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Blood, Vol. 94 No. 11 (December 1), 1999:
pp. 3872-3882
Spontaneous Apoptosis in Lymphocytes From Patients With
Wiskott-Aldrich Syndrome: Correlation of Accelerated Cell Death and
Attenuated Bcl-2 Expression
By
Stephen L. Rawlings,
Gay M. Crooks,
David Bockstoce,
Lora W. Barsky,
Robertson Parkman, and
Kenneth I. Weinberg
From the Division of Research Immunology and Bone Marrow
Transplantation, Childrens Hospital Los Angeles, Los Angeles, CA.
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ABSTRACT |
Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder
characterized by thrombocytopenia, eczema, and a progressive deterioration of immune function. WAS is caused by mutations in an
intracellular protein, WASP, that is involved in signal transduction and regulation of actin cytoskeleton rearrangement. Because immune dysfunction in WAS may be due to an accelerated destruction of lymphocytes, we examined the susceptibility to apoptosis of resting primary lymphocytes isolated from WAS patients in the absence of
exogenous apoptogenic stimulation. We found that unstimulated WAS
lymphocytes underwent spontaneous apoptosis at a greater frequency than
unstimulated normal lymphocytes. Coincident with increased apoptotic
susceptibility, WAS lymphocytes had markedly attenuated Bcl-2
expression, whereas Bax expression did not differ. A negative correlation between the frequency of spontaneous apoptosis and the
level of Bcl-2 expression was demonstrated. These data indicate that
accelerated lymphocyte destruction by spontaneous induction of
apoptosis may be one pathogenic mechanism by which the progressive immunodeficiency in WAS patients develops.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
WISKOTT-ALDRICH SYNDROME (WAS) is a rare
X-linked recessive disorder.1,2 Affected males are
clinically characterized by eczema, thrombocytopenia with platelets of
reduced size, immunodeficiency, and an increased susceptibility to
hematopoietic malignancy and autoimmunity (Remold-O'Donnell et
al3 and references therein). WAS patients suffer from
chronic and recurrent opportunistic and viral infections as a
consequence of immunologic abnormalities. A progressive deterioration
of T-lymphocyte function in affected children accompanies the
development of T lymphopenia by 6 years of age.4
Cell-mediated immunity is further compromised by poor macrophage and
neutrophil motility and chemotactic responses.4-7 WAS
patients commonly have low isohemagglutinin titers and a depressed response to polysaccharide antigens3; but, their impaired
humoral immunity has been at least partially attributed to T-lymphocyte dysfunction.8 WAS T lymphocytes have diminished
proliferative responses to mitogens, some specific antigens, and
allogeneic stimulation.3 Moreover, WAS T lymphocytes fail
to proliferate or upregulate interleukin-2 gene expression in response
to treatment with immobilized anti-CD3 antibody.9,10
Deformation and deregulation of the actin cytoskeleton is likely to be
the basis for many of the clinical defects associated with WAS,
particularly immune dysfunction.3,10,11 WAS
lymphocytes are frequently morphologically abnormal, with irregular and
bulbous cellular projections,10,12 a paucity of
microvilli,13 and a poorly delineated actin
cortex.10 CD3-mediated stimulation of WAS T-lymphoblastoid
cell lines results in abnormal actin polymerization, marked by the
absence of specific cytoskeleton rearrangements that occur in normal
T-lymphoblastoid cell lines.10 Epstein-Barr
virus-transformed B-cell lines from WAS patients also have marked
abnormalities in actin distribution and polymerization.11
The WAS gene was mapped to the short arm of the X chromosome at
Xp11.2214 and identified in 1994.15 The gene
encodes a 66-kD intracellular protein, WASP, that is expressed
exclusively in blood cells15,16 throughout
hematopoiesis.17 More than 138 unique mutations to WASP
have been described,18 which include frameshifts and
substitutions that generally nullify expression by causing mRNA
instability and protein truncation.11,19-21 There is
considerable variation in WAS severity; even members of the same
kindred may have markedly different phenotypes,22 possibly indicating polygenic or environmental contributions. WAS severity may
be partially determined by the level at which WASP is stably expressed.11,19-21
The function of WASP is unknown; however, it has been surmised that
WASP is involved in the regulation of actin polymerization and actin
cytoskeleton organization from observations that WAS lymphocytes,
platelets, granulocytes, and monocytes are defective in regulating the
cortical actin cytoskeleton (Remold-O'Donnell et al3 and
references therein). In support of this presumption, N-WASP, a
ubiquitous mammalian homolog of WASP, has been demonstrated to directly
induce actin depolymerizaton and actin cytoskeleton reorganization in
nerve growth factor-stimulated cells.23 Although WASP
apparently lacks catalytic activity, the protein colocalizes with actin
and interacts with WIP24 and PSTPIP,25 2 proteins that regulate actin polymerization. The Rho family small
guanosine triphosphatases (GTPases), Cdc42 and Rac, which have been
shown to specifically control actin cytoskeleton
reorganization,26 interact with WASP.27-30 WASP
also associates with numerous other proteins, including various
tyrosine kinases (Btk, Itk, Tec, Fyn, and c-Src), adaptor proteins (Nck
and Grb2), and phospholipase C 1, all of which are involved in
lymphoid signal transduction.31-36 Therefore, it has been
proposed that WASP functions as a molecular scaffold28 that
docks and aligns other proteins for more specific interaction.37 Thus, WASP may link components of the
cytoskeleton with key signal transduction elements to integrate
signaling in response to various intrinsic or extrinsic stimuli to
regulate actin cytoskeleton reorganization.
A WASP deficiency or the expression of dysfunctional WASP is probably
detrimental to the development and survival of hematopoietic cell
lineages.3 A uniformly nonrandom pattern of X-chromosome inactivation in all blood cells from WAS obligate heterozygous carriers and platelet loss in WAS patients suggests that
mutations to WASP probably confer a growth and/or survival disadvantage to affected cells.3 Consequently, we reasoned that the
development of lymphopenia and other progressive immune defects
associated with WAS may be caused by an inherently decreased potential
for survival and the accelerated destruction of peripheral lymphocytes.
In this study, we compared the levels of spontaneous apoptosis in
unstimulated resting lymphocytes isolated from WAS patients and from
normal, healthy controls. We found that WAS lymphocytes underwent
spontaneous apoptosis at a greater frequency than did normal
lymphocytes. Bcl-2 family members regulate the onset of apoptosis,
acting as either agonists or inhibitors of programmed cell
death.38 Because the deregulation of apoptosis in WAS
lymphocytes might be caused by aberrant expression of one or more of
the Bcl-2 family members, we determined that the level of Bcl-2
expression in WAS lymphocytes was attenuated. We suggest that the
accelerated destruction of lymphocytes by spontaneous apoptosis could
account for the progressive deterioration of immune function in WAS patients.
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MATERIALS AND METHODS |
Patient samples.
Samples from 5 male patients previously diagnosed with WAS were used in
this study. At the time of analysis, patients no. 1 and 2 were 2.5 and
4 years of age, respectively. Patients no. 3 and 4 are siblings and
were 4.5 and 14 years of age, respectively. Patient no. 5 was 14 months
of age. Diagnosis of WAS was initially based on family history and
presentation of immunodeficiency with recurrent infection,
thrombocytopenia, platelets with reduced volume, and eczema. To define
the WAS phenotype of the patients, clinical scores were assigned to
each according to published recommendations.18,21 Patients
no. 1, 2, 3, 4, and 5 were assigned clinical scores of 4, 4, 3, 3, and
3, respectively. The genotypes of 4 of 5 patients have been determined.
Patient no. 1 has a point mutation, C290T, resulting in an amino acid
substitution of R86C in exon 2. The genotype of patient no. 2 was not
determined. Patients no. 3 and 4, the siblings, have a deletion
mutation in exon 8 (John Bastian, personal communication, July
1999). Patient no. 5 has a splice site mutation (4-bp
deletion in intron 8), resulting in the deletion of exon 8; patient no.
5 does not express WASP. Patients no. 1, 2, and 5 were treated at
Childrens Hospital (CHLA) in Los Angeles, CA; each has since received
bone marrow transplantations. Patients no. 3 and 4 were treated at
Children's Hospital in San Diego, CA. These studies were performed in
accord with protocols approved by the Committee on Clinical
Investigations of the Institutional Review Board at CHLA. None of the
patients had been splenectomized. Normal controls (8) were obtained
from either healthy volunteer adult donors (5) or healthy pediatric
patients (3) at CHLA. No significant difference between the normal
pediatric and normal adult means was noted (P = .978). Whenever possible, patient and normal blood was collected and
processed under paired conditions. For the comparison of normal
pediatric and normal adult blood, specimens were collected and
processed under paired conditions.
Isolation and in vitro treatment of peripheral blood lymphocytes
(PBL).
Peripheral blood was collected from WAS patients or normal, healthy
individuals in heparinized Vacutainer tubes (Becton Dickinson, Franklin
Lakes, NJ). Within 8 hours, mononuclear cells were isolated from whole
blood by Ficoll-Hypaque (Pharmacia Biotech, Piscataway, NJ) density
gradient centrifugation. Mononuclear cells were washed in Hanks'
balanced salt solution (HBSS; BioWhittaker, Walkersville, MD) and
resuspended in R-10 medium (RPMI-1640 supplemented with 10%
heat-inactivated human A+ serum [CHLA Blood Bank, Los
Angeles, CA], 2 mmol/L L-glutamine [Gemini BioProducts, Calabasas,
CA], 1 × 10 6 mol/L 2-mercaptoethanol [Sigma,
St Louis, MO], and 1× penicillin-streptomycin [Gemini
BioProducts]). Cells were plated at a density of 5 × 106 cells/mL in 100-mm tissue culture dishes (Corning,
Corning, NY) and placed in a 37°C humidified incubator conditioned
with 5% CO2 for 1 hour. Nonadherent PBL were collected and
resuspended in R-10. PBL were plated at a density of 0.5 × 106 cells/mL in 24-well flat-bottomed dishes (Costar,
Cambridge, MA) and maintained in a humidified atmosphere with 5%
CO2 at 37°C for up to 10 days. Some experiments were
performed after maintaining PBL in R-10 containing 10%
heat-inactivated fetal calf serum (Summit Biotechnology, Fort Collins,
CO) instead of human serum, but no differences in the apoptotic
frequencies of PBL were apparent. PBL were not stimulated in vitro.
Detection of apoptosis.
Either of 2 methods, terminal deoxynucleotidyl transferase-mediated
dUTP nicked end labeling (TUNEL) or annexin V-labeling, was used to
mark apoptotic cells. For TUNEL,39 cells were harvested and
washed in 3 mL phosphate-buffered saline (PBS; Sigma). At least
105 cells were stained on ice and in the dark with
fluorochrome-conjugated antihuman antibodies recognizing specific cell
surface markers to be used in immunophenotyping. Incubations with the
antibodies were performed in PBS containing 0.1% human intravenous Ig
(Sandoz Pharmaceuticals, East Hanover, NJ) for 15 minutes. All
allophycocyanin- and phycoerythrin-conjugated antibodies were used
according to the supplier's recommendations (Becton Dickinson
Immunocytometry Systems [BDIS], San Jose, CA). Stained cells were
washed in 3 mL PBS and fixed in 0.5 mL 2% paraformaldehyde (Sigma) on
ice for 15 minutes. Cells were washed in 3 mL PBS and permeabilized in
0.5 mL PBS containing 0.5% Tween-20 (Sigma) and 0.2% bovine serum
albumin (BSA; fraction V; Sigma). Samples were mixed gently and placed
on ice for 15 minutes. Cells were washed in 3 mL PBS and resuspended in
50 µL TUNEL reaction mix containing 5 U terminal deoxynucleotidyl
transferase (TdT; Promega, Madison, WI), 1× TdT reaction buffer
(Promega; containing 100 mmol/L cacodylate buffer [pH 6.8], 1 mmol/L
cobalt chloride, and 0.1 mmol/L dithiothreitol), and 10 µmol/L
biotin-16-dUTP (Boehringer Mannheim Biochemicals, Indianapolis, IN).
Cells were incubated in the reaction mix at 37°C for 30 minutes.
Cells were washed in 3 mL PBS and resuspended in 100 µL labeling
solution containing 4× SSC, 0.1% Triton X-100 (Sigma), 10%
nonfat dry milk (Carnation, Glendale, CA), and 0.1% sodium azide (Sigma). Ten microliters of avidin-fluorescein (BDIS) was
mixed with the cells. Samples were kept at room temperature for 30 minutes in the dark. Cells were washed in 3 mL PBS containing 0.1%
Triton X-100. Cells were resuspended in 0.5 mL PBS containing 5 µg/mL
propidium iodide (PI; Sigma) and 0.1% DNase heat-inactivated RNase A
(Sigma). Negative control samples were treated in an identical manner
except that TdT was omitted from the TUNEL reaction mix. Labeled cells
were analyzed promptly by flow cytometry.
Annexin V-labeling was used to confirm the results of
TUNEL.40 Briefly, cells were harvested and washed in 3 mL
PBS. Cells were stained with immunophenotyping antibodies as described
above. Cells were then washed in 3 mL PBS and resuspended at a
concentration of 1 × 106 cells/mL in 1 × annexin V binding buffer (all reagents are supplied with the
Apoptosis Detection Kit; R&D Systems, Minneapolis, MN). One hundred
microliters (105) of cells was transferred to another tube.
Ten microliters of fluorescein-conjugated annexin V (10 µg/mL) and 10 µL PI (50 µg/mL) were mixed with the cells. Samples were kept at
room temperature for 15 minutes in the dark. Four hundred microliters
of 1× binding buffer was used to dilute the labeled cells and
analysis was performed by flow cytometry within 1 hour. Controls were
prepared according to the recommendations of the supplier.
Determination of relative Bcl-2 expression levels.
The relative level of Bcl-2 expression in PBL was determined by
intracellular staining using a hamster antihuman monoclonal antibody,
an assay similar to that used by von Freeden-Jeffry et
al.41 Briefly, at least 105 cells were
harvested and stained with immunophenotyping antibodies, as described
above. Cells were washed in 3 mL PBS and fixed in 0.5 mL 2%
paraformaldehyde for 15 minutes on ice. Cells were washed in 3 mL PBS
and permeabilized in 200 µL PBS containing 0.3% saponin (Sigma) and
0.2% BSA. All subsequent steps were performed in this buffer. Samples
were placed on ice for 15 minutes. Two micrograms of anti-Bcl-2
antibody (clone 6C8; Pharmingen, San Diego, CA) was mixed with the
cells. The negative control for measuring background fluorescence was
prepared in a similar manner, except that 2 µg hamster IgG isotype
control polyclonal antibody (Pharmingen) was added to the cells.
Samples were placed on ice for 20 minutes. Cells were washed in 3 mL
PBS and resuspended in 100 µL of the buffer. One microgram of
fluorescein-conjugated mouse antihamster IgG antibody (clone G70-204;
Pharmingen) was mixed with the cells. Samples were placed on ice in the
dark for 20 minutes. Cells were washed in 3 mL PBS and resuspended in
0.5 mL 2% paraformaldehyde. Samples were stored at 4°C until
analysis was performed by flow cytometry.
Flow cytometry and data analysis.
Analysis of TUNEL samples was performed using a FACSVantage flow
cytometer (BDIS) equipped with an argon laser tuned to 488 nm and a
helium-neon (HeNe) laser tuned to 633 nm. A 610 nm short-pass splitter
was used to divert PI fluorescence to FL-3. After electronic compensation, FL-3 fluorescence was measured using both linear and
logarithmic amplification; FL-1 (fluorescein), FL-2 (phycoerythrin), and FL-4 (allophycocyanin) fluorescences were measured using
logarithmic amplification. The flow rate was not permitted to exceed
200 events per second. The data from at least 5 × 104
events were collected for analysis of freshly isolated cells. For all
other experiments, the data from at least 104 events were
collected. The data were analyzed using CELLQuest software (BDIS). For
analysis of TUNEL data, fragmented cells and debris were electronically
excluded. Another region was set to electronically eliminate multiplet
events from the analysis. A negative control sample (no TdT added) was
used to establish TUNEL-negative and -positive regions, such that at
least 99% of the events were in the lower TUNEL-negative region.
Background events were then subtracted from the TUNEL-positive events.
All other flow cytometry was performed using a FACSCalibur instrument
(BDIS) fitted with both argon and HeNe lasers. The data from at least
104 events were collected. Analysis of fluorescein-annexin
V-labeled cells was performed according to the recommendations provided with the Apoptosis Detection Kit (R&D Systems). For analysis of annexin
V binding data, fragmented cells and debris were electronically excluded. For analysis of Bcl-2 expression, a region was set to include
the enriched lymphocyte population, excluding all other cells and
debris. The same region was used for analysis of both normal and WAS
lymphocytes so that differences in cell size and granularity, and
possible differences in mitochondrial copy number on a per cell basis,
were not reflected in the values reported. Background fluorescence was
measured using a negative isotype control. The mean fluorescence
intensity (MFI) reported for a given sample is the difference between
the sample value and the background value.
Statistical analysis of results.
Number Crunching Statistical Systems software (Dr Gerry L. Hintze,
Kayszille, UT) was used for the statistical analysis of data. Where
applicable, an unbalanced repeated measures analysis of variance
(ANOVA) was used to determine the significance of differences between
normal and WAS sample values. This method eliminates bias possibly
associated with analysis of unbalanced repeated measures. The TUNEL
data were also analyzed as a set of randomly chosen duplicate repeated
measures to test the validity of the first method of analysis. This
analysis showed that the first method was indeed valid, confirming the
statistical significance of the data. A probability level (P)
of  .05 is termed significant. An analysis of correlation between
Bcl-2 expression and level of apoptosis was performed using logarithmic
transformation. A correlation coefficient (r2) was
determined using the nonparametric Spearman method. Increases in the
frequency of apoptosis over time were determined by linear regression.
| |
RESULTS |
WAS PBL undergo spontaneous apoptosis in vitro at a greater rate than
normal PBL.
The levels of spontaneous apoptosis in unstimulated, resting PBL were
determined in this study. To compare the frequency of apoptosis in WAS
PBL and normal PBL, we determined the fraction of cells undergoing
apoptosis by 2 quantitative methods. TUNEL39 and annexin
V-labeling40 measure oligonucleosomal DNA degradation and
externalization of phosphatidylserine, respectively, 2 hallmarks of
apoptosis.42,43 Flow cytometry was used to quantify marked cells undergoing apoptosis immediately after PBL isolation or after a
period of in vitro incubation. Importantly, PBL were not treated with
cytokines or any other mitogenic or antigenic stimuli and neither was
an extrinsic stimulus used to induce apoptosis.
Representative TUNEL data shown in Fig 1
show that a larger fraction of cells isolated from a WAS patient were
undergoing spontaneous apoptosis compared with cells from a normal
control at the time of analysis. Another prominent characteristic of
apoptotic cells detectable by flow cytometry is cytoplasmic shrinkage,
resulting in decreased forward light scatter and increased side light
scatter, caused by increases in cellular density during volume
reduction.44 A direct comparison of the light scattering
properties of normal PBL with those isolated from a WAS patient showed
that a relatively larger subpopulation of WAS PBL had apoptotic
characteristics (Fig 1A). The results of an analysis of freshly
isolated PBL performed immediately after isolation are shown in Fig 1C.
The mean levels of apoptotic DNA fragmentation in freshly isolated PBL
from the group of 5 WAS patients and the group of 8 normal individuals (5 adults and 3 children) differed significantly (P = .0123). WAS PBL underwent spontaneous apoptosis at a 10-fold greater frequency immediately after isolation. Considerable variation in the levels of
spontaneous apoptosis in freshly isolated PBL from different WAS
patients was evident, possibly reflecting genotypic differences. However, differences between the siblings are also noted and suggest that polygenic factors may determine the susceptibility to apoptosis in
WAS PBL. Therefore, these data suggest that WAS PBL, as compared with
normal PBL, may have a lower threshold of susceptibility to apoptogenic
signals.
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| Fig 1.
Analysis of apoptotic lymphocytes isolated from WAS
patients. The levels of apoptosis were measured using TUNEL immediately
after isolation or after incubation in vitro. PBL were not stimulated.
(A) WAS lymphocytes had increased side scatter and decreased forward
scatter, characteristics of apoptosis, after 4 days of incubation in
vitro relative to normal lymphocytes. (B) Representative data acquired
after TUNEL indicate that a higher fraction of WAS lymphocytes were
undergoing apoptosis relative to normal, healthy donor lymphocytes
after 4 days of incubation in vitro. The fraction of TUNEL-positive
cells is indicated in the upper right corner. (C) WAS lymphocytes
underwent apoptosis at a greater frequency than normal lymphocytes.
Levels of apoptosis were measured immediately after isolation of PBL
from WAS patients or from normal individuals using the TUNEL assay. N,
the mean value for a group of 8 different normal controls (3 children
and 5 adults). W, the mean value for a group of 5 different WAS
patients. 1 through 5, the mean values for each individual WAS patient.
Patient no. 5 was analyzed twice in 2 independent experiments (with 6 and 3 replicates); all other patients were analyzed once in single
experiments (with 6 replicates). An unbalanced repeated measures
analysis of variance (ANOVA) showed that the values for patients no. 2, 3, 4, and 5 differed significantly from the mean normal value
(P .0001). The mean value for the group of 5 WAS patients
(W) also differed significantly from the mean normal (P = .0123). n, the number of different samples; *, statistical
significance. (D) WAS lymphocytes were more susceptible to apoptosis
than normal lymphocytes after in vitro incubations of 24, 48, and 96 hours. Considerable variability in the apoptotic susceptibility of PBL
from different WAS patients was apparent. Mean levels of apoptosis are
reported in Table 1. Patient no. 1 was analyzed once in a single
experiment, patient no. 2 was analyzed 3 times in 3 independent
experiments, and patients no. 3, 4, and 5 were analyzed twice in 2 independent experiments. Results from 8 different normal controls (3 children and 5 adults) are shown. The number of repeated measures made
in each experiment was varied, ranging from 2 to 6 (generally 4),
depending on the number of PBL isolated from the blood samples. An
unbalanced repeated measures ANOVA showed that differences between the
groups, WAS and normal, are significant at 24, 48, and 96 hours
(P = .000181, .00283, and .000190, respectively).
*Statistical significance.
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We examined the effects of maintaining isolated PBL in vitro for
varying periods in medium containing 10% human A+ serum,
which minimizes lymphocyte activation. The levels of spontaneous apoptosis in WAS PBL (5 patients) and normal PBL (5 adults and 3 children) differed significantly after incubations of 24, 48, and 96 hours (Fig 1D). We observed a steady increase in the fraction of WAS
PBL undergoing spontaneous apoptosis over time. In contrast, the
frequency of spontaneous apoptosis in normal PBL leveled off after 48 hours in vitro. Over the entire duration of the incubation, the
difference between the increases in WAS and normal PBL tendency to
undergo apoptosis was statistically significant (P < .0001). We observed that most of the cells undergoing apoptosis were resting in
either G0 or G1 of the cell cycle (data not shown).
Annexin V-labeling confirmed and extended the results obtained using
TUNEL to mark cells undergoing spontaneous apoptosis. Apoptotic cells
are distinguished by flow cytometry from live and dead cells by
fluorescein-conjugated annexin V binding and exclusion of the vital
dye, PI, respectively (Fig 2A). As shown in
Fig 2B, over the entire duration of incubation, WAS PBL from the 4 patients (no. 1 through 4) analyzed underwent spontaneous apoptosis at
a significantly greater frequency than normal PBL (P = .025).
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| Fig 2.
Annexin V staining confirmed that WAS lymphocytes
underwent apoptosis at a greater frequency than normal lymphocytes.
Representative data indicate that a higher percentage of WAS
lymphocytes had externalized phosphatidylserine and bound annexin V
compared with normal lymphocytes. Furthermore, a higher percentage of
lymphocytes from WAS patients were inviable, no longer excluding PI,
relative to lymphocytes from normal individuals. Apoptotic cells are
represented by events in the lower right-hand quadrant, with viable and
dead cells depicted by events in the lower left-hand and upper
right-hand quadrants, respectively. The fraction of cells undergoing
apoptosis and the fraction of dead cells are indicated at the right of
each plot. (B) Both methods of analysis, annexin V staining and TUNEL,
indicated that WAS lymphocytes underwent apoptosis at a greater
frequency than normal lymphocytes. Samples from 4 different WAS
patients (no. 1 through 4) and 6 different normal controls were
analyzed. Statistical analysis by an unbalanced repeated measures ANOVA
showed that the differences between WAS and normal levels of apoptosis
as measured using annexin V were significant after in vitro incubations
of 48 and 96 hours, as indicated by the asterisks (P = .0013 and .0181, respectively). TUNEL data (shown in Fig 1) is duplicated for
easy comparison.
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Both WAS B and T lymphocytes are more susceptible to apoptosis than
normal B and T lymphocytes.
WAS patients have defects in both humoral and cell-mediated immunity;
thus, we sought to determine if both T and B lymphocytes have increased
susceptibility to spontaneous apoptosis. We measured the frequencies of
spontaneous apoptosis in resting T and B lymphocytes isolated from WAS
patients (no. 1 through 4) and normal controls after incubation in
vitro. Figure 3A shows that unstimulated
WAS CD3+ T lymphocytes undergo spontaneous apoptosis at a
greater rate than do unstimulated normal CD3+ T
lymphocytes. In vitro incubation augmented the level of spontaneous apoptosis in unstimulated T lymphocytes. Over time, the frequency of
spontaneous apoptosis in WAS T lymphocytes increased more rapidly than
occurred normally (P = .024). Unstimulated WAS
CD19+ B lymphocytes also underwent spontaneous apoptosis at
an increasing frequency during in vitro incubation (Fig 3B). The mean
level of spontaneous apoptosis in WAS B lymphocytes was higher than in
normal B lymphocytes at each time point. This trend was consistent, although statistical significance could not be demonstrated due to the
presence of very low numbers of circulating B lymphocytes in our WAS
patients. In vitro incubation also augmented spontaneous apoptosis in
unstimulated WAS B lymphocytes by 2-fold over normal.
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| Fig 3.
Both T and B lymphocytes from WAS patients are more
susceptible to apoptosis than T and B lymphocytes from normal
individuals. Analysis of apoptosis was performed using TUNEL after
staining with immunophenotyping cell surface markers. Four different
WAS patients (no. 1 through 4) and 4 different normal controls were
analyzed. (A) CD3+ T lymphocytes from WAS patients
underwent apoptosis at a greater frequency than did normal lymphocytes.
Statistical significance was shown after 96 hours of incubation in
vitro (P = .0239) by an unbalanced repeated measures ANOVA.
(B) WAS B lymphocytes (CD19+) are more susceptible to
apoptosis than were normal B lymphocytes. The mean levels of apoptosis
were clearly different, although, because of small cell numbers, the
standard errors were too high to show statistical significance. The
scales used in (A) and (B) are identical.
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Bcl-2 expression is attenuated in WAS PBL.
Because Bcl-2 family members have been shown to regulate apoptosis in
hematopoietic cells, the accelerated spontaneous apoptosis in WAS PBL
could result from deregulated expression of regulatory factors, such as
Bcl-2,45 that repress apoptosis. We examined the relative
levels of Bcl-2 expression in WAS and normal PBL by measuring the MFI
of cells stained indirectly with anti-Bcl-2 antibody in situ.
Measurements were made either immediately after isolation of PBL
(Table 1) or after in vitro incubation
(Table 2). WAS PBL from 5 different
patients had a significantly reduced level of Bcl-2 compared with
normal PBL from 8 different individuals (Fig 4). The relative levels of Bcl-2
expressed in isolated PBL did not change significantly over time during
in vitro incubation. Wide ranges in Bcl-2 expression are evident, both
in PBL isolated from WAS patients and from normal controls. Analysis
showed that PBL from patients no. 2 and 5 had Bcl-2 levels that
differed significantly from normal (P = .00477 and .0254, respectively) at the initial time point. There was no significant
difference between the levels of Bax46 expression in WAS
and normal PBL (data not shown). Therefore, we have demonstrated that
Bcl-2, an inhibitor of apoptosis, but not Bax, an activator of
apoptosis, is aberrantly expressed in WAS PBL relative to normal PBL.
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Table 1.
Comparing the Frequency of Apoptosis and the Level
of Bcl-2 Expression in Freshly Isolated WAS Lymphocytes From
Different Patients
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Table 2.
Comparing the Frequency of Apoptosis and the Level of
Bcl-2 Expression in WAS and Normal Lymphocyte Populations
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| Fig 4.
Bcl-2 expression in WAS lymphocytes is attenuated as
compared with Bcl-2 expression in normal lymphocytes. MFI of the Bcl-2
signal was measured either immediately after isolation or after in
vitro incubation for 24 or 48 hours. Samples from 5 different WAS
patients and 7 different normal controls were analyzed. An unbalanced
repeated measures ANOVA showed that the differences in Bcl-2 expression
levels between the 2 groups, normal and WAS, at each time point (0, 24, and 48 hours) are significant (P = .0233, .00985, and .0192, respectively).
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Correlation between the level of Bcl-2 expression and susceptibility
to induction of spontaneous apoptosis in PBL.
The relationship between Bcl-2 expression and susceptibility to
induction of spontaneous apoptosis was studied in freshly isolated PBL
from 5 different WAS patients and from 4 normal, healthy individuals.
Relative Bcl-2 expression was measured immediately after isolation
without an extended in vitro incubation. At the same time and using the
same sample, we measured the level of apoptosis in the PBL population
after TUNEL. As illustrated in Fig 5, there
was an inverse relationship between Bcl-2 expression and the
susceptibility to induction of spontaneous apoptosis
(r2 = .744). Unstimulated normal PBL that expressed
relatively high levels of Bcl-2 were less susceptible to induction of
spontaneous apoptosis; the frequency of apoptosis in normal populations
was reduced when compared with the frequency of apoptosis in WAS
populations. In contrast, unstimulated WAS PBL that expressed
relatively low levels of Bcl-2 tended to be more susceptible to
induction of spontaneous apoptosis than were normal PBL.
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| Fig 5.
Correlation between the relative level of Bcl-2
expression and the susceptibility to spontaneous apoptosis in PBL
isolated from WAS patients and normal controls. The mean value for
replicate measures (2 to 3) of the MFI of the Bcl-2 signal was plotted
on the y-axis. The mean fraction of PBL undergoing spontaneous
apoptosis was determined using TUNEL and was plotted on the x-axis.
Both measurements were made immediately after isolation of PBL using
the same sample. Samples from 5 different WAS patients were analyzed;
patient no. 5 was analyzed twice in 2 independent experiments. The data
were fitted logarithmically. There is an inverse correlation between
Bcl-2 expression and the level of susceptibility to apoptosis
(r2 = .744).
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| |
DISCUSSION |
We have shown that, in the absence of extrinsic apoptotic stimuli,
resting WAS PBL undergo spontaneous apoptosis at a significantly greater frequency than resting normal PBL. We also have found that the
expression of Bcl-2 is attenuated in WAS PBL. Because Bcl-2 inhibits
the onset of apoptosis, insufficient levels of Bcl-2 expression could
diminish the survival potential and shorten the life span of
lymphocytes. We suggest then that peripheral T and B lymphocytes in WAS
patients undergo accelerated spontaneous apoptosis as a consequence of
a partial Bcl-2 deficiency. The accelerated destruction of peripheral T
lymphocytes by spontaneous apoptosis could account for the progressive
deterioration of T-lymphocyte function and lymphopenia in WAS patients.
Moreover, a poor humoral response to certain specific antigens by WAS
patients may not be strictly a consequence of T-lymphocyte
abnormalities, as has been previously suggested.3,8 The
accelerated destruction of memory B lymphocytes with diminished
survival potential may preclude secondary immune
responses.47 Therefore, chronic and recurrent infection in
WAS patients may result from a failure to develop immunologic memory
caused by accelerated spontaneous apoptosis of peripheral T and B lymphocytes.
Marked by DNA degradation, phosphatidylserine externalization, and
cytoplasmic shrinkage, the abnormally high incidence of apoptosis in
WAS lymphocytes suggests that inherent abnormalities of the actin
cytoskeleton and cell surface glycoproteins3 may engender
their self-destruction by programmed cell death (PCD). We found that
the frequency of WAS PBL apoptosis was augmented to a greater extent
than normal by in vitro incubation. This suggests that PCD was not
triggered before isolation or in a manner dependent on the removal of
lymphocytes from the periphery, where perhaps an extrinsic factor
enabled survival. Moreover, because an external stimulus was not
required to induce apoptosis in WAS PBL, we suggest that an internal
mechanism induces unstimulated, resting WAS PBL to undergo spontaneous
apoptosis at an accelerated frequency relative to normal PBL. The
necessary impetus to undergo spontaneous apoptosis might be provided by
the inherent abnormalities in WAS cells; these defects may conceivably
trigger intrinsic apoptogenic signaling by surveillance proteins that
monitor cellular damage.
Previous studies of WAS patients have demonstrated profound lymphopenia
in peripheral lymphoid organs, corroborating our results and suggesting
that our observations are clinically relevant. For example, the spleen
and lymph nodes of WAS patients were largely devoid of T
lymphocytes.48,49 Moreover, another case study of a WAS
patient reported that the germinal centers and follicles of the lymph
node and spleen were poorly developed or absent.49 Hypocellularity and abnormal tissue architecture of these secondary lymphoid tissues in WAS patients is consistent with the our observation that WAS lymphocytes undergo accelerated spontaneous apoptosis and
support the possible contention that WAS PBL have an inherently diminished potential for survival in vivo.
Studies of other affected blood cell lineages in WAS patients have also
provided data that are consistent with our observations. It has been
proposed that thrombocytopenia in WAS patients develops as a
consequence of the accelerated destruction of platelets.3 Studies of WAS heterozygotes have suggested that affected hematopoietic stem cells and their progeny that carry an active mutant X-chromosome are stringently selected against during development,3
conceivably due to a loss of a capacity to either proliferate or
survive. Affected blood cells in WAS heterozygotes may either fail to
renew or to expand in a manner competitive with their normal,
unaffected counterparts. Our study provides a unifying hypothesis that
may explain the mechanism of the accelerated destruction of platelets and lymphocytes in WAS patients and the strict exclusion of affected blood cells in WAS carriers. We propose that mutations to WASP cause
increased spontaneous apoptosis in all affected blood cells in both WAS
patients and WAS carriers.
Mutations to WASP may obstruct hematopoiesis, but clearly the
development of mature lymphocytes and platelets in WAS patients is not
strictly precluded. Instead, it seems that WASP mutations are a
detriment to the survival of mature blood cells in the periphery. In
this study, we found abnormally low levels of Bcl-2 in resting, unstimulated WAS PBL. Because other studies have demonstrated the
importance of Bcl-2 in regulating the survival of mature
lymphocytes,50-52 our results suggest that the increased
lability of WAS lymphocytes may be due to insufficient Bcl-2
expression. Interestingly, we note that the development and maintenance
of lymphocytes in WAS patients is mirrored by lymphopoiesis in
Bcl-2-deficient mice. Postnatally, it appears that lymphopoiesis in
Bcl-2-deficient mice is normal; however, older mice eventually develop
lymphopenia, as the thymus and spleen of these mice undergo massive
apoptotic involution.53 Furthermore, whereas
Bcl-2-deficient hematopoietic progenitor cells (HPC) in mouse chimeras
differentiate into phenotypically mature lymphocytes, their progeny
have markedly shortened life spans relative to the progeny of normal
HPC.54 It seems, therefore, that murine Bcl-2 is
dispensable for lymphocyte maturation but is required for the
maintenance of viability afterward. We now describe a similar phenomena
that occurs in WAS patients. Consistent with this observation,
WASP-deficient mice, despite having apparently normal lymphopoiesis,
have markedly decreased numbers of mature lymphocytes in the periphery
relative to wild-type mice.55
Considerable variation in the levels of susceptibility to spontaneous
apoptosis was found in PBL from different WAS patients. Despite
genotypic identity and presumably similar environments, PBL isolated
from the siblings, patients no. 3 and 4, had strikingly different
levels of susceptibility to spontaneous apoptosis. PBL from patient no.
4 were about 3 times more sensitive to intrinsic apoptotic stimuli than
were PBL from patient no. 3. Although both patients had grade 3 WAS,
patient no. 3 is several years younger than patient no. 4. Because
immunodeficiency associated with WAS is progressive, it is intriguing
to speculate that such phenotypic differences may arise with the
increasing age of the patient. Of course, there is the possibility that
multiple genetic factors determine the clinical severity of WAS.
Certainly, it is not uncommon to find that different members of the
same kindred have different manifestations of the
disease.18
We have observed considerable variation in Bcl-2 expression in freshly
isolated PBL from different WAS patients (Table 1). The level of
spontaneous apoptosis appears to be inversely related to the level of
Bcl-2 expression. For example, PBL from WAS patient no. 1, which
expressed relatively higher levels of Bcl-2 than PBL from the other
patients, were less susceptible to the induction of spontaneous
apoptosis. Because reduced Bcl-2 expression in WAS PBL could account
for the increased susceptibility to intrinsic apoptotic stimuli, we
speculate that WASP may be involved in signaling pathways that regulate
Bcl-2 expression. Many mutations entirely nullify WASP expression,
causing a clinically severe form of the disease.11,19-21
Other mutations cause only trace amounts of WASP to be stably expressed
and manifest a milder form of the disease. We suggest that WAS severity
could be determined by the variable susceptibility of different
patient's lymphocytes to spontaneous apoptosis, which, in turn, could
be determined by variable levels of Bcl-2 expression. If Bcl-2
expression is regulated by WASP-dependent signaling pathways, as we
have proposed, then insufficient WASP activity could cause a reduction
in the expression of Bcl-2 in WAS lymphocytes and thereby increase
their sensitivity to apoptogenic signals. It will be of interest to
examine the possible relationship between WASP insufficiency and the
expression level of Bcl-2 using a larger number of WAS patients in
future studies.
The function of WASP is not currently known; however, it has been
suggested that, because WASP lacks catalytic activity, the protein may
act as a molecular scaffold28 to coordinate the interactions of other signaling proteins that control the dynamic organization of the actin cytoskeleton. In support of this proposal, WASP interacts with Cdc42 and Rac,27-30 small GTPases of
the Rho family that regulate the formation of filopodia and
lamellipodia, respectively.26 Emerging now is the concept
that, along with the actin cytoskeleton, the Rho family GTPases have
the ability to coordinately regulate other cellular activities, such as
cell cycle progression, activation of mitogen-activated protein (MAP) kinase signaling cascades, and cell survival.26,56 Several studies have demonstrated that Rho family GTPase activity is essential, not only to actin cytoskeleton dynamics, but also to cell
survival.57-60 We suggest then that WASP might function as
a scaffold for Cdc42- or Rac-dependent effector complexes that
coordinately regulate actin cytoskeleton reorganization and cell
survival in lymphocytes. This WASP-dependent complex may facilitate the
activation of phosphoinositide 3-kinase (PI 3-kinase) by
Cdc42.61 PI 3-kinase has been shown to be involved in both
suppression of apoptosis62,63 and actin cytoskeleton
dynamics.64,65 Alternatively, Btk, a protein kinase that
regulates the expression of Bcl-XL in B
lymphocytes,66 might become activated by a Cdc42- or
Rac-dependent effector complex in a manner that requires its
interaction with WASP.34,67,68 Perhaps Itk, another Tec
kinase family member expressed in T lymphocytes and shown to interact
with WASP,33 will be found to have a role analogous to
Btk.69
If this is true, then the lack of WASP in WAS blood cells could result
in irregular signaling through Cdc42 or Rac. A WASP deficiency may
result in the random activation of other discrete pools of Rho family
GTPase, possibly by the mechanism discussed by Reif and
Cantrell.56 The nonspecific activation of different pools
of Cdc42/Rac-effector complexes could result in sustained signaling
through alternative pathways, such as the c-Jun N-terminal kinase or
stress-activated protein kinase (JNK/SAPK) MAP kinase cascades,70,71 which may lead to the induction of
apoptosis.72,73 In support of this idea, overexpression of
activated Rho family GTPases markedly increases
apoptosis.74-76 Consequently, in addition to the effect of
decreased Bcl-2 expression by WAS lymphocytes, erratic signaling
through Cdc42 or Rac, in the absence of sufficient WASP activity, could
possibly promote the onset of apoptosis.
In conclusion, this study implicates a pathogenic mechanism in WAS that
involves the accelerated apoptotic depletion of peripheral lymphocytes
and that may thereby cause immunodeficiency in WAS patients. We suggest
that the progressive immune dysfunction associated with WAS may develop
as the result of an inability of the thymus to generate new T
lymphocytes at a rate that is compensatory with their depletion in the
periphery. The deregulation of apoptosis has now been associated with
the pathogenesis of a variety of diseases.77,78 Moreover,
in those diseases associated with increased apoptosis, it is apparent
that the deficient expression of antiapoptotic regulatory factors, such
as Bcl-2 and Bcl-XL, may be responsible for the decreased
survival of affected cells. For example, in patients with X-linked
agammaglobulinemia (XLA), in which B-lymphocyte development is arrested
by mutations to the tyrosine kinase Btk,79 manifest
immunodeficiency might develop, in part, due to the attrition of
abnormally fragile pre-B lymphocytes. This was suggested by studies of
xid mice, an animal model for XLA, also carrying mutations in
Btk,80 that demonstrated that the poor survival of
peripheral B lymphocytes may be due to abnormal regulation of both
Bcl-2 and Bcl-XL.50,66 In human immunodeficiency virus
infection, CD4+ T lymphocytes undergo accelerated
destruction,81-83 possibly due to diminished expression of
Bcl-2,84,85 and immunodeficiency may then progress from
insufficient compensatory lymphopoiesis.86 Thus, together
with the results of this study, it would appear that deregulation of
Bcl-2 family members may be a common mechanism involved in the
pathogenesis of various immunodeficiencies. An analysis of larger
numbers of WAS patients, in an effort to correlate susceptibility to
spontaneous apoptosis and disease severity, will be important in
understanding the clinical course of WAS. Future studies will also
address the possible mechanisms by which WASP-dependent signaling
pathways may regulate the survival of peripheral lymphocytes.
| |
ACKNOWLEDGMENT |
The authors express our gratitude to Dr Leo Mascarenhas and Dr John
Bastian (Children's Hospital, San Diego, CA) for their assistance in
procuring blood samples from WAS patients, and Dr Hans Ochs for
providing the genotypic information. We appreciate the efforts of Earl
Leonard, our biostatistician. We thank Dr Donald Durden, Dr Jane
Fountain, and Brile Chung for the critical reading of this manuscript.
We are grateful to Flavia Thiemann and Matthieu DeClerck for their
technical assistance. We gratefully acknowledge the CHLA Research
Institute, and the Achievement Rewards for College Scientists
Foundation (Los Angeles Chapter) for the support of S.L.R., the
recipient of the John H. Richardson & Margaret Kersten Ponty Endowed
Fellowship through the Norris Comprehensive Cancer Center, University
of Southern California, Los Angeles, CA.
| |
FOOTNOTES |
Submitted January 18, 1999; accepted July 29, 1999.
Supported in part by Grants No. AI40581, HL54729, and HL54850
(Specialized Center of Research in Stem Cell Biology) from the National
Institutes of Health. S.L.R. is a fellow of the Achievement Rewards for
College Scientists Foundation and Childrens Hospital Los Angeles
Research Institute.
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 Kenneth I. Weinberg, MD, Division of
Research Immunology and Bone Marrow Transplantation, Mail Stop #62,
Childrens Hospital Los Angeles, 4650 Sunset Blvd, Los Angeles, CA
90027; e-mail: kweinberg{at}chla.usc.edu.
| |
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