Dexamethasone has pro-apoptotic effects on non-activated fresh peripheral blood mononuclear cells.

Abstract

Apoptosis is a physiological method of cell death commonly referred to as programmed cell death. However, non-apoptotic programmed cell death, such as autophagy and programmed necrosis, has been characterized by morphological criteria. In view of the human therapeutic use of DEX, and considering that no difference in the number and/or affinity of glucocorticoid receptors in activated and non-activated lymphocytes has been reported, we decided to evaluate the effect of DEX on fresh peripheral blood mononuclear cells (PBMC). Transmission electron microscopy showed that DEX can significantly induce apoptosis in non-activated PBMC. It was also observed by transmission electron microscopy that, independently of DEX treatment, PBMC also died by a process marked by extreme vacuolization and increase in cellular volume; these cells were erroneously classified as viable by flow cytometry using the 7-AAD assay. It is concluded that the DEX pro-apoptotic effect is not restricted to activated PBMC and, therefore, DEX-induced apoptosis could play either homeostatic (activated PBMC) or immunosuppressive (non-activated PBMC) roles.

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Dexamethasone has pro-apoptotic effects on non-activated fresh
peripheral blood mononuclear cells
Paulo Renato Rivas Totinoa, Evelyn Kety Pratt Riccioa, Suzana Corte-Realb,
Cla ´udio Tadeu Daniel-Ribeiroa, Maria de Fa ´tima Ferreira-da-Cruza,*
aDepartment of Immunology, WHO Collaborating Center for Research and Training in the Immunology of Parasitic Diseases,
Instituto Oswaldo Cruz, Fiocruz, RJ, Brazil
bDepartment of Ultrastructure and Cellular Biology, Instituto Oswaldo Cruz, Fiocruz, RJ, Brazil
Received 10 May 2005; revised 26 July 2005; accepted 7 September 2005
Abstract
Apoptosis is a physiological method of cell death commonly referred to as programmed cell death. However, non-apoptotic programmed cell
death, such as autophagy and programmed necrosis, has been characterized by morphological criteria. In view of the human therapeutic use of
DEX, and considering that no difference in the number and/or affinity of glucocorticoid receptors in activated and non-activated lymphocytes has
been reported, we decided to evaluate the effect of DEX on fresh peripheral blood mononuclear cells (PBMC). Transmission electron micros-
copy showed that DEX can significantly induce apoptosis in non-activated PBMC. It was also observed by transmission electron microscopy
that, independently of DEX treatment, PBMC also died by a process marked by extreme vacuolization and increase in cellular volume; these
cells were erroneously classified as viable by flow cytometry using the 7-AAD assay. It is concluded that the DEX pro-apoptotic effect is not
restricted to activated PBMC and, therefore, DEX-induced apoptosis could play either homeostatic (activated PBMC) or immunosuppressive
(non-activated PBMC) roles.
? 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.
Keywords: Dexamethasone; Apoptosis; Non-activated PBMC; Transmission electron microscopy
1. Introduction
Apoptosis is a physiological method of programmed cell
death associated with animal development (Kerr et al., 1972)
and physiopathological processes (Riccio et al., 2003). It is
well known that apoptosis can be induced by a variety of
agents, including dexamethasone (DEX), a synthetic glucocor-
ticoidendowed with anti-inflammatory andimmuno-
suppressive properties, which probably result from its ability
to induce apoptosis in immune cells (Schimidt et al., 2001;
Yoshimura et al., 2001). Because of these properties, DEX is
widely used in the treatment of autoimmune diseases (Ho
et al., 2001) and allergic disorders (Sun et al., 1998), as well
as in co-adjuvant drugs for cancer therapy (Herr et al., 2003)
and immunomodulation of transplant patients (Ribarac-Stepic
et al., 2001). Thus, it has been suggested that DEX can play
a homeostatic role inducing apoptosis in lymphocytes after ac-
tivation, and that this takes place through the interaction of
DEX with the glucocorticoid receptor that activates the cas-
pases by an intrinsic pathway (Brunetti et al., 1995).
Since the morphological characterization of apoptosis and
its differentiation from necrosis (accidental cell death) (Kerr
et al., 1972), apoptosis has been commonly referred to as pro-
grammedcelldeath.However,according tocellular
Abbreviations: DEX, dexamethasone; PBMC, peripheral blood mononu-
clear cells.
* Corresponding author. Instituto Oswaldo Cruz, Fiocruz, Pavilha ˜o Leo ˆnidas
Deane, Laborato ´rio de Pesquisas em Mala ´ria. Av. Brasil, 4365, Manguinhos.
Rio de Janeiro, RJ, Brasil, CEP: 21045-900. Tel.: C55 21 3865 8185; fax:
C55 21 3865 8145.
E-mail address: mffcruz@ioc.fiocruz.br (M.F. Ferreira-da-Cruz).
1065-6995/$ - see front matter ? 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.cellbi.2005.09.002
Cell Biology International 30 (2006) 133e137
www.elsevier.com/locate/cellbi

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morphology, three types of programmed cell death have been
described (Clarke, 1990): (i) apoptotic cell death, mainly
marked by nuclear condensation and shrinkage of the cellular
volume; (ii) autophagic cell death, characterized by numerous
autophagic vacuoles; and (iii) cytoplasmic cell death (non-ly-
sosomal vesiculate degradation), identified by dilation of organ-
elles and disintegration of cytoplasm. Besides morphological
criteria, the use of apoptosis inhibitors has shown that
programmed cell death can also occur through a necrotic
and caspase-independent pathway. For instance, an alternative
non-apoptotic programmed cell death showing a necrotic-like
morphology marked by cytoplasmic vacuolization has been
detected by the inhibition of the caspase activation cascade e
one of the most important biochemical pathways of apoptotic
death, clearly demonstrating a cellular intrinsic pathway dis-
tinct from that of apoptosis (Xiang et al., 1996; Deas et al.,
1998; Chautan et al., 1999).
Flow cytometry, fluorescent microscopy and gel electro-
phoresis have been used to identify apoptotic processes. How-
ever, these methodologies have some limitations as DNA
fragmentation, loss of mitochondrial transmembrane potential
and poly (ADP-ribose) polymerase cleavage have also been re-
corded in non-apoptotic cell death (Duriez and Shah, 1997;
Lecoeur et al., 2001; Van Cruchten and Van Den Broeck,
2002). Even using 7-aminoactinomycin D (7-AAD) or annexin
V/propidium iodide, classical apoptosis detection dyes, it is
not possible to distinguish apoptosis from oncosisdthe early
stage of classical necrosis (Lecoeur et al., 2001; Lecoeur
et al., 2002).
In view of the importance of DEX administration in the
treatment of several human immunological diseases, and
considering that no difference in the number and/or affinity
of glucocorticoid receptors in activated and non-activated lym-
phocytes has been reported (Brunetti et al., 1995), we decided
to evaluate the effect of DEX treatment in non-activated
PBMC using transmission electron microscopydthe gold
standard for identification of cell morphology.
2. Materials and methods
Venous blood was collected from ten healthy volunteers from the Labora-
tory of Malaria Research, Instituto Oswaldo Cruz. PBMC were isolated from
heparinized whole blood by Ficoll-Hypaque density gradient centrifugation.
The cells were then washed twice in RPMI-1640 medium (Sigma) containing
15 mM glutamine (Sigma), 10 mM Hepes, 200 U/ml penicillin (Sigma),
200 mg/ml streptomycin (Sigma), 3 mg/ml gentamycin (Sigma), and 2 g/l so-
dium bicarbonate (Grupo Quı ´mica). PBMC were resuspended in RPMI medium
supplemented with 10% inactivated fetal calf serum (W.L. Imunoquı ´mica) and
adjusted to contain 3 ! 106cells/ml. PBMC were cultured in 24-well culture
plates (Falcon) at 37?C in 5% CO2for 24 or 48 h in a final volume of 1 ml of
cellular suspension (3 ! 106cells/ml) plus 1 ml of culture medium. Cells
from ten volunteers were cultured in the presence (five individuals) or absence
(five individuals) of dexamethasone (1 mM) (Hipolabor).
The morphology and quantification of each cellular process were assessed
by transmission electron microscopy. The cultured PBMC were washed in
PBS and fixed with 2.5% glutaraldehyde in 0.1 M Na-cacodylate buffer for
1 h at 4?C, washed three times in the same buffer and fixed with 1% osmium
tetroxide in 0.1 M cacodylate buffer for 1 h at 4?C. The cells were then
washed in buffer, dehydrated in graded acetone, and embedded in Epon. Ultra-
thin sections were cut using a Reichert ultramicrotome OmU3, collected on
copper grids, stained with aqueous uranyl acetate and lead citrate and observed
in an EM 10C Zeiss transmission electron microscope. For the quantification
of cellular processes, at least 300 cells were scored in each sample.
The data were analyzed using the program GrapPad Instat version 2.05a.
Statistical differences were determined by the ManneWhitney test.
p ! 0.05 was considered significant.
3. Results
To evaluate the effect of DEX in fresh non-stimulated
PBMC, we have assessed, by transmission electron microscopy,
cells from ten clinically healthy individuals, cultured in the
presence or absence of DEX stimulus for 24 and 48 h. Inde-
pendently of DEX treatment and time of analysis, it was pos-
sible to identify in the same sample cells at four cellular stages:
viable cells, early and late apoptotic cells and vacuolated
cells (Fig. 1AeD).
Cells classified as viable presented well-preserved cyto-
plasm, conserved organelles and nuclear wrapper integrity,
with well-individualized euchromatin and heterochromatin
(Fig. 1A). Cells displaying extreme condensation of the
chromatin, fragmented or non-fragmented nuclei, preserved
cytoplasm organelles and nuclear wrapper integrity were
considered to be in early apoptosis (Fig. 1B). Cells in late ap-
optosis presented similar condensation of the chromatin, but
without organelles and nuclear wrapper (Fig. 1C). Vacuolated
cells showed normal nuclear morphology (in spite of its trans-
location to the cellular periphery due to extreme cytoplasmic
vacuolization), absence of cytoplasmic organelles, increase
in cellular volume and a great number of cytoplasmic vacuoles
(Fig. 1D). In these cells, the vacuoles usually displayed auto-
phagosome morphology with double membrane and cellular
contents (Fig. 1E) and their formation through endoplasmic re-
ticulum could be observed close to the nucleus (Fig. 1F).
After the identification, PBMC were then quantified and
separated into three groupsdviable, apoptotic and vacuolated
cellsdas summarized in Table 1. In this manner it was possi-
ble to distinguish an increase in the frequency of apoptotic
cells in thesamples cultured
(p ! 0.01) and 48 h (p ! 0.007) (Fig. 2). Independently of
the presence of DEX, vacuolated cells were also observed
and the number of vacuolated cells was somewhat increased
in samples cultured without DEX, but this difference was
not statistically significant (p O 0.09) (Fig. 2). Interestingly,
in spite of the period of culture and DEX treatment, both the
total number of PBMC that died (apoptosis plus vacuolated
cells) and the total number of viable PBMC were similar in
24 or 48 h cultures (p O 0.5) and the predominant type of
cell death was by the vacuolization process.
withDEXafter 24 h
4. Discussion
Independent of DEX treatment, we observed a process of
cell death characterized by extreme vacuolization and increase
of cellular volume. This non-apoptotic death probably results
from a lack of survival stimuli, since programmed necrosis
and autophagy have also been induced by growth factor
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deprivation (Sperandio et al., 2000; Lum et al., 2005). However,
our results showed that DEX treatment could also rescue cul-
tured PBMC from the vacuolation process through its apopto-
sis-inducing effect, since the total numbers of dead and viable
PBMC were similar.
Programmed necrotic death, previously designated as an in
vitro phenomenon and observed under circumstances in which
caspase activity was experimentally inhibited, has recently
been reported in physiological conditions, such as viral
infection and DNA damage in growing cells (Edinger and
Thompson, 2004). Although programmed necrosis and au-
tophagy could be triggered by common inducers, autophagic
PCD in mammalian cells can represent a survival strategy in
cells under environmental changes (Edinger and Thompson,
2004). In fact, an autophagic process has been reported after
interleukin-3 (IL-3) withdrawal in IL-3-dependent bone mar-
row cells lacking the apoptosis pathway (Lum et al., 2005).
In our study cells showing a phenotype compatible to the
Fig. 1. Electron micrographs of cellular processes observed in non-activated PBMC treated (or not) with DEX-stimulus for 24 or 48 h. (A) Viable cell (!5600),
(B) early apoptotic cell (!7500), (C) late apoptotic cell (!8860) and (D) vacuolated cell (!6100). Arrowheads show autophagosomes with double membrane and
cellular contents in (E) (!32,500) and their formation in (F) (!24,700).
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autophagic oneddouble membrane vacuoles containing rec-
ognizable cellular contents by electron microscopydwere
classified as viable using flow cytometric analysis with 7-
AAD (data not shown). In view of these facts, together with
the absence of classical necrotic death patterns (such as kar-
yolysis and cellular membrane permeability), we suggest
that the vacuolization-induced process observed in cultured
PBMC may have occurred by an autophagic process.
Lymphocytes as well as PBMC that seem to be largely in-
sensitive to DEX induction of apoptosis are able to undergo
apoptosis only when activated before DEX treatment. In the
same way, prednisolone and methylprednisolone glucocorti-
coids can only induce apoptosis in mitogen-activated PBMC,
suggesting that apoptosis could operate after cell activation
as a homeostatic regulatory mechanism (Brunetti et al.,
1995; Horigome et al., 1997). In fact, an increase in the apo-
ptosis susceptibility of lymphocytes from systemic lupus er-
ythematosus patients after DEX treatment has already been
reported (Ho et al., 2001). Here we demonstrate for the first
time that DEX stimulus can induce apoptosis in non-activated
PBMC. Although we have not phenotyped which cell
population was involved, the use of electron microscopy
enabled us to demonstrate that both lymphocytes and
monocytesdthemainimmuneeffectorcellsdwereundergoing
apoptosis.IfthePBMCpopulationsthatunderwentapoptosisin
vitro have not been previously activated in vivo, we conclude
that DEX can induce apoptosis in both activated and non-
activated PBMC. Taking into account the fact that DEX is
also able to induce apoptosis in non-activated PBMC, its thera-
peutic use could also generate an adverse biological effect by
causing death in non-activated cells, leading, therefore, to an
immunosuppressive pathological process when a homeostatic
therapeutic function is required.
In summary, we have demonstrated that DEX can have
a pro-apoptotic effect in non-activated PBMC, an effect that,
until now, seemed to be restricted to activated cells. We pro-
pose that, independently of the DEX stimulus, non-activated
PBMC die in culture, mainly by a programmed cell death pro-
cess similar to autophagy. Moreover, the autophagic-like pro-
grammed cell death can only be identified by ultrastructural
analysis, showing the limitations of 7-ADD analysis when
a precise identification of cell death type is needed.
Acknowledgements
This work was supported by Instituto Oswaldo Cruz, Fioc-
ruz and Conselho Nacional de Desenvolvimento Cientı ´fico e
Tecnolo ´gico, CNPq.
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ViableApoptotic Vacuolated Total death
DEXC
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
65/57
68/57
69/53
66/56
63/55
9/15
11/16
10/13
12/25
6/11
26/28
21/27
21/34
22/19
31/34
35/43
32/43
31/47
34/44
37/45
DEX?
Sample 6
Sample 7
Sample 8
Sample 9
Sample 10
63/60
75/59
65/68
51/40
67/54
4/4
4/5
2/7
8/4
2/6
33/36
21/36
33/25
41/56
31/40
37/40
25/41
35/32
49/60
33/46
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