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Recognition of Granzyme B-generated autoantigen fragments in scleroderma patients with ischemic digital loss.

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Vol. 46, No. 7, July 2002, pp 1873–1884
DOI 10.1002/art.10407
© 2002, American College of Rheumatology
Recognition of Granzyme B–Generated Autoantigen Fragments
in Scleroderma Patients With Ischemic Digital Loss
Lionel Schachna, Fredrick M. Wigley, Steven Morris, Allan C. Gelber, Antony Rosen,
and Livia Casciola-Rosen
Objective. To examine whether autoantibodies
recognizing granzyme B (GB)–cleaved autoantigens are
associated with ischemic digital loss (IDL) in limited
systemic sclerosis (SSc).
Methods. Fifteen of 19 patients with limited SSc
and IDL were matched by age, sex, race, and duration of
disease to controls with limited SSc but without IDL.
The sera were used to immunoblot HeLa cell lysates and
chromosome preparations that had been incubated in
vitro in the absence or presence of GB. Anticentromere
antibodies (ACAs) were assayed by immunofluorescence
and immunoprecipitation of in vitro–translated centromere proteins (CENP-B and CENP-C). Immunoprecipitation of GB-cleaved CENPs was also performed.
Results. GB-cleaved autoantigens were immunoblotted by 16 of 19 IDL sera (84.2%) compared with 6 of
15 non-IDL sera (40.0%) (odds ratio 8.0, 95% confidence
interval 1.6–40.0). This association persisted after adjustment for ACA status. Furthermore, the presence of
antibodies to centromere proteins as well as to GBcleaved antigens was highly specific for IDL, occurring
in 12 of 19 IDL patients (63.2%) and in none of 15
controls (P < 0.0001). An identical 60-kd GB-generated
fragment was recognized by 5 of 16 IDL sera (31.3%)
and was demonstrated to arise through GB-mediated
cleavage of CENP-C. GB-cleaved CENP-C fragments
were recognized preferentially over the intact CENP-C
molecule by antibodies from patients with IDL.
Conclusion. The striking recognition of GBgenerated autoantigen fragments by sera from patients
with limited SSc and IDL constitutes the first in vivo
evidence that antibodies against GB-generated centromeric peptide fragments identify a distinct clinical
Systemic sclerosis (SSc; scleroderma) is a complex inflammatory, fibrogenic disorder in which vascular
dysfunction underlies the major clinical manifestations
(1). In common with other connective tissue disorders,
the autoantibodies elaborated in this disease are predictive of characteristic disease phenotypes (2). Wellrecognized associations include anticentromere antibodies (ACAs) with the CREST (calcinosis, Raynaud’s
phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias) variant of SSc, and anti–topoisomerase I
antibodies with the diffuse subset of SSc and pulmonary
fibrosis (for review, see refs. 3–5).
Recent studies on the connective tissue disorders
have implicated modification of autoantigen structure
during cell death in the selection of target molecules of
the autoimmune response (6,7). Relevant pathways include those occurring during both caspase-dependent
and caspase-independent modes of cell death. One
property increasingly recognized as unifying many
autoantigens across the spectrum of autoimmunity (including SSc, systemic lupus erythematosus, Sjögren’s
syndrome, inflammatory muscle disease, and Rasmussen’s encephalitis) is susceptibility to modification by
proteases in the cytotoxic lymphocyte granule pathway.
In this regard, numerous autoantigens are susceptible to
proteolytic cleavage by granzyme B (GB) during cyto-
Supported by the Scleroderma Research Foundation. Dr.
Schachna’s work was supported by a Virginia Engalitcheff Postdoctoral Fellowship award from the Maryland Chapter of the Arthritis
Foundation and by the Don & Nancy Powell, Alan & Goldijean
Turow, and Mary Lou Gilbane Carolan gift funds. Dr. Gelber’s work
was supported by an Arthritis Investigator award from the Arthritis
Foundation. Dr. Rosen’s work was supported by NIH grant DE-12354
and by a Burroughs Wellcome Fund Translational Research award.
Dr. Casciola-Rosen’s work was supported by NIH grant AR-44684.
Lionel Schachna, MBBS, FRACP, Fredrick M. Wigley, MD,
Steven Morris, MS, Allan C. Gelber, MD, MPH, PhD, Antony Rosen,
MBChB, BSc (Hons), Livia Casciola-Rosen, PhD: Johns Hopkins
University School of Medicine, Baltimore, Maryland.
Address correspondence and reprint requests to Livia
Casciola-Rosen, PhD, Johns Hopkins University School of Medicine,
720 Rutland Avenue, Ross 733, Baltimore, MD 21205. E-mail:
Submitted for publication September 21, 2001; accepted in
revised form March 25, 2002.
toxic lymphocyte granule–mediated cell death, generating unique fragments not observed during other forms of
cell death (8–10). A smaller number of autoantigens
targeted in systemic autoimmunity are also directly
cleaved by granzyme A (11–13).
While several autoantigens are cleaved with similar efficiencies (but at different sites) by both GB and
caspases during cell death, other autoantigens are exclusively or preferentially cleaved by GB (9). Prominent
among the latter molecules are autoantigens targeted in
scleroderma, including CENP-B and fibrillarin (which
are cleaved by GB but not by caspases) as well as
topoisomerase I (which is cleaved by GB ⬃100-fold
more efficiently than by caspases). Interestingly, in the
one instance in which this has been studied, autoantibodies reactive against uniquely modified forms of
autoantigens appear to be predictive of clinical phenotype. Specifically, recognition of the caspase-cleaved
form of U1–70 kd by autoantibodies was associated with
lupus skin disease (14). Since different modes of apoptotic cell death generate distinct modified forms of
autoantigens, the demonstration of an association between reactivity against specifically modified autoantigens and unique clinical phenotypes may implicate a
particular apoptotic cell death pathway in generating
disease expression.
Of particular interest in this regard is the SSc
phenotype of severe digital ischemia. Although vascular
abnormalities are prominent and universal features in
SSc patients (15,16), only a distinct subset develops
critical tissue ischemia resulting in ischemic digital loss
(IDL) (17). There are 3 recognized predictors of this
unique phenotype (17–23): limited cutaneous involvement, anti–endothelial cell antibodies (AECAs), and
Since CENP-B (a major component of the centromere antigen [24]) is cleaved by GB but not by
caspases, we addressed whether autoantibodies from
patients with limited SSc and digital loss preferentially
immunoblot GB-cleaved forms of autoantigens. We
demonstrated a striking association between recognition
of GB-cleaved autoantigens and IDL in this population.
Interestingly, the presence of both ACAs and antifragment antibodies was highly specific for IDL. In several
cases, autoantibodies preferentially recognized the
cleaved fragment over the intact form of the antigen.
Moreover, in 1 subject with serum available prior to the
onset of IDL, these autoantibodies preceded digital loss,
suggesting that antibodies recognizing GB-generated
fragments may predict subsequent phenotype. Taken
together, these studies strongly implicate the cytotoxic
lymphocyte granule pathway in the generation of the
immune response associated with this specific clinical
Study design and patient selection. Nineteen patients
with limited SSc were selected based on a history of IDL and
availability of serum, from among patients attending
The Johns Hopkins and University of Maryland Scleroderma
Center between 1991 and 2000. We attempted to match each
patient individually with a control subject with limited SSc but
without IDL. Matching variables included age (by decade of
life), sex, race, and duration of disease (within 5 years). A
single control subject was randomly selected when more than
1 matched control was available. For 4 patients, we were
unable to find matched controls by our age and disease
duration criteria. Overall, we therefore evaluated sera from 19
patients and 15 controls. In addition, serum samples from a
single patient with IDL collected sequentially over the course
of 9 years were available for analysis. The initial sample for this
patient was obtained 3 years prior to the first episode of digital
All study participants satisfied the American College
of Rheumatology (formerly, the American Rheumatism Association) criteria for SSc (25) and had skin thickening limited to
the hands, forearms, feet, legs, and/or face (26). The extent of
cutaneous involvement was also dichotomized as follows:
subjects with type I disease had sclerodactyly with or without
face or neck involvement; those with type II disease had type
I disease plus skin thickening over the forearm or leg. IDL was
defined as the loss of all or part of a digit secondary to an
ischemic event. In all but 1 patient, the first episode of digital
loss occurred prior to collection of serum samples.
Clinical and laboratory evaluation. Clinical data were
obtained in a uniform manner at the initial evaluation in the
Scleroderma Center. The presence of pulmonary hypertension, interstitial lung disease, and renal insufficiency was
determined using standard criteria (27). Risk factors for
vascular disease were recorded as dichotomous variables.
These included hypertension (defined as systolic blood pressure ⬎140 mm Hg and/or diastolic blood pressure ⬎90 mm Hg
on at least 2 separate occasions) or the use of antihypertensive
medication. Since calcium channel blockers were almost universally prescribed for the management of Raynaud’s phenomenon, only non–calcium channel blocker antihypertensives
were considered within this definition. Diabetes mellitus was
defined as at least 2 random blood glucose measurements
yielding levels ⬎200 mg/dl. The smoking status of subjects was
dichotomized as ever smoked (current or former smokers) and
never smoked. Duration of disease was calculated from the
onset of the first scleroderma feature (other than Raynaud’s
phenomenon) to the date of serum collection. In addition,
disease duration was determined from the onset of Raynaud’s
phenomenon and from the date of physician diagnosis of SSc
to the date of serum collection.
Following initial consultation in the Scleroderma Center, serum was obtained for routine hematologic and biochem-
ical measurements and conventional serologic assays and
stored at –80°C for future analyses. Conventional serologic
assays included determination of antinuclear antibody (ANA)
titer and immunofluorescence pattern using HEp-2 cells as
substrate. ANA titers ⱖ1:160 were considered positive. The
presence of anti–topoisomerase I antibodies was determined
by standard immunodiffusion techniques.
Immunoblotting of HeLa lysates to detect cleavage of
endogenous antigens. HeLa cells were cultured using standard
conditions. Lysates for in vitro reactions were prepared in
buffer A, containing 10 mM HEPES/KOH pH 7.4, 2 mM
EDTA, 1% Nonidet P40, and the protease inhibitors phenylmethylsulfonyl fluoride, antipain, pepstatin, and leupeptin
(28). In vitro incubations were performed by incubating these
lysates at 37°C for 1 hour in the absence or presence of granule
contents (an amount equivalent to 42 nM purified GB), 42 nM
purified GB, or 50 nM caspase 3. Identical fragments were
detected by immunoblotting when cleavage was performed
using granule contents or purified GB. Granule contents and
GB were prepared from YT cells as previously described (8),
and caspase 3 was a kind gift from Dr. N. Thornberry (Merck,
Rahway, NJ). GB (and granule content) cleavage reactions
were performed in the presence of 10 mM iodoacetamide
(IAA) to inhibit activation of endogenous caspases by GB.
Dithiothreitol (DTT; 5 mM) was added to reactions performed
with caspase 3 and the IAA was omitted, to enable the added
caspase 3 as well as endogenous caspases to be active.
All reactions were terminated with sodium dodecyl
sulfate (SDS)–sample buffer, and 50-␮g aliquots were electrophoresed on 10% SDS–polyacrylamide gels. Proteins were
transferred to nitrocellulose, and intact antigens and cleaved
fragments were immunoblotted (29) using patient sera at
1:2,000 dilution. Blotted proteins were detected with horseradish peroxidase–labeled secondary antibody diluted at 1:10,000
(Jackson ImmunoResearch, West Grove, PA) and chemiluminescence (Pierce, Rockford, IL).
Preparation of chromosomes. Crude chromosomes
were prepared from mitotic K562 cells as described (30), with
the following modifications. K562 cells were blocked in mitosis
by adding nocodazole at 0.4 ␮g/ml to the cultures for 16 hours.
The cells were swollen, lysed, and homogenized, and the nuclei
and debris were removed by centrifugation (250g for 5 minutes
at 4°C). Chromosomes were centrifuged (1,300g for 20 minutes
at 4°C), resuspended in buffer A, and incubated in the absence
or presence of GB for 60 minutes at 37°C. CaCl2 (2 mM) and
micrococcal nuclease (50 ␮g/ml) were added to the reactions
(for 20 minutes at 4°C) before addition of gel buffer and
boiling. The samples were immunoblotted with a rabbit polyclonal antibody raised against CENP-C (a kind gift from Dr.
Ann Pluta) (31,32) (1:2,000 dilution) or patient sera, as
described above.
Cleavage and immunoprecipitation of 35 Smethionine–labeled CENP-B and CENP-C. 35S-methionine–
labeled CENP-B and CENP-C were generated by coupled in
vitro transcription/translation (IVTT) using the appropriate
full-length complementary DNA (kind gifts from Dr. Ann
Pluta). For in vitro cleavage reactions, the radiolabeled CENPs
were diluted in buffer A and incubated for 60 minutes at 37°C
in the presence of purified caspases 3, 6, 7, or 8 (each at 50 nM)
or purified GB (at concentrations indicated in the figures). All
purified proteases were gifts from Dr. N. Thornberry (Merck,
Rahway, NJ). DTT (5 mM final concentration) was added to
the reactions containing caspases. In some cases, cleavage of
S-methionine–labeled CENP-C was performed in the presence of added unlabeled HeLa lysate (45 ␮g/reaction) and 10
mM IAA. In vitro cleavage reactions were either terminated by
adding gel buffer and boiling or were used for immunoprecipitations. In the latter case, 70 mM chymostatin was added to
stop GB activity, and all further processing was performed at
4°C. Immunoprecipitations to test which of the sera recognized
intact 35S-methionine–labeled CENP-B and CENP-C, or the
fragments generated by GB cleavage, were performed as
described (33). Gel samples were electrophoresed on 10%
SDS–polyacrylamide gels, and intact proteins and their cleaved
fragments were detected by fluorography.
Statistical analysis. Demographic, clinical, and serologic variables for the patients with IDL and for the control
patients without IDL were summarized as the mean ⫾ SD and
as proportions. Differences in these variables between patients
and controls were analyzed using Student’s t-test for continuous variables and chi-square test or Fisher’s exact test (if n ⱕ
5) for categorical variables. We performed simple logistic
regression to estimate the odds ratio (OR) and 95% confidence interval (95% CI) for recognition of GB-cleaved HeLa
lysates in patients compared with controls. Using multiple
logistic regression to evaluate for possible confounding, we
adjusted for ACA status. The relationship between recognition
of GB-cleaved HeLa lysates and IDL was also examined for
the 15 matched patients and controls using McNemar’s
matched-pairs analysis. All statistical analyses were performed
using Stata Statistical Software (Release 7.0 [2001]; Stata
Corporation, College Station, TX), and reported P values are
2-tailed with ␣ ⫽ 0.05.
Patient characteristics. The demographic, clinical, and serologic characteristics of study participants at
the time of serum collection are summarized in Table 1.
Thirty-two of the 34 study participants were women
(94.1%) and 30 were white (88.2%). Their mean ⫾ SD
age was 57.1 ⫾ 12.0 years and their mean ⫾ SD duration
of disease from SSc onset to the date of serum collection
was 13.4 ⫾ 11.3 years. The duration of disease from the
onset of Raynaud’s phenomenon was 17.7 ⫾ 13.5 years
and from the date of physician diagnosis of SSc, 10.1 ⫾
9.6 years. A higher proportion of patients with IDL had
type I skin involvement and a history of smoking,
although the difference was not statistically significant.
None of the study participants had diabetes mellitus, and
the frequencies of systemic hypertension, pulmonary
hypertension, interstitial lung disease, and renal insufficiency did not differ between patients and controls. All
subjects were ANA positive; a centromere immunofluorescence pattern was seen in 15 of 19 patients with IDL
Table 1. Demographic, clinical, and serologic characteristics among 19 SSc patients with and 15 control patients without
ischemic digital loss*
Age, mean ⫾ SD years
Female, no. (%)
Caucasian, no. (%)
Duration of SSc, mean ⫾ SD years
Skin involvement, no. (%)
Type I
Type II
Ever smoked, no. (%)
Hypertension, no. (%)
Major organ involvement, no. (%)
Pulmonary hypertension†
Interstitial lung disease‡
Renal insufficiency§
Autoantibodies, no. (%)
ANA titer ⱖ1:160
Centromere ANA pattern
All study
(n ⫽ 34)
Patients with
digital loss
(n ⫽ 19)
Controls without
digital loss
(n ⫽ 15)
57.1 ⫾ 12.0
32 (94.1)
30 (88.2)
13.4 ⫾ 11.3
59.1 ⫾ 13.9
17 (89.5)
17 (89.5)
14.2 ⫾ 11.8
54.5 ⫾ 9.0
15 (100)
13 (86.7)
12.3 ⫾ 10.9
21 (61.8)
13 (38.2)
22 (64.7)
8 (23.5)
14 (73.7)
5 (26.3)
15 (78.9)
4 (21.1)
7 (46.7)
8 (53.3)
7 (46.7)
4 (26.7)
6 (17.6)
5 (14.7)
2 (5.9)
2 (10.5)
3 (15.8)
0 (0)
4 (26.7)
2 (13.3)
2 (13.3)
34 (100)
20 (58.8)
4 (11.8)
19 (100)
15 (78.9)
1 (5.3)
15 (100)
5 (33.3)
3 (20.0)
* SSc ⫽ systemic sclerosis. ANA ⫽ antinuclear antibody.
† Estimated by right ventricular systolic pressure ⬎35 mm Hg on Doppler echocardiography.
‡ Forced vital capacity ⬍80% of predicted, or diffuse interstitial changes on chest radiography.
§ Serum creatinine concentration ⬎1.3 mg/dl.
(78.9%) compared with 5 of 15 controls (33.3%) (P ⫽
Novel fragments generated in GB-cleaved HeLa
lysates are recognized by sera from patients with digital
loss. Sera from the 19 patients with IDL were immunoblotted against control HeLa cell lysates. Of these sera,
18 detected distinct bands (for representative examples,
see left lanes of panels in Figure 1A). A robust signal
was detected using serum dilutions of 1:2,000 and enhanced chemiluminescence exposure times of 10–30
seconds. When HeLa cell lysates were incubated in vitro
with GB-containing granule contents, 16 of the 19 sera
immunoblotted novel fragments (for representative examples, see right lanes of panels in Figure 1A; open
arrows denote novel fragments). Identical results were
obtained when immunoblots were performed on lysates
made from human umbilical vein endothelial cells incubated in the absence or presence of GB (data not shown)
and when purified GB was used instead of GBcontaining granule contents (data not shown). Since in
vitro incubations were performed in the presence of 10
mM IAA to prevent activation of endogenous caspases
by GB, the new bands that were generated represent
novel fragments resulting specifically from GB cleavage
of intact antigens. Of note, the generation of identical
fragments after cleavage with GB or granule contents
demonstrates that the cleavage products observed with
the latter are specifically produced by GB rather than by
another granule component.
When sera from the 15 matched control patients
without IDL were assayed using the same approach as
that shown in Figure 1, the results were strikingly
different. Three sera did not immunoblot any bands (for
a representative example, see FW-143 serum in Figure
1B). Four sera detected topoisomerase I and/or U1–70
kd (for a representative example, see serum FW-100 in
Figure 1B). Both of these antigens are known to be
specifically cleaved by GB to generate novel fragments
(9). Six sera immunoblotted antigens in HeLa cell
lysates, but these antigens were not cleaved by GB after
in vitro incubation with the protease (for representative
examples, see sera RFW-158, RFW-148, and FW-83 in
Figure 1B). The final 2 sera immunoblotted antigens
which were cleaved by GB, generating novel fragments.
We therefore determined that 16 of 19 sera from
patients with IDL (84.2%) immunoblotted novel fragments generated in HeLa lysates by GB treatment
compared with 6 of 15 control sera (40.0%) (OR 8.0
[95% CI 1.6–40.0], P ⫽ 0.012). This association persisted after adjusting for ACA status (OR 21.5 [95% CI
Figure 1. Immunoblotting of novel fragments in granzyme B (GB)–cleaved HeLa cell lysates by sera from systemic sclerosis patients with ischemic
digital loss. HeLa lysates containing 10 mM iodoacetamide were incubated for 60 minutes in the absence or presence of GB-containing granule
contents. The samples were subsequently immunoblotted with A, sera from patients with digital loss, B, sera from patients without digital loss, and
C, sera, obtained longitudinally on the indicated dates from a patient with digital loss (patient S-43). The migration of molecular weight marker
standards (in kd) is indicated on each panel, and open arrows indicate new fragments detected after GB cleavage. In B, the identities of the bands
denoted “topo 1” and “U1-70 kDa” that were immunoblotted by serum FW-100 were confirmed by comigration with bands blotted by reference sera
and by generation of the signature 62-kd and 40-kd fragments produced by GB and caspase cleavage, respectively (in the case of U1–70 kd), and
the 95-kd fragment generated by GB cleavage (topo I) (data not shown). Topo I ⫽ topoisomerase I.
2.0–229.9], P ⫽ 0.011). In a McNemar matched-pairs
analysis involving only the 15 matched patients and
controls, GB-cleaved autoantigens were again associated
with an increased risk of IDL (OR 8.0 [95% CI 1.1–
355.0], P ⫽ 0.039).
Development of phenotype preceded by recognition of cleaved antigens in longitudinal study of sera
from patient S-43. Sera had been collected from patient
S-43 for 3 years prior to the first IDL episode and for 6
years thereafter. To address the temporal sequence of
Figure 2. Recognition, after granzyme B (GB) cleavage, of a new 60-kd fragment by 5 of 19 sera
from patients with digital loss. Lanes 1–11 and 13, HeLa lysates containing 10 mM iodoacetamide
were incubated for 60 minutes at 37°C in the absence or presence of GB-containing granule
contents. Lane 12, HeLa lysate containing 50 nM purified caspase 3 (C3) and 5 mM dithiothreitol
was incubated for 60 minutes at 37°C. The samples were immunoblotted with the indicated sera
from patients with digital loss. The migration of molecular weight marker standards (in kd) is
indicated; solid arrows denote the 60-kd fragment generated by cleavage with GB.
reactivity to GB-cleaved HeLa cells and digital loss,
these sera were used to immunoblot HeLa cell lysates
incubated in the absence or presence of GB (Figure 1C).
The sera were all used at a 1:2,000 dilution, and the
lysates were electrophoresed on a single gel and immunoblotted simultaneously to facilitate direct comparison of
the profiles. Although the first digit loss event in this
patient occurred in 1994, antibodies detecting intact
antigens and GB-cleaved fragments were present in 1991
(the first serum sample available) (Figure 1C). The sera
obtained from 1991 to 1996 immunoblotted intact antigens of ⬃250 kd (unidentified) and topoisomerase I,
both of which are cleaved by GB. In addition to antibodies against these two antigens, the sera obtained in
1999 and 2000 contained antibodies against a 44-kd
antigen, which was not cleaved by GB. Therefore, in this
patient, recognition of cleaved antigens preceded the
development of the unique associated phenotype.
Identical 60-kd fragment immunoblotted by sera
from several patients with digital loss. Interestingly, 5 of
the 16 sera that immunoblotted novel fragments in
GB-cleaved HeLa lysates recognized the identical 60-kd
fragment (solid arrow, lanes 2, 4, 6, 8, and 10 in Figure
2). In contrast to previously described GB-cleaved
autoantigens in which the intact molecule and fragment
are recognized similarly by autoantibody (8,9), this 60-kd
fragment was much more strongly recognized than any
of the high molecular weight proteins from which it may
have potentially arisen. This was particularly evident for
sera S-04, FW-14, and FW-121. This preferential immunoblotting of a GB-generated fragment is of great
interest because it was enriched in sera from patients
with IDL. When HeLa lysates were treated with caspase
3, the 60-kd fragment was not observed (compare lanes
12 and 13 in Figure 2). Caspase 3 activity in these lysates
was verified by immunoblotting with a patient serum
containing antibodies to U1–70 kd; the 40-kd fragment
arising from caspase 3 cleavage was detected (data not
shown). It is noteworthy that of the 2 sera from patients
without IDL which immunoblotted antigens cleaved by
GB, neither detected 60-kd fragments in GB-cleaved
HeLa lysates.
Recognition of CENP-B and/or CENP-C, in addition to GB-cleaved antigens, is highly specific for
digital loss in limited SSc. Since an ACA immunofluorescence pattern was also associated with IDL, the
presence or absence of antibodies to CENP-B and
CENP-C in patient and control sera was confirmed by
immunoprecipitation using 35S-methionine–labeled antigens generated by IVTT. As expected, the results were
similar to ACA determination by immunofluorescence.
Thus, 14 of 19 patient sera (73.7%) recognized CENP-B,
compared with 4 of 15 control sera (26.7%) (P ⫽ 0.006),
while 13 of 19 patient sera (68.4%) recognized CENP-C,
compared with 4 of 15 control sera (26.7%) (P ⫽ 0.016).
Four patient sera recognized non–60-kd fragments generated by GB cleavage of HeLa cells and did not
immunoblot CENP-B or CENP-C. Importantly, sera
Figure 3. Granzyme B (GB) cleavage in vitro of 35S-methionine–labeled CENP-B and CENP-C.
S-methionine–labeled CENP-B and CENP-C (generated by in vitro transcription/translation) are
cleaved by GB in vitro, generating unique fragments. Lanes 1–15, CENP-B (lanes 1–6) or CENP-C
(lanes 7–15) was incubated without added proteases (Minus) or with purified caspases (Casp) 3, 6,
7, or 8 (each at 50 nM) or GB (at the indicated concentrations). Dithiothreitol (5 mM) was added
to the caspase reactions. Lanes 16–18, Unlabeled HeLa lysate (45 ␮g) was added to reactions
containing 35S-methionine–labeled CENP-C, 10 mM iodoacetamide (to inhibit activation of
endogenous lysate caspases by GB), and the indicated amounts of purified GB. Note that higher
concentrations of GB were used in the latter reactions because the HeLa lysate itself provided a
pool of alternate (unlabeled) GB substrates (compare lanes 13 and 18). All lanes, After incubating
reactions for 60 minutes at 37°C, the samples were electrophoresed on 10% sodium dodecyl
sulfate–polyacrylamide gels, and radiolabeled CENP-B, CENP-C, and their cleaved fragments
were visualized by fluorography. The radiolabeled CENP-C fragments generated in the presence of
HeLa lysate (lanes 17 and 18) were identical to, but detected much better than, those seen in the
absence of added lysate (lanes 12 and 13).
from 12 of 19 patients with IDL (63.2%) had reactivity
to GB-cleaved fragments as well as to one or both CENP
antigens, compared with none of 15 control subjects
(OR 51.7 [95% CI 2.6–1,002.2], P ⬍ 0.0001). Therefore,
detection of antibodies reactive both against GB-cleaved
antigens and against a CENP antigen was a very specific
finding for subjects with IDL in this limited SSc population.
CENP-B and CENP-C are cleaved by GB, generating novel fragments not observed during other forms
of apoptotic death. The susceptibility of CENP-B and
CENP-C to cleavage by GB and by a panel of caspases
was tested in vitro. 35S-methionine–labeled CENP-B was
not cleaved by caspases 3, 6, 7, and 8, but was cleaved by
purified GB, generating fragments of 55, 40, 28, and 25
kd (kcat/Km ⫽ 1.1 ⫻ 104M⫺1 䡠 s⫺1) (lanes 1–6 in Figure
3). In contrast, CENP-C was cleaved by each of the
caspases tested (lanes 8–11 in Figure 3) and was very
efficiently cleaved by GB (kcat/Km ⫽ 7.2 ⫻ 104M⫺1 䡠 s⫺1)
(lanes 7, 12, and 13 in Figure 3). It is noteworthy that
cleavage of the radiolabeled CENP-C by purified GB
resulted in loss of intact antigen, without good visualization of the fragments generated, despite careful titration
of the amount of protease used (compare lanes 7, 12,
and 13 in Figure 3).
Detection of these fragments was optimized by
adding unlabeled HeLa lysate to the in vitro cleavage
reactions in the presence of 10 mM IAA (which inactivates the endogenous caspases in HeLa lysate). Under
these conditions, distinct CENP-C fragments of ⬃90 kd
(doublet), 60 kd (doublet), 40 kd, and 21 kd were
observed (lane 18 in Figure 3). Radiolabeled CENP-C
migration on SDS–polyacrylamide gel electrophoresis
(PAGE) was much more discrete when HeLa lysate was
added (compare lanes 14 and 16 in Figure 3), suggesting
that interactions with components in the lysate alter
CENP-C conformation. It is also possible that components in the lysate may be influencing the activity and/or
specificity of the cationic granzyme. Future studies will
further define the mechanisms of this effect, since they
may provide important insights into the molecular interactions in vivo that ultimately result in generation of the
cleavage fragments preferentially recognized by the patient sera.
Immunoblotted 60-kd fragment detected in GBcleaved cell lysates is derived from CENP-C. Several
previous studies have shown that autoantibody recognition of centromere proteins by immunoblotting is enhanced in mitotic cells, possibly due to mitosis-specific
posttranslational modifications (34,35). To facilitate detection of CENP-C by immunoblotting, crude chromosomes, enriched in centromere proteins, were prepared
from mitotic K562 cells. These were incubated in the
absence or presence of GB and immunoblotted with
serum S-396 or a polyclonal antibody raised against
After cleavage by GB, a novel 60-kd fragment
was detected by serum S-396; this comigrated with the
band detected by the polyclonal anti–CENP-C antibody
(Figure 4). (Note that identical sets of samples were
electrophoresed in adjacent gel lanes for this comparison.) Interestingly, both sera detected a slight alteration
in SDS-PAGE migration of CENP-C (a loss of ⬃1–2 kd)
after incubation for 60 minutes at 37°C in the absence of
GB, as compared with unincubated samples, probably
due to phosphorylation of the CENP-C in mitotic cells
which is removed by phosphatases during in vitro incubation of lysates. As noted in Figure 2, sera from
patients with IDL recognized intact CENP-C poorly by
immunoblotting, yet recognized the 60-kd fragment very
well. Interestingly, 4 sera from the control group recognized CENP-C by immunoprecipitation, but none of
these sera immunoblotted the 60-kd CENP-C fragment.
GB-cleaved form of CENP-C protein is preferentially detected by autoantibodies recognizing CENP-C.
The ability of sera from patients with IDL to detect
Figure 4. Cleavage by granzyme B (GB) of CENP-C in crude chromosomal preparations. CENP-C in crude chromosomal preparations is
cleaved by GB, generating a 60-kd fragment which comigrates with
that immunoblotted by S-396 serum. Crude chromosomes, prepared
from mitotic K562 cells, were incubated for 1 hour at 37°C in the
absence or presence of 56 nM purified GB. A third sample in each set
was not incubated; gel buffer was added directly to the freshly
prepared chromosomes. All reactions contained identical amounts of
crude chromosomes (equivalent to an amount prepared from 2.5 ⫻ 106
mitotic K562 cells), and the reactions were performed in a volume of
25 ␮l. Duplicate sample sets were electrophoresed adjacent to each
other on 10% sodium dodecyl sulfate–polyacrylamide gels and immunoblotted with a polyclonal antibody raised against CENP-C or patient
serum S-396.
intact or GB-cleaved CENP-B or CENP-C was tested
directly by immunoprecipitation. IVTT-generated
CENPs were incubated in the absence or presence of
GB, prior to immunoprecipitation with the sera. Patient
sera containing antibodies to CENP-B recognized both
the intact antigen and the GB-cleaved fragments, with a
preference for the intact antigen (compare the profile of
the cleaved CENP-B in lane 2 of Figure 5 with that of
the cleaved, precipitated CENP-B in lane 4 of Figure 5).
In contrast, patient sera containing antibodies to
CENP-C recognized the GB-cleaved CENP-C fragments well but detected the intact protein poorly (compare the profile of the cleaved CENP-C in lane 6 of
Figure 5 with that of the cleaved, precipitated CENP-C
in lane 8 of Figure 5), indicating that these sera are
fragment-specific/preferential. This is consistent with
location will require determination of the GB cleavage
sites in CENP-C by mutation analysis.
Figure 5. Preferential detection of granzyme B (GB)–cleaved form of
CENP-C. Patient sera recognizing CENP-C preferentially detect the
GB-cleaved form of this molecule. 35S-methionine–labeled CENP-B
(generated by in vitro transcription/translation [IVTT]) was incubated
in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 40 nM
purified GB for 60 minutes at 37°C. 35S-methionine–labeled CENP-C
(generated by IVTT) was added to 45 ␮g unlabeled HeLa lysate
containing 1 mM iodoacetamide and incubated in the absence (lanes 5,
7, and 9) or presence (lanes 6, 8, and 10) of 50 nM purified GB for 1
hour at 37°C. An aliquot of each of the cleavage reaction mixtures was
used to prepare a gel sample to assess the extent of cleavage (lanes 1,
2, 5, and 6). The remainder of each cleavage reaction mixture was used
for immunoprecipitation (Ipptn) with patient serum. The following
sera were used: S-04 (lanes 3 and 4), S-396 (lanes 7 and 8), and FW-121
(lanes 9 and 10). Antibodies to CENP-B recognized the intact antigen
better than the cleaved fragments. In contrast, antibodies to CENP-C
preferentially recognized the CENP-C fragments generated by GB
cleavage. Note that the additional bands (generated in IVTT reactions) that are smaller than the intact antigen arise from less efficient,
alternate initiation sites and/or premature termination of mRNA
the results observed with immunoblotting (Figure 2),
and is the first description of an autoantibody that
preferentially recognizes the cleaved form of its cognate
antigen. Of note, the patient sera recognized the prominently labeled 21-kd fragment generated by GB cleavage. It is likely that this fragment partially overlaps the
60-kd fragment observed by immunoblotting. The exact
Modification of autoantigen structure during different forms of cell death has been implicated in the
selection of molecules as targets of the autoimmune
response. By demonstrating a striking association between recognition of GB-cleaved autoantigens and a
specific clinical phenotype (IDL in limited scleroderma),
this study strongly implicates the cytotoxic lymphocyte
granule pathway in the generation of this phenotype and
its associated autoantibody response. Furthermore, this
is the first direct demonstration that a subgroup of
phenotype-associated autoantibodies preferentially recognizes the cleaved form of autoantigens, indicating that
GB-mediated cleavage plays an important role in unmasking cryptic B cell epitopes. Finally, since there are
very few predictors of the development of digital loss in
patients with limited scleroderma, these findings have
potential clinical relevance for identifying patients who
are at particularly high risk for developing IDL.
GB is a serine protease expressed in cytoplasmic
granules of cytotoxic T lymphocytes (CTLs) and natural
killer (NK) cells. It induces apoptosis in target cells
during granule exocytosis-induced cytotoxicity by catalyzing the cleavage and activation of several caspases, as
well as through caspase-independent pathways. Unlike
most forms of apoptotic death, which occur in noninflammatory contexts and actively suppress the initiation
of primary immune responses (36), the GB pathway has
a major function at the proinflammatory host–pathogen
interface, where it plays a central role in clearance of
intracellular infections (37–39).
The following findings of several recent studies
have strongly implicated the GB pathway in the pathogenesis of systemic autoimmune diseases. First, CTLs
are present and express an activated phenotype with
markedly up-regulated granzyme expression in several
diseases, including scleroderma (40), Sjögren’s syndrome (41,42), and autoimmune myositis (43). Second,
GB is expressed at high levels by several cell types
(including T cells and NK cells) within the synovium in
rheumatoid arthritis (RA) (44,45). Third, levels of GB in
joint fluid and serum appear to be predictive of the
subsequent development of erosive joint disease in RA
(46,47). Fourth, GB specifically cleaves most autoantigens at sites not recognized by caspases (9), potentially
revealing cryptic T cell epitopes. While this last property
is strongly associated with a molecule’s autoantigen
status, previous studies have not demonstrated that
recognition of GB-cleaved autoantigens associates with
particular phenotypes, nor has there been any demonstration of preferential recognition by autoantibodies of
GB-cleaved forms of antigens over the intact molecules
This study demonstrates that autoantibody recognition of GB-cleaved antigens is strongly associated with
IDL in limited SSc. The mechanism underlying this
association is not yet known, but may involve a direct
role for GB in altering the immunogenicity of these
molecules (see below) or may identify another structural
feature that influences immunogenicity.
Although they frequently coexist with ACAs, the
antibodies that recognize cleaved antigens contribute
independently to the prediction of digital loss. Approximately one-third of sera from patients with IDL recognized a 60-kd fragment arising from GB-mediated cleavage of CENP-C. Of note, antibodies to CENP-C
preferentially recognized the fragment rather than the
intact form of the antigen. Although both CENP-B and
CENP-C are susceptible to cleavage by GB, proteolysis
of these two proteins differed in 3 distinct ways. First,
CENP-C was susceptible to efficient cleavage by several
caspases as well as by GB, with generation of distinct
fragments, while CENP-B was exclusively cleavable by
GB. Second, GB cleaved CENP-C significantly more
efficiently than CENP-B. Third, GB-generated fragments of CENP-C were preferentially recognized over
the intact form by sera from patients with IDL, while
fragments of CENP-B were not preferentially bound.
Thus, among the numerous autoantigens that are
cleaved by GB, CENP-C represents the first autoantigen
in which fragment-preferential autoantibodies have
been demonstrated (9).
Fragment-specific autoantibodies may arise when
cleavage generates or reveals novel structure that is
cryptic in the native molecule. This might include exposure of the new termini generated by cleavage, or other
conformational modifications distant from the cleavage
site. The presence of such fragment-specific antibodies
argues strongly that the cleaved form of the antigen is
responsible for driving the immune response in patients
with this phenotype. The fact that the majority of
autoantibodies targeted across the spectrum of autoimmune diseases do not preferentially recognize cleaved
forms of their target molecules strongly suggests that the
B cell epitopes in these molecules are not hidden in the
intact molecule, while the relevant T cell epitopes are
(and hence might require cleavage in order to be
revealed). By demonstrating preferential recognition of
GB-induced fragments in some individuals manifesting a
unique clinical phenotype, this study shows the first in
vivo evidence that the cytotoxic lymphocyte granule
pathway plays a role in the selection of targets for an
autoantibody response. Demonstration that a similar
principle applies to the generation of T cell epitopes for
many other autoantigens is a high priority.
The fact that most patients with limited scleroderma and without digital loss did not recognize GBcleaved antigens, but did manifest other clinical features
of limited SSc, suggests that the GB pathway plays a role
in generating some aspects of this phenotype, but that it
is not a universal component of pathogenesis in this
disease. Previous studies have demonstrated that vascular cell apoptosis may be an early event in SSc (21), but
the inducers of this cell death in vivo are not yet known.
Potential candidates include cytotoxic lymphocytes (including NK cells and CTLs), antibody-mediated cytotoxicity (e.g., AECAs), and recurrent ischemia-reperfusion.
The potential for additional autoantigen modifications
(e.g., metal-catalyzed oxidation, glutathiolation) during
the last two of these processes has been emphasized
(48,49). It will be important to determine whether the
autoantigens that are shared between patients with and
without IDL and that also are not cleaved by GB are
otherwise modified during apoptotic cell death. Defining
such a unifying process will focus attention on additional
pathogenic pathways of universal importance in this
disease spectrum. In this regard, it is noteworthy that
pyruvate dehydrogenase E2, the major autoantigen in
primary biliary cirrhosis, which is sometimes associated
with limited SSc, is oxidatively modified during apoptotic death in a cell type–specific manner (49).
In summary, we have demonstrated a striking and
(in some cases) preferential recognition of GBgenerated autoantigen fragments in sera from patients
with limited SSc and the unique phenotype of IDL.
These findings constitute the first in vivo evidence that
antibodies against GB-generated centromeric peptide
fragments identify a distinct clinical subset. Furthermore, the identification of serologic risk factors for IDL
in limited scleroderma may allow the opportunity for
early targeted intervention in at-risk patients.
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