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Selective cleavage of nucleolar autoantigen B23 by granzyme B in differentiated vascular smooth muscle cellsInsights into the association of specific autoantibodies with distinct disease phenotypes.

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Vol. 50, No. 1, January 2004, pp 233–241
DOI 10.1002/art.11485
© 2004, American College of Rheumatology
Selective Cleavage of Nucleolar Autoantigen B23 by
Granzyme B in Differentiated Vascular Smooth Muscle Cells
Insights Into the Association of Specific Autoantibodies With
Distinct Disease Phenotypes
Danielle B. Ulanet,1 Nicholas A. Flavahan,2 Livia Casciola-Rosen,1 and Antony Rosen1
Conclusion. These data demonstrate that the
cleavage of B23 by GB in intact cells is dependent upon
both cell type and phenotype. The susceptibility of this
autoantigen (which is associated with a distinct pulmonary vascular phenotype in scleroderma) to GBmediated proteolysis selectively in vascular smooth
muscle cells suggests that the GB-cleavable conformation of autoantigens may occur selectively in the target
tissue, and may play a role in shaping the phenotypespecific autoimmune response.
Objective. To investigate the association of specific autoantibodies with distinct disease phenotypes.
The association of autoantibodies to nucleophosmin/
B23 with pulmonary hypertension in scleroderma, and
the susceptibility of autoantigens to cleavage by granzyme B (GB), provided a focus for these studies.
Methods. Intact cells were subjected to cytotoxic
lymphocyte granule–induced death, and the susceptibility of autoantigens to cleavage by GB was addressed by
immunoblotting and/or by a novel immunofluorescence
Results. B23 was cleaved efficiently by GB in vitro,
but was highly resistant to cleavage by GB during
cytotoxic lymphocyte granule–mediated death of many
intact cell types. In contrast, this molecule was highly
susceptible to GB-mediated proteolysis exclusively in
differentiated vascular smooth muscle cells. Topoisomerase I and several other GB substrates did not show
this striking change in cleavage susceptibility in different cell types.
The hallmarks of systemic sclerosis (scleroderma)
include widespread vascular abnormalities (1), excess
tissue fibrosis (2), and a high-titer autoantibody response to ubiquitously expressed nuclear/nucleolar proteins (3). While the etiology of the autoimmune response and accompanying vascular pathology is
currently unknown, clues may be derived from the
observation that different scleroderma-associated autoantibodies serve as markers for specific clinical phenotypes within the disease. For example, antibodies to
centromere proteins are characteristic of limited scleroderma (4,5), while antibodies to topoisomerase I are
most commonly found in diffuse scleroderma in association with pulmonary fibrosis (6,7).
Recently, antibodies to the nucleolar phosphoprotein, nucleophosmin/B23, were identified in scleroderma patient sera, and were found to occur in association with pulmonary hypertension (8), similar to
antifibrillarin antibodies (9,10). Interestingly, the antiB23 autoantibodies could be detected in patient sera
prior to detection of this pulmonary phenotype. It has
been suggested that autoantibodies may be acting as
“immunologic imprints” of the events that induced their
generation (11). The striking association of different
Supported by the Scleroderma Research Foundation, an
institutional grant from the Maryland Chapter of the Arthritis Foundation, and by the Donald Stabler Foundation. Dr. Casciola-Rosen’s
work was supported by a grant from the NIH (AR-44684). Dr. Rosen’s
work was supported by a Burroughs Wellcome Fund Translational
Research Award and by grants from the NIH (DE-12354 and HL56091).
Danielle B. Ulanet, PhD (current address: University of
California–San Francisco Diabetes Center), Livia Casciola-Rosen,
PhD, Antony Rosen, MD: Johns Hopkins University School of Medicine, Baltimore, Maryland; 2Nicholas A. Flavahan, PhD: Heart and
Lung Institute, Ohio State University, Columbus.
Address correspondence and reprint requests to Antony
Rosen, MD, Johns Hopkins University School of Medicine, 720 Rutland
Avenue, Ross 1059, Baltimore, MD 21205. E-mail: or
Submitted for publication October 3, 2002; accepted in revised form September 11, 2003.
autoantibodies with distinct phenotypes implies that
ubiquitously expressed autoantigens may exhibit specific
features in the microenvironment in which initial injury
and subsequent disease propagation occurs. Hence, defining the specific alterations that self-proteins may
display in the disease-relevant target tissues may shed
some light on important pathogenic events.
One property that unifies most autoantigens targeted across the spectrum of autoimmunity, which is not
shared by non-autoantigens, is susceptibility to cleavage
by the cytotoxic lymphocyte granule protease, granzyme
B (GB) (12–18). During cytotoxic lymphocyte granule–
mediated cell death, proteolysis by GB allows for the
generation of autoantigen fragments not observed during other forms of cell death. Of note, all of the
scleroderma autoantigens examined to date (topoisomerase I, NOR-90, RNA polymerase II, CENP-B, fibrillarin) are efficiently cleaved by GB in vitro. Since
autoantibodies to these molecules are associated with
distinct clinical phenotypes in scleroderma, we have
hypothesized that this association may be a function of
altered susceptibility of autoantigens to unique fragmentation by GB in the disease-relevant microenvironment.
In scleroderma, this microenvironment likely includes
the small vessels that become damaged during disease
initiation and propagation (19).
The association of anti-B23 autoantibodies with
pulmonary vascular disease (8) focused our attention on
the differential susceptibility of this autoantigen to
cleavage by GB in cells of the vasculature. We found that
while B23 was efficiently cleaved by GB in vitro, its
cleavage by GB was not observed in most intact cell
types undergoing cytotoxic lymphocyte granule–induced
death. In contrast, B23 cleavage by GB was highly
efficient in differentiated vascular smooth muscle cells
(VSMCs) undergoing cytotoxic lymphocyte granule–
induced death. This differential susceptibility to cleavage was not a feature of several other scleroderma
autoantigens. We propose that unique features of autoantigens in the disease-relevant microenvironment may
influence their susceptibility to cleavage by GB and
thereby favor their targeting by the autoimmune response.
Isolation of granule contents from the natural killer
(NK) cell line, YT, and purification of GB were as previously
described (20). The polyclonal rabbit antibody raised against
intact B23 (R3434) was generated as previously described (8)
and affinity-purified against recombinant B23 that was electrophoresed and transferred onto Immobilon-P (Millipore,
Bedford, MA) according to the method of Olmsted (21). The
B23 conformation-specific antibody (R3956) was generated in
rabbits by immunization with an 8–amino acid peptide of B23
(comprising the LAAD161 GB cleavage site) conjugated to
keyhole limpet hemocyanin (KLH) via an N-terminal cysteine
residue (KLH-KKVKLAAD). The R3434 antibody preferentially recognizes intact B23 by immunoblotting (with some
recognition of GB-induced fragments), while R3956 specifically immunoblots the 22-kd N-terminal GB cleavage fragment
of B23 generated by cleavage after Asp161.
In vitro cleavage of endogenous nucleolar autoantigens by GB in HeLa lysates. HeLa cells were grown using
standard tissue culture procedures in 10% heat-inactivated
newborn calf serum. HeLa cells were lysed in Nonidet P40
(NP40) lysis buffer (1% NP40, 20 mM Tris [pH 7.4], 150 mM
NaCl, 1 mM EDTA) containing the protease inhibitors pepstatin, antipain, leupeptin, and phenylmethylsulfonyl fluoride
and incubated at 37°C for 60 minutes with either purified GB
or YT cell granule contents (as indicated in the various figure
legends) and 2 mM iodoacetamide (IAA). The amount of
granule contents used was based on the GB activity (1 ␮l
granule contents contained approximately the same activity as
1 ␮l [3.7 pmoles] of purified GB). Reactions were electrophoresed on 12% sodium dodecyl sulfate (SDS)–polyacrylamide
gels and transferred to nitrocellulose for immunoblotting with
a patient antiserum recognizing topoisomerase I, or with the
polyclonal rabbit antibody recognizing B23 (R3434). Proteins
were detected using horseradish peroxidase–labeled secondary
antibodies (Pierce, Rockford, IL) and chemiluminescence.
Cleavage of endogenous nucleolar autoantigens after
in vivo incubation of intact cells with YT cell granule contents
or lymphokine-activated killer (LAK) cells. LAK cells were
prepared and incubated with K562 Fas-deficient target cells (at
a 3:1 effector:target ratio), as previously described (20). Fibroblasts were obtained from human skin biopsies and grown,
using standard tissue culture procedures, in Dulbecco’s modified Eagle’s medium (high glucose) with 20% heat-inactivated
fetal calf serum. Human umbilical vein endothelial cells
(HUVECs) and aortic smooth muscle cells (AoSMCs) were
grown with Clonetics EGM-2 and SmGM-2 BulletKits, respectively (BioWhittaker, Walkersville, MD). AoSMCs were differentiated in culture by growth in serum-free medium containing an insulin–transferrin–selenium-X supplement (Gibco
BRL, Gaithersburg, MD) for 3 days, followed by growth for an
additional 3 days in the same medium supplemented with 25
␮M MnTMPyP (a superoxide dismutase mimic) and 2 mM
N-acetylcysteine (Alexis Biochemicals, Florence, Italy), as
previously described (22).
For granule content experiments, cells were washed
with Ca⫹⫹-free Hanks’ balanced salt solution (HBSS; Gibco
BRL), lifted off 10-cm dishes, and resuspended at 8 ⫻ 106
cells/ml in Ca⫹⫹-free HBSS containing 10 mM HEPES (pH
7.4), 0.5M MgCl2, and 0.4M MgSO4. Cells were incubated in
the absence or presence of granule contents for 5 minutes on
ice. A final concentration of 1.5 mM CaCl2 was then added to
each reaction and cells were incubated at 37°C for 2 hours.
Autoantigen cleavage was monitored by immunoblot analysis
following SDS–polyacrylamide gel electrophoresis and transfer
to nitrocellulose. Patient antisera were used to immunoblot
poly(ADP-ribose) polymerase (PARP), nuclear mitotic appa-
ratus protein (NuMA), La, U1–70 kd, Ufd-2, fibrillarin, and
Calculation of relative catalytic constant (kcat/Km)
values. The percent cleavage of each substrate was determined
by densitometry. Together with the known enzyme concentration and incubation time, these values were fitted to the
first-order rate equation,
% substrate cleaved ⫽ 100 ⫻ (1 ⫺ e⫺kcat 䡠 [E]/Km 䡠 time),
to calculate nominal kcat/Km. To calculate the difference in
cleavage efficiency of different substrates in undifferentiated
and differentiated SMCs, kcat/Km values in differentiated cells
were divided by those in undifferentiated cells.
Site-directed mutagenesis to identify GB cleavage site.
A complementary DNA (cDNA) clone encoding B23 was used
as a template to generate clones with a selected Asp3 Ala
substitution in the putative GB substrate P1 position. Mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Incorporation of the
desired point mutation without additional changes to the
original sequence was confirmed by sequencing. The mutated
construct was subjected to in vitro transcription/translation in a
rabbit reticulocyte lysate-based system (Promega, Madison,
WI) to generate 35S-methionine–labeled protein, and the
S-methionine–labeled polypeptide was treated with GB as
described above to measure for inhibition of cleavage. For
detection of oligomeric B23, in vitro–translated samples were
electrophoresed on 10% SDS-polyacrylamide minigels in sample buffer containing 0.1% SDS and 5 mM dithiothreitol
without prior boiling.
Detection of GB-induced B23 fragments by immunofluorescence on AoSMCs treated with granule contents.
AoSMCs were plated on coverslips (#1 thickness; Bellco,
Vineland, NJ) and grown in either normal growth medium or
serum-free medium for differentiation. Cells were washed
several times in Ca⫹⫹-free HBSS, and coverslips were incubated in the absence or presence of granule contents in
Ca⫹⫹-free HBSS containing HEPES and Mg⫹⫹ for 10 minutes at 4°C. An equal volume of the same buffer, supplemented
with 3 mM CaCl2, was then added to the coverslips (for 1.5 mM
final concentration) and cells were incubated in a humidified
chamber at 37°C for 40 minutes. For immunofluorescence,
cells were fixed in 4% paraformaldehyde for 5 minutes at 4°C
and permeabilized in cold acetone for 20 seconds. The affinitypurified R3956 antibody was used at 10 ␮g/ml and visualized
with a fluorescein isothiocyanate–conjugated secondary antibody (Jackson ImmunoResearch, Avondale, PA). Coverslips
were mounted on glass slides with Permount (Lipshaw, Pittsburgh, PA) containing DABCO (Sigma, St. Louis, MO) prior
to viewing on an Axioskop fluorescent microscope (Zeiss,
Thornwood, NY) and photography with a DC290 Zoom digital
camera (Kodak, Rochester, NY).
Cleavage of endogenous B23 and topoisomerase I
by GB in HeLa lysates and during cytotoxic lymphocyte
granule–mediated death of intact cells. The susceptibility of B23 and topoisomerase I to cleavage by GB in
Figure 1. Demonstration, by immunoblotting, that B23 is not cleaved
during granule-induced death of intact target cells, but is cleaved in
cell lysates. A, HeLa cell lysates containing 2 mM iodoacetamide were
incubated in the absence or presence of granzyme B (GB) or YT
granule contents (GC) as follows: topoisomerase I (topo I), 17.2 nM
GB, B23, 1.0 ␮l granule contents (⬃172 nM GB). Protein (50 ␮g) was
electrophoresed in each gel lane, and autoantigens were detected by
immunoblotting using R3434 (for B23 detection) and monospecific
patient serum (topo I). B, Intact HeLa cells were incubated in the
absence or presence of 2.5 ␮l YT granule contents for 2 hours at 37°C,
as described in Materials and Methods. After termination of the
reactions, 2 ⫻ 105 cells were electrophoresed in each lane and the
samples were immunoblotted as described in A. Solid arrows indicate
intact antigens; open arrows indicate GB-cleaved fragments.
HeLa cell lysates was addressed by incubating lysates in
the absence or presence of GB or YT cell granule
contents (containing GB), and autoantigen cleavage was
detected by immunoblotting. HeLa lysates were supplemented with 2 mM IAA to inhibit the activity of
endogenous caspases. B23 was well cleaved by GB in this
system, with the production of 2 major fragments of 20
and 22 kd (Figure 1A). To address the fate of B23 in
intact cells undergoing cytotoxic lymphocyte granule–
induced death, the cleavage of B23 was examined in
HeLa cells exposed to YT cell granule contents (Figure
1B). B23 was highly resistant to cleavage in this system,
as demonstrated by a lack of/loss of intact B23 in cleaved
samples, and a complete absence of B23 fragments. An
overexposed image is shown to highlight this (Figure
1B). This was in marked contrast to topoisomerase I,
which is efficiently cleaved by GB under these conditions.
PARP cleavage in the K562 target cells could be readily
assessed. In contrast to B23, PARP was well cleaved in
the target cells (Figure 2, upper panel).
To precisely define the GB cleavage site(s) in
B23, the B23 cDNA was used as a template for sitedirected mutagenesis of potential P1 aspartic acid residues in B23 that contained the GB tetrapeptide consensus sequence recently described by Thornberry and
colleagues (23,24). Cleavage of B23 by GB was completely abolished when the aspartic acid residue D161
was mutated to alanine (Figure 3), defining LAAD161 as
the GB cleavage site.
Cleavage of B23 in VSMCs during cytotoxic
lymphocyte granule–mediated death. Since autoantigens
targeted across the spectrum of systemic autoimmune
diseases are GB substrates and are expressed ubiquitously in most cell types, while only subsets of these
autoantigens are targeted by the autoimmune response
in a particular disease, we hypothesized that the susceptibility of autoantigens to cleavage by GB may be
favored in the disease-relevant microenvironment. The
cleavage of B23 and other autoantigens was examined
during the cytotoxic lymphocyte granule–mediated
death of several different cell types that are of potential
relevance in scleroderma. The cell types chosen included
the major cellular constituents of blood vessels (fibroblasts, endothelial cells, and SMCs), potential sites of
disease initiation and propagation in scleroderma. Ex-
Figure 2. Lymphokine-activated killer (LAK) cell–induced death of
K562 cells. LAK cells were incubated with K562 target cells, as
described in Materials and Methods. After termination of the reactions, 3 ⫻ 105 LAK cells, 1 ⫻ 105 K562 cells, or 3 ⫻ 105 LAK cells plus
1 ⫻ 105 K562 cells were electrophoresed in each gel lane. B23 and
poly(ADP-ribose) polymerase (PARP) were detected by immunoblotting with R3434 (for B23) and a monospecific patient serum (for
B23 cleavage in a cell–cell killing assay was also
evaluated using K562 target cells incubated with LAK
cells. Since K562 cells are Fas-negative, killing of these
cells occurs exclusively via the granule exocytosis pathway. In this intact cell-killing assay, B23 cleavage was
entirely absent (Figure 2, lower panel). The proteolysis
of PARP was also assayed to confirm that the K562
target cells were indeed undergoing apoptosis. Since
PARP is not expressed in LAK cells, the efficiency of
Figure 3. Cleavage of B23 by granzyme B (GB) at LAAD161. Wildtype (wt) and mutated B23 (D1613 A) 35S-methionine–labeled
polypeptides, containing a point mutation in the P1 position(s) of the
predicted GB cleavage site (LAAD161), were generated. The polypeptides were incubated in the absence or presence of granule contents
(GC) for 60 minutes at 37°C. After termination of the reactions, the gel
samples, containing 0.1% sodium dodecyl sulfate, were electrophoresed. Polypeptides were detected by fluorography. Depicted is the
oligomeric form of B23, in which cleavage by GB occurs exclusively
after D161.
Figure 4. Generation of B23 fragments during cytotoxic lymphocyte granule–mediated death of
vascular cell types. A, Intact fibroblasts and undifferentiated (U) or differentiated (D) aortic
smooth muscle cells (AoSMCs) were incubated in the absence or presence of 2.5 ␮l YT granule
contents (GC) for 2 hours at 37°C. After termination of the reactions, 2 ⫻ 105 cells were
electrophoresed in each lane, and the samples were immunoblotted with R3434 to detect
cleavage of B23 or with a patient antiserum recognizing topoisomerase I (topo I). B, Undifferentiated or differentiated AoSMCs were incubated in the absence or presence of granule
contents as described above. Cleavage of autoantigens was detected by immunoblotting with
patient antisera. NuMA ⫽ nuclear mitotic apparatus protein.
periments were performed by incubating intact cells with
granule contents and evaluating autoantigen cleavage by
immunoblotting. In fibroblasts, B23 remained refractory
to cleavage upon treatment with granule contents, while
topoisomerase I was well cleaved (Figure 4A). Minimal
cleavage of B23 could be detected both in HUVECs
(results not shown) and in undifferentiated AoSMCs, as
demonstrated by the generation of the 22-kd GB fragment (Figure 4A) and a decrease in levels of the intact
protein (clearly noted on lighter autoradiograph exposures; results not shown).
To more closely replicate an in vivo microenvironment, AoSMCs were induced to undergo differentiation in culture using a method that serves to increase
oxidant activity and the expression of several differentiation proteins (see Materials and Methods and ref. 22).
The differentiation status of these cells was confirmed by
the expression of high levels of smooth muscle ␣-actin as
determined by immunoblotting (results not shown).
These differentiated cells were treated with cytotoxic
lymphocyte granules, and the cleavage of a panel of
autoantigens was assessed. In striking contrast to the
complete resistance of B23 to cleavage in other cell
types, B23 was efficiently cleaved in these differentiated
SMCs (Figure 4A) and was accompanied by the concomitant generation of the signature 22-kd GB cleavage
fragment. B23 was cleaved ⬃38-fold more efficiently in
differentiated cells (Table 1). Examination of B23 cleavage during cytotoxic lymphocyte granule–induced cell
death of keratinocytes that had been similarly grown in
a serum-free medium for 6 days did not lead to an
increased susceptibility to proteolysis by GB (results not
Cleavage of numerous additional autoantigens
in undifferentiated and differentiated SMCs was also
evaluated. Several findings were noteworthy. Although
equal protein amounts were loaded in each gel lane,
expression levels of several autoantigens were diminished in the differentiated SMCs (e.g., NuMA, U1–70
kd, and Ufd-2), while levels of other autoantigens were
unchanged in the undifferentiated and differentiated
cells (e.g., Mi-2). While B23 was resistant to cleavage in
undifferentiated SMCs but was very efficiently cleaved
in the differentiated cells, all these other autoantigens
were efficiently cleaved even in undifferentiated cells,
with little difference in cleavage efficiency being observed in differentiated and undifferentiated cells
(Figure 4B and Table 1).
Table 1. Susceptibility of autoantigens to cleavage by GB in undifferentiated and differentiated AoSMCs*
Topo I
U1–70 kd
Cleaved by GB in
SSc overlap
Cleaved by GB in
Fold increase in cleavage
efficiency (kcat/Km
diffentiated cells ⫼ kcat/Km
undifferentiated cells)
* GB ⫽ granzyme B; AoSMCs ⫽ aortic smooth muscle cells; SSc ⫽ systemic sclerosis; topo I ⫽ topoisomerase I; NuMA ⫽ nuclear mitotic apparatus
Interestingly, our previous studies have shown
that autoantibodies recognizing B23 are frequently associated with autoantibodies to fibrillarin (8). Fibrillarin
was also completely resistant to proteolysis during cytotoxic lymphocyte granule–induced death of undifferentiated VSMCs, but was much more efficiently proteolyzed (⬃19-fold) during granule-induced death of
differentiated VSMCs (Figure 4B). Unfortunately, all
available antibodies exclusively recognized the intact
form of fibrillarin and not the GB-induced fragments,
precluding definitive analysis of the mechanism of proteolysis at this time. However, the data indicate that
fibrillarin may well behave similarly to B23 under these
circumstances. The differential susceptibility of another
nonscleroderma autoantigen, PARP, to cleavage by GB
in undifferentiated versus differentiated AoSMCs could
not be assessed due to the minimal expression of this
autoantigen in the differentiated cells (results not
shown). It is of interest that the cleavage of U1–70 kd in
differentiated SMCs exclusively generates the GB cleavage fragment of this molecule, and not the caspasegenerated 40-kd fragment, which is more prominent in
undifferentiated cells (Figure 4B).
As an additional readout of the cleavage of B23
by GB in intact cells, HeLa and AoSMCs (undifferentiated and differentiated) were grown on coverslips,
treated with granule contents, and cleavage was assayed
by immunofluorescence with an antibody that selectively
recognizes GB-cleaved B23 (R3956). While this antibody was highly specific for the GB fragment of B23 by
immunoblotting of denatured protein (results not
shown), faint nucleolar staining of AoSMCs could be
detected with R3956, even in the absence of treatment
with granule contents (Figure 5). Consistent with the
finding that B23 was poorly cleaved in cytotoxic lymphocyte granule content–treated undifferentiated AoSMCs,
no significant difference was seen between R3956 staining of untreated versus granule content–treated undifferentiated cells. Similar findings were observed in HeLa
cells exposed to the same experimental protocol (results
not shown). In contrast, identical treatment of the
differentiated AoSMCs resulted in a large increase in
Figure 5. Selective detection of granzyme B (GB) fragment of B23 by
immunofluorescence in nucleoli of differentiated aortic smooth muscle
cells (AoSMCs) treated with YT cell granule contents (GC). Undifferentiated or differentiated AoSMCs were incubated in the absence
(top) or presence (middle and bottom) of YT cell GC. Generation of
GB fragments of B23 was detected by staining with the R3956 antibody
and examination by fluorescence microscopy. Bar ⫽ 70 ␮m. (Original
magnification of the images in the bottom row is 2⫻ that in the top and
middle rows.)
the intensity of nucleolar staining with R3956 in ⬃30%
of cells after a 40-minute incubation, again consistent
with the biochemical findings of selective B23 cleavage
by GB in these differentiated cells. Staining of the above
cell types with a polyclonal antibody to full-length B23
(R3434) did not reveal any alterations in the distribution
of B23 within the different cell types either in the
absence or the presence of granule content treatment
(results not shown).
In these studies, we have begun to investigate
whether unique features of ubiquitously expressed autoantigens are specifically observed in the microenvironment in which ongoing tissue damage occurs in distinct
autoimmune phenotypes. We chose to study the nucleolar autoantigen B23, which is a target of a high-titer
autoantibody response in patients with scleroderma, and
which is strongly associated with pulmonary hypertension in this disease (8). B23 is cleaved by GB in vitro;
such susceptibility to cleavage at a specific site by GB
appears to be an exclusive feature of autoantigens (12).
While most of these potential GB substrates are ubiquitously expressed, and cytotoxic lymphocyte granule–
mediated apoptosis occurs routinely during immunosurveillance by NK and cytotoxic T cells, autoantibody
responses to specific self-molecules are associated with
distinct clinical phenotypes. We have thus hypothesized
that in vivo, GB-mediated cleavage of phenotypespecific autoantigens may be regulated and potentially
favored in the relevant disease microenvironment.
We demonstrate in this study that B23 exhibits a
selective susceptibility to cleavage by GB during the
cytotoxic lymphocyte granule–mediated death of
VSMCs induced to undergo differentiation in culture.
The procedure used for differentiation of these SMCs
has previously been shown to induce a selective increase
in smooth muscle differentiation markers (smooth muscle forms of ␣-actin, SM1/SM2 myosin, and calponin)
and a resumption of the contractile activity of the cells
(22). Thus, these cells are phenotypically similar to
contractile VSMCs, which are present in the media of
blood vessels. In contrast, cultured proliferating SMCs
have low expression of differentiation markers and do
not display contractile activity (22). Despite the fact that
B23 is efficiently cleaved by GB in vitro, and GB has
been demonstrated to rapidly accumulate in the nucleolus of target cells (25), B23 is refractory to cleavage by
GB during the cytotoxic lymphocyte granule–mediated
death of all other intact cell types studied to date. In
addition to being cell-type specific, the striking enhancement of GB-mediated proteolysis of B23 and fibrillarin
in differentiated SMCs is also autoantigen specific,
because the efficiency of cleavage of several other
autoantigens studied was not dramatically changed in
these cells.
The differential susceptibility of B23 to cleavage
by GB during cytotoxic lymphocyte granule–mediated
death suggests that GB proteolysis of autoantigens can
be regulated. It is notable that SMCs are major components of vessels, a proposed site of primary injury in
scleroderma. Reversible vasospasm (19), resulting in
ischemia–reperfusion injury, is thought to play a role in
the development of the vascular lesions in scleroderma
(including endothelial cell apoptosis, and a thickening of
the intima due to the proliferation of SMCs and excess
collagen deposition) (26). The underlying vasospastic
activity in scleroderma has been proposed to be caused
in part by increased contractility of VSMCs to ␣2adrenergic receptor stimulation (27). Thus, alterations
in the phenotype and proliferation status of SMCs likely
contribute to the underlying vascular defects in scleroderma, including the vasoconstriction and vascular remodeling that occur in pulmonary hypertension. It is
therefore particularly interesting that cleavage of B23 is
enhanced in VSMCs demonstrating contractile activity,
and that the specific immune response for this molecule
is a marker of a subset of scleroderma patients with
pulmonary hypertension (8).
It is tempting to speculate that during the initial
injury leading to the dysregulation of these cells, an
active inflammatory response promotes the unique fragmentation of B23 by GB, allowing for the selection of
this molecule by the autoimmune response, and hence
the association of antibodies against B23 with the pulmonary hypertension phenotype. Fibrillarin is another
nucleolar autoantigen in scleroderma which is a GB
substrate, and which is also the target of autoantibodies
in patients with isolated pulmonary hypertension (9,10).
It is therefore particularly interesting that fibrillarin
appears to behave similarly to B23 in terms of its
enhanced sensitivity to proteolysis during granuleinduced death of differentiated SMCs. It will be important to define whether the striking correlation between
exclusive expression of a cleavable form of an autoantigen in relevant cells of the target tissue and autoantibodies to that antigen is also observed in other autoimmune rheumatic diseases.
The finding of GB fragments of autoantigens
specifically in the relevant diseased tissue would help
verify that GB proteolysis of autoantigens is of potential
relevance in vivo. The R3956 anti-B23 antibody may be
a valuable tool in this endeavor. By immunoblotting, this
antibody exclusively recognizes an N-terminal GB cleavage fragment of B23 (but not the intact molecule). Most
notably, R3956 staining of granule content–treated cultured differentiated SMCs, but no other cell types
examined, revealed a dramatic increase in the intensity
of nucleolar staining compared with untreated cells. This
antibody may thus be a useful probe for selectively
staining cells in tissue sections obtained from relevant
diseased human tissue. Since GB cleavage of autoantigens may be particularly pertinent during the initial
phase of the autoimmune response, such in vivo studies
may prove challenging in scleroderma, where the relevant microenvironment (i.e., pulmonary vessels) is quite
inaccessible, and tissue biopsy samples are frequently
only available from patients with later or end-stage
Similar studies may hold more promise in elucidating the etiology of the autoimmune response in
diseases such as cancer, in which the immunizing tissue
is self-renewing as the tumor grows, and from which
biopsy samples are frequently obtained. Of note, several
of the nucleolar scleroderma autoantigens (B23, fibrillarin, and NOR-90) can also be targeted by the autoimmune response in ⬃10% of patients with hepatocellular carcinoma (HCC) (28). In this disease, the onset of
HCC is preceded by chronic hepatitis and cirrhosis.
Also, work by Zhang et al (29) has demonstrated that
during the transition from chronic liver disease to HCC,
patients can develop autoantibodies that were not
present during the preceding chronic liver disease phase.
Since the stages of disease progression of HCC are well
defined, the use of this model will allow for the study of
changes in the GB cleavage susceptibility of nucleolar
autoantigens during tumorigenesis.
The mechanism underlying the selective susceptibility of B23 to cleavage by GB in differentiated SMCs
is currently unclear. It is possible that the uptake of GB
and/or trafficking of GB to the nucleolus could vary in
the different cell types (30,31). Preliminary comparison
of undifferentiated and differentiated AoSMCs in terms
of their levels of expression of the cation-independent
mannose-6–phosphate receptor by immunoblotting did
not reveal increased levels of this receptor for GB in the
differentiated cells (results not shown). Further, since
other autoantigens such as topoisomerase I and La were
well cleaved in the same cells in which B23 was resistant,
it is highly unlikely that the uptake and/or activity of GB
is compromised in these cell types. It will be interesting
to monitor the localization of GB during cytotoxic
lymphocyte granule–mediated cell death of the different
cell types as well as the relative sensitivity of these cells
to this form of apoptosis. It is also possible that undifferentiated cells express a GB-interacting serpin, perhaps enriched in the nucleolus. Immunoblotting studies
with an antibody against PI-9 (a proteinase inhibitor of
GB in human cells) demonstrated that PI-9 expression
was not detectable in either undifferentiated or differentiated VSMCs, while it was expressed at high levels in
HUVECs and cytotoxic lymphocytes (results not
Other antigen-specific features that might serve
to obscure the GB cleavage site (e.g., posttranslational
modifications, complex formation) could also be influencing the GB cleavage susceptibility of B23 in the
different cell types. Defining the changes in B23 binding
partners or modifications between undifferentiated and
differentiated SMCs may also provide significant insights. Additionally, understanding how the cleavage site
is obscured in intact cells and how this affects processing
of B23 may provide important insights into the relevance
of the GB cleavage site and/or cleavage in determining
the immunogenicity of this molecule. Finally, while
these studies have analyzed the cleavage of B23 in
distinct differentiation states of VSMCs, they did not
address whether B23 was differentially susceptible to
cleavage in different populations of vascular endothelial
cells. Future studies addressing this question are clearly
These studies demonstrate that B23, which is
targeted by patient autoantibodies in scleroderma (in
association with pulmonary hypertension), is selectively
susceptible to GB-mediated proteolysis in differentiated
VSMCs. The observation that phenotype-specific autoantigens express unique susceptibility to cleavage by GB
in cells of the potential target tissue focuses attention on
tissue-specific antigen conformation and protease cleavability as potentially important parameters in the selection of targets for high-titer, phenotype-specific autoantibody responses. From these data taken together, we
propose that the immunizing microenvironment might
play a central role in shaping the specific autoimmune
response in human autoimmune diseases.
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