germany) diluted 1:5,000 in PBS-T, 2% dried milk powder, for 1
hour at room temperature, and were subsequently washed 5
times with PBS-T. Immune complexes finally were visualized
using the enhanced chemiluminescence detection system (Amersham) and documented on x-ray films.
RESULTS
Human L7 fragments (peptides I-IX) used for
ELISA. To address the fine specificity of the anti-17
response, we established an ELISA with 9 overlapping
fragments of L7, designated peptides I-IX. The fragments were expressed as fusion proteins with GST in E
coli and were purified by affinity chromatography on
glutathione agarose (21). Figure 1 shows the positions of
peptides I-IX in L7.
Specificity of anti-17 autoantibodies. The pattern of recognition of peptides I-IX on protein L7 in
sera from 82 patients with systemic autoimmune diseases was analyzed by ELISA. Plates were coated with
peptides I-IX fused to GST or, as a control, with GST
alone. In analogy to the previously described ELISA, the
screening was carried out with sera diluted 1:400, which
NEU ET AL
Patient
(Dx)
Patient
Date
I
II
111
IV
V
VI
VII
Vlll
IX
20.03.90
23.07.90
17.06.92
0.027
0.014
0.093
17.11.94
16.12.94
02.02.95
13.02.95
03.03.95
24.03.95
0,000 0.334 0.099 0.010 0,000
0.000 0.376 0.084 0.000 0.009
0.101 0.092
0.038 0.382
0.015 0.468 0.135 0.000 0.006
0.075
0.128 0.079 0.008
0.026 0.374 0.117 0.011 0.079
29.07.91
08.08.91
22.10.91
05.11.91
09.01.92
10.03.92
03.04.92
26.05.92
14.07.92
03.09.92
27.1 1.92
29.01.93
06.04.93
17.06.93
18.08.93
06.10.93
05.04.94
12.01.9s
07.04.95
0.029
0.036
0.089
0.089
0.068
0.061
0.030
0.028
0.071
0.043
0.060
0.014
0.008
0.084
0.005
0.004
0.030
0.000
0.099
27.09.91
06.12.91
23.01.92
27.03.92
14.05.92
26.06.92
05.08.92
14.04.93
19.04.93
21.04.93
13.08.93
03.11.93
01.12.94
0.014
0.021
0.001
0.095
0.003
0.056
0.028
0.004
0.008
0.056
0.098
0.000
0.062
(SLE)
11.07.94
18.08.94
27.09.94
0.056 0.819
0.058 0.091 0.067 U f 8 0 . 0 7 9 0.069
0.049
0.128 0.109 0.049 0.091
0.159 0.007
0.101
0.091 0.019 0.102 0.004 0.082 0.082 0.103
21
(SLE)
10.11.93
13.09.94
0.041
0.113 0.022 0.021 0.019 0.049 0.016 0.022
a 8 9 0.468 0 . 3 0 4 0.036 0.115 0.051 0.121 0.044
EMT
05.02.87
17.04.90
12.12.90
06.11.91
14.08.92
04.05.94
08.11.94
0.055
0.046
0.193
0.033 0.642 0.160
0.052 0.764
0.052 0.713 0.195
0.274
0.109
0.051 0.747 0.234
0.039
0.030
0.018
0.031
0.030
0.103
0.029
04.07.90
28.01.91
23.09.92
03.02.93
13.02.95
0.036 0.719 0.191
0.185
0.062
0.000 0.753 0.180
0.182
_0.782
0.016
0.093 8.693 0.097
0.036 0.053 0.004 0.031
0.058 0.071 0.010 0.036
0.022 0.034 0.000 0.022
0.015 0.036 0,000 0,025
0.069 0.096 0.057 0.092
EVK
(RA)
ETE
(RA)
MLH
fSLE1
\
,
RMD
(SLE)
0.249
0.349
0.147 0.092 0.047 0.018 0.106 0.019 0.114
0.179 0,088 0.088 0.128 0.125 0.123 0.169
0.091 0.029 0.092 0.049 0.003 0.028 D.289
0.020 0.056
0.040 0.062
0.097 0.045
0.090
0.052 0.085
0.071 0.133
9;21Lz
0.036 0.063
0.050 0.119
0.075 0.038
0.060 0.053
0.035 0.050
0.019 0.017
0.002 0.035
0.022 0.015
0.016 0.013
0.025 0.016
0.013 0.010
0.007 0.006
0,000 0.042 0.082 0.174
0.004
0,100
0.035
0.048
0.006
0.057
0.028
0.009
0.026
0.018
0.036
0.049
0.057
0.058
0.054
0.093
0.094
0.122
0.436 0.144
0.398
0.115
9,489 0. I46
0.307 0.119
-
0.006
0.000
0.057
0.014
0.008
0.009
0.013
0.021
0.008
0.094
09.04.90
13.05.92
0.016
0,011
nsN
(RA)
02.09.92
16.09.92
28.10.92
16.12.92
10.02.93
22.11.93
26.01.94
10.03.94
0.002
0.000
0.000
0.013
0.025
0.019
0.006
0.028
0.005 0.003
0.011 0.009
0.011 0.008
0.031 0.025
0.015 0.030 0.019
0.002 0.002 0.021
0.052
0.052
0.046
0.094
0.078
0.029
12.07.88
19.12.88
01.10.90
12.11.90
26.09.91
i9.05.92
15.12.92
25.05.93
08.12.93
0.035 0.623 0.136 0.030 0.027 0.021 0.071 0.044
0.038
0.153 0.046 0.026 0.018 0 . 0 ~ 3 0.060
0.063 0 689 O.t51 0.052 0.055 0.018 0.081 0.066
0.092
0.278 0.091 0.001 0.078 0.102 0.018
0.006
0.259 0.009 0.007 0.000 0.076 0.043
0.027
0.182 0.032 0.024 0.019 0.095 0.042
0,018 0.758 O . Z l Q O . O l 8 0.027 0.006 0.091 0.057
0.101 0.686 0.091 0.108 0.084 0.061 0.002 0.091
0.013 0.631 0.152 0.014 0.022 0.002 0.067 0.046
0.108
SDR
(SLE)
10.03.88
12.11.90
22.01.92
0.066 Q,?12 0.144 0.010 0.024 0,000 0.042 0.026 0.054
0,055 0.276 0.116 0.007 0.019 0,000 0.049 0.039 0.066
0.132 0.015 0.028 0.046 0.054 0.027 0.050
0.080 Q&Q
RJR
(SLE)
23.06.93
17.03.94
14.07.94
13.12.94
0.084 0.309
0 s ~ 0.003 0.008
__ 0.....
0.084 0.704 0.195 0.016 0.036
0.104 0.589 0.098 0.086 0.008
0.061 n.701 0.182 0.015 0.031
IKB
09.02.90
01.10.90
30.10.90
15.11.90
24.01.91
24.07.91
06.08.92
10.08.92
13.08.92
02.1 1.92
13.10.93
0.007
0.027
0.012
0.005
0.003
0.025
0.026
0.093
0.003
0.024
0.01 1
23.11.90
12.04.91
10.12.91
12.08.92
17.08.92
29.06.93
08.03.94
12.04.95
0.020
0.058
0.008
0.000
0.076
0.008
0,000
0.013
(RA)
ADE
(RA)
e698
9.701.
0.072
0.068
0.043
0.065
0.059
0.093
0.057
13
(SLE)
27.06.94
06.07.94
09.02.95
0.040 0.761 0.111 0.032 0.038 0.021 0.045 0.039 0.039
0.035
0.022 0.067 0.038 0.104 0.035 0.159
0.126 0.038 0.037 0,028 0.056 0.051 0.050
0.057
18
(SLE)
14.12.94
22.12.94
0.031 9.372 0.126 0.064 0.082 0.088 0.092 0.101 0.096
0.093 0.391 0.268 0.091 0.000 0.039 0.081 0.039 0.002
RRA
05.11.91
0.038 0.689 0.198 0.010 0.037 0.059 0.123 0.065 0.103
0.101 0.034 0.057 0.079 0.132 0.079 0.125
0.045
II
111
VI
VII
Vlll
0.086 0.021 0.024 0,000 0.025 0.047 0.040
0.446 0.107 0.009 0.029 0.017 0.057 0.036 0.126
0 . 4 2 ~aoxs
0.0kZ
0.069
0.069
0.073
0.416 0.126
0.003
0,000
0.000
0.025
0,012
0.009
0.118 0.006
0.093 0.031
Q&J
&&5J
0.492
0.003
0.005
0.000
0.015
0.002
0.003
0.009
0.028
0.000 0.018 0.018 0.068
0.000 0.014 0.012 0.055
0.000
0.012
0.009
0,001
0.011
0.015
0.106
0.066
0.030
0.038
0.007
0.016
0.030 0.168
0.015 0.105
0.031 0.140
0.018 0.105
0.014 0.073
0.035 0.068
o.ono 0.094
0.000 0.075
0.006 0.092
0.098 0.085
0.095 0.082
ECA
(SLE)
0.118 0.159
0.127
0.010
0.087
0.052
0.086
0.074
0.061
0.069
0.068
0.031
0.099
0.068
0.139
0.083
0.107
0.134
0.097
0.099
0.073
0.067
0.000
0.007
0.053
0.004
0.003
0.006
0.009
0.000
0.089
0.107
0.054
0.074
0.068
0.057
0.067
0.076
0.116
0.065
0.084
0.000
0.098
0.092
0.094
0.086
0.094
0.089
0.110
(SLE)
0.126
0.056
0.136
0.127
0.157
0.146
&&6
M
0.094
0.097
0.107 0.119
AHN
(SLE)
5
IX
VWT
(RA)
0.128
0.099
0.145
0.J51 0.074 0.026 0.014 0.005 0.067 0.096 0.095
0.014
0.032
0.024
0.025
0.024
0.007
0.009
0.024
0.014
0.091
v
I
!U&!
0.335 0.085 0.043 0.023 0.018 0.083 0.100 0.174
_0.368 0.029 0.018 0.019 0.021 0.090 0.113 0.157
0.074
0.496 0.119
0.079
0.368 0.086
IV
Date
(Ox)
0.140
0.039
0.033
0.046
0.029
0.103
0.097
0.091
0.133
0.122
0.156
0.122
0.007
0.099
0.105 0.091
0.093
0.082
0.096
0.088
0.029
0.006
0.078
0.054
0.083
0.074
0.095
0.129
0.039
0.057
0.029
0.034
0.006
0.079
0.090
0.069
0.069
0.002
( M O )13.01.93
0.000
0.057
0.009
0.003
0.000
0.008
0.017
0.019
0.021
0.008
0.000
0.095
0.021
0.01 1
0.031
0.001
0.035
0.017
0.013
0.013
0.036
0.022
0,000
0.017
0.022
0.056
0.433 0.101 0.017
0.023
0.043
0.013
0.000
0.010
0.009
0.017
0.015
0.095
0.091
0.100
0.123
0.076
0.097
0.132
0.084
0.092
0.079
0.077
0.298 0.120
0.181
0 2 9 5 0.075
0.106
0.078
0.470 0.098
0.125
b.448
a
a
0.000
0.001 0.000
0.006
0.016
0.129
0.016
0.054
0.092
0.002
0.082
0.058
0.069
0.103
0.061
0.101
0.120
0.058
0.104
0.103
0.117
0.003
0.107
0.014
0.072
0.002
0.073
0.000 0.018
0.000 0.008
0.067 0.001
0.023 0.004
o.oon 0.032
0.009 0.012
0.044 0.056
0.008 0.021
0.000 0.021
0.015 0.027
0.009 0.035
0.01 1 0.101
0.010 0.046
0.003
0.019
0.014
0,000
0.013
0.012
0.032
0.034
0.065
0.074
0.005
0.059
0.068
o.oio
0.065
0.107
0,081
0.043
0.078
0.098
0.125
0.096
0.000
0.024
0.007
0.002
0.031
0.002
0.005
0.020
0.031
0.057
0.069
0.170
0.064
0.064
0.022
0.030
0.045
0.081
0.086
0.107
0.119
0.048
0.114
0.149
0.111 0.153
0.05s 0.095
0.706
Figure 3. Epitope-recognition pattern of anti-17-positive sera from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE),
and mixed connective tissue disease (MCTD) at different time points, as shown by optical density (OD) values on enzyme-linked immunosorbent
assay (1:400 dilution). Cut-off for positivity was OD,,, nm 0.200 units, as described elsewhere (20). Positive values are shown in bold and are
underlined. Date is given as day.month.year. Dx = diagnosis.
were regarded as positive when the difference between
the OD,,2 nm measured on the specific coat and the
OD492nm
on the GST coat was >0.2 units (20).
Anti-L7-positive patients were selected from a
group of 64 RA, 60 SLE, and 110 SSc patients. Among
the 82 anti-L7-positive patients, 16 had RA, 20 had SLE,
and 46 had SSc. Sera from 50 healthy individuals served
as controls. Sera positive for peptide I1 displayed ODs
homogeneously distributed in the range of 0.2 to 0.9.
Sera positive for the other peptides (peptides I, 111,
IV, V, VI, VII, VIII, and IX) displayed ODs homogeneously distributed in the range of 0.2 to 0.4. Sera with
significant anti-GST titers (OD,,, nm 20.150) have
been excluded from our analysis. It should be mentioned
that <2% of the autoimmune sera showed anti-GST
titers (20).
As shown in Figure 2, every peptide was recognized by at least 1 autoimmune serum. Peptide I1 was
recognized by all sera, and 25 sera were positive for
peptide 111, irrespective of the patient's diagnosis. In
HUMORAL AUTOIMMUNE RESPONSE TO PROTEIN L7
A
I
epitope rccngnition pattern
665
B
SLE activity (ECLAM)
peptides
Ii
-
+
+
v
in N
VI vii vm IX
MW (k
+ . - . . . .
+
-
+ +
-
+
.
-
-
+
+
t
+ - + + +
.
.
+
.
Herpes
.
- 97
ZOSler
+ - - - + - +
+
t
+ + - + + +
+ . . . . . .
1-
~
.
.
.
.
.
.
ogenes
*
+
+
+
+
- 40
. . . .
. . . .
. + . .
. . . . .
. + . .
. . . . .
. +
. . . . . . .
.
.
.
- 55
"a
+
. . . . . . .
. . . . .
. . . . .
. .
. .
I"" 93
Oct
- 31
93
94
Jan 95
95
GST I
I1
111
IV V VI VII VIII
IX
- 21
Figure 4. A, Epitope-recognition pattern of anti-17 autoantibodies, as detected by enzyme-linked immunosorbent assay, and the clinical course of
systemic lupus erythematosus (SLE) activity in patient MLH between July 1991 and April 1995. Positive reactions (+) represent OD,,, nm >0.200
units (see Figure 3). SLE activity was assessed according to the European Consensus Lupus Activity Measurement (ECLAM) index (0 = inactive
and 10 = very active) (27). Arrows indicate concomitant infections. B, Immunoblotting analysis to detect reactivities to peptides I-IX in serum
(diluted 1:1,000) from patient MLH obtained November 5, 1991. Glutathione-S-transferase (GST) was blotted as a control. MW = molecular weight
markers.
addition to the recognition of peptide I1 andlor peptide
111, 7 of 16 sera from RA patients reacted with peptide
IX, but none of them recognized peptides IV-VIII.
Compared with the sera from the RA patients, the
epitope-recognition pattern of 20 SLE patients' sera was
more heterogeneous. Peptides V-IX were recognized by
5 sera. In contrast, of 46 sera from SSc patients, none
contained antibodies to peptide IX, which may thus be
useful as a serologic marker for discriminating between
SSc and other rheumatic diseases. None of the 50 sera
from healthy persons had a titer of >0.2 units with any
of the 9 peptides.
The argument could be raised that autoimmune
sera recognize the junction between GST and the L7derived peptide. This is very unlikely, particularly in the
case of the recognition of GST-fused peptide 11. Of the
82 sera selected here for their reactivity against GSTL7,51 exclusively reacted with GST-peptide 11. Because
these fusion proteins have junction sequences which
differ from each other, and because GST alone was not
detected, it can be concluded that the common structure
recognized by autoantibodies on GST-L7 and GSTpeptide I1 lies on peptide 11.
Intramolecular epitope spreading. Blood samples taken at different time points from 10 SLE patients,
7 RA patients, and 1 MCTD patient were analyzed for
changes in the epitope-recognition pattern of the autoimmune anti-17 response during the course of the
disease. The samples were taken during a time period of
up to 7 years. The results of this analysis are summarized
in Figure 3. All patients produced autoantibodies against
peptide I1 at all time points examined. In SLE patient
MLH, the anti-17 autoimmune response was characterized by intramolecular epitope spreading. The response
was primarily restricted to peptide I1 (sample from July
29, 1991), but additional antibodies directed against
peptides 111, IV, V, VII, VIII, and IX were detected in
the 6 samples collected during the following 8 months
until April 3, 1992. Thereafter, only antibodies directed
against peptides I1 and I11 were detected, and the 8
serum samples collected between January 29, 1993 and
April 7, 1995 showed a response that was restricted to
peptide 11. Sera from SLE patients RMD and 21 and
from RA patients EVK and ETE recognized peptide I1
before peptide IX. The autoimmune B cell response to
protein L7 seemed to spread from the recognition of the
canonical peptide I1 to other less-frequently recognized
peptides.
Correlation of epitope spreading with disease
activity. In SLE patient MLH, we examined whether
epitope spreading during the course of the disease was
correlated with disease activity. Disease activity was
assessed by means of the European Consensus Lupus
Activity Measurement (ECLAM) index, which represents a standardized index for the measurement of SLE
disease activity (27). During the time period between
October 1991 and April 1992, epitope spreading and
high scores on the ECLAM index clearly coincided
(Figure 4A). IgG levels were in the normal range (8-18
gm/liter) during this time period (data not shown). To
rule out a potential recognition of bacterial contami-
NEU ET AL
666
-.-
A
B
MLH
EVK
-AETE
-v- AHN
4- HGN
-0RMD
-ACGS
-0- JKB
-. 0- competitions
with GST
-0-
100
80
60
0
k
m
I
80
Z 60
I-
T
m
I
z
$? 40
&? 40
20
20
0
0
10.'
106
1o 8
10-6
MOLARITY OF THE COMPETITOR (PEPTIDE 11)
C
-0-
-A-
-v-
-*-0-
I
1o - ~
10-8
I 0-9
OF THE COMPETITOR (PEPTIDE IX)
.
MLH
EVK
ETE
AHN
HGN
RMD
0.- competitions
with GST
r
40
MOLARITY
-.-
1001
$?
MLH
EVK
-AETE
-v- AHN
4- HGN
-0RMD
--0--competitions
with GST
-0-
0
z
z
-.-
100
-
20 -
10-6
Io
-~
10-8
Io - ~
MOLARITY OF THE COMPETITOR (PEPTIDE 11)
Figure 5. Competitive binding-inhibition enzyme-linked immunosorbent assay (ELISA) of anti-17 autoantibodies from serum MLH
(October 22, 1991), EVK (June 17, 1992), ETE (February 2, 1995),
AHN (March 8,1994), HGN (March 16,1992), RMD (April 21,1993),
CGS (June 16,1993), and JKB (November 15,1990) (see Figure 3). A,
Competition of binding to glutathione-S-transferase (GST)-fused
peptide 11-coated ELISA plates with soluble GST-fused peptide 11. B,
Competition of binding to GST-fused peptide IX-coated ELISA
plates with soluble GST-fused peptide IX. C, Competition of binding
to GST-L7-coated ELISA plates with GST-fused peptide 11.
HUMORAL AUTOIMMUNE RESPONSE TO PROTEIN L7
A
667
B
C
1234567
1234567
- 5.0
-5.0
-6.5
1234567M
-8.0
Figure 6. Isoelectric focusing analyses of anti-17 autoantibodies from 6 patients with systemic
autoimmune diseases: lane 1, patient RMD (August 13, 1993); lane 2, patient ETE (February 2,
1995); lane 3, patient RJR (June 23,1993); lane 4,patient RRA (November 5,1991); lane 5, patient
MLH (October 22, 1991); lane 6, patient JKB (November 15, 1990) (see Figure 3); and from 1
healthy subject (lane 7). A, Coomassie brilliant blue-stained gel. Serum proteins were transferred
to nitrocellulose membranes, which were developed with biotinylated glutathione-S-transferase
(GST)-L7 (B) and biotinylated GST (C). M = pH markers.
nants, we also performed immunoblotting with patient
serum MLH obtained November 5, 1991. This serum
detected only the GST-peptide fusion proteins (Figure
4B). Moreover, ELISA and immunoblotting analysis
clearly showed the same epitope-recognition pattern.
It should be mentioned that during this time,
patient MLH had bacterial and viral infections that may
have contributed to eliciting the response to minor L7
epitopes. For SLE patients RMD and 21 (Figure 3), we
cannot safely correlate epitope spreading with disease
activity because the disease parameters had not been
assessed under standardized conditions. Nevertheless, in
these patients too, epitope spreading was observed at
time points when the disease apparently was active (data
not shown). Infections were not reported in these
patients.
Relative affrnity and frequency of antipeptide I1
and antipeptide IX autoantibodies. To characterize
autoantibodies that targeted distinct peptides on protein
L7, we evaluated the quality of anti-17 autoantibodies
by determining their relative affinities for GST-fused
peptides I1 and IX in a competitive binding-inhibition
analysis. The binding of antipeptide I1 and antipeptide
IX autoantibodies to their immobilized target fragments
was inhibited when GST-fused peptide I1 and peptide
IX, respectively, were used as soluble competitors. As a
control, GST alone was used as a competitor (Figure 5).
The average relative affinities (I&) of 8 sera for
peptide I1 were in the range of 1-3.5 X lO-'M. The
steep slopes of the competition curves and the narrow
range of IC,, values indicated homogeneous antipeptide
I1 responses (Figure 5A). In contrast, the responses to
peptide IX were characterized by flat competition curves
and a wide range of low average affinities, indicating
heterogeneity (Figure 5B).
To analyze the frequency of autoantibodies targeting peptide I1 in anti-17-positive sera, we employed
ELISA plates coated with full-length protein L7 (GSTL7) and used GST-fused peptide I1 as a soluble competitor. The inhibition curves reached plateaus between
70% and 90% inhibition (Figure SC), indicating that
antibodies targeting peptide I1 represent 70-90% of the
autoimmune anti-17 response. These antibodies have
IC,, values of 0.8-3.5 X lO-'M, thus confirming the
results of the analysis shown in Figure 5A. GST alone
did not compete.
Inhibition of GST-L7 recognition by GSTpeptide TI, again, argues strongly against the theoretical
point that autoantibodies cross-react with the junction
sequence between GST and peptide 11. The peptide I1
part and not the junction should be the competitor,
because peptide I1 and GST are the only common
structures shared between GST-L7 and GST-peptide 11,
and GST alone does not compete.
Isoelectric focusing of anti-17 autoantibodies.
Anti-L7 autoantibodies were subjected to IEF to investigate the heterogeneity of the autoimmune B cell
response to the L7 protein. The spectrotype, as defined
by the IEF banding pattern, is an overall measure of
antibody heterogeneity (28,29). Anti-L7 autoantibodies
were focused, blotted onto nitrocellulose membranes,
and visualized using biotinylated GST-L7. As a control,
biotinylated GST was used.
Figure 6 shows 6 autoimmune sera (18 tested)
NEU ET AL
668
and 1 healthy serum (5 tested) that were focused in the
pH range of 5-8. All patient sera analyzed for this study
showed a restricted banding pattern. IEF is shown within
the range of 3 pH units because focusing within the
range of pH 3-9 did not improve the resolution of the
banding pattern (data not shown). Increasing the volume of the focused samples also failed to increase
spectrotypic complexity. Each patient serum showed a
restricted, but distinct, pattern of anti-17 autoantibody
spectrotypes, whereas sera from healthy individuals did
not react with biotinylated GST-L7 (Figure 6B). No
serum reacted with biotinylated GST (Figure 6C). We
assume that the banding pattern obtained here represents autoantibodies targeting peptide I1 (compare serum ETE in Figure 5C versus Figure 6 [lane 21) because
they have high affinity and represent 70-90% of the
autoimmune anti-17 response. The serum of a rabbit
repeatedly immunized with human protein L7 in adjuvant showed an unresolved smear of bands over the
entire pH range (results not shown). Since previous
analyses have shown that the autoimmune anti-17 response is restricted to the IgG isotype (20), we presume
that the banding pattern obtained here represents IgG
antibodies. Moreover, the conditions used for IEF in this
study very likely would not focus IgM.
IEF and the binding-inhibition analyses strongly
suggest that the autoimmune B cell response to protein
L7 is dominated by few clones. The clonality of the
response cannot be precisely determined by IEF because
1 B cell clone can be represented by up to 5 bands (29).
A similar limited heterogeneity is observed in antiribosoma1 P peptide autoantibodies (30) and anti-La/SS-B
autoantibodies (8).
DISCUSSION
One purpose of the present study was to investigate in patients with systemic autoimmune diseases
whether there is a correlation between the recognition of
distinct epitopes on the autoantigen L7 and their clinical
setting. To this end, we developed an ELISA using 9
fragments of L7 and screened the epitope-recognition
profile of 82 anti-17 autoantibody-positive patients with
RA, SLE, and SSc. Regardless of the diagnosis, sera
from all anti-17-positive patients analyzed here recognized 1 common epitope residing in the N-terminal part
of protein L7, namely peptide 11. So far, we have
encountered only 2 exceptions to the canonical recognition of this peptide (see below). Some patients with RA
and SLE also developed autoantibodies that targeted
epitopes located closer to the C-terminus. These auto-
antibodies have low affinity and do not simply occur as
a result of a general increased IgG level. We have shown
that the anti-17 response is not correlated with elevated
levels of IgG (20). The more heterogeneous epitoperecognition pattern found in SLE sera is consistent with
previous findings obtained by immunoblotting (21). In
contrast to SLE and RA patients, sera from the SSc
patients apparently do not recognize the epitope on
peptide IX. This is evidence of a disease-specific
epitope-recognition pattern.
Disease-specific epitope-recognition patterns
have been described for other autoantigens, such as the
60-kd SS-A/Ro protein (31), 52-kd RolSS-A protein (7),
human nuclear lamin B2 (32), and fibrillarin (11). Our
finding could be useful for the clinician, since a better
distinction between clinical subsets and overlap syndromes may result in a more precise diagnosis and
prognosis for the patient. In the case of the anti-17
response, the recognition of peptide IX could be useful
since its recognition may contribute to the exclusion of
the diagnosis of SSc. The fact, that peptide IX is targeted
by antibodies from SLE and RA, but not SSc, sera
implies that different mechanisms are involved in the
anti-17 autoimmune response in these diseases.
N-terminal peptide I1 obviously represents the
immunodominant region of L7. In a previous immunoblotting study (21), we analyzed the epitope-recognition
pattern of anti-17 autoantibodies in the sera of 6 SLE
and 3 MCTD patients. These sera, except for serum
MLH (November 5, 1991) and serum JKB (October 1,
1990), are not included in the present ELISA-based
study. The results obtained in the previous and in the
present study differ from each other in that a higher
frequency of complex recognition patterns is detected by
immunoblotting: 6 sera recognized peptide I1 plus other
peptides, 3 sera recognized only peptide 11. Sera MLH
and JKB recognized peptides that are also detected by
ELISA. There is apparently no qualitative difference
between recognition patterns defined either by immunoblotting or by ELISA. We think that the higher frequency of complex patterns in the immunoblotting analysis of relatively few sera is due to the fact that the
samples for this study were collected from patients
during active phases of their diseases. In these samples
one would expect epitope spreading.
Two RA sera, neither of which is available for
further testing, were included in the immunoblotting
study (21). They recognized only peptide V and did not
react with peptide 11. We have not seen this particular
recognition pattern in other sera. In the present study,
which included 176 individual sera, we found 2 SLE and
HUMORAL, AUTOIMMUNE RESPONSE TO PROTEIN L7
2 SSc sera that contained autoantibodies against peptide
V together with antibodies against the immunodominant
peptide I1 and other peptides (see Figures 2 and 3 ) .
Antibodies targeting the epitope on peptide V are
therefore rare and seem to appear during epitope
spreading.
There is evidence that the target structure of
peptide I1 is apparently not a linear epitope, but rather,
is a conformational epitope that involves residues at
positions 12-26 and 57-64, since only peptide 11, and not
the overlapping fragments (peptide I, peptide 111, peptide IV, and peptide V), was recognized. We have
reported elsewhere that peptide I1 very likely assumes
the conformation of an a helix and that the sequence
,,VPEXXXKKR,, is critical for the recognition by
autoantibodies, but we did not exclude the possibility
that other sequences of peptide I1 also play a role (21,
33). The present analysis strongly suggests that the
sequence shown above and a sequence located between
positions 57 and 64 form a conformational epitope. The
renaturation of this epitope so that it would be detected
by immunoblotting is consistent with the partial folding
and renaturation of other proteins after SDS-PAGE and
transfer to nitrocellulose (34-36).
Moreover, peptide I1 overlaps with the messenger RNA-binding domain of protein L7 (21,33), thus
supporting the view that autoantibodies predominantly
seem to target the functional domain of an autoantigen
(17). We also showed that autoantibodies targeting the
BZIP-like region of L7 interfere with its ability to inhibit
cell-free translation (37). In this context it should be
mentioned that peptide IX overlaps with the second
RNA-binding domain of protein L7 (Hemmerich et al:
unpublished observations).
Results of binding-inhibition analyses and isoelectric focusing strongly suggest that anti-17 autoantibodies stem from a few dominant B lymphocyte
clones that carry receptors with high affinity for peptide
11. We do not know what elicits the response to this
peptide. It can be speculated that “antigenic mimicry”
(for review, see ref. 38) plays a role, but we do not have
experimental evidence to support this hypothesis. It has
been suggested that complexes of cytoplasmic and nuclear proteins released from apoptotic cells are a source
of autoantigen (39). Since protein L7 is capable of
inducing apoptosis in lymphoid cells (40), it is possible
that there is a connection between this function of L7
and its autoantigenic properties. It is tempting to speculate that L7 from apoptotic blebs is autoimmunogenic,
but it has not been verified.
Spreading of autoantigen recognition in autoim-
669
munity has recently been reported for B and T cells. The
initial response is limited to confined regions of the
autoantigen and later spreads intra- and/or intermolecularly to additional determinants (12-15,41-47). Most
reports on epitope spreading are based on results obtained from animals immunized with autoantigens. For
example, initially cryptic determinants of myelin basic
protein were shown to become immunogenic in the
course of murine experimental allergic encephalitis
(44,45). Other investigators reported the spontaneous
loss of T cell tolerance to confined regions of glutamic
acid decarboxylase in murine insulin-dependent diabetes, with subsequent intramolecular spreading to additional determinants (46).
The intramolecular epitope spreading we observed in the anti-17 response of the SLE and RA
patients could result from stimulation of additional B
cell clones with low-affinity receptors for minor L7
epitopes by autoantigenic L7 released from as-yetunidentified sources. There are hints that this occurs
during active phases of SLE as a result of acute tissue
damage, albeit epitope spreading in correlation with
active SLE is unambiguously documented for only 1case
so far. An involvement of infections in triggering epitope
spreading remains to be determined. However, it should
be emphasized that epitope spreading is not due to a
microbial polyclonal B cell activation, since anti-17
autoantibodies are of the IgG class and still show limited
heterogeneity during periods of epitope spreading (compare Figure 5C and Figure 6 lanes 1,2, and 5). Evidence
for epitope spreading as part of the pathology of disease
relapse has also been described by McRae et a1 (47) in
murine experimental autoimmune encephalomyelitis.
The process of epitope spreading could be initiated by the recognition of a “triggering” epitope, which
may mimic a foreign antigen. In the series of evolving
autoepitopes, only 1 may have significance for the
disease, and its recognition would consequently correlate with disease parameters (6). As for the autoimmune
anti-17 response, peptide I1 may contain the triggering
epitope and peptide IX may contain the epitope significant for disease. An example of an epitope with significance for the disease is stem-loop IV of U1 RNA in
patients with SLE-overlap syndrome. Some patients
have antibodies to both epitopes of U1 RNA, but only
the antibodies to stem-loop IV correlate with the severity of the disease (48).
In conclusion, our data show a restricted diseasespecific autoantibody profile directed against protein L7.
There is one immunodominant epitope residing in the
N-terminal region of L7. Recognition of additional
NEU ET AL
670
epitopes located in the C-terminal region of L7 by
low-affinity autoantibodies is observed in some cases of
SLE and RA, and seems to indicate a state of the disease
during which autoantigenic protein L7 becomes accessible to the immune system.
ACKNOWLEDGMENTS
We gratefully acknowledge the help of Sabine Weber
(Universitatsklinikum Freiburg), who runs our serum bank.
W e thank Thomas Dick (Rheumaklinik Aachen) for kindly
providing SSc patient sera, and P. von Wussow and A. AlMasri (Medizinische Hochschule Hannover) for donating SLE
patient sera.
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