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Cell Biology International (2006) 30, 133–137 (Printed in Great Britain)
Dexamethasone has pro-apoptotic effects on non-activated fresh peripheral blood mononuclear cells
Paulo Renato Rivas Totinoa, Evelyn Kety Pratt Riccioa, Suzana Corte‑Realb, Cláudio Tadeu Daniel‑Ribeiroa and Maria de Fátima 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


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.


Keywords: Dexamethasone, Apoptosis, Non-activated PBMC, Transmission electron microscopy.

*Corresponding author. Instituto Oswaldo Cruz, Fiocruz, Pavilhão Leônidas Deane, Laboratório de Pesquisas em Malária. Av. Brasil, 4365, Manguinhos. Rio de Janeiro, RJ, Brasil, CEP: 21045-900. Tel.: +55 21 3865 8185; fax: +55 21 3865 8145.


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 glucocorticoid endowed with anti-inflammatory and immuno-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 activation, and that this takes place through the interaction of DEX with the glucocorticoid receptor that activates the caspases 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 programmed cell death. However, according to cellular 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-lysosomal vesiculate degradation), identified by dilation of organelles 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 – one of the most important biochemical pathways of apoptotic death, clearly demonstrating a cellular intrinsic pathway distinct from that of apoptosis (Xiang et al., 1996; Deas et al., 1998; Chautan et al., 1999).

Flow cytometry, fluorescent microscopy and gel electrophoresis have been used to identify apoptotic processes. However, these methodologies have some limitations as DNA fragmentation, loss of mitochondrial transmembrane potential and poly (ADP-ribose) polymerase cleavage have also been recorded 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 oncosis—the 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 lymphocytes has been reported (Brunetti et al., 1995), we decided to evaluate the effect of DEX treatment in non-activated PBMC using transmission electron microscopy—the gold standard for identification of cell morphology.

2 Materials and methods

Venous blood was collected from ten healthy volunteers from the Laboratory 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 15mM glutamine (Sigma), 10mM Hepes, 200U/ml penicillin (Sigma), 200μg/ml streptomycin (Sigma), 3mg/ml gentamycin (Sigma), and 2g/l sodium bicarbonate (Grupo Química). PBMC were resuspended in RPMI medium supplemented with 10% inactivated fetal calf serum (W.L. Imunoquímica) and adjusted to contain 3×106 cells/ml. PBMC were cultured in 24-well culture plates (Falcon) at 37°C in 5% CO2 for 24 or 48h in a final volume of 1ml of cellular suspension (3×106 cells/ml) plus 1ml of culture medium. Cells from ten volunteers were cultured in the presence (five individuals) or absence (five individuals) of dexamethasone (1μM) (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 0.1M Na-cacodylate buffer for 1h at 4°C, washed three times in the same buffer and fixed with 1% osmium tetroxide in 0.1M cacodylate buffer for 1h at 4°C. The cells were then washed in buffer, dehydrated in graded acetone, and embedded in Epon. Ultrathin 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 Mann–Whitney 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 48h. Independently of DEX treatment and time of analysis, it was possible to identify in the same sample cells at four cellular stages: viable cells, early and late apoptotic cells and vacuolated cells (Fig. 1A–D).


Fig. 1

Electron micrographs of cellular processes observed in non-activated PBMC treated (or not) with DEX-stimulus for 24 or 48h. (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).


Cells classified as viable presented well-preserved cytoplasm, 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 apoptosis presented similar condensation of the chromatin, but without organelles and nuclear wrapper (Fig. 1C). Vacuolated cells showed normal nuclear morphology (in spite of its translocation 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 autophagosome morphology with double membrane and cellular contents (Fig. 1E) and their formation through endoplasmic reticulum could be observed close to the nucleus (Fig. 1F).

After the identification, PBMC were then quantified and separated into three groups—viable, apoptotic and vacuolated cells—as summarized in Table 1. In this manner it was possible to distinguish an increase in the frequency of apoptotic cells in the samples cultured with DEX after 24h (p<0.01) and 48h (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>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 48h cultures (p>0.5) and the predominant type of cell death was by the vacuolization process.


Table 1.

Percentage of viable, apoptotic and vacuolated PBMC from ten clinically healthy individuals cultured for 24 and 48 h in the presence (DEX+) or absence (DEX−) of dexamethasone, as assessed by transmission electron microscopy


Fig. 2

Mean PBMC death frequencies in samples from ten clinically healthy individuals cultured for 24 or 48h in the presence (DEX+) or absence (DEX−) of dexamethasone, as assessed by transmission electron microscopy. Independent of the period of culture, differences between DEX+ and DEX− were statistically significant for apoptotic processes (p<0.01/24h; p<0.007/48h), but not for vacuolated cells cultured for either 24 or 48h (p>0.09).


ViableApoptoticVacuolatedTotal death
DEX+
Sample 165/579/1526/2835/43
Sample 268/5711/1621/2732/43
Sample 369/5310/1321/3431/47
Sample 466/5612/2522/1934/44
Sample 563/556/1131/3437/45

DEX−
Sample 663/604/433/3637/40
Sample 775/594/521/3625/41
Sample 865/682/733/2535/32
Sample 951/408/441/5649/60
Sample 1067/542/631/4033/46


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 deprivation (Sperandio et al., 2000; Lum et al., 2005). However, our results showed that DEX treatment could also rescue cultured PBMC from the vacuolation process through its apoptosis-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 autophagy 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 marrow cells lacking the apoptosis pathway (Lum et al., 2005). In our study cells showing a phenotype compatible to the autophagic one—double membrane vacuoles containing recognizable cellular contents by electron microscopy—were 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 karyolysis 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 insensitive to DEX induction of apoptosis are able to undergo apoptosis only when activated before DEX treatment. In the same way, prednisolone and methylprednisolone glucocorticoids 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 apoptosis susceptibility of lymphocytes from systemic lupus erythematosus 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 monocytes—the main immune effector cells—were undergoing apoptosis. If the PBMC populations that underwent apoptosis in 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 therapeutic 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 propose that, independently of the DEX stimulus, non-activated PBMC die in culture, mainly by a programmed cell death process similar to autophagy. Moreover, the autophagic-like programmed 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, Fiocruz and Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq.

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Received 10 May 2005/26 July 2005; accepted 7 September 2005

doi:10.1016/j.cellbi.2005.09.002


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)