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Blood, Vol. 94 No. 9 (November 1), 1999:
pp. 3067-3076
Expression of CD10 by Human T Cells That Undergo Apoptosis Both In
Vitro and In Vivo
By
Giovanna Cutrona,
Nicolò Leanza,
Massimo Ulivi,
Giovanni Melioli,
Vito L. Burgio,
Giovanni Mazzarello,
Giovanni Gabutti,
Silvio Roncella, and
Manlio Ferrarini
From the Servizio di Immunologia Clinica and Servizio di Citometria
CBA, Istituto Nazionale per la Ricerca sul Cancro, IST Genoa; the
a Fondazione Andrea Cesalpino, Istituto di 1 Clinica Medica,
Università "La Sapienza," Rome; the Ia Clinica
Malattie Infettive, and the Istituto di Igiene, Università
di Genova, Genoa; the Servizio di Istologia e Anatomia Patologica,
Ospedale Sant'Andrea, La Spezia; and the Dipartimento di Oncologia,
Biologia e Genetica, Università di Genova, Genoa, Italy.
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ABSTRACT |
This study shows that human postthymic T cells express CD10 when
undergoing apoptosis, irrespective of the signal responsible for
initiating the apoptotic process. Cells from continuous T-cell lines
did not normally express CD10, but became CD10+ when
induced into apoptosis by human immunodeficiency virus (HIV) infection
and exposure to CD95 monoclonal antibody, etoposide, or staurosporin.
Inhibitors of caspases blocked apoptosis and CD10 expression. Both
CD4+ and CD8+ T cells purified from normal
peripheral blood expressed CD10 on apoptotic induction. CD10 was newly
synthesized by the apoptosing cells because its expression was
inhibited by exposure to cycloheximide and CD10 mRNA became detectable
by reverse transcription-polymerase chain reaction in T cells cultured
under conditions favoring apoptosis. To show CD10 on T cells apoptosing
in vivo, lymph node and peripheral blood T cells from
HIV+ subjects were used. These suspensions were composed
of a substantial, although variable, proportion of apoptosing T cells
that consistently expressed CD10. In contrast, CD10+ as
well as spontaneously apoptosing T cells were virtually absent in
peripheral blood from normal individuals. Collectively, these observations indicate that CD10 may represent a reliable marker for
identifying and isolating apoptosing T cells in vitro and ex vivo and
possibly suggest novel functions for surface CD10 in the apoptotic
process of lymphoid cells.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
CD10 (NEUTRAL endopeptidase peptidase
24.11 [NEP]) is a 100-kilodalton kD, type II integral
membrane protein characterized by a single hydrophobic sequence that
functions as a signal peptide and a transmembrane
region.1-4 CD10 was initially discovered on the surface of
acute lymphoblastic leukemia cells and was considered to be a
tumor-specific antigen.5 Later, it became clear that a
variety of normal cells at particular maturational or functional stages
are able to express CD10.3,4 These include cells of nonhematopoietic origin such as epithelial cells from kidney, liver,
breast, and lung, as well as fibroblasts and cells of the central
nervous system.6-13 Among the hematopoietic cells, CD10 is
expressed by immature T and B cells,14-18 by the B cells of the germinal centers of lymphoid follicles,19 by
granulocytes,8 and by the cells of a number of lymphoid
malignancies.20
Although the neutral endopeptidase activity of CD10 is well
documented,3,4 its function in the physiology of
CD10-expressing lymphoid cells is poorly understood. However, some
evidence, obtained primarily on mature B cells, suggests that CD10
expression may be related to apoptosis. For example, CD10 is found on
germinal center B cells that are particularly prone to apoptosis and
absent on other subsets of mature B cells that do not spontaneously
undergo apoptosis.21-23 In addition, Burkitt's lymphoma
(BL) cells, which readily undergo spontaneous apoptosis both in vivo
and in vitro, express abundant surface CD10.24,25
Furthermore, B cells from lymphoblastoid B-cell lines transfected with
c-myc-carrying episomes, concomitantly acquire the capacity to express
CD10 and an increased propensity to spontaneous apoptosis in
vitro.26,27 Finally, B cells induced into apoptosis by
human immunodeficiency virus (HIV) infection in vitro express
CD10.28
In the present study, we investigated whether the correlation between
apoptosis and CD10 expression noticed in B cells was also true for T
cells. Indeed, we found that mature T cells induced into apoptosis in a
variety of manners in vitro, or spontaneously undergoing apoptosis in
vivo, invariably expressed CD10. These findings indicate that CD10 may
represent a valuable marker for apoptosing T cells and suggest a number
of hypotheses for the physiological function of CD10.
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MATERIALS AND METHODS |
Patients and tissue or peripheral blood specimens.
Peripheral blood was obtained from 10 HIV-seropositive patients. Three
were A2, 3 B2, 2 B3, and 2 C3 stage according to the Centers for
Disease Control and Prevention staging
system.29 Ten normal volunteers matched for sex and age
were used as controls. Mononuclear cells (MNC) were separated by
Ficoll-Hypaque density gradients and used for surface marker
analysis.22 A fragment of a lymph node was obtained from an
HIV+ patient undergoing biopsy for a suspected lymphoma.
Subsequent histologic examination excluded the presence of malignant
tissue. The specimen was minced to a fine suspension22 and
analyzed by immunofluorescence. Informed consent for the use of tissue specimens was obtained from all patients.
Cells and cell cultures.
The H9 cell line was used for HIV infection. These CD4+ T
cells were infected with HIV according to the method of Tersmette et
al.30 T cells were purified from the peripheral blood of normal individuals by rosetting with neuraminidase-treated sheep red
blood cells.22 CD4-positive cells were purified by removing CD8- and CD16-positive cells from these suspensions with immunomagnetic beads.23 Purified CD4+ T cells were stimulated
with SEB (Staphylococcal Enterotoxin B; Sigma Aldrich, Milan, Italy)
and/or CD4 MoAb according to the method of Westendorp et
al.31 Briefly, CD4+ T cells were exposed to CD4
monoclonal antibodies (MoAb) (Leu3a; Becton Dickinson, Sunnyvale, CA)
(1 µg/106 cells) in the cold for 30 minutes. The cells
were washed, rosetted with goat anti-mouse immunoglobulin G (IgG) in
the cold for 30 minutes and subsequently incubated in culture
(5 × 105 cells/mL). SEB (50 ng/mL) was added to the
cultures after 3 hours and the cells obtained at intervals. In control
preparations an irrelevant MoAb (anti-CD19, Leu12; Becton Dickinson)
was substituted for the anti-CD4 MoAb or SEB was omitted from the
cultures. LAM was a BL cell line stabilized from an HIV
patient.32
Continuous T-cell lines were generated from peripheral blood MNC of
normal subjects. 1 × 106 MNC were stimulated with
phytohemagglutinin (PHA) (1 µg/mL; Dakopatts, Glostrup,
Denmark). Interleukin-2 (IL-2) (50 U/mL; Glaxo, Geneva, Switzerland)
was added after 24 hours, and the cells were expanded in vitro with
subsequent additions of IL-2 at the concentration of 20 U/mL. The
culture medium used throughout was RPMI 1640 (Seromed, Biochrom
KG, Berlin, Germany) supplemented with 10% fetal calf serum (FCS)
(Seromed, Biochrom KG).
Immunofluorescence.
The following MoAb were used for immunofluorescence staining: anti-CD3
(Leu-4); anti-CD4 (Leu-3a); anti-CD8 (Leu-2a); anti-CD16 (Leu-11b);
anti-CD25 (anti-IL-2 R); anti-HLA-DR (Becton Dickinson); anti-CD10
(J5) (Coulter Corp, Hialeah, FL); and anti-gp120 (NEN Life Science
Products, Boston, MA). All of these MoAb were used in indirect
immunofluorescence. The second fluorescein isothiocyanate (FITC)- or
phycoerythrin (PE)-conjugated antibodies to the appropriate murine Ig
isotype were from Southern Biotechnology (Birmingham, AL). The cells
were analyzed by flow cytometry (Becton Dickinson) and no fixative was
added to the stained cells. In the case of HIV-seropositive patients,
the flow cytometer was washed extensively with sodium hypochlorite
solution after the analyses. Triple staining was performed using the
following reagents: CD3-ECD (Coulter Corp), CD10-Pe (J5) (Coulter
Corp), and Annexin-V conjugated with FITC (1 µg/mL) (ApolertTM
Apoptosis Kit; Clontech Laboratories Inc, Palo Alto, CA) or CD10 (J5)
followed by a second step reaction antibody (anti-mouse IgG2A-PerCP;
Becton Dickinson), together with Annexin-V-FITC and propidium iodide
(PI) (Sigma Aldrich) 50 µg/mL in isotonic solution
(phosphate-buffered saline). The cells were analyzed by flow cytometry
(Epics-Elite flow cytometer; Coulter Corp). CD10-positive and -negative
cells were physically separated by cell sorting (Epics-Elite). The 2 populations were gated on the basis of CD10 expression and forward
light scatter parameter.
Reverse transcription-polymerase chain reaction (RT-PCR).
CD10 and glucose 3-phosphate dehydrogenase (GAPDH) transcripts were
detected using RT-PCR as previously reported.22 The following synthetic primers33 were used: CD10 sense:
5'-TTGTAAGCAGCCTCAGCCG-3'; CD10 antisense: 5'-TTGTCCACCTTTTCTCGGAG-3'
(94°C for 1 minute, 50°C for 1 minute, 72°C for 1 minute; 35 cycles); GAPDH sense: 5'-ACATCgCTCAgAACACCTATgg-3'; GAPDH antisense:
5'-gggTCTACATggCAACTgTgAg-3' (94°C for 1 minute, 59°C for 1 minute,
72°C for 2 minutes; 27 cycles).
After amplification, 45 µL of the PCR sample were run on a 2%
agarose gel in the presence of the appropriate molecular-weight markers. The PCR products were detected by ethidium bromide staining and the gel was photographed with T55 film (Polaroid Corp,
Cambridge, MA).
Amplification products were digested with specific restriction
endonucleases (Alu I, TIB MOLBIOL; Genoa, Italy) as
recommended by the manufacturer and the fragments were electrophoresed
on 2.5% agarose gel (Metaphor; FMC Bioproduct, Rockland, ME).
The PCR products were purified by precipitation with ethanol and
sequenced by the dideoxy-chain termination method, automatically using
fluorescent-labeled ddNTP and Taq polymerase (PE Applied Biosystem,
Foster City, CA). Data obtained were compared with the CD10 sequence
obtained from the European Molecular Biology Laboratory (EMBL) database.
Apoptosis assays.
Cells were induced into apoptosis by exposure to either CD95 MoAb
(200 ng/5 × 105 cells/mL) (FAS Immunotech, Marseille,
France), etoposide (50 µmol/L) (VEPESID; Bristol-Myers Squibb, Rome,
Italy), staurosporin (10 µmol/L) (Boehringer Mannheim, BmbH,
Mannheim, Germany), SEB and CD4 MoAb, IL-2 starvation or infection with
HIV depending on the design of the individual experiments. Apoptosis
was measured by staining with Annexin-V conjugated with FITC (1 µg/mL) (Apolert Apoptosis Kit; Clontech Laboratories Inc) or by PI
staining (Sigma Aldrich) and analyzed by flow cytometry (Becton Dickinson).
To inhibit protein synthesis during induction of apoptosis by CD95
MoAb, H9 T cells were preincubated for 2 hours with cycloheximide (50 µg/mL) (Sigma Aldrich).
To test the effect of caspase inhibitors on CD10 expression, H9 T
cells were exposed to CD95 MoAb in the presence of VAD-FMK (10 µmol/L final concentration, Apolert ICE family protease inhibitor; Clontech Laboratories Inc). The inhibition of caspases was shown by using the CPP32/Caspase 3 Colorimetric Assay Kit (Clontech Laboratories Inc).
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RESULTS |
Expression of CD10 by apoptotic H9 T cells.
H9 T cells, chronically infected with HIV, were analyzed for CD10
expression. These cultures were composed of a substantial number of
CD10+ cells (approximately 50%) and of an equivalent
proportion of apoptotic cells. Both CD10+ cells and
apoptotic cells were gated within the same cell subset (gate 1, Fig
1A) characterized by FSC and
side scatter light (SSC) profiles distinct from those of
the remaining CD10 and nonapoptotic cells (gate 2, Fig
1A). CD10-positive cells as well as apoptotic cells were not observed
in the control (non-HIV-infected) cells (Fig 1B). These experiments
suggested that cells undergoing apoptosis concomitantly expressed
surface CD10. This correlation was investigated further in the
experiments where H9 T cells were induced into apoptosis by exposure to
CD95 MoAb for 24 hours. Again, 2 cell subsets with different FSC and
SSC profiles were observed in the cell suspensions exposed to CD95
MoAb. One of these (gate 1, Fig 1C), which was composed of the majority
of the cells, was enriched in both apoptotic and CD10-positive cells. CD10-positive or apoptotic cells were virtually absent in the other
cell subset gated in 2 (Fig 1C). No CD10-positive or apoptotic cells
were seen in the control cultures exposed to an irrelevant MoAb and the
flow cytometry profile of these cells was identical to that of
untreated cells (not shown). In another set of experiments, H9 T cells
were exposed to staurosporin, etoposide, or CD95 MoAb, and both CD10
expression and apoptosis were measured after 24 hours. Again, after
these treatments, there was an accumulation of apoptotic and
CD10-positive cells in gate 1 (Fig 1D). Collectively, these experiments
show that apoptosing H9 T cells expressed CD10, irrespective of the
nature of the apoptotic signal.
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| Fig 1.
CD10 expression by H9 T cells after HIV
infection (A), exposure to CD95 MoAb (C), and staurosporin or etoposide
(D) for 24 hours. The cells induced into apoptosis by one of these
treatments were separated from nonapoptotic cells based on their FSC
and SSC. Apoptotic and CD10+ cells were observed in the
same gate (gate 1). Apoptosis was measured by PI staining of
permeabilized cells (A through D) or Annexin-V staining (D) and flow
cytometry. (D) Only the percentage of CD10+ and apoptotic
cells gated in 1 is reported. Control cells (B), incubated with medium
or with an irrelevant MoAb, were composed of a homogeneous cell
population that did not express CD10 and was not apoptotic. The inset
in A shows the flow cytometry profile for gp120 staining to document
the ongoing HIV infection of the cells in vitro.
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The above observations were checked in a number of control experiments.
First, H9 T cells were induced into apoptosis by exposure to CD95 MoAb
for 18 hours, stained with CD10 MoAb and analyzed by flow cytometry.
Both large CD10+ and large CD10 cells that
differed for SSC features (gates B and C in Fig
2A) were sorted and reanalyzed for their
SSC features and for apoptosis by PI staining. Small cells were
excluded from the analyses because they were composed of some cells
that become necrotic after an "early" apoptosis. Sorted
CD10+ cells were composed of a large number of apoptotic
cells. In contrast, in the sorted CD10 cells there were few apoptotic
cells. Second, H9 T cells were exposed to medium or CD95 MoAb for 48 hours, obtained and triple stained in suspension with PI, CD10 and
Annexin-V-FITC. Because the cells were stained in isotonic medium, PI
staining discriminated between viable and nonviable cells with damaged
membranes. The viable cells were gated and analyzed for CD10 and
Annexin-V-FITC staining. As shown in Fig 2B, 50% of the viable cells
present in the cultures exposed to CD95 MoAb double stained for
Annexin-V and CD10, whereas only 8% of these cells were double stained
in the control cultures. This finding rules out the hypothesis that
both the CD10 and Annexin-V staining was because of nonspecific binding
of the reagents by damaged (possibly necrotic) cells, which accumulate
in culture after exposure to CD95 MoAb for a prolonged period. Finally,
H9 T cells were exposed to CD95 MoAb for 24 hours in the presence of
cycloheximide. Under these conditions, apoptosis was not blocked, as
previously reported34; however, CD10 expression was
inhibited, indicating that CD10 was synthesized by the cells induced
into undergoing apoptosis.
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| Fig 2.
Control tests to ascertain that CD10 is synthesized and
expressed by apoptotic T cells. (A) Cells were exposed to CD95 MoAb for
18 hours, gated as indicated, and sorted. The sorted cells were
analyzed for SSC and FSC or apoptosis by PI staining. (B) Cells were
exposed to CD95 MoAb or medium for 48 hours and triple stained in
isotonic medium with PI, Annexin-V-FITC, and CD10 MoAb-PerCP. The cells
that excluded PI were gated and analyzed. (C) Cells were exposed to
CD95 MoAb in the presence or absence of cycloheximide (50 µg/mL) and
analyzed for CD10 expression and apoptosis by Annexin-V FITC
staining.
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Treatment of the cells with caspase inhibitors prevents CD10
expression.
The earliest event in apoptosis is the activation of caspases as
documented by the finding that the inhibition of these enzymes results
in the concomitant inhibition of their downstream events.35 Caspase blockage can be achieved with synthetic cell-permeable peptides
that are noncleavable analogs of their substrates and function as
noncompetitive inhibitors.36 To test whether inhibition of
caspases also resulted in downregulation of CD10 expression, we used
VAD-FMK.37
H9 T cells were cultured with an irrelevant MoAb, an anti-CD95 MoAb, or
an anti-CD95 MoAb in the presence of VAD-FMK at a final
concentration of 10 µmol/L. This concentration was selected based on
the results of preliminary tests. The cells were cultured for 24 or 48 hours, obtained and double stained with Annexin-V-FITC and CD10 MoAb.
As shown in Fig 3, a substantial proportion
of cells had already undergone apoptosis after exposure to CD95 MoAb after 24 hours. At 48 hours, most of the CD95 MoAb-treated cells were
apoptotic. Apoptotic cells coexpressed CD10. This coexpression was
particularly evident in the 2-color staining tests performed at 48 hours, whereas the proportion of Annexin-V-positive cells somewhat
exceeded that of CD10-positive cells at 24 hours, possibly indicating
that the expression of phosphatidyl serine residues preceded that of
CD10 (see Fig 1D). Indeed, Annexin-V binds to the phosphatidyl serine
residues, which are expressed from the early stages of
apoptosis.38,39 In the presence of VAD-FMK there were very
few CD10-positive and Annexin-V-positive cells after 24 hours of
culture and their percentage was virtually identical to that observed
in the cultures with medium alone. Interestingly, the intensity of
staining for CD10 of the few positive cells present in these 2 preparations was higher than that seen in CD95 MoAb-treated cells. This
finding does not have an apparent explanation. At 48 hours, the
proportion of cells staining for CD10 and Annexin-V in the culture
exposed to CD95 MoAb and VAD-FMK was slightly increased compared with
the 24-hour cultures. These values, although substantially lower than
those observed in the cultures exposed to the CD95 MoAb alone, may
indicate an inferior efficiency of VAD-FMK in preventing apoptosis in
cultures extended over relatively long periods. By 48 hours, a large
proportion of cells exposed to CD95 MoAb alone expressed both CD10 and
Annexin-V binding sites, whereas a small minority of cells bound
Annexin-V but were CD10-negative or only weakly positive. This
minority, however, acquired the capacity to express CD10 on prolonged
culture periods (72 hours, not shown).
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| Fig 3.
Inhibition of CD10 expression by treatment with VAD-FMK.
H9 T cells were cultured with CD95 MoAb in the presence or absence of
VAD-FMK (10 µmol/L, final concentration) for 24 or 48 hours. Control
suspensions were cultured with medium alone. At the end of the culture
period, the cells were obtained and stained with CD10 MoAb and
Annexin-V-FITC or an irrelevant MoAb and Annexin-V-FITC.
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When H9 T cells were exposed to etoposide for 48 hours in the presence
of VAD-FMK, inhibition of both CD10 and apoptosis
(Annexin-V-FITC-positive cells) was noticed (data not shown). Because
chemically induced apoptosis is initiated primarily via the activation
of executioner caspases,40,41 these observations indicate
an involvement of such caspases in CD10 expression.
Expression of CD10 by peripheral blood CD4+ T cells
after apoptosis induction.
Peripheral blood CD4+ T cells were cultured with SEB in the
presence or absence of a CD4 MoAb under cross-linking conditions. As
already observed by other investigators,31,42 T cells
exposed to SEB or CD4 MoAb alone failed to undergo apoptosis, whereas substantial apoptosis was detected in the cells exposed to the combination of the 2 reagents (Fig 4A). T
cells that underwent apoptosis also expressed CD10 (Fig 4A). In these
experiments, CD10 mRNA was also measured by RT-PCR in the T cells
exposed to the various stimuli. It was only detected in the T cells
exposed to both CD4 MoAb and SEB (Fig 4B), indicating that, after the induction of apoptosis, there was de novo CD10 synthesis. The CD10 mRNA
band from the T cells was of identical size to that observed in BL
cells that express CD10 constitutively,24,32 and also
displayed the same restriction pattern after treatment with Alu 1 esonuclease (Fig 4C). Moreover, the nucleotide sequence of CD10
cDNA amplified from both T and BL cells was identical to that obtained
from the EMBC databases (not shown).
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| Fig 4.
Expression of CD10 by CD4+ T cells induced
into apoptosis in vitro. (A) Purified CD4+ T cells were
exposed to SEB and/or CD4 MoAb under cross-linking conditions in
different combinations as indicated. The cells were obtained after 24 hours and CD10 expression and apoptosis (PI staining) were measured by
flow cytometry. (B) RT-PCR analysis of CD10 mRNA expression in
CD4+ T cells exposed to the indicated stimuli for 24 hours in vitro. Burkitt's lymphoma B cells (LAM cell line) were used
as positive control. (C) Alu I esonuclease digestion and analysis of
the RT-PCR fragments extracted from the indicated cells. These results
represent a typical experiment of the 3 performed.
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Expression of CD10 by CD8+ T-cell blasts on
induction of apoptosis.
In these experiments, we investigated whether CD8+ T cells
also expressed CD10 after the induction of apoptosis. Continuous cell
lines of normal peripheral blood T cells stimulated with PHA and
maintained in culture with IL-2 were used. These cell lines, which were
composed of both CD4+ and CD8+ cells (not
shown), were induced into apoptosis by culturing in the absence of
IL-2.43 After 48 hours of IL-2 deprivation (which represented the optimal timing for detecting apoptosis), the cells were
obtained and double stained with CD8 and CD10 MoAb or with CD8 MoAb and
Annexin-V. As shown in Fig 5, which reports
the results of a typical experiment out of the 3 performed on different
cell lines, virtually all of the CD8+ cells also expressed
CD10 in the absence of IL-2. Likewise, the large majority of
CD8+ cells were Annexin-V-positive, thus indicating that
CD8+ cells were capable of expressing CD10 on induction of
apoptosis. As expected, analogous double-staining tests showed that the
CD4-positive cells present in the same cell lines were able to express
CD10 on induction of apoptosis (not shown).
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| Fig 5.
Expression of CD10 by apoptosing CD8-positive T-cell
blasts. T-cell blasts from IL-2-dependent continuous T-cell lines were
cultured in the presence or absence of IL-2 (20 U/mL) for 48 hours. The
cells were obtained, double stained as indicated, and analyzed by flow
cytometry. The quadrants were drawn based on the analysis of negative
controls stained with an irrelevant (isotype-matched) MoAb or analyzed
in the absence of Annexin-V-FITC staining.
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Expression of CD10 by T cells that undergo apoptosis in vivo.
In these experiments, we investigated whether T cells apoptosing in
vivo had the capacity to express CD10. The depletion of T lymphocytes,
which occurs in HIV+ individuals, seems to be related to a
large extent to apoptosis.44-47 Hence, tissues and
peripheral blood from HIV+ subjects were likely to be
composed of a sufficient number of apoptosing T cells to allow an
assessment of their CD10 expression.48-51
The initial experiment was performed on cell suspensions prepared from
a lymph node of an HIV+ patient undergoing a diagnostic
biopsy. These cells were double stained for CD3, Annexin-V, CD4, or CD8
in various combinations. As shown in Fig 6, a substantial proportion of
CD3+ cells also stained for CD10 or Annexin-V. Triple
staining showed that the majority of CD3+ cells that
expressed CD10 also bound Annexin-V, thus indicating that CD10
represents a marker for apoptosing T cells in vivo. The apoptosing
(CD10+) cells were found among CD4+ and
CD8+ cells (Fig 6). From the
flow cytometry profiles shown, it is evident that the intensity of
staining for CD10 was inversely correlated with that for CD3, CD4, and
CD8, consistent with the notion that apoptosing T cells downregulate a
number of surface markers, including CD3, CD4, and
CD8.52,53
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| Fig 6.
Expression of CD10 by apoptosing T cells in vivo. Lymph
node cell suspensions from an HIV-seropositive individual were double
or triple stained as indicated and analyzed by flow cytometry.
CTR (control) indicates that the cells were exposed to an
irrelevant MoAb.
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Subsequent experiments were performed on the peripheral blood MNC from
10 HIV-seropositive patients and 10 normal controls. As summarized in
Fig 7, in normal controls the proportions of CD3+ cells
that also expressed CD10 were 5% or less (3.3 ± 0.95). Values
within the normal range were observed in only 3 of the 10 HIV+ individuals, whereas in the others there was a
substantial, although variable, proportion of T cells that expressed
CD10 (Fig 7). The CD10+ cells
belonged to both the CD4+ and CD8+ cell subsets
(Fig 7). Staining with Annexin-V showed that CD10-positive cells were
apoptosing cells because in normal controls and in the 3 HIV-seropositive patients with low percentages of circulating CD10+ T cells, the proportion of Annexin-V+
cells was also 5% or less (not shown). Moreover, double-staining tests
confirmed that CD10 was on cells that bound Annexin-V. Figure 8 shows 3 different flow cytometry profiles
from 1 normal control and 2 HIV-seropositive individuals with different
proportions of CD10+ cells. Triple-staining tests on the
cells from patient no. 10 showed that the majority of cells that
coexpressed CD10 and CD3 also stained with Annexin-V.
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| Fig 7.
Expression of CD10 by T cells from the peripheral blood
of HIV-seropositive individuals. Summary of the data obtained on the
peripheral blood T cells from 10 HIV-seropositive patients and the
average values obtained in 10 control individuals.
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| Fig 8.
Expression of CD10 by apoptosing peripheral blood T cells
from HIV-seropositive patients. Peripheral blood MNC from 1 normal
control or 2 HIV-seropositive patients were double or triple stained as
indicated in Fig 6, and analyzed by flow cytometry. The quadrants were
drawn based on the analysis of negative controls stained with an
irrelevant (isotype-matched) MoAb or in the absence of
Annexin-V-FITC.
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DISCUSSION |
The present study shows a close correlation between CD10 expression and
apoptosis of T cells. Evidence supporting this conclusion was obtained
from in vitro tests and from observations on T cells obtained from
HIV+ patients. Collectively, the experiments show that
apoptosing cells of both the CD4+ and CD8+ cell
subsets synthesize and express CD10, irrespective of the nature of the
stimuli causing apoptosis.
As alluded to above, CD10 has a neutral endopeptidase activity.
Structural studies have determined that the catalytic activity as well
as the regulatory, zinc-binding moiety of this type II membrane protein
is located in the extracellular segment near the COOH
terminus.3,4 Therefore, CD10 hydrolyzes polypeptide molecules in the fluids that surround the apoptosing cells. Given the
fact that CD10 is selectively expressed during apoptosis, its function
may be that of cleaving proteins with inflammatory or proinflammatory
activity released by the dying T cells. Hence, CD10 expression would
represent a safety device intended to limit a potential inflammatory
reaction triggered by the apoptosing cells. An alternative, and not
mutually exclusive, function of CD10 might be that of degrading
cytokines that reach the cell when the apoptotic process has already
started. Because a variety of cytokines have a protective effect on
T-cell apoptosis, CD10 expression may be seen as a means for
potentiating the cell's apoptotic ability by inhibiting the protective
signal(s). Notably, CD10 is able to hydrolyze a variety of biologically
active peptides, including growth and chemotactic
factors.54-59 The latter of the 2 mechanisms described
above might also apply to germinal center B cells, which have the
propensity to undergo apoptosis and upregulate CD10 expression on
apoptotic induction.19,21,22 Conceivably, CD10 expression
makes the process of selection in the germinal center more stringent by
increasing the threshold of cytokines required to prevent B-cell
apoptosis. In a separate study, we found that those thymocytes that
undergo apoptosis in vivo also express CD10 (Cutrona et al, manuscript
in preparation). Because processes of positive and
negative selection also occur within the thymus,60 one
could speculate that CD10 augments the stringency of selection in this
case, too. The present and previous studies suggest a strong
correlation between CD10 expression and apoptosis in both T and B
cells. Although an extrapolation of findings on lymphoid cells to other
cells is not immediate, it is possible that the correlation also exists
in other cell types.
A number of observations can be interpreted, at least in part, in light
of the involvement of CD10 in cell apoptosis. These include the finding
that inhibition of CD10 enzyme activity results in an increased
capacity of CD10+ murine B-cell progenitors to proliferate
in vitro and to reconstitute the B-cell compartment of irradiated mice
in vivo,61 as well as the observation that the
bombesin-dependent growth of the cells from small-cell lung cancers is
enhanced after inhibition of CD10 activity.62 The
expression of CD10 by the cells of certain malignant T-cell lines
growing in culture may also be related to the number of cells
undergoing apoptosis in vitro.63 Although the issue of
whether inhibition of CD10 enzyme activity also results in an impaired
apoptotic capacity of the cells will be the subject of a separate
study, preliminary data have shown that inhibition of the neutral
endopeptidase activity does not affect the intrinsic capacities of the
cells to undergo apoptosis, a finding which is in line with the concept
that CD10 expression may regulate cell apoptosis by interfering with
the negative or positive signals delivered to the cell from the environment.
The present observations confirm that apoptosing cells have the ability
to synthesize and transport into the cell membrane a new set of
molecules, some of which may serve to facilitate recognition of the
apoptotic cell by macrophages causing its subsequent elimination.64 Whether CD10 also has such recognition
function must still be clarified.
T cells from HIV+ individuals were found to express CD10
(see Figs 6, 7, and 8). Two-color flow cytometry disclosed that these CD10+ T cells bound Annexin-V, thus showing that CD10 may
represent a marker for T cells apoptosing in vivo. In line with the
results of previous studies, we found substantial variations in the
number of spontaneously apoptosing T cells in different individuals. These variations may be related to the clinical features of individual patients (ie, clinical stage and/or viral burden).48,49,51 Although this aspect was not specifically investigated in this study,
it may be worth mentioning that we have found correlations between
proportions of CD10-expressing T cells, viral burden, and clinical
stage of HIV infection in the course of preliminary tests. The method
selected to measure apoptosis may also account for some of the existing
discrepancies44-51 on the reported numbers of spontaneously
apoptosing T cells in HIV-seropositive patients.65
To be a useful marker for apoptosing T cells, CD10 has to be detected
simultaneously with another T-cell marker (eg, CD3) in double staining
tests. This may pose some difficulties as apoptosing T cells tend to
downregulate the expression of a number of surface molecules. Perhaps
for this reason, we found that flow cytometry is the most suitable
method for detecting apoptosing CD10+ T cells, possibly
owing to its great sensitivity. In contrast, we faced a number of
difficulties with immunohistochemistry techniques in tissue sections,
probably because of the impossibility of detecting minute amounts of
CD3 on apoptosing T cells (V.L.B., unpublished data, 1998).
Although there is general consensus that the in vitro apoptosis of
cells from HIV-harboring cell lines is directly related to the
infection,66,67 the mechanisms that cause the in vivo apoptosis of T cells in HIV+ individuals are more complex,
one particular feature being that the large majority of apoptosing
CD4+ or CD8+ T cells are non-HIV-infected
cells.44-48 Indeed, in these experiments we found that a
substantial fraction of T cells apoptosing in vivo was represented by
CD8+ T cells that usually do not harbor HIV (see Figs 6 and
7). Several mechanisms have been proposed to explain the apoptosis of
uninfected T cells, including surface CD4 cross-linking by gp120,
autocrine interaction of surface Fas with its ligand, and IL-2
starvation31,42,68; however, the phenomenon remains poorly
understood. CD10 expression provides a new tool for identifying and
possibly isolating apoptosing T cells ex vivo and, hence, for exploring
the problem further. Moreover, the proportion of CD10+ T
cells may possibly represent an additional parameter for evaluating HIV-related disease progression.
| |
ACKNOWLEDGMENT |
We thank M. Ulivi for revising the manuscript, Drs F. Fais and S. Zupo
for helpful discussion, and T. Tavilla for excellent assistance in the
preparation of this manuscript.
| |
FOOTNOTES |
Submitted October 6, 1998; accepted June 29, 1999.
Supported by grants from Istituto Superiore di Sanità (ISS) (AIDS
Project), Associazione Italiana per la Ricerca sul Cancro (AIRC), and
Fondazione Andrea Cesalpino (to V.L.B.); N.L. is a fellow of the
Associazione Italiana Leucemie (AIL).
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 Giovanna Cutrona, PhD,
Servizio di Immunologia Clinica, Istituto Nazionale per la Ricerca sul
cancro, IST, Largo Rosanna Benzi, n. 10, 16132 Genova GEItaly;
e-mail: gcutrona{at}hp380.ist.unige.it.
| |
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ABSTRACT
INTRODUCTION