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Does the cellular localization of antigens in or on apoptotic blebs influence the pathogenicity or benefit of cognate antibodies Comment on the article by Dieud┬й et al.

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2080
A, Takei S, Sadeghi S, Stout A, Shaham B, Bernstein B, et al.
Etanercept therapy in children with treatment-resistant uveitis.
Arthritis Rheum 2001;44:1411–5). In that article we reported
that etanercept injected subcutaneously twice weekly had a
beneficial effect in 10 children (18 eyes) with treatmentresistant chronic uveitis. Within 3 months, 10 of 16 affected
eyes (63%; P ⫽ 0.017) showed a rapid decrease in anterior
chamber cell density, with remission achieved in 4 eyes.
Among children with a visual acuity of ⬍20/25, 4 of 10 eyes
(40%) improved. An exacerbation of uveitis during treatment
with etanercept occurred in only 1 child (1 of 14 eyes) (7%).
Other ocular outcome parameters, such as intraoccular pressure, synechiae formation, and lens clarity, remained unchanged. An increase in the dosage of etanercept to an average
of 1.1 mg/kg after 3 months in 7 children failed to produce
further improvement after 6 months of treatment.
Since the completion of our original data accumulation, 2 years have passed. In the meantime, multiple studies
describing the use of tumor necrosis factor antagonists in
uveitis treatment have been published. Of particular interest
are the results of a small double-blind placebo-controlled trial
of etanercept in the treatment of pediatric uveitis, reported as
an abstract at the 2002 American College of Rheumatology
Annual Scientific Meeting (Smith JA, Smith S, Whitcup SM,
Suhler E, Clarke G, Thompson D, et al. The treatment of
JRA-associated uveitis with etanercept [abstract]. Arthritis
Rheum 2002;46 Suppl 9:S482). In that study 12 children (mean
age 11 years) were treated with either etanercept 0.4 mg/kg
(maximum dosage 25 mg twice weekly) or placebo. After 6
months the authors were unable to detect any improvement in
anterior chamber cell density in either patient group. In
contrast, the beneficial effect of the treatment has been
maintained in a majority of our patients. Of the 10 children
originally enrolled, 2 withdrew within the first 6 months for
reasons unrelated to the study drug. Both children had initially
improved, and had flares after etanercept was discontinued.
Four children did not respond to treatment, and 3 of them
discontinued etanercept within 12 months. One child continues
to have uncontrolled uveitis but still takes etanercept for
control of his arthritis.
Of the 4 children with sustained improvement, 1 girl
continued to show improvement for 18 months, and her vision
normalized. The family has since moved and the child was lost
to followup. One girl who initially had a favorable response
discontinued etanercept after 14 months of treatment when
she experienced a grand mal seizure. Results of a diagnostic
evaluation, including magnetic resonance imaging of the brain,
lumbar puncture, and electroencephalography, were inconclusive for demyelinating disease. She was diagnosed as having
benign childhood epilepsy (rolandic seizures) and started on
gabapentin treatment. One month after discontinuation of
etanercept she experienced a severe uveitis flare in both eyes.
Etanercept was restarted 3 months later, and the uveitis again
improved. She still has occasional mild seizure activity, while
the uveitis in her left eye remains fully controlled and her right
eye has minor inflammation. One girl continued to show
improvement and had minimal uveitis but chose to discontinue
etanercept after 22 months. She experienced a significant
disease flare 2 months later. She was initially unable to restart
etanercept due to a worldwide shortage of the drug. However,
she was able to resume etanercept therapy after 15 months,
LETTERS
and her uveitis has improved significantly ever since. Lastly,
one girl continues to improve, currently has no active uveitis,
and her vision has significantly improved.
In summary, in 6 of 10 children enrolled in this small,
prospective open-label trial, etanercept had a beneficial effect
for at least 3 months, and 4 children exhibited sustained benefit
for at least 1 year. Etanercept may represent a viable alternative for children with active uveitis who have been treated
unsuccessfully with traditional disease-modifying antirheumatic drugs.
Andreas Reiff, MD
University of Southern California
Childrens Hospital Los Angeles
Los Angeles, CA
DOI 10.1002/art.11139
Does the cellular localization of antigens in or on
apoptotic blebs influence the pathogenicity or benefit
of cognate antibodies? Comment on the article by
Dieudé et al
To the Editor:
Given the flood of attention directed toward understanding the affector arm in autoimmune diseases, studies
that address the effector arm to study the pathogenicity
of the perpetrator are of great interest. In certain circumstances the effects of an autoantibody may even be beneficial.
This was in fact the situation being studied by Dieudé et al,
who sought the mechanism by which autoantibodies to nuclear lamin B1 abrogate the strong prothrombotic risk associated with the lupus anticoagulant (1). The authors tested
the hypothesis that circulating anti–lamin B1 antibodies
block the procoagulant effect of apoptotic blebs by binding to
lamin B1 displayed at the external surface of the blebs.
However, by both biochemical and morphologic criteria, their
extensive studies convincingly negated this notion. Lamin B1,
in contrast to SSB/La, is not present on the surface of the
apoptotic blebs but rather remains buried within the bleb,
thereby being inaccessible to external anti–lamin B1 antibodies. Although these findings were considered negative in one
context, they are indeed informative when considering another
context.
As discussed by the authors, although apoptotic blebs
serve as an important target for certain autoantibodies, it
is now apparent that this is not generalizable to all autoantigens. With regard to the pathogenicity of anti-SSA/Ro
and anti-SSB/La in the development of congenital heart
block, unambiguous demonstration of antibody binding to
the surface of apoptotic cardiomyocytes was a critical link that
provided a plausible explanation of how antibodies were
accessible to otherwise sequestered intracellular antigens (2).
If intracellular trafficking of sequestered nuclear antigens to
the membrane surface is not universally applicable to all such
antigens, this may help explain the specificity of one antibody
system versus another in the pathogenesis of disease, in
particular tissue damage in the developing fetal heart.
LETTERS
2081
Figure 1. Cellular topology of SSA/Ro–SSB/La and lamin B1 in permeabilized and nonpermeabilized apoptotic
cultured human fetal cardiomyocytes. For all fields shown, cultured human fetal cardiomyocytes were incubated
with staurosporine to induce apoptosis (round condensed nuclei; Hoechst stained [blue]). A–C, Cells were
permeabilized prior to staining. A, Apoptotic blebs emerging from the surface were stained by a combination of
affinity-purified human antibodies to 48-kd SSB/La, 52-kd SSA/Ro, or 60-kd SSA/Ro (red fluorescence). B,
Similar results were observed in permeabilized apoptotic cells stained with anti–lamin B1 antibodies (green
fluorescence). In this same field, anti–lamin B1 antibodies stained only the nucleus in the nonapoptotic cell. C,
Neither the 3 apoptotic cells nor the single nonapoptotic cell stained with either normal human serum or mouse
isotype control. D, Apoptotic cells were not permeabilized prior to staining. Strong red fluorescence indicates
surface staining by the affinity-purified anti-SSA/Ro–anti-SSB/La antibodies. In contrast, there is no staining by
the anti–lamin B1 antibody. Hoechst crosses intact membranes, accounting for the blue staining in these
nonpermeabilized cells.
In parallel with the studies presented by Dieudé et al
(1), we have confirmed and extended the observation that
lamin B1 is redistributed during apoptosis but, unlike SSA/Ro
or SSB/La, is not bound by cognate antibodies. Induction of
apoptosis included not only staurosporine, the protein kinase
C inhibitor used by Dieudé et al, but also the redox cycling
quinone, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). A
third method of inducing apoptosis exploited culturing of the
cells on poly-(2-hydroxyethyl methacrylate) (poly-HEMA),
which results in apoptosis because cells are unable to adhere
due to the loss of anchoring signals (3). The target cells were
human cardiomyocytes, isolated and cultured from fetuses of
gestational age 16–24 weeks. Cells were separately incubated
with 0.5 ␮M staurosporine for 7 hours, 0.2 mM DMNQ for 7
hours, or plated on poly-HEMA for 12 hours. Cells were then
incubated in the absence or presence of mouse anti–lamin B1
antibody (Chemicon, Temecula, CA) and a combination of
affinity-purified human antibodies to 48-kd SSB/La, 52-kd
SSA/Ro, and 60-kd SSA/Ro for 30 minutes at 37°C. The media
were removed, and the cells were washed twice in phosphate
buffered saline with calcium. Cells were fixed with 4% paraformaldehyde for 20 minutes at 22°C. In a second set of
experiments the apoptotic cells were first permeabilized with
100% acetone for 60 minutes at 22°C prior to addition of the
primary antibodies. The permeabilized and nonpermeabilized
cells were subsequently incubated with fluorescein
isothiocyanate–conjugated rabbit anti-mouse IgG (for localization of lamin B1) or CY3-conjugated goat anti-human IgG (for
localization of SSA/Ro and SSB/La) for 30 minutes at 22°C.
Matched isotype controls were negative.
In permeabilized nonapoptotic cardiomyocytes, lamin
B1 and SSA/Ro–SSB/La antigens were localized to the nucleus
2082
LETTERS
with minor cytoplasmic staining, consistent with previous reports (1,4,5, and data not shown). With regard to the latter
antigens, it is acknowledged that the topology observed cannot
be ascribed uniquely to 52-kd or 60-kd SSA/Ro or to 48-kd
SSB/La, given the affinity-purified antisera used for detection.
After induction of apoptosis by all 3 methods, lamin B1 as well
as SSA/Ro–SSB/La translocated to apoptotic blebs (Figure 1A
and B). However, lamin B1, in contrast to SSA/Ro–SSB/La,
was not detected at the cell surface (Figure 1D).
Taken together, our observations and those of
Dieudé et al support discordance in the final cellular destination of translocated nuclear autoantigens during the process
of apoptosis. In the case of lamin B1, physiologic noninflammatory clearance of apoptotic cells should proceed uneventfully even in the presence of circulating cognate antibodies. However, in congenital heart block, the maternal
anti-SSA/Ro–anti-SSB/La antibodies result in opsonization
and inflammatory/fibrotic sequelae. Even if it turns out that
SSA/Ro–SSB/La are not unique in this regard, there may be
other factors, such as complement binding of certain antigens
or degradation of antigens, that facilitate clearing without
further sequelae. Establishing the fact that at least one other
nuclear autoantigen is not surface-bound during apoptosis of
human fetal cardiomyocytes is a step forward.
Supported by NIH grant AR-42455. Ms. Chandrashekhar’s work was supported by the Arthritis Foundation, New York
chapter.
Robert M. Clancy, PhD
Hospital for Joint Diseases
and New York University School of Medicine
New York, NY
Edward K. L. Chan, PhD
University of Florida
Gainesville, FL
Sarayu Chandrashekhar, BS
Jill P. Buyon, MD
Hospital for Joint Diseases
and New York University School of Medicine
New York, NY
1. Dieudé M, Senécal JL, Rauch J, Hanly JG, Fortin P, Brassard N, et
al. Association of autoantibodies to nuclear lamin B1 with thromboprotection in systemic lupus erythematosus: lack of evidence for
a direct role of lamin B1 in apoptotic blebs. Arthritis Rheum
2002;46:2695–707.
2. Miranda-Carús ME, Dinu Askanase A, Clancy RM, Di Donato F,
Chou TM, Libera MR, et al. Anti-SSA/Ro and anti-SSB/La autoantibodies bind the surface of apoptotic fetal cardiocytes and
promote secretion of TNF-␣ by macrophages. J Immunol 2000;165:
5345–51.
3. Clancy RM, Askanase AD, Kapur RP, Chiopelas E, Azar N,
Miranda-Carus ME, et al. Transdifferentiation of cardiac fibroblasts, a fetal factor in anti-SSA/Ro-SSB/La antibody-mediated
congenital heart block. J Immunol 2002;169:2156–63.
4. Chan EKL, Andrade LEC. Antinuclear antibodies in Sjögren’s
syndrome. Rheum Dis Clin North Am 1992;18:551–70.
5. Veldhoven CH, Pruijn GJ, Meilof JF, Thijssen JP, van der Kemp
AW, van Venrooij WJ, et al. Characterization of murine-monoclonal antibodies against 60 kD Ro/SS-A and 48 kD La/SS-B
autoantigens. Clin Exp Immunol 1995;101:45–54.
DOI 10.1002/art.11176
Reply
To the Editor:
We thank Clancy et al for their interest in our work and
for providing us with an opportunity to speculate regarding
why some autoantigens are confined within apoptotic blebs
while others are expressed at the bleb surface. In a large,
multicenter study, we demonstrated the association between
the presence of autoantibodies to nuclear lamin B1 in patients
with systemic lupus erythematosus (SLE) and protection
against thrombosis (thromboprotection). We then aimed to
elucidate the mechanism by which autoantibodies to nuclear
lamin B1 cause thromboprotection in vivo. Because a number
of autoantigens in SLE have been localized specifically to the
external surface of apoptotic blebs (1–3), we hypothesized that
circulating autoantibodies to nuclear lamin B1 may block the
procoagulant effect of apoptotic blebs by binding to lamin B1
displayed at the external bleb surface. Therefore, with biochemical and morphologic studies, we determined the localization of lamin B1 in apoptotic cells and blebs. We used
Jurkat cells, a well-studied cell type model for apoptosis, and
human umbilical vein endothelial cells. Apoptosis was induced
using 2 agents: staurosporine (to induce the mitochondrial
apoptotic pathway) and anti-Fas antibody (to trigger the cell
death receptor apoptosis pathway). In all cases, lamin B1 was
shown to be translocated into surface blebs during apoptosis
but was entirely enclosed within the apoptotic bleb membrane.
Clancy et al, with microscopy studies, now extend our
results to another cell type, cardiomyocytes, which were induced into apoptosis using DMNQ and poly-HEMA. In the
figure provided by Clancy et al, corresponding phase-contrast
images and scale bars would have been helpful to better
identify morphologic structures, especially for the abnormal
nuclear morphology observed in Figure 1D, which appears
quite different from the nuclear morphology observed in
Figures 1A–C. Nevertheless, Clancy et al confirm our results
by showing that during apoptosis lamin B1 is relocalized to the
blebs but is not recognized by anti–lamin B1 antibodies under
nonpermeabilized conditions.
Our finding that lamin B1 is not expressed at the
surface of blebs is in striking contrast with the finding that
other autoantigens, such as Ro and La, are expressed at the
surface of blebs. As discussed by Clancy et al, the demonstration of antibody binding to the surface of apoptotic cells is a
potentially critical link to the physiopathologic mechanisms
applicable to anti-Ro and anti-La, but obviously this is not
generalizable to all autoantigens such as lamin B1.
These data raise the important question of the predictability of autoantigen expression at the surface versus the
interior of apoptotic blebs (i.e., why are certain autoantigens
expressed at the surface while others are not?). Autoantigens
such as Ro and actin (4) that are translocated at the external
surface of the blebs are cytoplasmic. Even the La nuclear
antigen is known to shuttle between nucleus and cytoplasm,
playing a role in the biogenesis of RNA polymerase III
transcripts and translation (5). Indeed, La is predominantly,
but not exclusively, immunolocalized to the nucleus in nonapoptotic cells (6). Furthermore, the signal for La to reenter
the nucleus, located in the La C-terminus, is cleaved during
early apoptosis, causing La accumulation in the cytoplasm (7).
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