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Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4230-4237
Immunophenotypic Analysis of Peripheral Blood Mononuclear Cells
Undergoing In Vitro Apoptosis After Isolation From Human
Immunodeficiency Virus-Infected Children
By
Thomas W. McCloskey,
Saroj Bakshi,
Soe Than,
Parisa Arman, and
Savita Pahwa
From the North Shore University Hospital-New York University School
of Medicine, the Department of Pediatrics, the Division of
Allergy/Immunology, Manhasset, NY.
 |
ABSTRACT |
Lymphocytes of human immunodeficiency virus (HIV)-infected
individuals undergo accelerated apoptosis in vitro, but the subsets of
cells affected have not been clearly defined. This study examined the
relationship between lymphocyte phenotype and apoptotic cell death in
HIV-infected children by flow cytometry. Direct examination of the
phenotype of apoptotic lymphocytes was accomplished using a combination
of surface antigen labeling performed simultaneously with the Tdt
mediated Utp nick end-labeling (TUNEL) assay. In comparison to live cells, apoptotic lymphocytes displayed an
overrepresentation of CD45RO and HLA-DR expressing cells, while CD28
and CD95 expressing cells were underrepresented. Lymphocytes expressing
CD4, CD8, and CD38 were equally represented in apoptotic and live
populations. When percent lymphocyte apoptosis follow- ing
culture was examined independently with lymphocyte subsets in fresh
blood, apoptosis was negatively correlated with the percentage of CD4
cells, but not with specific CD4 T-cell subsets. Although not
correlated with the percentage of total CD8 cells, apoptosis was
positively correlated with specific CD8 T-cell subsets expressing
CD45RO and CD95 and negatively correlated for CD8 T cells expressing CD45RA. These results provide direct evidence that a population of
activated lymphocytes with the memory phenotype lacking the costimulatory molecule CD28 are especially prone to undergo apoptosis. The findings related to CD95 expression in fresh and apoptotic cells
implicate Fas-dependent and Fas-independent pathways of apoptosis in
HIV disease in children.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
INFECTION WITH human immunodeficiency
virus (HIV) results in accelerated lymphocyte apoptosis, identifiable
in peripheral blood mononuclear cells (PBMC) in adults1-3
and children.4-6 The mechanism of HIV-induced cell death is
still a subject of controversy and its role in disease pathogenesis
remains unclear. An unresolved issue stems from the observation in an
in vitro HIV infection system that apoptosis was limited to
productively infected cells,7 while in vivo examination of
lymphoid tissue suggested that uninfected bystander cells were
dying.8 Apoptotic cell death was recently described in both
productively infected cells and bystander cells by two different
mechanisms.9 The large percentage of lymphocytes that
undergo apoptosis in vitro in comparison to the low percentage of
productively infected cells in peripheral lymphocytes argues in favor
of bystander cell death as the major contributor to cell loss. One
potential mechanism for apoptosis induction in HIV-infected individuals
is attributable to signaling through the CD4 molecule10,11
initiated by gp120/anti-gp120-induced cross-linking.12
Another potential mechanism to explain the increased apoptosis is
chronic immune activation due to the persistent nature of this viral
infection. Lymphocyte progression to a memory phenotype has been
associated with a decrease in expression of Bcl-213,14 and
a similar phenomenon has been noted to occur in lymphocytes of
HIV-infected adults.15 In concordance with these
observations, Boudet et al16 described a subset of CD8 lymphocytes with low expression of Bcl-2 in conjunction with the activation markers CD45RO, HLA-DR, and CD38 in HIV seropositive donors.
In addition, a significant correlation of anti-CD3-induced apoptosis
with expression of CD45RO and HLA-DR has been reported in cells of
HIV-infected subjects.17
Although the above findings support the notion that in vivo activated
lymphocytes are the prime candidates for cell deletion in HIV-infected
persons, direct proof of phenotypic identity of cells undergoing
apoptosis is lacking. In this study, we used flow cytometry to directly
identify the phenotypic expression of lymphocyte subsets undergoing
apoptosis, as detected by the Tdt mediated Utp nick end-labeling
(TUNEL) assay, in a cohort of HIV-infected children. We confirmed that
both CD4 and CD8 lymphocytes undergo apoptosis, and that the dying
subset is enriched for cells expressing HLA DR and CD45RO, while the
live population is enriched for CD28 and CD95 expressing cells. These
results provide direct evidence that apoptosis, which occurs during
pediatric HIV infection, predominantly involves cellular activation
together with a loss of costimulatory signaling. Concomitant studies of
T-cell phenotypes in fresh blood in relation to apoptosis in cultured
cells implicate Fas-dependent and Fas-independent mechanisms of cell
death in this patient population.
| |
MATERIALS AND METHODS |
Study population.
This work is based on a cross-sectional study of HIV-infected children
(n = 62) during visits to North Shore University Hospital between
September 1996 and November 1997 for routine clinical testing as per
Institutional Review Board approved protocols. Median age
of the children in this study was 8 years (25th to 75th percentile 6 to
12 years; range, 2 months to 17 years) with a median absolute CD4 count
of 388 (25th to 75th percentile 67 to 869) and a median virus load of
39,095 RNA copies/mL (25th to 75th percentile 12,020 to 87,183). None
of the children in this cohort experienced an opportunistic infection
during the study period.
Thirty-five children were tested at more than one time point. The time
interval between testing varied from less than 1 month to 12 months
with a median of 4 months. Twenty-two of these 35 children had advanced
clinical and/or immunologic suppression at the time of testing,
placing them in the most severe of clinical (category C) or immune
(category 3) classifications. Three of the 35 children were not
receiving any antiretroviral medications; all others at first testing
had received several months of therapy with the longest duration being
83 months, and only four children had received less than 12 months of
treatment. The treatment assignment consisted of reverse transcriptase
inhibitor monotherapy in nine children and combination of two or more
in 23 children. None of the children was receiving protease inhibitors
at first testing, while four did at last testing.
A similar group of 27 unselected children had testing for apoptosis
done at one time point only. Fourteen of these children had severe
clinical disease or severe immunosuppression at the time of testing.
Five subjects in this group were not receiving any treatment, one was
treated with protease inhibitor, while all others were receiving
combinations of two or more reverse transcriptase inhibitors. The
duration of treatment was greater than 12 months for all but seven of
these children.
Concurrent analysis of phenotype and apoptosis in cultured PBMC.
Blood was drawn after informed consent had been obtained and PBMC were
isolated by Ficoll-Hypaque (Lymphoprep; Nycomed, Oslo, Norway) density
gradient centrifugation. Cells were cultured for 3 to 5 days based on a previous time course study3 in RPMI 1640 (Whittaker Bioproducts, Walkersville, MD), 10% fetal calf serum
(Hyclone Laboratories, Logan, UT), 2 mmol/L L-glutamine (Whittaker),
100 U/mL penicillin G, and 100 µg/mL streptomycin (Whittaker). At termination of culture, PBMC were labeled with allophycocyanin (APC)-conjugated monoclonal antibody directed against
either CD4, CD8, HLA DR, CD95, and with APC isotype
control (Chromaprobe, Mountain View, CA) or with phycoerythrin
(PE)-conjugated monoclonal antibody to either CD28, CD38,
CD45RO, and PE isotype control (Becton Dickinson, San Jose, CA).
Samples were then fixed with Permeafix reagent (Ortho, Raritan, NJ) for
40 minutes at room temperature, after which cells were incubated with
the TUNEL labeling solution as per the manufacturer's (Phoenix Flow
Systems, San Diego, CA) directions. Negative and positive control cells were prepared with each experiment. Samples were stored at 4°C in
the dark until flow cytometric analysis (Fig 1) on an Epics Elite ESP
flow cytometer (Coulter Corp, Miami, FL). For measurement of CD95
expression on apoptotic lymphocytes following short-term culture,
annexin labeling was used to detect surface phosphatidylserine. PBMC
were incubated overnight and then labeled with anti-CD95 FITC
(Immunotech, Westbrook, ME) and annexin biotin (R & D Systems, Minneapolis, MN) followed by the secondary reagent streptavidin allophycocyanin (Molecular Probes, Eugene, OR).
Immunophenotyping of fresh PBMC.
A whole blood method was used to immunophenotype fresh PBMC using a
previously described three-color panel.18 Briefly, samples were incubated with appropriate concentrations of monoclonal antibodies for 10 minutes at room temperature in the dark. Samples were lysed with
a commercially available lysing reagent (Coulter lyse) and fixed in 1%
paraformaldehyde until flow cytometric analysis. Monoclonal antibodies
labeled with fluorescein isothiocyanate (FITC)/PE/peridinin chlorophyll
protein (perCP) directed against the following
combinations of antigens were used: CD45/CD14/CD3 to optimize the
lymphocyte gate and determine purity, CD4/CD8/CD3 to quantitate helper
and suppressor T-cell subsets, HLA DR/CD28/CD8 to measure activation and costimulation markers, HLA DR/CD38/CD4 or CD8 to determine levels
of activation/maturation antigens, CD95/CD45RO/CD4 or CD8 to detect the
apoptosis-associated marker Fas and memory cells.
Determination of virus load.
Virus load was measured by determining HIV RNA levels19 by
quantitative reverse transcription-polymerase chain reaction assay
(Roche Molecular Systems, Branchburg, NJ). The lower limit of detection
for this assay was 200 HIV RNA copies/mL.
Statistical analysis.
The percentage of apoptotic cells expressing a particular marker as
compared with the percentage of live cells expressing that marker was
first checked for normality of distribution by the Kolmogorov-Smirnov
test and then compared in a paired manner using either the Student's
t-test or the Wilcoxon Signed Rank test as appropriate
(Sigmastat, Jandel Scientific, San Rafael, CA). Relationship of
apoptosis to virus load and phenotypic profile of fresh PBMC was
determined using Spearman's Correlation Coefficient.
| |
RESULTS |
Characterization of phenotype of live and apoptotic cells.
Our analysis determined whether a particular phenotype was
differentially represented in either the live or dying cell population by comparing its percentage in the viable gate with its percentage in
the apoptotic gate in a paired manner (Fig
1). Based on the fluorescence pattern, cells that were TUNEL positive
were identified as apoptotic, while those that were TUNEL negative were
designated as "live" cells. CD4 and CD8 T cells were equally
represented in the apoptotic and viable lymphocyte populations
(Fig 2). Although the data were suggestive
of an enhancement of CD38 negative cells undergoing apoptosis (median
CD38+ in the live population = 68%, median
CD38+ in the dead population = 48%, P = .07),
there was no significant difference in the percentage of live or dead
cells expressing CD38 (Fig 2). The apoptotic lymphocyte population was
significantly enriched for cells expressing HLA-DR (median
HLA-DR+ in the live population = 14%, median
HLA-DR+ in the dead population = 44%, P < .001)
and CD45RO (median CD45RO+ in the live population = 23%,
median CD45RO+ in the dead population = 34%, P = .03) as compared with the viable population (Fig 2). In addition, there
was a disproportionate representation of cells lacking CD28 in the
apoptotic population (median CD28+ in the live
population = 59%, median CD28+ in the dead population = 15%, P < .001).
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| Fig 1.
Representative histograms showing simultaneous
measurement of apoptosis and immunophenotype. Histograms shown
represent samples from four different HIV-infected children. PBMC were
labeled with anti-CD4 APC (log red fluorescence, y-axis) and then
subjected to the TUNEL procedure (log green fluorescence, x-axis) to
enumerate apoptotic cells. The percentage of cells in each region is
shown.
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| Fig 2.
Differential phenotype representation in live and
apoptotic lymphocytes from HIV-infected children. Populations of live
and apoptotic cells (as assessed by the TUNEL method) were examined for
their expression of the indicated surface antigens. Data are presented
as median with 25th and 75th percentile. Preferential selection of
certain populations is indicated (*) and was calculated by comparing
the percentage of a particular phenotype in the apoptotic population
with the percentage in the viable population by the paired Student's
t-test or the Wilcoxon Rank Sign test with P < .05 (*) considered significant.
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In an attempt to determine the role of Fas-dependent apoptosis, the
expression of CD95 was examined in the apoptotic and live populations.
The percentage of CD95 expressing cells in the live population was
significantly greater (34% v 22%, P = .001) than that
in the apoptotic population (Fig 2). To rule out the possibility that
lymphocytes may have been dying rapidly by a Fas-dependent pathway
during the duration of culture, apoptosis, as detected by annexin
labeling, and CD95 expression were simultaneously examined after
overnight culture of PBMC. These results confirmed that CD95 was
overrepresented in the live population (median CD95+ in the
live population = 43%, median CD95+ in the dead population = 27%, P = .016).
Relationship of phenotype expression in fresh cells with apoptosis.
Results of an extended three-color immunophenotyping panel performed on
fresh lymphocytes in a subset of patients were analyzed in relation to
independent analysis of spontaneous lymphocyte apoptosis measured after
3 to 5 days culture of PBMC. The percentage of CD4 cells was negatively
correlated with the percentage of lymphocytes undergoing apoptosis
(r =  .302, P = .01, n = 67, Fig 3). Percentages of total T cells or of
CD8+ cells did not show a correlation with apoptosis. Among
specific lymphocyte subsets, no particular subset of the CD4 population showed a correlation with apoptosis. However, expression of specific markers, CD95 (r = .587, P = .0001, n = 30) and CD45RO
(r = .532, P = .002, n = 30) on fresh CD8 T
cells correlated with the percentage of cells undergoing apoptosis,
whereas the expression of CD45RA on fresh CD8 lymphocytes (r = .426, P = .02, n = 29) was negatively correlated with
apoptosis (Fig 3).
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| Fig 3.
Correlation of percent apoptosis after culture and
expression of specific lymphocyte surface antigens. Apoptosis is
plotted against the percentage of CD4+ cells and the
percentage of CD95, CD45RA, and CD45RO+ cells in the CD8
subset measured in fresh lymphocytes. Spearman's test was used to
calculate the correlation coefficient.
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Relationship of apoptosis to virus load, antiretroviral therapy, age,
and disease stage.
Plasma HIV RNA determinations were performed in a subset of patients on
the same sample used to quantify apoptosis. We compared the percentage
apoptosis with virus load at one time point for each individual and
found no correlation (r = .220, P = .197, n = 36). We
next tested for a relationship between the change in virus load in
those patients assessed at multiple time points and the corresponding
change in percentage apoptosis, but were unable to detect a
correlation. There was also no correlation between the change in
percentage apoptosis and the change in percentage CD4 or absolute CD4
count. An additional analysis focused on patients with significant
changes in virus load (defined as >0.5 log), CD4 percentage (defined
as >10% CD4), or CD4 count (defined as >100 CD4 cells/µL) again
showed no correlation with change in apoptosis. We also found no
significant differences in the change in percentage apoptosis when
children were classified into broad treatment categories (one reverse
transcriptase inhibitor, two or more reverse transcriptase inhibitors,
or protease inhibitor). Because T-cell numbers and subset distribution
vary with age, a group of young children was compared with an older
group. The subset of younger children (<72 months, n = 18) exhibited
less apoptosis (median 25% v 36%, P = .046)
when compared with the older children (>120 months, n = 24). The
majority of the older children were classified with severe
immunosuppression (17 of 24 were immune category 3), while fewer of the
younger group fell into this category (7 of 18). Also of potential
interest is the observation that the three children with clinically
stable disease receiving no treatment who were assayed at two different
time points showed absolutely no change in percentage lymphocyte
apoptosis.
| |
DISCUSSION |
It is well established that accelerated lymphocyte apoptosis occurs in
HIV-infected individuals, but its role in disease pathogenesis and the
underlying mechanisms remain unclear. In this study, a direct
identification of phenotypic expression of lymphocytes undergoing
apoptosis was performed in HIV-infected children. The major finding of
this study was that the lymphocytes undergoing apoptosis consist
primarily of activated cells that lack CD28, suggesting that inability
to receive costimulatory signaling may play a major role in this
process.
The presence of activated lymphocytes expressing HLA-DR20
and a shift toward the CD45RO phenotype21,22 are
characteristic findings in HIV-infected persons. Studies in
HIV-infected children have indicated that increased lymphocyte
apoptosis is indirectly correlated with HLA-DR expression4
and constant cellular differentiation from resting naive cells to
primed memory cells during infection in these children increases the
propensity of T lymphocytes to undergo apoptosis.23 In
agreement with these findings, we observed that expression of CD45RO on
CD8 cells in fresh blood correlates with induction of apoptosis. A
direct examination of apoptotic cells indicated that they
preferentially expressed CD45RO and HLA-DR. These results conflict with
a previous report, which directly measured the phenotype of apoptotic
cells in HIV-infected adults,24 including detection of the
surface antigens HLA-DR and CD45RO, and concluded that lymphocyte cell
death was not confined to a specific subset. These conclusions were
based on analysis of a small number of adults, some of whom were in
primary infection. Potentially, differences in disease stage or age of
the subjects studied could explain these contradictory observations.
However, the current findings demonstrate that lymphocyte activation
plays a major role in apoptotic cell death during HIV infection. In fact, lack of chronic immune activation, as defined by low HLA-DR and
CD45RO expression, in HIV-infected chimpanzees is correlated with
resistance to apoptosis and absence of disease
progression.25 These findings lead to the concept that the
host response, resulting in chronic immune activation, may be the
driving force behind pathogenesis of apoptosis in HIV disease. Death of
activated lymphocytes has been suggested as a means of rapidly removing
those cells which have served their roles in the immune
response.26 Thus, this mechanism of death may represent a
normal physiologic process during resolution of an infection. However,
the chronic course of HIV infection may allow this process to occur in
the absence of viral clearance, setting up a vicious cycle, which
ultimately leads to lymphopenia, a contention supported by our
observation of a correlation between loss of CD4 cells and increased
apoptosis and by our finding that a group of older children, most of
whom had severe disease, manifested higher levels of lymphocyte cell death than a group of younger children with less severe immune suppression.
The percentage of both CD427 and CD820 cells
expressing CD38 is increased during HIV infection in adults. Expression
of CD38 on CD8 cells has been implicated as an adverse prognostic marker for disease progression.28 Increases in the
percentage of CD8 cells, which coexpress CD38, have also been
demonstrated for a group of HIV-infected children under 2 years of
age,29 as well as older infected children.30
Lymphocytes expressing CD38 in children may represent two populations:
(1) newly recruited immature lymphocytes and (2) mature, activated
lymphocytes31; thus, our finding indicates that CD38
expression per se does not specifically detect cells primed to undergo
apoptosis.
Another major feature of disease progression in HIV infection is that
of a substantial increase in T cells lacking the costimulatory molecule
CD28.32,33 The potential role of costimulation in protecting cells from undergoing apoptosis derives from the
observations that ligation of the CD28 molecule influences long-term
T-cell survival by upregulating the antiapoptotic protein
Bcl-xL34 and prevents T-cell death in response
to TCR stimulation, Fas cross-linking, or interleukin-2 (IL-2)
withdrawal.35 Furthermore, apoptosis of CD8 T lymphocytes
was shown to be related to loss of CD28 in patients with
HIV36 and herpes virus37 infections. In the
present study, we provide direct evidence that lymphocytes lacking CD28
preferentially undergo apoptosis in HIV-infected children. Although the
basis for the progressive loss of CD28 in HIV infection is unclear,
cytotoxic T-cell function has been ascribed to the CD8+
CD28- subset38; preferential apoptosis of this
subset thus may contribute to effector cell depletion during HIV
disease progression.
The finding that CD95+ cells were enriched in the live
population was unexpected. CD95 (Fas)-mediated death signals have been strongly implicated in the accelerated lymphocyte apoptosis occurring in HIV disease.39-43 Supporting this idea are the findings
that the percentage of T cells expressing CD95 is increased in
HIV-infected adults44 and children,5,6 and that
these cells are increasingly susceptible to anti-CD95-mediated
apoptosis.45 Blocking the CD95 interaction with its ligand
in vitro has been reported by some investigators to reduce HIV-mediated
lymphocyte apoptosis,39,42 others have been unable to do
so.7,46,47 Expression of CD95 alone, however, is not
sufficient for susceptibility to apoptosis, as Fas can transduce
activation signals in normal T lymphocytes,48 and cells
only become sensitive to Fas signaling after they have been primed
after repeated antigenic stimulation49-52 or after CD4
cross-linking.11,53,54 Thus, in our study, in the direct phenotypic analysis of live and apoptotic cells, the preferential expression of CD95 in live cells may be representative of lymphocytes that had not been primed for apoptosis in vivo and possibly were further protected by a rescue signal generated through CD28. The CD95
expressing cells in the apoptotic cell population most likely represent
cells that were primed for apoptosis in vivo. The association between
degree of apoptosis and percentage of CD95 expressing CD8 lymphocytes
in fresh blood supports the participation of Fas-dependent apoptosis
contributing to the death of CD8 T cells.
The presence of CD95 negative cells in the apoptotic population
suggests that Fas-independent mechanisms are also involved in
exaggerated lymphocyte apoptosis seen in HIV-infected patients. Mechanisms other than Fas/Fas ligand signaling are supported by the
observations that monocytes of HIV+ patients are deficient
in Fas ligand55 and that blocking CD95 in patient
samples47 or during in vitro infection7,46
fails to inhibit apoptosis. A recent report noted that contact of
uninfected CD4 T lymphocytes with HIV envelope glycoprotein expressing
cells56 led to death of both infected and bystander cells
not mediated by CD95. Many new death receptors have recently been
described.57,58 One candidate for the death effector
molecule in HIV disease is TRAIL, a tumor necrosis factor (TNF) family
member, which has been implicated in HIV-induced
apoptosis.59,60 However, the death effector mechanisms may
differ by disease stage, intensity of the host immune response, or the
lymphocyte subset involved; these factors merit further consideration.
Therapy-induced reduction in virus load has been shown to result in
increases in lymphocyte cell number61,62 and reductions in
virus load, including the amount of virus in lymphoid
tissue.63 Although we were unable to detect a change in
lymphocyte apoptosis due to the effect of concurrent antiretroviral
therapies, highly active therapy has been shown to reduce the
proportion of HLA-DR expressing T cells concomitant with a trend toward
normalization of CD28 expression.64 The current study was
not designed to address this issue, with pretreatment values for
apoptosis not available for this cohort and changes to combination
therapy not necessarily coinciding with cell death determinations.
However, data from our laboratory from a longitudinal study of
well-defined adult treatment groups suggest that reduced lymphocyte
apoptosis occurs subsequent to therapy-induced reduction of virus load
(S. Chavan and S. Pahwa, submitted). It is reasonable to
suggest, with highly effective combination therapies now being used to treat HIV-infected children, that dramatic changes in virus load might
be accompanied by decreases in lymphocyte apoptosis. In light of our
findings that HIV-mediated lymphocyte apoptosis appears to be
predominantly activation-induced cell death, decreased levels of
antigen may serve to decelerate this process.
| |
ACKNOWLEDGMENT |
We thank Regina Kowalski and Maria Marecki for technical assistance and
Caroline Nubel for assistance with patient information.
| |
FOOTNOTES |
Submitted May 11, 1998;
accepted July 22, 1998.
Supported by Grants No. AI28281 and DA05161 from the National
Institutes of Health.
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 Savita Pahwa, MD, North Shore University
Hospital, 350 Community Dr, Manhasset, NY 11030; e-mail:
spahwa{at}nshs.edu.
| |
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Abstract
Introduction