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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 4 (February 15), 1999:
pp. 1264-1270
Enhanced Endothelial Cell Apoptosis in Splenic Tissues of
Patients With Thrombotic Thrombocytopenic Purpura
By
Chau T. Dang,
Margret S. Magid,
Babette Weksler,
Amy Chadburn, and
Jeffrey Laurence
From the Laboratory for AIDS Virus Research, Division of
Hematology-Oncology, Department of Medicine, and Department of
Pathology, Cornell University Medical College, New York, NY.
 |
ABSTRACT |
Idiopathic thrombotic thrombocytopenic purpura (TTP) is a thrombotic
microangiopathy of obscure etiology. The fundamental pathologic lesion
is a hyaline thrombus composed of platelets and some fibrin accompanied
by endothelial cell proliferation and detachment, in the absence of an
inflammatory response. We have previously demonstrated that plasmas
from patients with both idiopathic TTP and a related disorder, sporadic
hemolytic-uremic syndrome (HUS), induce apoptosis and expression of the
apoptosis-associated molecule Fas (CD95) in vitro in those lineages of
microvascular endothelial cells (MVECs) that are affected
pathologically. We now demonstrate the presence of enhanced MVEC
apoptosis in splenic tissues from patients with TTP, documented by
terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling
(TUNEL) and morphology. This is accompanied by elevated Fas expression.
It contrasts with the absence of apoptosis in splenic tissues obtained
after splenectomy for trauma or immune thrombocytopenic purpura.
TUNEL-positive cells, identified by immunohistochemistry as MVECs or
macrophages, presumably engulfing apoptotic ECs, are noted in numerous
areas, including those apart from microthrombi. Thus, it is unlikely
that EC apoptosis is simply a sequela of thrombus formation. Based on
these data, we propose that MVEC apoptosis is of pathophysiologic
significance in idiopathic TTP/sporadic HUS.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
THROMBOTIC thrombocytopenic purpura (TTP)
is classically a pentad of thrombocytopenia, microangiopathic hemolytic
anemia, neurologic abnormalities, renal dysfunction, and
fever.1 Hemolytic-uremic syndrome (HUS) is part of the
spectrum of TTP-related disorders, defined by a triad of
thrombocytopenia, microangiopathic hemolytic anemia, and renal
dysfunction.2 Both of these entities are thrombotic
angiopathies. Idiopathic TTP and sporadic HUS, but not diarrhea,
malignancy, or most drug-associated forms of TTP/HUS, are closely
related in terms of response to therapeutic plasmapheresis and
distribution of pathologic lesions.2 The fundamental
pathologic lesion in idiopathic TTP/sporadic HUS is a hyaline thrombus,
composed of platelets and some fibrin, that is accompanied by localized endothelial cell (EC) proliferation and detachment in the distinct absence of inflammation.1,3-5 These thrombi are limited to the microvasculature and found in all tissues, with the notable exception of the lungs.5
Several observations suggest that EC damage may be the initial event in
TTP/sporadic HUS, mediated by components in the plasma of these
patients. Electron microscopy studies of early lesions have shown
multiple cytoplasmic vacuoles, lysosomes, and swollen mitochondria in
the microvascular endothelial cells (MVECs).1,6 These
changes, classically associated with early apoptotic events, have been
noted even in the absence of overt thrombi in these vessels, indicating
that they are not simply sequelae of thrombosis. Morphologic evidence
of overt apoptosis has also been recognized in renal glomerular cells
of probable MVEC origin in one HUS patient who has been extensively
studied.7 In addition, Lefevre et al8 have
demonstrated the presence of ECs in the whole blood of patients with
TTP, consistent with the detachment of apoptotic ECs and their entry
into the circulation. Finally, our group has documented the ability of
plasmas from TTP and sporadic HUS patients to induce apoptosis of
cultured MVECs, an activity that resolves after therapeutic
plasmapheresis.9-11
We now document the presence of enhanced apoptosis and expression of
the activation and apoptosis-associated molecule Fas (CD95) in MVECs in
sinusoidal areas of splenic tissues from patients with TTP. We compare
the incidence of these changes with that in control spleens, removed
after trauma or as a therapeutic intervention in immune
thrombocytopenic purpura (ITP). In conjunction with our prior in vitro
data, this suggests that MVEC apoptosis is a fundamental event in
idiopathic TTP/sporadic HUS and that the use of apoptotic inhibitors
should be considered in the experimental treatment of the thrombotic microangiopathies.
| |
PATIENTS, MATERIALS, AND METHODS |
Patients.
Cases of TTP were selected by screening surgical pathology records from
New York Hospital-Cornell University Medical Center (New York, NY) for
the period of 1975 through 1997. Paraffin blocks of splenic tissues
were accessible from 8 splenectomized patients with TTP. Patient charts
were reviewed for the validity of the diagnosis, duration of disease,
treatment, and outcome. The diagnosis of TTP was made according to the
following criteria: fever, neurologic dysfunction, renal dysfunction
with serum creatinine level of greater than 1.5 mg/dL or greater than
50% of the previous baseline value, thrombocytopenia with platelet
count less than 150,000/µL, and evidence of microangiopathic
hemolytic anemia on peripheral blood smear.
Control paraffin blocks of splenic tissues were obtained from 2 patients with ITP, 3 patients with splenic trauma, and 1 patient with
littoral cell angioma.
Tissues.
All specimens, fixed in formalin and embedded in paraffin, were cut in
5-µm sections. Selected slides, stained with hematoxylin and eosin
(H&E) and periodic-acid-Schiff (PAS), were examined for the presence of
hyaline subendothelial deposits (SED), endothelial cell reactive
changes, arteriolar thrombi, periarteriolar concentric fibrosis (PAF)
or "onion-skinning," and germinal centers. These changes have all
been described in TTP, but none is considered pathognomonic for the
disease.12
TUNEL.
A TUNEL (terminal deoxynucleotidyl-transferase-mediated dUTP nick-end
labeling) assay was used to identify double stranded DNA fragmentation,
characteristic of DNA degradation by apoptosis. An ApopTag in situ
apoptosis detection kit (Oncor, Gaithersburg, MD) was used according to
the manufacturer's directions. Briefly, tissue slides were
deparaffinized, treated with proteinase K (20 µg/mL) for 15 minutes
at room temperature, and then quenched in 2% hydrogen peroxide. After
rinsing in phosphate-buffered saline (PBS), pH 7.4, specimens were
incubated in 1× Equilibration Buffer (Oncor) for 10 to 15 seconds. Slides were next incubated with terminal deoxynucleotidyl
transferase (Tdt) for 1 hour at 37°C, blocked with Stop/Wash Buffer
(Oncor), and then incubated with peroxidase-conjugated antidigoxigenin
antibody for 30 minutes at room temperature. Finally, slides were
developed using diaminobenzidine (DAB; Sigma, St Louis, MO) and
counterstained with methyl green.
Immunohistochemical analysis.
An immunohistochemical analysis for CD34- (endothelial cell, stem cell
marker) and CD68- (macrophage-specific marker) expressing cells was
performed on all 4 TUNEL-positive slides using the Chem Mate Secondary
Detection Kit Alkaline Phosphatase (Ventana Medical Systems, Inc,
Tucson, AZ). These samples were stained initially via TUNEL assay, as
described above, without the methyl green counterstaining.
One set of slides was heated in 10 mmol/L sodium citrate buffer, pH
6.0, in a pressure cooker (Prestige; Gebrauchsanweisung Beachten,
London, UK) for 10 minutes and then allowed to cool in cold water.
Slides were then washed in distilled water 4 times and in PBS 3 times.
Next, samples were quenched in Blocking Antibodies, a mixture of sodium
azide, and proprietary ingredients (Ventana Medical Systems, Inc) and
then incubated in mouse anti-CD34 MoAb (Biogenex, San Ramon, CA) at
1:100 dilution for 15 minutes at room temperature.
Another set of slides was incubated in 0.2% trypsin for 14 minutes at
37°C, washed in distilled water and PBS, and then quenched in
Blocking Antibodies before incubation with mouse anti-CD68 MoAb (DAKO,
Carpinteria, CA) at 1:300 dilution for 15 minutes at room temperature.
Slides were washed in PBS and then incubated in a mixture of
biotinylated goat antimouse IgG and IgM and goat antirabbit IgG (3Ab-AB2 Biotin; Ventana Medical Systems, Inc). Slides were next incubated in Avidin-Alkaline Phosphatase complex (Ventana Medical Systems, Inc) for 30 minutes, washed in PBS, developed with BT Red
chromogen components (Ventana Medical Systems, Inc) for 10 minutes, and
then counterstained with Mayers hematoxylin.
Fas expression.
Slides from 7 of 8 TTP patients and 5 of 6 control patients were
available to compare Fas (CD95) expression in splenic tissues. The
Immunocruz Staining System FAS (C-20) K:sc-715-K (Santa Cruz Biotechnology, Inc, Santa Cruz, CA) was used according to the manufacturer's directions. Slides were deparaffinized, treated with
proteinase K (20 µg/mL) for 30 minutes at 37°C, heated in 10 mmol/L sodium citrate buffer, pH 6.0, for 5 minutes at 95°C, and
then cooled for 20 minutes. Specimens were washed with distilled water,
incubated in peroxidase for 5 minutes and prediluted goat serum block
for 20 minutes, and then incubated in prediluted anti-Fas rabbit
polyclonal Ig for 2 hours. Samples were washed in PBS and incubated in
biotinylated goat antirabbit polyclonal Ig for 30 minutes and in
horseradish peroxidase (HRP)-streptavidin complex for 30 minutes. Specimens were then washed in PBS, developed with HRP
substrate DAB for 10 minutes, and then counterstained with methyl green.
Quantitation of apoptotic cells and Fas expression.
The percentage of apoptotic cells on each slide was determined with a
Cell Analysis System (CAS) 100 (Cell Analysis Systems, Inc, Lombard,
IL), a software-controlled analyzer.13 Images from each
slide were screened at 400× and stored, permitting measurements of the number of cells per image and analysis of the percentage of
apoptosis in any given population of cells. For each slide, eight
random images of the splenic red pulp and eight random images of
arterioles were analyzed and averaged for the presence of apoptosis. All cell types were examined within these fields. The significance of
the difference in apoptosis between TTP and control samples was
measured using two-tailed variance analysis.
The same principle was used to detect for the percentage of Fas
expression in the splenic red pulp of available slides. For trauma-related splenectomy samples, areas away from lacerations were studied.
| |
RESULTS |
Patients.
The clinical findings of all patients are summarized in
Table 1. There were 7 females and 1 male
with TTP, ranging from 10 to 48 years of age. Only 2 of 8 patients
presented with the full pentad of fever, thrombocytopenia,
microangiopathic hemolytic anemia, neurologic abnormality, and renal
dysfunction. However, all individuals had moderate to severe
schistocytosis on peripheral blood smear, thrombocytopenia, and fever.
Renal abnormalities were the least common finding, present in only 2 of
8 patients. Patients' platelet counts ranged from 9,000 to
83,000/µL, with an average of 31,000/µL (Table 1). The lactate
dehydrogenase (LDH) levels ranged from 834 to 2,000 IU/L, with a mean
of 1,312 IU/L (Table 1). Neurologic abnormalities ranged from headache to confusion, hemiparesis, seizures, and coma. Treatment consisted of
plasma infusion and plasma exchange in 5 patients (Table 1). Other
treatments included aspirin, dipyridamole, steroids, and vincristine
(Table 1). All patients eventually underwent splenectomy due to a
relapsing course or refractory TTP. Remission was achieved in 7 of 8 patients. One patient died on hospital day 59 of cardiopulmonary arrest.
Control patients included 2 females with ITP (14 and 41 years of age),
2 males and 1 female with splenic trauma (30, 35, and 60 years of age),
and 1 female with littoral cell angioma (27 years of age).
Gross examination of the spleens showed weights of 100 to 289 g in TTP
patients. There was no splenomegaly in the ITP and trauma patients.
Gross splenomegaly was present in the patient with littoral cell
angioma, with a spleen weighing 1,087 g (Table 1).
Histology.
The histologic findings in TTP and control splenic tissues are
summarized in Table 2. In our review of the
selected pathology slides, we identified 2 TTP spleens with evidence of
arteriolar thrombi (Fig 1A). In a review of
the accompanying surgical pathology reports, we found additionally 3 more TTP spleens with arteriolar thrombi. No thrombi were noted in the
control samples. All 8 TTP splenic tissues had evidence of
subendothelial deposits associated with the presence of focally
reactive ECs with hyperchromatic nuclei (Fig 1B). However,
subendothelial deposits were also seen in 2 patients with splenic
trauma and the patient with littoral cell angioma. Only 1 TTP patient
and 1 ITP patient had periarteriolar concentric fibrosis. Variable
numbers of germinal centers were present in 3 TTP spleens but also in
all control samples, including 2 ITP spleens. There was no evidence of
vasculitis in any of the specimens.
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Table 2.
Pathologic Findings and CAS 100 Analysis of Degree of
Apoptosis in the Splenic Red Pulp and Arterioles and Fas Expression in
Splenic Red Pulp of TTP and Control Patients
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| Fig 1.
Photomicrographs of microthrombus, SED, TUNEL staining,
and immunohistochemical staining for CD34- and CD68-expressing cells on
TUNEL-positive slides. Microthrombus (A) and SED with associated
reactive endothelial cells (B) are seen in the splenic section of a
representative TTP patient. H&E stain and PAS stain, respectively.
TUNEL is positive in the splenic red pulp of a TTP patient (C) and
negative in a control patient (D). Methyl green counterstain. Double
staining for CD34- or CD68-expressing cells on TUNEL-positive TTP
slides demonstrates the presence of apoptotic ECs (E) and apoptotic
bodies in the cytoplasm of macrophages (F). Hematoxylin counterstain.
Original magnification × 400 for all sections.
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Identification of apoptotic cells.
TUNEL assay demonstrated apoptosis in the splenic red pulp in 4 of 8 TTP patients (representative section, Fig 1C). In marked contrast there
was virtual absence of detectable apoptosis ( 1%) in all control
samples (representative section, Fig 1D). CAS 100 (Table 2) showed an
apoptotic range of 4.3% to 12.8% (average, 7.3%) in the splenic red
pulp of the 4 TTP patients with apoptosis versus 0.0% to 1.0% in all
of the control samples (P < .0001). Furthermore, 2 of the 4 TTP patients with apoptosis in the red pulp had apoptosis in their
arterioles (2.0% and 4.1%, respectively), whereas no control patient
demonstrated any measurable apoptosis of their arterioles.
Many ECs in sinusoidal regions of the red pulp of TTP, but not control,
spleens were TUNEL positive. The ECs were identified initially based on
their location and structure. These cells had flattened nuclei and
lined the lumen of capillaries and sinuses. Their identity was further
confirmed by immunohistochemical analysis. Concurrent TUNEL and
immunohistochemical stainings demonstrated the colocalization of TUNEL
positivity and either CD34 (EC) or CD68 (macrophage) expression in the
4 TTP samples. This confirmed the presence of apoptotic ECs (Fig 1E)
and showed apoptotic bodies, presumably of endothelial origin, within
neighboring macrophages (Fig 1F).
Fas expression.
All 7 TTP and all 5 control samples available for analysis showed some
Fas expression in the splenic red pulp (Table 2 and Fig 2). CAS 100 showed Fas expression in
TTP samples ranging from 1.5% to 18.7% of the cells, with a mean of
9.1%. The other 3 non-ITP control specimens demonstrated Fas
expression from 2.2% to 5.4%, with a mean of 3.9%. In addition, the
intensity of staining was much greater in the TTP versus control
specimens (Fig 2). One of the two ITP control spleens showed levels of
Fas staining comparable to that seen in TTP (Table 2).
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| Fig 2.
Photomicrographs of Fas expression in representative TTP
and control slides. Fas expression of TTP-8 (A) is enhanced and more
intense in appearance than that of Control-6 (B). Original
magnifications × 400 for all sections.
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DISCUSSION |
The pathogenesis of TTP is unknown. Previously, our laboratory has
demonstrated that plasmas from idiopathic TTP and sporadic HUS patients
induce apoptosis in cultured human MVECs of dermal, renal, cerebral,
and tonsillar, but not in those of pulmonary origin or in ECs of large
vessels.9-11 This finding is consistent with
restriction of in vivo pathologic change to the microvessels of certain
tissues5 and raises the possibility that enhanced EC
apoptosis, initiated by plasma factors, is etiologic in idiopathic TTP/sporadic HUS. Using the TUNEL assay, we now demonstrate apoptosis in the splenic red pulp in 4 of 8 TTP patients and in arterioles of 2 of these patients but not in control samples. Using immunohistochemical analysis, we have identified the TUNEL-positive cells in the splenic red pulp of TTP patients as either ECs or macrophages. These
TUNEL-positive macrophages, which are in the perivascular distribution,
are presumed to have engulfed apoptotic bodies, most of which are
likely to be endothelial in origin.
Although only half of our TTP specimens show evidence of apoptosis, we
believe that this is a significant finding and that the extent of
apoptosis in these specimens, an average of 7.3%, is of pathologic
importance. There are several explanations for the presence of EC
apoptosis in only 50% of the TTP specimens. First, a minor population
of scattered apoptotic cells would not be detectable in situ. Such
cells are rapidly phagocytosed by macrophages and by
"nonprofessional" phagocytes, obviating an inflammatory
response.14,15 Thus, a significant number of cells must be
undergoing an accelerated apoptotic process to see any evidence in a
tissue section at one point in time. This phenomenon is consistent with
other disorders associated with apoptosis in vitro and in vivo,
including human immunodeficiency virus (HIV) and CD4+
T-lymphocyte apoptosis.16-18 Thus, the rapid process of
removal of apoptotic cells may account for the presence of apoptosis
seen in only one half of our TTP specimens. Secondly, MVEC apoptosis may not occur at all stages of TTP/sporadic HUS, or the syndrome may
involve more than one pathologic mechanism. Indeed, plasma-based MVEC
apoptosis has been previously recognized in vitro with only about 75%
of the TTP/sporadic HUS plasmas that we have screened.10,11 Thirdly, the TUNEL assay cannot demonstrate the early phase of cellular
apoptosis, such as cell shrinkage and membrane blebbing. Thus, some
evidence of apoptosis will not be recognized by the TUNEL system.
There are certain precautions that must be taken when using the TUNEL
technique to detect apoptosis. In a variety of experimental systems, it
has been demonstrated that internucleosomal cleavage of DNA is a late
event in the apoptotic process, whereas chromatin condensation and
reduction in cell volume occur much earlier.19,20 Furthermore, DNA fragmentation, similar to that seen in apoptosis, can
be present in necrosis.21 The fact that our tissues were obtained at surgery and fixed quickly obviate much of the later problems reported in autopsy series using the TUNEL assay.
Additionally, in agreement with our previous in vitro data of enhanced
expression of the apoptosis-associated molecule Fas in restricted
lineages of MVECs exposed to plasmas from TTP/sporadic HUS
patients,10 we are able to demonstrate increased Fas
expression in available splenic tissues of TTP (mean, 9.1%) versus 3 non-ITP control patients (mean, 3.9%). Because of the autoimmune
process in ITP, we suspect that Fas expression would be elevated in
ITP, but the small number of specimens available is insufficient to reach a conclusion about Fas expression in the setting of ITP. We have
found no correlation between levels of Fas expression and degree of
apoptosis in the current pathologic specimens or in our in vitro MVEC
model.10,11 This finding is consistent with our in vitro
data that MVEC apoptosis is only partially inhibited by soluble
anti-Fas MoAb.9 One possible explanation for the discordance between the degree of TUNEL positivity and Fas expression in some of our tissue samples is that enhanced EC apoptosis in TTP may
occur via a Fas-independent mechanism, and enhanced Fas expression is
simply a marker for activated cells in TTP.
Classic pathologic examinations of TTP/sporadic HUS lesions have been
unable to establish whether EC damage with exposure of subendothelial
surfaces is a primary event, preceding deposition of platelets and
fibrin within the vessel lumen, or if EC damage surrounding these
thrombi, including apoptosis, is a secondary response to platelet
aggregation resulting in microvascular occlusion.22 However, some electron micrograph-based studies of TTP have
demonstrated that MVECs develop such preapoptotic changes as multiple
cytoplasmic vacuoles and lysosomes, swollen mitochondria, and other
evidence for intense EC activation before EC detachment and platelet
plugging.1,6 This finding is consistent with our hypothesis
that apoptosis is an initiating event in TTP. Similarly, we have noted
TUNEL-positive cells in areas apart from microthrombi (Fig 1C), making
it unlikely that apoptosis is simply an event of prior MVEC thrombus
formation. Indeed, apoptotic ECs have been demonstrated by our
group10 and others23,24 to be procoagulant and
thus more likely to predispose to platelet aggregation and fibrin
formation.25,26 Finally, Karpman et al27 have
demonstrated marked apoptosis of epithelial, but not endothelial, cells
from tubuli and glomeruli from diarrhea-associated HUS patients and
from mice inoculated with Shiga-like toxin-2, which is thought to be
the causative factor of this disorder. The fact that EC apoptosis is
not a prominent feature of this form of HUS in humans further suggests
that EC apoptosis is not simply a response to thrombotic events. This finding is consistent with our in vitro work, which fails to
demonstrate plasma-induced EC apoptosis using samples from patients
with diarrhea-associated HUS.10,11
To our knowledge, this is the first reported series of TTP patients
with evidence of significantly enhanced apoptosis of ECs in an involved
tissue. Based on our results with the TUNEL assay and
immunohistochemistry, we propose that EC apoptosis plays an important
role in the pathophysiology of idiopathic TTP and its related disorder,
sporadic HUS. The cause of enhanced EC apoptosis in idiopathic
TTP/sporadic HUS is unknown. One possible explanation is the loss of
extracellular matrix (ECM) proteins, which are important to cell
survival. This process is known as anoikis or "without a home."
Hynes28 has demonstrated that ECM-cell interactions are
mediated largely by integrins, and Meredith et al29 have shown that the loss of ECM proteins may block integrin-mediated signals, resulting in EC apoptosis in vitro. Based on this background, we are currently studying the expression of several ECM proteins, such
as thrombospondin-1 and fibronectin, in splenic tissues of TTP and
control patients. Further work pertaining to the pathophysiology of
enhanced EC apoptosis in idiopathic TTP/sporadic HUS may provide clues
to the treatment of this syndrome. Finally, recent reports of the use
of apoptosis inhibitors in our in vitro TTP model30 and in
in vivo murine models for other apoptotic disorders31 also
suggest possible avenues for the experimental therapeutics of disorders
linked to accelerated programmed cell death, perhaps including
idiopathic TTP/sporadic HUS.
| |
ACKNOWLEDGMENT |
The authors thank Liang Ying for excellent technical support.
| |
FOOTNOTES |
Submitted July 15, 1998; accepted October 16, 1998.
Supported by National Institutes of Health Grants No. HL55646, AI41327,
and DE11348 to J.L.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Jeffrey Laurence, MD, Cornell University
Medical College, 411 E 69th St, New York, NY 10021.
| |
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Abstract
Introduction