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Blood, Vol. 95 No. 4 (February 15), 2000:
pp. 1283-1292
HEMATOPOIESIS
Actin cytoskeletal function is spared, but apoptosis is
increased, in WAS patient hematopoietic cells
Ramesh Rengan,
Hans D. Ochs,
Leonard I. Sweet,
Michael L. Keil,
William T. Gunning,
Neil A. Lachant,
Laurence A. Boxer, and
Geneva M. Omann
From the Departments of Surgery, Biological Chemistry, and
Pediatrics and Communicable Diseases, University of Michigan Medical
School, Ann Arbor, MI; the Veterans Administration Medical Center, Ann
Arbor, MI; the Barbara Ann Karmanos Cancer Institute and Department of
Medicine, Wayne State University School of Medicine, Detroit, MI; the
Department of Pediatrics, University of Washington School of Medicine,
Seattle, WA; and the Department of Pathology, Medical College of Ohio,
Toledo, OH.
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Abstract |
Mutations in the Wiskott-Aldrich syndrome protein (WASP) have been
hypothesized to cause defective actin cytoskeletal function. This
resultant dysfunction of the actin cytoskeleton has been implicated in
the pathogenesis of Wiskott-Aldrich syndrome (WAS). In contrast, it was
found that stimulated actin polymerization is kinetically normal in the
hematopoietic lineages affected in WAS. It was also found that the
actin cytoskeleton in WAS platelets is capable of producing the
hallmark cytoarchitectural features associated with activation. Further
analysis revealed accelerated cell death in WAS lymphocytes as
evidenced by increased caspase-3 activity. This increased activity
resulted in accelerated apoptosis of these cells. CD95 expression was
also increased in these cells, suggesting an up-regulation in the FAS
pathway in WAS lymphocytes. Additionally, inhibition of actin
polymerization in lymphocytes using cytochalasin B did not accelerate
apoptosis in these cells. This suggests that the accelerated apoptosis
observed in WAS lymphocytes was not secondary to an underlying defect
in actin polymerization caused by mutation of the WAS gene.
These data indicate that WASP does not play a universal role in
signaling actin polymerization, but does play a role in delaying cell
death. Therefore, the principal consequence of mutations in the
WAS gene is to accelerate lymphocyte apoptosis, potentially
through up-regulation of the FAS-mediated cell death pathway. This
accelerated apoptosis may ultimately give rise to the clinical
manifestations observed in WAS.
(Blood. 2000;95:1283-1292)
© 2000 by The American Society of Hematology.
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Introduction |
Wiskott-Aldrich syndrome (WAS) is an X-linked recessive
disorder characterized by recurrent bacterial infection,
thrombocytopenia, eczema, and lymphoreticular malignancy. The disease
affects most hematopoietic lineages, including platelets, lymphocytes,
monocytes, and neutrophils.1,2 The platelets are among the
most severely affected cells in that they are abnormally small, and
circulating counts can be 10% of normal or lower. Lymphocytes are also
notably affected in that the antibody responses to polysaccharide and protein antigens are compromised, and these cells are typically deficient in surface sialoglycoproteins and microvilli. Lymphopenia is
evident in patients with WAS by 6 years of age.1,2
The gene responsible for WAS has been cloned and subsequently shown to
be allelic with X-linked thrombocytopenia (XLT), a clinically milder
disorder.3,4 Analysis of the WAS protein (WASP) structure
has revealed several domains, including a GTPase binding domain and
domains homologous to actin-regulating proteins. WASP has been shown to
bind the small guanosine triphosphate-binding protein
Cdc42.5,6,7 Cdc42 is a member of the Ras superfamily of
small GTPases and has been implicated as playing a role in signaling
cytoskeletal reorganization and specifically in the formation of
filopodia.8,9 Furthermore, in vivo overexpression of WASP
increased polymerized actin, and this increase was dependent on WASP
binding Cdc42.5 In addition, recombinant Cdc42 has been
shown to induce actin polymerization in lysates from neutrophils (which
express WASP).10
These observations have raised the possibility that the underlying
problem in WAS is related to defects in the Cdc42/WASP-mediated signaling pathways that regulate actin polymerization. This hypothesis is consistent with the observation that lymphocytes from patients with
WAS exhibit cytoarchitectural defects, most notably the paucity of
microvilli. Microvilli are slender surface projections that cover
normal blood lymphocytes. Because actin-binding proteins and filaments
have been found at the base of these structures, microvillus formation
is thought to be dependent on proper microfilament organization.11 The observation that impaired microvillus
formation was present in newborns with WAS suggests that it occurs as a direct consequence of mutations in the WAS gene.11
However, 2 recent observations are not consistent with this proposed
pathogenesis. Deletion of the WASP binding domain from Cdc42 does not
impair the ability of cells to produce filopodia.12 In
addition, coexpression of WASP and active Cdc42 does not result in
filopodia formation.13 One alternative explanation for WASP
function may lie in the observation that activated Cdc42 induces
apoptosis in Jurkatt T-lymphocytes.14 This suggests that
WASP, through its interactions with Cdc42, might play a role in the
regulation of cell death. Accelerated lymphocyte death could explain
the progressive T-cell lymphopenia observed in patients with
WAS.1,2
The purpose of this study was to determine the consequence of a mutated
or absent WASP for leukocyte and platelet function in primary cells
isolated directly from patients rather than in cell lines expressing
mutant WASP. The principle of the model tested was that mutations in
WASP impair the ability of hematopoietic cells to polymerize actin in
response to relevant stimuli. In healthy persons, platelets and
leukocytes are capable of producing an actin
polymerization/depolymerization response on activation by a variety of
physiologically relevant stimuli.15-19 Therefore, any
defect in WASP-dependent signaling to the actin cytoskeleton would be
readily identified from detailed comparison of the actin polymerization
response in platelets and leukocytes from patients with classic WAS and
XLT and from controls. Additionally, we examined apoptosis and
caspase-3 activity in lymphocytes isolated from patients with WAS and
XLT and from controls.
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Patients and methods |
Materials
N-formyl-methionyl-leucyl-phenylalanine (FMLP), 9,11-dideoxy-11 ,
9-epoxymethano-prostaglandin F2 (PGF2,
U46 619), and adenosine diphosphate (ADP) were purchased from Sigma
Chemical (St. Louis, MO). Phorbol-12-myristate-13-acetate (PMA) was
purchased from Calbiochem (LaJolla, CA). Fluorescein isothiocyanate
(FITC)-anti-CD3 and phycoerythrin (PE)-anti-CD14 antibodies were
purchased from Becton Dickinson Immunocytometry Systems (San Jose, CA).
FITC-anti-CD95 and Cy5-anti-CD3 antibodies were purchased from
PharMingen (San Diego, CA). Monoclonal anti-CD3 receptor antibody
(OKT3) was a generous gift from Dr Richard A. Kroczek (Robert Koch
Institut, Berlin, Germany). Monoclonal anti-FCRIII receptor antibody
3G8 was purchased from Medarex (Annandale, NJ). Monoclonal anti-CD18 receptor antibody (IB4) was purchased from Ancell (Bayport, MN). Polyclonal goat antimouse antibody was purchased from Jackson Immunoresearch (West Grove, PA). Apoptosis detection kit was purchased from R&D Systems (Minneapolis, MN). Caspase-3 assay kit was purchased from OncoImmunin (College Park, MD). zVAD-fmk
(benzyloxycarbonyl-Val-Ala-Asp(Ome)-fluoromethylketone) was
purchased from Enzyme Systems (Livermore, CA).
Patients and controls
Five patients with XLT were studied, 4 of whom had missense
mutations in the plekstrin homology domain of WASP (2 siblings-L39P, T48I, V75 M). The fifth had a splice site mutation in intron
6 (nt593 + 5 g a), resulting in multiple products. Of the 2 patients with WAS, 1 had a single nucleotide deletion in exon 2 (211delT), resulting in a frameshift mutation and stop at amino acid
75; the other had a splice site mutation in intron 3 (nt395-1
ga), resulting in insertion of intron 3, frameshift, and stop
at amino acid 200 of WASP. Patients with XLT were defined as those who exhibited platelet abnormalities (small platelets, thrombocytopenia) but did not have significant immunologic defects. Patients with WAS
were defined as those who exhibited significant immune defects (eg,
recurrent bacterial infection) in addition to the platelet abnormalities observed in the patients with XLT. One patient with WAS
underwent bone marrow transplantation after the initiation of this
project and was therefore removed for the remainder of the study.
Western blot analysis of cell lysates from B-lymphoblastoid cell lines
using a polyclonal anti-WASP antibody revealed decreased amounts of
normal-sized WASP in cells from patients with missense mutations (4 with XLT), but it failed to detect WASP expression in the others (1 with XLT and 2 with WAS).20 Peripheral blood leukocytes and
platelets were isolated from at least 5 different volunteers to serve
as controls for actin polymerization and viability studies. Informed
consent was obtained from all patients and volunteers, as prescribed by
the Human Studies Review Committee at the University of Washington and
the Institutional Review Board at the University of Michigan.
Leukocyte and platelet isolation
Platelets and leukocytes were isolated essentially as previously
described.21-23 Briefly, whole blood was partially purified by dextran sedimentation to remove red blood cells. The leukocytes in
the partially purified whole blood were separated into 2 bands, 1 consisting primarily of neutrophils and the other of monocytes and
lymphocytes, by centrifugation on a 42% to 60% Percoll gradient. The
neutrophil band was resuspended for 1 minute in an ammonia buffer to
lyse red blood cells. Subsequent to this lysis step, neutrophils were
resuspended at a concentration of 1 × 108 cells/mL
and stored at 4°C in a modified Gey's buffer (MGB) without calcium
(10 mmol/L HEPES, 5 mmol/L KCl, 0.22 mmol/L
KH2PO4, 1.1 mmol/L
Na2HPO4, 5.5 mmol/L glucose, 0.3 mmol/L MgSO4, 1 mmol/L MgCl2, 147 mmol/L NaCl, pH 7.4).
The monocyte/lymphocyte band was removed and used for these studies. In
addition to using their light-scattering properties to distinguish the
monocyte population from the lymphocytes, we labeled cells with
PE-anti-CD14 antibody and fluorescein-isothiocyanate (FITC)-anti-CD3
antibody and analyzed them on a Becton Dickinson FACScan (Figure
1). This analysis was carried out for cells isolated immediately after venipuncture (Figures 1A, 1B) and for cells isolated
from 24-hour-old blood (Figures 1C to 1F), allowing for identification
of the monocyte and lymphocyte populations. An additional population of
necrotic lymphocytes that had unique scattering properties was
identified in cells isolated from 24-hour-old blood. We could not
detect a distinct necrotic monocyte population, and the necrotic
lymphocyte population did not contain monocytes. This analysis allowed
for unambiguous identification of the monocyte and T-lymphocyte
populations. Because T and B lymphocytes have identical scattering
properties, the scattering parameters of the FITC-anti-CD3-labeled
cell population were used to gate electronically for all lymphocytes.
The scattering parameters of the PE-anti-CD14-labeled cell population
were used to gate electronically for all monocytes. For the
purposes of the actin and caspase-3 assays, the necrotic lymphocyte population was excluded.
Cell stimulation and quantification of F-actin content
Platelets were stimulated as previously described.21 The
sole modification was the addition of 1% Triton X-100 to the staining cocktail to enhance uptake of the fluorescent probe
N-(7-nitrobenz-2-oxa-1, 3-diazole 4-yl) (NBD)-phallicidin.
Stimulus was added directly to stirred platelet-rich plasma at
37°C. An aqueous stock of stimulus was prepared on the day of the
experiment as a 1:100 or 1:10 dilution in MGB of a frozen, concentrated
stock solution originally made in dimethyl sulfoxide (DMSO). This
aqueous stock was added as a 1:100 dilution to platelet-rich plasma.
Thus, the final concentration of DMSO added to the cells was 0.1% or less.
Lymphocytes, monocytes, and neutrophils were stimulated essentially as
previously described.24 Briefly, cells were resuspended at
a concentration of 2 × 106 cells/mL in MGB in the
presence of calcium (10 mmol/L HEPES, 5 mmol/L KCl, 0.22 mmol/L
KH2PO4, 1.1 mmol/L
Na2HPO4, 5.5 mmol/L glucose, 0.3 mmol/L MgSO4, 1 mmol/L MgCl2, 1.5 mmol/L CaCl2, 147 mmol/L NaCl, pH 7.4) and stirred at
37°C for 10 minutes before the addition of stimulus prepared as
described for platelets. Stimulus was added as a 1:100 dilution to
stirred cells in suspension. Thus, the final concentration of DMSO
added to cells was 0.1% or less. After stimulation, aliquots of cells
were removed and fixed in 3.7% formalin at various time points during
the course of the response. These samples were subsequently stained for
F-actin with NBD-phallicidin and quantified by flow cytometry.
For analysis of the CD3 receptor-mediated actin polymerization response
in lymphocytes, cells were incubated at a concentration of
4 × 106 cells/mL in MGB containing 8 µg/mL OKT3
anti-CD3 antibody for 30 minutes at 4°C. After this incubation,
cells were stirred at 37°C for 10 minutes before stimulation. Cells
were then stimulated with 80 µg/mL goat antimouse antibody to
cross-link the primary antibody bound to the receptor, which resulted
in the initiation of actin polymerization.
For analysis of the FCRIII receptor- and CD-18 receptor-mediated
actin polymerization responses in neutrophils, cells were incubated at
a concentration of 5 × 107 cells/mL in MGB
containing the appropriate antibody (3G8 and IB4, respectively) for 30 minutes at 4°C. Neutrophils were incubated with 3G8 at a
concentration of 5 µg/mL and with IB4 at a concentration of 10 µg/mL. Subsequent to this incubation, cells were suspended at a
concentration of 2 × 106 cells/mL in MGB and
stirred at 37°C for 10 minutes before stimulation. Stimulation of
these cells was achieved using 50 µg/mL goat antimouse antibody to
cross-link the primary antibody bound to the receptor, which resulted
in the initiation of actin polymerization.
Identification of apoptotic cells
Viable apoptotic and necrotic lymphocytes were identified as
directed using the apoptosis detection kit (R&D Systems, Minneapolis, MN). Briefly, cells were labeled with FITC-annexin-V and propidium iodide and analyzed on a Becton Dickinson FACScan. An excitation wavelength of 488 nm was used; emissions were read at 530 nm for the FITC-annexin-V and at more than 650 nm for the propidium iodide. Necrotic cells were defined as those that bound annexin-V and propidium
iodide. Apoptotic cells were defined as those that bound annexin-V but
excluded propidium iodide. Viable cells were defined as those that did
not bind either stain.
Determination and inhibition of caspase-3 activity
Caspase-3 activity was measured as previously described using the
caspase-3 assay kit.25 Briefly, cells were incubated in 10 µmol/L PhiPhiLux-G2D2 substrate solution.
This substrate is cleaved to a fluorescent product by caspase-3. After
incubation for 1 hour in this solution, cells were analyzed using a
Becton Dickinson FACScan. An excitation wavelength of 488 nm was used, and emission was read at 585 nm. Non-necrotic lymphocytes were selected
using light-scattering parameters, and the fluorescence of this
population was measured. This fluorescence was reflective of caspase-3 activity.
To inhibit caspase-3, whole blood drawn from patients with WAS was
mixed with 50 µmol/L zVAD-fmk immediately after venipuncture. This
concentration of inhibitor was used because it has been shown to
inhibit significantly the signature events seen in
apoptosis.26 The blood was then incubated at room
temperature for 24 hours before lymphocytes were isolated.
Determination of CD95 receptor expression
Lymphocytes isolated from peripheral blood as described above were
labeled with FITC-anti-CD95 and Cy5-anti-CD3 (to identify T
lymphocytes) or FITC-anti-CD95 and PE-anti-CD14 (to identify monocytes) and were analyzed by flow cytometry to quantify CD95 expression. Briefly, 1 × 106 cells were suspended
in 50 µL MGB. To this suspension, 20 µL FITC-anti-CD95 antibody
and 5 µL Cy5-anti-CD3 (to identify T lymphocytes) or 20 µL
FITC-anti-CD95 antibody and 10 µL PE-anti-CD14 (to identify monocytes) antibody were added and maintained on ice for 20 minutes. Cells were washed and resuspended in MGB and analyzed on a Becton Dickinson FACScan.
Kinetics of WAS and XLT lymphocyte apoptosis
To determine the kinetics of apoptosis in WAS, XLT, and patient
lymphocytes, cells were resuspended at a concentration of 1 × 106 cells/mL in T-cell media (RPMI 1640, 8%
fetal bovine serum, 2% human serum) and maintained at 37°C. Cells
were then removed at indicated time intervals and analyzed for
viability as determined by annexin-V and propidium iodide exclusion as
described above.
Cytochalasin-B incubation of lymphocytes
After purification, lymphocytes and monocytes were incubated at a
concentration of 1 × 106 cells/mL with the
indicated concentration of cytochalasin B, cytochalasin B
and zVAD-fmk, or vehicle control at room temperature for the indicated
time intervals. Lymphocytes were then analyzed for apoptosis and
caspase-3 activity as described above.
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Results |
Quantitative and qualitative assessments of stimulated actin
polymerization response in platelets isolated from patients with
WAS and XLT
Platelets isolated from patients with WAS and XLT and from controls
were stimulated with either PGF2 or ADP. These
compounds, which activate G-protein-dependent signaling
pathways,27 have been shown to induce rapid actin
polymerization and depolymerization in platelets isolated from healthy
volunteers.21 The kinetics of actin polymerization in
platelets isolated from patients with WAS and XLT, in response to
stimulation with these compounds, was determined by fixing and staining
the F-actin with NBD-phallacidin during the time course of the response
(Figures 2A, 2B). No notable difference in the kinetics
of actin cytoskeletal remodeling of patient platelets relative to
platelets from healthy volunteers was observed. After taking into
account their smaller size, it was found that resting F-actin content
in WAS platelets was not lower than that in control platelets (data not
shown).
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| Fig 1.
Identification of lymphocyte and monocyte subpopulations
in cells isolated from WAS and control blood immediately and 24 hours
after venipuncture.
(A, C, E) Identification of unlabeled, FITC-anti-CD3-labeled, and
PE-anti-CD14-labeled populations in cells isolated from WAS patient
blood immediately (A) and 24 hours (C) after venipuncture and in cells
isolated from control blood 24 hours (E) after venipuncture. Four
different subpopulations were identified in these cells: T lymphocytes
(CD3+/CD14-), monocytes (CD14+/CD3-), unlabeled (UN), and
necrotic lymphocytes (CD3+/CD14-(N)-seen only in panel
C). These graphs are representative of data collected
from 2 patients with WAS and at least 5 control donors. (B, D, F) Dot
plots showing forward and side-scatter data for cells isolated from WAS
patient blood immediately (B) and 24 hours (D) after venipuncture and
in cells isolated from control blood 24 hours (F) after venipuncture.
Three different subpopulations were identified in these cells:
lymphocytes, necrotic lymphocytes (only in C), and monocytes. These
graphs are representative of data collected from 2 patients with WAS
and at least 5 control donors.
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To assess qualitatively the actin cytoskeletal function in platelets
isolated from patients with WAS and XLT, scanning electron micrograph
images of these cells were obtained during the time course of
stimulation with PGF2 (Figures 2C to 2F).28
Although the patient platelets (Figures 2E, 2F) were clearly smaller
than platelets isolated from healthy volunteers (Figures 2C, 2D), they produced cytoskeletal extensions strikingly similar to those produced by control platelets once stimulated. It is important to
note that in the patients with classic WAS included in these studies, we found no detectable levels of WASP.
Quantification of stimulated actin polymerization in neutrophils and
monocytes isolated from patients with WAS and XLT
Neutrophils and monocytes isolated from patients with WAS and XLT
were stimulated by different pathways known to result in actin polymerization in these cells (Figures
3, 4). FMLP is a potent chemoattractant that activates actin polymerization in neutrophils and monocytes.16,17 It has been shown to
activate actin polymerization in neutrophils through a pertussis
toxin-sensitive G-protein-dependent pathway.16 PMA
activates actin polymerization in neutrophils through a membrane
receptor-independent pathway.29 Ligation of the FCRIII
receptor and of the  2-integrin receptor has been shown to result in
actin polymerization in neutrophils, likely by way of pathways
important for phagocytosis and adhesion.30,31 Activation of
neutrophils isolated from patients with WAS and XLT with these
compounds resulted in an actin polymerization response that was
indistinguishable from the response in control neutrophils (Figure 3).
There were no notable differences in either the kinetics or the
magnitude of the actin polymerization response between patient and
control neutrophils stimulated with these compounds. Additionally, the
stimulation of monocytes isolated from WAS and XLT cells in patients
with FMLP resulted in an actin polymerization response that was the
same as the response seen in control cells (Figure 4). It is also
important to note that resting levels of F-actin were identical in WAS,
XLT, and control neutrophils and monocytes (data not shown).
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| Fig 2.
Actin polymerization in platelets from patients with WAS
and XLT and from controls.
(A, B) Platelets isolated from whole blood and maintained at 37°C
in autologous platelet-rich plasma were stimulated with either
PGF2, ADP, or buffer ( )at the indicated
concentrations.21 Data reflect the typical responses from
each patient group and the control group. Error bars represent the SEM
for duplicate simultaneous determinations for a single donor (control,
WAS, or XLT). These graphs are representative of data collected from 2 patients with WAS ( ), 2 patients with XLT ( ), and 5 control
donors ( ). (C-F) Representative scanning electron micrographs of
platelets fixed before (C, E) and 180 seconds after stimulation (D, F)
with 1 µmol/L PGF2 from a patient with
classic WAS (E, F) and control (C, D). Images are 8-µm fields, and
samples were prepared as previously described.28 Platelets
isolated from patients with WAS were consistently smaller, as
previously reported.2 Images are representative of data
collected from 2 patients with WAS and 3 control donors.
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| Fig 3.
Kinetics of actin polymerization in WAS and XLT
neutrophils.
Actin polymerization in WAS (), XLT(), and control
()neutrophils in response to stimulation with FMLP (A), IB4 (B), 3G8
(C), PMA (D), or buffer control (A-D, ). Error bars represent SEM of
duplicate simultaneous determinations for a single donor (control, WAS,
or XLT). These graphs are representative of data collected from 2 patients with WAS, 3 patients with XLT. and at least 5 control
donors.
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Quantification of stimulated actin polymerization in lymphocytes
isolated from patients with WAS and XLT
The ability of patient lymphocytes to dynamically remodel
their actin cytoskeleton in response to stimuli was examined.
Activation of protein kinase C has been shown to induce shape changes
in lymphocytes.16 Two potent stimuli of lymphocytes by
protein kinase C-mediated pathways, PMA and bryostatin, were used, and F-actin levels were detected by binding of
NBD-phallacidin.16,19,24 Additionally, lymphocytes were
stimulated through ligation of the CD3 receptor, which has previously
been shown to induce actin polymerization in lymphocytes.32
PMA and bryostatin induced actin polymerization responses in
lymphocytes from patients with WAS and XLT that were indistinguishable
from those of control lymphocytes (Figures
5A, 5B). Activation of the CD3 receptor
resulted in actin polymerization in lymphocytes isolated from healthy
volunteers (Figure 5C). Ligation of the CD3 receptor also induced an
actin polymerization response in lymphocytes isolated from patients with WAS and XLT that was similar to that of control. The kinetics of
the actin polymerization response in the WAS and XLT lymphocytes was
essentially indistinguishable from that of the control lymphocytes. Furthermore, though the magnitude of the actin polymerization response
in XLT lymphocytes was indistinguishable from that of control
cells, the magnitude of the response in WAS lymphocytes appeared
diminished. This diminishment, however, was not statistically significant as determined by paired Student t test
analysis. It is also important to note that resting levels of F-actin
were identical in control and patient lymphocytes (data not shown).
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| Fig 4.
Kinetics of actin polymerization in WAS, XLT, and control
monocytes.
Actin polymerization in WAS (), XLT (), and control ()
neutrophils in response to stimulation with 100 nmol/L FMLP. (Buffer,
.) Error bars represent SEM of duplicate simultaneous determinations
for a single donor (control, WAS, or XLT). These graphs are
representative of data collected from 2 patients with WAS, 3 patients
with XLT, and at least 5 control donors.
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Identification of lymphocyte and monocyte subpopulations in
cells isolated from WAS patient blood immediately and 24 hours
after venipuncture
To determine the feasibility of using blood from patients with WAS
that had been shipped overnight for our studies, whole blood was stored
at room temperature for 24 hours before the isolation of lymphocytes
and monocytes (Figure 1). After
purification, the lymphocyte/monocyte mixture was labeled with
FITC-anti-CD3 and PE-anti-CD14 antibodies and was analyzed by flow
cytometry to identify each subpopulation. In cells isolated from WAS
whole blood immediately after venipuncture (Figures 1A, 1B), 3 distinct subpopulations of cells were identified. The
CD3+/CD14-population was identified as T lymphocytes
because these cells express the CD3 antigen.33 Similarly,
the CD14+ population was identified as monocytes because these cells
express the CD14 antigen.34 There was also an unlabeled
subpopulation of cells that consisted primarily of B lymphocytes. These
3 subpopulations were also present in cells isolated from WAS blood 24 hours after venipuncture (Figures 1C, 1D). However, an additional
subpopulation was identified (CD3+/CD14-(N)). This subpopulation was
not present in cells isolated from control blood 24 hours after
venipuncture (Figures 1E, 1F). Because nonactivated monocytes do not
significantly shed CD14 within 24 hours,35 this new
subpopulation was identified as lymphocytes. Therefore, this
subpopulation of T lymphocytes was present in cells isolated from WAS
blood 24 hours after venipuncture (Figures 1C, 1D), but was not present
in cells isolated from 24-hour-old control blood (Figures 1E, 1F).
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| Fig 5.
Kinetics of actin polymerization in WAS, XLT, and control
lymphocytes.
(A, B) Actin polymerization in lymphocytes in response to stimulation
with 100 nmol/L PMA, 100 nmol/L bryostatin (Bryo), and a buffer control
(). Error bars represent SEM of duplicate simultaneous
determinations for a single donor (control, ; WAS, ; or XLT,
). These graphs are representative of data collected from 2 patients
with WAS, 5 patients with XLT, and 5 control donors. (C) Actin
polymerization in lymphocytes in response to stimulation with OKT3
anti-CD3-receptor antibody and a buffer control. Error bars represent
SEM of duplicate simultaneous determinations for a single donor
(control, WAS, or XLT). These graphs are representative of data
collected from 1 patient with WAS, 1 patient with XLT, and 2 healthy
volunteers.
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Viability of lymphocytes isolated from 24-hour-old blood from
patients with WAS and XLT
To better understand the effect of storing blood at room temperature
for 24 hours before lymphocyte isolation, the viability of cells
isolated from 24-hour-old WAS, XLT, and control blood was determined.
We used an assay that identified viable, apoptotic, and necrotic cells
(see "Patients and Methods" for greater detail). There were no
statistically significant differences in the percentages of viable,
apoptotic, or necrotic lymphocytes isolated from 24-hour-old XLT and
control blood (Figure 6). However, the
24-hour incubation had a significant effect on the viability
of WAS lymphocytes, which on average was 42%, whereas control and XLT
lymphocyte viability rates were significantly (P < .05)
higher at 65% and 74%, respectively (Figure 6). After 24 hours, there
was a significant increase in apoptotic lymphocytes in cells isolated
from the blood of patients with WAS compared with control blood (10%
compared with 4%). This trend was also apparent when comparing the
percentage of apoptotic lymphocytes isolated from WAS and XLT blood
(10% compared with 8%); however, the difference was not statistically
significant (Figure 6). There was a significantly higher percentage of
necrotic WAS lymphocytes (46%) compared with control (29%) and XLT
lymphocytes (17%) isolated from 24-hour-old blood (Figure 6).
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| Fig 6.
Viability, apoptosis, and necrosis in WAS, XLT, and
control lymphocytes isolated 24 hours after venipuncture.
Percentages of viable, apoptotic, and necrotic lymphocytes isolated
from 24-hour-old WAS, XLT, and control blood were determined by
annexin-V binding/propidium iodide exclusion. Lymphocytes were purified
24 hours after venipuncture by gradient centrifugation of partially
purified whole blood. WAS data represent the average of 5 separate
determinations performed in 2 patients with WAS. Control data represent
the average of 5 experiments in 4 volunteers run concurrently with
experiments in the patients with WAS. XLT data represent the average of
3 experiments in 3 patients. Error bars represent SEM.
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Quantification of stimulated actin polymerization in non-necrotic
lymphocytes isolated from 24-hour-old blood from patients with WAS and
XLT
To better characterize the CD3+/CD14-
lymphocyte population isolated from 24-hour-old WAS, XLT, and
control blood (see Figures 1C, 1D), the actin polymerization response
to stimulation with PMA in these cells was examined. This population of
cells was identified as containing primarily viable and some apoptotic
lymphocytes and will heretofore be referred to as the non-necrotic
lymphocyte population. Whereas control and XLT non-necrotic lymphocytes
were able to produce a robust actin polymerization response on
stimulation with 100 nmol/L PMA (Figure 7),
WAS non-necrotic lymphocytes were essentially incapable of responding
to stimulation (Figure 7).
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| Fig 7.
Actin polymerization in WAS, XLT, and control lymphocytes
isolated 24 hours after venipuncture.
Actin cytoskeletal remodeling in response to stimulation with 100 nmol/L PMA or buffer () in WAS ( ), XLT (), and control ()
lymphocytes isolated from whole blood incubated at room temperature for
24 hours after venipuncture. Aliquots of cells were removed and
analyzed for F-actin content at the indicated times. Error bars reflect
SEM of duplicate simultaneous determinations from 1 donor (control or
WAS). The graph is representative of data collected from 2 patients
with WAS, 3 patients with XLT, and 5 control donors.
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Determination of caspase-3 activity and effect of caspase-3
inhibition in non-necrotic lymphocytes isolated from 24-hour-old blood
from patients with WAS
To test the hypothesis that the non-necrotic lymphocytes isolated
from 24-hour-old WAS patient blood were in the early stages of
apoptosis, we determined the caspase-3 activity in this population of
cells. Caspase-3 activation is among the earliest signature events in
apoptosis in lymphocytic cell lines, and it has been shown to
occur before the expression of phosphatidylserine on the outer
membrane.36-38 Therefore, this assay allows for
identification of early apoptotic cell populations that would escape
detection by annexin-V binding. As shown in Figures
8A and 8B, caspase-3 activity was higher in
the 24-hour-old non-necrotic WAS lymphocytes than in control
lymphocytes isolated from 24-hour-old blood.
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| Fig 8.
Caspase-3 activity and actin polymerization response in
zVAD-fmk-treated and untreated WAS lymphocytes isolated 24 hours after
venipuncture.
(A, B) Activity of caspase-3 in lymphocytes isolated from whole blood
that had been incubated at room temperature for 24 hours. Data are
expressed as a histogram with fluorescence intensity on the x-axis and
cell number on the y-axis. Mean fluorescence intensities of the control
and patient lymphocyte populations were 116 and 290, respectively.
Graphs are representative of data collected from a single WAS patient
and a control. (C, D) Caspase-3 activity in lymphocytes isolated from
whole blood (from a single patient with WAS) that had been treated for
24 hours with and without 50 µmol/L zVAD-fmk. (C) Caspase-3 activity
in untreated lymphocytes. (D) Caspase-3 activity in treated cells. Note
the marked decrease in fluorescence in the treated lymphocytes. Mean
fluorescence intensities in untreated and zVAD-fmk-treated WAS
lymphocytes were 403 and 223, respectively. Because the instrumental
settings of the flow cytometer varied from day to day, these data
cannot be compared directly with those shown in A and B. (E)
Percentages of viable, apoptotic, and necrotic lymphocytes from WAS
lymphocytes isolated from 24-hour-old blood incubated with and without
50 µmol/L zVAD-fmk as defined previously. Graphs are representative
of data taken from a single WAS patient and a single control. (F) Actin
polymerization response in WAS lymphocytes isolated from 24-hour-old
blood incubated with and without 50 µmol/L zVAD-fmk. The actin
polymerization response of WAS lymphocytes was stimulated with 100 nmol/L PMA for 40 minutes at 37°C. Error bars represent SEM of
duplicate determinations from a single donor (WAS). This graph is
representative of data collected from a single patient with WAS.
|
|
To determine whether the elevated caspase-3 activity had an
effect on lymphocyte viability, we added the caspase-3 inhibitor zVAD-fmk to WAS patient whole blood before the 24-hour incubation. At a
concentration of 50 µmol/L, zVAD-fmk decreased caspase-3 activity in
non-necrotic WAS lymphocytes (Figures 8C, 8D), and this resulted in a
15% increase in WAS lymphocyte viability (Figure 8E). As expected,
this inhibitor also decreased the number of apoptotic and necrotic
lymphocytes in cells isolated from patient blood (Figure 8E). Addition
of this inhibitor had no notable effect on the viability of control
lymphocytes (2% increase in viability). Additionally, lymphocytes
isolated from zVAD-fmk-treated whole blood exhibited a more robust
actin polymerization response to stimulation with PMA than that
observed in lymphocytes isolated from untreated WAS patient blood
(Figure 8F).
CD95 expression and kinetics of lymphocyte apoptosis
The kinetics of lymphocyte cell death in WAS lymphocytes was also
determined (Figure 9). When compared with
control lymphocytes, WAS lymphocytes displayed approximately a 2-fold
greater cell death rate. Although control lymphocytes exhibited a 9%
lymphocyte death rate per day, WAS lymphocytes succumbed at a rate of
19% per day as measured out to 48 hours (Figure 9A). WAS lymphocytes also exhibited 2-fold higher apoptosis and necrosis (10% and 10% per
day, respectively) than control lymphocytes (4% and 5% per day,
respectively) (Figures 9B, 9C). XLT lymphocytes did not exhibit increased apoptosis or necrosis when compared with control (5% and 5%
per day, respectively). That WAS lymphocyte necrosis exhibited the same
kinetics as the apoptosis of these cells indicates that the necrosis is
likely secondary to lymphocyte apoptosis.39,40
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| Fig 9.
Kinetics of cell death in WAS and control lymphocytes.
Viability (A), apoptosis (B), and necrosis (C) were examined in WAS and
control lymphocytes. Lymphocytes were incubated at 37°C. Error bars
represent SEM of triplicate determinations from a patient with WAS and
a control.
|
|
Ligation of the CD95, or Fas, receptor has been shown to initiate
lymphocyte-programmed cell death.41 Therefore, Fas receptor expression on lymphocytes isolated from patients with WAS was determined by flow cytometry (Figures 10A to
10C). WAS T-lymphocytes were found to
express CD95 at almost 2-fold higher levels than those of control cells
and XLT lymphocytes. This increased Fas receptor expression was not
observed in WAS monocytes (Figures 10D to 10F).
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| Fig 10.
CD95 receptor expression in WAS, XLT, and control T
lymphocytes and monocytes.
CD95 receptor expression as measured by flow cytometry on control (A,
D), XLT (B, E), and WAS (C, F) lymphocytes (A-C) and monocytes (D-F).
Data are expressed as a histogram with fluorescence intensity on the
x-axis and cell number on the y-axis. Mean fluorescence intensities of
the control and patient lymphocyte populations were 20 and 43, respectively. Graphs are representative of data collected from a
patient with WAS and a control.
|
|
Effect of cytochalasin B treatment on lymphocyte viability,
apoptosis, and necrosis
Because cytochalasin B has been shown to inhibit actin
polymerization,42 we determined the effect of treatment
with lymphocytes isolated from healthy volunteers with this compound on
the viability, apoptosis, and necrosis of these cells (Figure
11). After a 24-hour incubation with
varying concentrations of cytochalasin B, there were no notable
differences in the viability, apoptosis, or necrosis of control
lymphocytes and those treated with 0.2 µmol/L and 2 µmol/L
cytochalasin B. There was a slight decrease in viability and increase
in necrosis and apoptosis observed in lymphocytes treated with 20 µmol/L cytochalasin B and in cells treated with 20 µmol/L
cytochalasin B and 50 µmol/L zVAD-fmk when compared with control;
however, these differences were not statistically significant.
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| Fig 11.
Effect of cytochalasin B treatment on viability,
apoptosis, and necrosis of lymphocytes.
Percentages of viable, apoptotic, and necrotic lymphocytes in cells
isolated from healthy volunteers and incubated for 24 hours with
indicated concentrations of cytochalasin B (CYTO B), zVAD-fmk, or
vehicle control. Error bars reflect SEM of duplicate simultaneous
determinations from 2 healthy control donors.
|
|
| |
Discussion |
Platelets are affected in all patients who have mutations of the
WAS gene, regardless of clinical phenotype. To determine whether these platelet abnormalities are related to an inability to
polymerize actin on activation of signaling pathways, platelets isolated from patients with WAS and XLT were stimulated with ADP and
PGF2. The observation that platelets
isolated from patients with WAS and XLT are capable of producing a
normal actin polymerization response subsequent to stimulation
suggested that dynamic actin cytoskeletal function was not universally
impaired in these cells. Further, the observation that resting F-actin
content in platelets isolated from patients with WAS and XLT was not
significantly different from that of control platelets suggested that
mutation of the WAS gene did not affect the resting F-actin
pool. Because platelets isolated from healthy volunteers produce
cytoskeletal extensions such as filopodia and microspikes on
stimulation,43 we assessed the ability of WAS platelets to
produce these cytoarchitectural features subsequent to stimulation. As
Figures 2C to 2F show, platelets isolated from WAS patients were
smaller than control platelets, but on stimulation they were capable of
producing the same cytoskeletal extensions produced by platelets
isolated from a healthy volunteer. These data suggested that mutation
of the WAS gene did not universally impair stimulated platelet
actin polymerization or the ability of these cells to exhibit
morphologic changes on stimulation.
One of the primary distinctions between classic WAS and XLT is that
patients with WAS have pronounced cellular immune dysfunction. For this
reason, we examined the actin polymerization response in leukocytes
isolated from patients with WAS and patients with XLT. Because WASP is
expressed in neutrophils,3 we conducted detailed
examinations of the actin polymerization response in these cells
through the activation of different pathways known to result in actin
polymerization. We examined the actin polymerization response in
neutrophils to stimuli that activate pathways important for chemotaxis
(FMLP), phagocytosis, 3G8 adhesion (IB4), and receptor-independent pathways (PMA). The observation that neutrophils from patients with WAS
and XLT were capable of producing a robust actin polymerization response on stimulation with any of these compounds suggests that WASP
mutation does not result in any universal defect in the pathways regulating actin polymerization in these cells. Additionally, the
observation that monocytes from patients with WAS and XLT stimulated
with FMLP were capable of producing an actin polymerization response
that was indistinguishable from that of control cells suggests that
WASP mutation does not universally affect the ability of these cells to
polymerize actin.
Because cytoarchitectural abnormalities have been identified in
lymphocytic cell lines expressing mutant WASP,11 we
examined the ability of lymphocytes isolated from patients with WAS and XLT to produce an actin polymerization response. The observation that
lymphocytes isolated from patients with WAS and XLT were able to
produce a robust actin polymerization response on stimulation with
either PMA or bryostatin suggests that WASP mutation does not
universally impair this response in these cells. Furthermore, lymphocytic cell lines expressing mutant WASP have been shown to be
incapable of polymerizing actin in response to CD3 receptor stimulation.44 Therefore, we examined CD3
receptor-stimulated actin polymerization in lymphocytes isolated from
patients with WAS and XLT. In contrast to the observations in T-cell
lines, lymphocytes isolated from patients with WAS and XLT were capable of producing an actin polymerization response to CD3 receptor stimulation (Figure 5D). Interestingly, in accordance with previously published observations,44 WAS lymphocytes did not
proliferate in response to CD3 receptor stimulation (data not shown).
This suggests that though certain aspects of T-cell receptor signaling are defective in WAS lymphocytes, the receptor is still capable of
signaling actin cytoskeletal reorganization.
To summarize, we examined the actin polymerization response in the
hematopoietic lineages most significantly affected in patients expressing mutant WASP, namely platelets, neutrophils, monocytes, and
lymphocytes. We were unable to identify any detectable defect in the
actin polymerization response in any of these cells on stimulation of a
variety of different pathways with a variety of different ligands. We
also examined the resting F-actin content in these cells and found that
there was no difference in resting F-actin levels between cells
expressing mutant WASP and control cells. This strongly suggested that
mutation of the WAS gene did not universally affect the ability
of these cells to polymerize actin. Further, the observation that WAS
platelets were able to produce hallmark cytoarchitectural features on
stimulation suggested that dynamic actin cytoskeletal function was
preserved in these cells.
To include additional patients with WASP in our study, we examined the
effect of overnight incubation of whole blood before lymphocyte
isolation on these cells. This incubation resulted in significant
lymphocyte death in cells isolated from patients with WAS (Figures 1,
6). That this cell death was not observed in patients with XLT is
consistent with the fact that these patients do not suffer from the
profound immune dysfunction that patients with WAS exhibit. Further,
the increase in necrotic lymphocytes observed in cells isolated from
24-hour-old WAS blood may be a result of secondary necrosis subsequent
to apoptosis of these cells.39,40 Additionally, we examined
the actin polymerization response in the non-necrotic lymphocyte
population isolated from 24-hour-old WAS and XLT blood (Figure 7).
These cells produced a significantly attenuated actin polymerization
response when compared with XLT and control lymphocytes. A possible
explanation for this attenuation is that the WAS lymphocytes were in
the earliest stages of apoptosis. Such a mechanism would be compatible
with the observation that apoptotic cells have been shown to have
impaired functional responses when compared with non-apoptotic
cells.45,46 The observation of elevated caspase-3 activity
in lymphocytes isolated from 24-hour-old WAS blood suggested that these
cells may indeed have been in the early stages of apoptosis. Further, the observation that inhibition of caspase-3 in WAS lymphocytes resulted in increased viability and decreased apoptosis and necrosis in
these cells strongly suggested that apoptosis was accelerated in these
lymphocytes (Figure 8). Finally, the observation that the inhibition of
caspase-3 in lymphocytes isolated from 24-hour-old WAS blood resulted
in enhancement of the actin polymerization response to
stimulation with PMA indicated that apoptosis was the underlying cause
of the observed attenuation of the actin polymerization response
in these cells (Figure 7).
To better characterize the accelerated apoptosis observed in WAS
lymphocytes, we determined the kinetics of cell death in WAS, XLT, and
control lymphocytes. WAS lymphocytes exhibited an apoptosis rate 2-fold
greater than control or XLT lymphocytes (Figure 9). To determine the
underlying mechanism driving the accelerated apoptosis observed in WAS
lymphocytes, we quantified CD95 receptor expression in these cells
(Figure 10). The fact that WAS lymphocytes had roughly a 2-fold higher
membrane density of CD95 receptor than control or XLT lymphocytes
indicated that this might have been the underlying cause of the
increased programmed cell death observed in WAS lymphocytes. This also
suggested that the Fas-mediated cell death pathway might have been
up-regulated in WAS.
Finally, we addressed whether an underlying defect in actin
polymerization could have led to the accelerated apoptosis we observed
in WAS lymphocytes. To address this, we treated lymphocytes with
cytochalasin B, a potent inhibitor of actin
polymerization,42 and examined the effect of this treatment
on the viability of these cells. The observation that treatment of
lymphocytes with cytochalasin B (0.2 µmol/L-20 µmol/L) did not
result in a significant decrease in lymphocyte viability or increase in
apoptosis or necrosis of these cells suggests that the accelerated
apoptosis we observed was not caused by an underlying defect in actin
polymerization (Figure 11).
Based on these observations, we hypothesize that WASP plays a role in
delaying apoptosis and that WASP mutation interferes with the
regulation of the apoptotic response. Accelerated apoptosis could
explain many of the clinical features that have been observed in WAS.
Immune dysfunction, a characteristic feature of classic WAS, is partly
a consequence of the progressive T-cell depletion observed in these
patients over time.47 This progressive lymphopenia may be a
direct result of the accelerated apoptosis and secondary cell clearance
of WAS lymphocytes. The fact that XLT lymphocytes did not exhibit
increased CD95 receptor, but did exhibit an increased rate of apoptosis
compared with control, also correlates with the clinical observation of
significant immune deficiency in patients with WAS but not with XLT.
Finally, the association of WASP with the actin cytoskeleton may have
little significance for the structural function of cytoskeleton itself
but may have consequences for the regulation of cell death. There is
increasing evidence linking cytoskeletal-associated proteins such as
gelsolin with key roles in regulating apoptosis.48,49
Similarly, the potential role of WASP in delaying apoptosis may be
mediated through its cytoskeletal attachments. Thus, the cytoskeletal
abnormalities that have been observed in cells from patients with WAS
and in cells expressing mutant WASP likely reflect the cytoskeletal
changes that are associated with apoptosis.50
Our data indicated that a defect in the dynamic function of the actin
cytoskeleton plays a minor role, if any, in the pathogenesis of WAS and
XLT because mutations of WASP do not appear to affect the ability of
hematopoietic cells to polymerize actin in response to stimulation. In
addition, platelets from patients with WAS were able to produce the
cytoarchitectural features associated with the stimulation of normal
platelets. A possible explanation for the complex abnormalities
observed in WAS lies in the observation that Fas receptor expression
and apoptosis were increased in lymphocytes isolated from patients with
WAS. This observation suggests the possibility that the primary
consequence of mutations in WASP is defective regulation of apoptosis
and not abnormal cytoskeletal function.
| |
Acknowledgments |
We thank Dr A. Al-Katib for generously donating bryostatin (synthesized
by Dr G. Petit) and Dr Anu Srinivasan for helpful suggestions during
the course of the project. We also thank Dr T. Futatani for determining
the CD3-induced proliferation response in lymphocytes. Finally, we
thank E. Calomeni, M. Young, and A. Tantri for excellent
technical assistance.
| |
Footnotes |
Submitted October 1, 1998; accepted October 19, 1999.
Supported by the Office of Research and Development, Medical Research
Service, Department of Veteran Affairs, and by grants from the
Arthritis Foundation, the National Science Foundation (BES 9410403),
and the National Institutes of Health (AI20065; training grants T32
GM07767/Pharmacological Sciences Training Program and T32
GM07863/Medical Scientist Training Program). Supported also by grants
from the National Institutes of Health, the March of Dimes Birth
Defects Foundation, and the DiJoria Wiskott-Aldrich Research Fund for
that part of the study conducted at the Clinical Research Center,
University of Washington, Seattle, WA.
Reprints: Geneva M. Omann, Research Services (11R), Room G1,
Building 22, Veterans Administration Medical Center, 2215 Fuller Rd,
Ann Arbor, MI 48105-2399; e-mail: gmomann{at}umich.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Top
Abstract
Introduction