In vitro activity of labdane diterpene from Alomia myriadenia (Asteraceae): immunosuppression via induction of apoptosis in monocytes

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International Immunopharmacology 3 (2003) 383 – 392

In vitro activity of labdane diterpene from Alomia myriadenia (Asteraceae): immunosuppression via induction of apoptosis in monocytes Elaine M. Souza-Fagundes a, Giovanni Gazzinelli b,c, Gleydes Gambogi Parreira d, Olindo A. Martins-Filho e, Gustavo P. Amarante-Mendes f, Rodrigo Correˆa-Oliveira b, Carlos L. Zani a,* a

Laborato´rio de Quı´mica de Produtos Naturais, Centro de Pequisas Rene´ Rachou-Fiocruz, Av. Augusto de Lima 1715 Barro Preto, Belo Horizonte 30190-002, Brazil b Laborato´rio de Imunologia Celular e Molecular, Centro de Pequisas Rene´ Rachou-Fiocruz, Brazil c Laborato´rio de Imunologia Celular e Molecular do Instituto de Ensino e Pesquisa da Santa Casa de Belo Horizonte, Brazil d Departamento de Morfologia, Instituto de Cieˆncias Biolo´gicas, UFMG, Brazil e Laborato´rio de Doencßa de Chagas, Centro de Pequisas Rene´ Rachou-Fiocruz, Brazil f Departamento de Imunologia, Instituto de Cieˆncias Biome´dicas, Universidade de Sa˜o Paulo e Instituto de Investigacßa˜o em Imunologia, Instituto do Mileˆnio, Brazil Received 16 July 2002; received in revised form 24 November 2002; accepted 16 December 2002

Abstract A screening program in Brazilian flora was carried out to detect the presence of immunosuppressive compounds by using the in vitro phytohemagglutinin A (PHA)-induced human peripheral blood mononuclear cell (PBMC) proliferation assay. In this screening, we isolated from Alomia myriadenia Schultz-Bip. ex. Baker (Asteraceae), a labdane-type diterpene named myriadenolide. Incubation of human PBMC with this compound reduced significantly the percentage of CD14+ cells, but it has no effect on the relative amount of CD3+CD4 CD8+ and CD3+CD4+CD8 T lymphocyte subpopulations. Neither viability nor proliferative competence of T lymphocytes was significantly affected by myriadenolide. The toxic effect on monocytes (CD14+ cells) may explain the inhibitory effect observed on PHA-induced lymphocyte proliferation. The cytotoxic effect of myriadenolide on monocytes was determined by measuring the percentage of hypodiploid nuclei content by propidium iodide staining, electron microscopy and simultaneous detection of CD14 and annexin V binding by flow cytometry. The results showed that myriadenolide induces a dose-dependent apoptosis in monocytes and thus explain the immunosuppressive effect observed. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Alomia myriadenia; Diterpene labdane; Phytohemagglutinin; Apoptosis; Immunosuppression; Natural products

1. Introduction * Corresponding author. Tel.: +55-31-3295-3566; fax: +55-313295-3115. E-mail address: [email protected] (C.L. Zani).

The integrity and efficiency of the immune system is important for the success of many chemo-

1567-5769/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1567-5769(02)00296-5


E.M. Souza-Fagundes et al. / International Immunopharmacology 3 (2003) 383–392

therapeutic interventions [1]. Several primary and secondary plant metabolites have been described to interfere with different immune system functions [2,3]. The cells involved in the immune response, including lymphocytes and monocytes, can be modulated by various types of agents including bacterial, fungal, plant and synthetic products [4,5]. It is known that many current immunosuppressive drugs were discovered by random screening of plant extracts [6], so our group has been involved in the identification of new immunosuppressive compounds obtained from the Brazilian flora. The extract of Alomia myriadenia was one of the most active among 300 plant extracts tested, completely inhibiting the in vitro lymphoproliferation [7]. The ethanol extract of A. myriadenia is known to contain the cytotoxic labdane-type diterpene myriadenolide (12S,16-dihydroxy-ent-labda-7,13-dien-15,16-olide) [8]. This rare diterpene, known to occur only in the Asteraceae species A. myriadenia and Acritopappus hagei [9], was shown to present significant activity against some human cancer cell lines [8]. In a lymphocyte proliferation assay, the myriadenolide completely inhibited the blastogenesis at a low micromolecular level (submitted for publication). Labdane-type diterpenes are known to occur in terrestrial plants and marine organisms. They display interesting biological activities such as antibacterial, antifungal, anti-inflammatory, anti-leishmanial, cardiotonic and cytotoxic, among other activities, showing potential tools for the development of new drugs [10]. Recent papers have described new labdanes with cytotoxic [1], anti-inflammatory [2] and immunomodulatory [3] activity, somehow corroborating this potential. We reported herein the investigation of a specific target cell within myriadenolide-treated PBMC and diterpene apoptosis induction in monocytes.We also studied the viability and proliferative activity of lymphocytes after treatment with this natural product.

from Sigma (St. Louis, USA). Percoll and Ficoll– Hypaque (Ficoll – Paque) were obtained from Pharmacia (Piscataway, NJ). RPMI-1640 and L-glutamine were obtained from Gibco (Grand Island, NY). The antibody clones, Leu-4 FITC (CD3), Leu-3a FITC (CD4), Leu-2a FITC (CD8) and Leu-M3 PE (CD14), were purchased from Becton Dickinson (San Jose, CA, USA). [3H]-Thymidine (0.56 Tbq/mmol) was obtained from New England Nuclear (USA). Heatinactivated, pooled AB sera were obtained from Flow Laboratories (Royaune, UK). DMSO, agar –agar, glutaraldehyde, sodium cacodylate, sodium citrate and uranyl acetate were purchased from Merck (Deutschland, Germany); Epon 812 were obtained from Polyscience (USA). 2.2. Myriadenolide Myriadenolide was isolated from an ethanol extract of aerial parts of A. myriadenia Schultz-Bip. ex. Baker (Asteraceae) (voucher code BHCB 42865) as previously described [8]. All spectral data (MS, IR, 1H- and 13 C-NMR) of the crystals obtained for this study (77.5 mg) were in agreement with that published for myriadenolide (Fig. 1). Myriadenolide was dissolved in DMSO, diluted in RPMI and added to the culture so as to attain the desired final concentrations. The control experiment used was performed using DMSO (0.05%).

2. Materials and methods 2.1. Materials Phytohemagglutinin A (PHA), propidium iodide and antibiotic/antimicotic solution were purchased

Fig. 1. Chemical structure of myriadenolide.

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2.3. Isolation of PBMC Peripheral blood mononuclear cells (PBMC) were prepared using the protocol previously described [11]. Briefly, PBMC were obtained from healthy adult volunteers of both sexes by centrifugation of heparinized venous blood over Ficoll – Hypaque cushion. Mononuclear cells were collected from the interphase after Ficoll separation and washed three times in RPMI-1640 before further processing. The cell suspensions were adjusted to 1.5  106 cells/ml. All cultures were carried out in RPMI-1640 medium, supplemented with 5% (v/v) heat-inactivated, pooled AB sera and 2 mM L-glutamine. An antibiotic/ antimicotic solution containing 1000 U/ml penicillin, 1000 Ag/ml streptomycin and 25 Ag/ml fungisone was added to control fungal and bacterial contamination (complete medium). 2.4. Lymphocyte –monocyte separation Macrophage-enriched fractions were obtained using the following procedure. PBMC were separated into lymphocytes and monocytes using a hypotonic Percoll density gradient (1.129 g/ml). Monocytes were collected from the upper interphase and washed three times with PBS. The monocytes were adjusted to 1.0  106 cell/ml, placed in 24-well plates (Costar 3513, Corning, NY, USA) and further enriched by a 90-min adherence to culture plates. Adherent cells were incubated with cold PBS and dislodged by careful homogenization with a pipette [12]. Lymphocytes were purified from PBMC by using the above Percoll density gradient, and the cells were collected from the inferior interphase. Then, the lymphocytes were washed three times with PBS to obtain a suspension with at least 95% purity, as assessed by flow cytometry analysis. 2.5. Immunophenotyping-flow cytometry We used the CDC-recommended two-color monoclonal antibody panel protocol [14]. PBMC (1.5  106 cells/ml) were cultured in polypropylene tubes (Falcon, 2063) in the presence of 7.5 AM myriadenolide for 72 h. They were subjected to immunotyping using the following antibody clones (Becton


Dickinson): Leu-4 FITC (CD3), Leu-3a FITC (CD4), Leu-2a FITC (CD8) and Leu-M3 PE (CD14). The isotype controls used were IgG1 conjugated to phycoerythrin and IgG2 conjugated to FITC. Data acquisition and analysis were performed in a single-laser flow cytometer (FACscan, Becton Dickinson) using the CellQuest software. Ten thousand events were counted for each determination. Laser scatter dot plots (size versus granularity) were used to identify and select the PBMC subpopulations. Bidimensional fluorescence dot plots (FL1 versus FL2) were used to identify labeled cell subpopulation after staining with fluorochrome-conjugated monoclonal antibodies. 2.6. PBMC proliferation assay and evaluation of CD14 subpopulation The in vitro cellular proliferation assay (blastogenesis) was performed as previously described [11]. Briefly, 1.5  106 PBMC/ml (1.5  105 cells per well) were treated with myriadenolide (7.5 AM) and DMSO for 18 h and washed three times with RPMI-1640 medium, then they were stimulated with 2.5 Ag/ml of PHA and incubated with complete medium in flat-bottomed microtiter plates (Costar, Tissue Culture Treated Polystyrene) for 72 h at 37 jC in a humidified atmosphere containing 5% CO2. Six hours before harvesting, 0.5 ACi [3H]-thymidine in 25 Al RPMI was added to each well. The cells were harvested by collection onto glass fiber filters with an automated cell harvester (model M.245, BRANDEL). The filters were air-dried and added to vials containing 3 ml scintillation fluid (0.26 g POPOP and 2.1 g PPO per liter toluene) for scintillation counting. The [3H]-thymidine incorporation was expressed as percent proliferation when compared to control proliferation. In parallel, treated PBMC and control as above were submitted to immunotyping using the antibody clone Leu-M3 PE (CD14), according to the procedure described on Section 2.5. 2.7. Cell viability The viability of the myriadenolide and DMSOtreated PBMC after 18 h culture was determined by staining with 2 Ag/ml propidium iodide (Sigma) for 10


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Table 1 Effect of myriadenolide on the percentage of different cell populations in human PBMCa Positive cells a

CD3 CD4a CD8 CD14a

Control (%)

Myriadenolide (%)


79.7 F 5.4 50.4 F 2.3 25.4 F 1.0 12.7 F 1.6

88.9 F 5.9 60.5 F 3.3 24.5 F 1.0 1.1 F 0.3

0.01 0.007 0.2 0.001

a PBMC were cultured for 72 h in the presence of myriadenolide at 7.5 AM. The subpopulations were labeled for the expression of T lymphocyte cell surface markers (CD3, CD4 and CD8) and monocytes (CD14). Data represent mean F S.E.M.

min, followed by flow cytometry analysis according to standard protocols [13]. 2.8. Autologous lymphocyte – monocyte co-culture A modification of the protocol described by Vries et al. [15] was used. The following experiments were ran with purified lymphocytes (1.35  105 cells) and purified autologous monocytes (1.5  104 cells): (A) untreated purified lymphocytes were mixed with untreated autologous monocytes, stimu-

lated with 2.5 Ag/ml PHA and incubated for 72 h; (B) purified lymphocytes were incubated with myriadenolide at 7.5 AM for 18 h. After medium exchange, they were transferred to a plate containing purified untreated autologous monocytes, stimulated with 2.5 Ag/ml PHA and incubated for additional 72 h; (C) in the reverse experiment, the monocytes were treated with myriadenolide for 18 h. After medium exchange, they were co-cultured with purified autologous lymphocytes, stimulated with 2.5 Ag/ml PHA and incubated for additional 72 h as above; (D) PBMC (1.5  105 cell) were treated with myriadenolide for 18 h, washed three times with RPMI-1640, stimulated with 2.5 Ag/ml PHA and incubated for additional 72 h. After these different procedures, proliferation was estimated by measuring [3H]-thymidine incorporation as described on Section 2.7. Control experiments were performed using DMSO (0.05%) in parallel. The experiments were run in triplicate and repeated five times using different blood donors. The results were expressed as percent proliferation when compared to control proliferation.

Fig. 2. Effect of myriadenolide on lymphocyte viability and CD14 expression. PBMC were treated with 7.5 AM myriadenolide for 18 h. Lymphocyte viability was measured by propidium iodide incorporation and flow cytometric analysis (A). The marker M2 corresponds to the viable lymphocytes. In (B), monocytes were labeled with anti-CD14 mAb Leu-M3 and analyzed by flow cytometry.

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2.9. DNA labeling and flow cytometry analysis In order to detect apoptotic nuclei, 2  105 monocytes were treated with myriadenolide (7.5 and 30 AM) and DMSO (0.05%) for 18 h. After incubation, the cells were resuspended in hypotonic solution (50 Ag/ml PI in 0.1% sodium citrate plus 0.1% Triton X100) [16]. The samples were incubated 4 h at 4 jC, and PI fluorescence of individual nuclei was measured using a FACScalibur flow cytometer (Becton Dickinson Immunocytometry Systems). The data were analyzed using the Lysis software (Becton Dickinson).


each experiment were analyzed by Student’s t-test. Statistical significance was considered when p was V 0.05.

3. Results 3.1. Effects of myriadenolide on human PBMC In the previous work, the labdane-type diterpene myriadenolide (Fig. 1) was shown to completely

2.10. Transmission electron microscopy Monocytes (1.5  106 cells/ml) were cultured in polypropylene tubes (Falcon, 2063) in the presence of 30 AM myriadenolide or DMSO (0.05%) for 18 h. After culture, treated and untreated monocytes were centrifuged, and the pellets immersed in 2% agar-agar mixture. This material was fixed in 1% glutaraldehyde/0.1 M sodium cacodylate (pH 7.4) buffer, postfixed in osmium 1%/ferrocyanide 1.5% mixture [17] for an hour and embedded in Epon 812. Ultrathin sections were mounted on 300 mesh grids and poststained with aqueous saturated uranyl acetate and 2% lead citrate before being analyzed on a EM-10 Zeiss electron microscope. 2.11. Detection of annexin V staining and CD14 expression Monocytes, treated as described above, were double-labeled with PE-conjugated Leu-M3 mAb (antiCD14) and annexin V-FITC in PBS for 1 h at room temperature in the dark. PE-conjugated murine IgG mAbs of unrelated specificity were used as control. After staining, the cells were washed twice in PBS and measured by flow cytometry [12]. 2.12. Statistical analysis Each experiment with PBMC and purified cells was ran in triplicate and repeated at least five times in different days using PBMC obtained from different individuals. The results were given as mean F standard error of means (S.E.M). All the data for

Fig. 3. Effect of different myriadenolide treatments on lymphocyte proliferative response: (A) control untreated PBMC incubated for 18 h, washed with RPMI and then stimulated with 2.5 Ag/ml PHA for 72 h; (B) PBMC treated with myriadenolide at 7.5 AM for 18 h, washed and then stimulated with 2.5 Ag/ml PHA for 72 h; (C) untreated purified lymphocytes mixed with untreated autologous purified monocytes, stimulated with 2.5 Ag/ml PHA and incubated for 72 h; (D) purified lymphocytes treated with myriadenolide at 7.5 AM. After 18 h they were washed with RPMI, mixed with purified untreated autologous monocytes and stimulated with PHA for 72 h; (E) monocytes treated with myriadenolide for 18 h, washed and cocultured with purified untreated autologous lymphocytes as above. Cell proliferation was determined by [3H]-thymidine incorporation. Statistical difference between (A) and (B) ( p = 0.03, n = 5) and (C) and (E) ( p < 0.05, n = 6) are shown.


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inhibit cellular proliferation (blastogenesis) at 7.5 AM when tested in a lymphocyte proliferation PHAinduced, 72-h assay. In this condition, the lymphocyte viability was not altered (submitted for publication). In view of this significant activity, we investigated here in which PBMC subpopulation was the target of this natural product. Flow cytometry immunophenotyping analysis was done using monoclonal antibodies, according to the CDC guidelines [14]. The experiment showed that after 72 h of treatment with 7.5 AM myriadenolide, the CD8+ cells were not significantly affected, while CD3+ and CD4+ lymphocytes subpopulations showed a slight increase (Table 1). In contrast, the proportion of monocytes (CD14+ cells) in PBMC was significantly reduced from 12.7 F 1.6% to 1.1 F 0.3% after myriadenolide treatment at 7.5 AM for 72 h. In order to allow the analysis of monocytes after incubation with the labdane, we performed a kinetic experiment using a range of doses of myriadenolide and different tie of incubation. Our data demonstrated that after 18 h of treatment at 7.5 AM, the percentual of CD14+ cells was significantly, but not totally, eliminated. Moreover, the percentages of CD3+, CD4+ and CD8+ cells were not significantly altered (data not shown). The flow cytometry analysis of propidium iodide staining of myriadenolide (7.5 AM) PBMC treated for 18 h showed that lymphocytes viability did not change significantly (Fig. 2A). On the other hand, the same treatment was sufficient to reduce monocyte population in f 54% (Fig. 2B) and lymphoproliferative response to PHA in 40% (Fig. 3B) when compared with control (Fig. 3A).

3.2. Effect of myriadenolide on isolated lymphocyte and monocyte populations obtained from human PBMC To determine whether the reduction of PHAinduced lymphoproliferative response in the presence of myriadenolide was due to the toxic effect of the drug on lymphocytes or monocytes, we carried out an autologous monocyte – lymphocyte co-culture experiment, as described in Section 2. The results are summarized in Fig. 3 and are expressed as percentage proliferation compared to PBMC control without myriadenolide. Fig. 3C shows that Percoll gradient did not alter the PHA-stimulated response. In a similar way, the proliferative response was not significantly reduced when treated lymphocytes were co-cultured with untreated monocytes (Fig. 3D). In contrast, untreated lymphocytes incubated with treated monocytes (Fig. 3E) proliferated significantly less than the control (Fig. 3C) ( f 40% reduction, n = 5, p < 0.05). The level of proliferation in this situation was similar to the one observed in the unfractionated PBMC treated with myriadenolide (Fig. 3B). 3.3. Cytotoxic effect of myriadenolide on human monocytes In order to characterize the nature of cell death caused by myriadenolide, purified monocytes from human PBMC were treated with cytotoxic doses of 7.5 or 30 AM for 18 h, and the changes in DNA content was measured by flow cytometry. The

Fig. 4. Analysis of DNA content in monocytes after myriadenolide treatment. Purified monocytes were treated with myriadenolide at 7.5 (B) and 30 AM (C) for 18 h. The cells were stained with propidium iodide and analyzed by flow cytometry. The percent F S.E.M hypodiploid nucleus is indicated. Untreated control cells are represented in (A). Representative data of four independent experiments are shown (*significantly different from (A)).

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profile of PI-stained events clearly distinguished nuclei with normal diploid DNA observed in control culture (3.9 F 0.05%), from the nuclei with hypo-

Fig. 6. Effect of myriadenolide on annexin V binding and CD14 expression after incubation with myriadenolide at 30 AM for 18 h. CD14 expression and apoptosis on monocytes in treated culture were measured by PE-labeled anti-CD14 mAb Leu-M3 (5) and staining with FITC-labeled annexin V (n). In the control experiment, monocytes were cultured in medium with DMSO. Representative data of three independent experiments are shown (*significantly different from control).

diploid DNA found in treated cultures (9.8 F 2.0% and 45.5 F 3.6% for 7.5 and 30 AM, respectively). Taken together, these results suggest that myriadenolide is capable of inducing a dose-dependent apoptosis in human monocytes (Fig. 4). To confirm that cell death measured by flow cytometry corresponded to apoptosis, we analyzed the morphology of macrophages 18 h after myriadenolide treatment (30 AM) by electron microscopy. Fig. 5A shows normal, typical control monocytes, where the nucleus shows a normal heterogeneous chromatin and the cytoplasmic organelles are clearly visible. After myriadenolide treatment, monocytes depicted typical signs of apoptosis, with dense nuclear condensation, cytoplasmic vacuolization, shrinkage, loss of plasma membrane microvilli and degeneration of organelles (Fig. 5B and 5C, respectively).

Fig. 5. Electron microscopic features of monocytes treated with myriadenolide. Monocytes were cultured or not with myriadenolide at 30 AM for 18 h before being prepared for TEM. In the control population (A), nearly all monocytes remained normal concerning morphology of nucleus and cytoplasm. After myriadenolide treatment, monocytes exhibited typical signs of apoptosis with dense nuclear condensation, hypervacuolization (B) and degeneration of organelles and cytoplasm (C). Magnification  24000.


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3.4. Apoptosis induced by myriadenolide is related with decrease on CD14 expression and increase in annexin V binding on monocytes in vitro To further support the hypothesis of a proapoptotic activity of myriadenolide, purified treated (7.5 AM) and nontreated monocytes were simultaneously labeled with anti-CD14-PE antibody and annexin VFITC, and then these were analyzed by flow cytometry. In the control sample, about 90% of the monocytes were labeled with anti-CDl4. After treatment, the staining decreased significantly to 8%. On the contrary, there is a significant increase in the annexin V-positive cells from 4% on the control to 66% after incubation with myriadenolide (Fig. 6).

4. Discussion Previous investigation of A. myriadenia extract demonstrated that the labdane diterpene myriadenolide is a compound with cytotoxic activity against human tumor cells [8]. Myriadenolide is also a compound responsible for the observed antiproliferative activity of the crude extract of this plant. It abolishes the lymphocyte proliferative response of human PBMC to PHA at 7.5 mM after 72 h of contact, without alter lymphocyte viability (submitted for publication). The data presented here demonstrates that the cellular targets involved on the inhibition of PHA-induced lymphoproliferative response were the CD14+ cells. While the percentage of CD3+, CD4+ and CD8+ T lymphocytes from PBMC preparations were not altered by myriadenolide treatment after 72 h (Table 1), the number of monocytes was drastically reduced. Although our results showed that the lymphocyte numbers were apparently not affected by treatment with the myriadenolide, we questioned if these cells retained their viability. As kinetic experiment showed that only percentage of CD14+ cells were significantly reduced after myriadenolide treatment for 18 h (data not shown), the correlation between CD14+ cell reduction with T cell viability and proliferative response was evaluated using this incubation time. Flow cytometry analysis using propidium iodide staining of PBMC preparations was ran in parallel with the lymphoproliferative experiment and strongly suggested that the lymphocytes viability

did not change significantly after an 18-h treatment with 7.5 mM myriadenolide. On other hand, this treatment reduces drastically the monocyte population from 11.98% to 5.47% ( f 50% drop) and f 40% of the lymphoproliferative response (Fig. 2). We investigated whether the reduction of PHA-induced lymphoproliferative response by myriadenolide was due to a toxic effect on lymphocyte or monocyte. To evaluate if the viable lymphocytes were still able to proliferate, treated and untreated T lymphocytes were mixed with untreated and treated monocytes, respectively. The results in Fig. 3 show clearly that myriadenolide-treated lymphocytes proliferated as the control when cultured with PHA in the presence of untreated monocytes, demonstrating that myriadenolide was not cytotoxic to T lymphocytes and did not interfere with their proliferative response to PHA. On the contrary, monocytes treated with myriadenolide and co-cultured with untreated lymphocytes induced a significant reduction in lymphoproliferative response, indicating a toxic effect of myriadenolide on monocytes. Numerous reports have shown the importance of the macrophages in the context of mitogen-induced lymphocyte proliferation. The induction of T lymphocyte proliferation by mitogens involves both interactions of macrophages – lymphocytes and molecular communication that can only be achieved with viable macrophages [15,18,19]. Our results are in agreement with these reports once myriadenolide depleted the functional accessory cell population necessary for T cell proliferative responses. Therefore, this toxicity of myriadenolide towards monocytes (CD14+ cells) explains its in vitro inhibitory activity on PHAinduced lymphocyte proliferation of human PBMC. Monocytes and macrophages belong to one of the main immune cell populations that are involved in a wide range of inflammatory disorders and responses to pathogens. The multifunctional CD14 receptor has been previously described as a useful marker to detect monocytes and macrophages diagnostically [12]. Furthermore, our results demonstrated that the significant reduction of CD14+ population by myriadenolide treatment after 18 h was due to a selective and dosedependent induction of apoptosis. In fact, a significant increase in the percentage of the monocytes with hypodiploid DNA was observed after treatment with myriadenolide at 30 AM. Later, by means of trans-

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mission electron microscopy (TEM), myriadenolidetreated monocytes revealed typical signs of apoptosis, such as nuclear condensation and cytoplasmic hypervacuolization. In addition, we evaluated the relationship between CD14 expression and apoptosis induced by myriadenolide on monocytes by simultaneously measuring membrane CD14 expression and annexin V binding as a marker of early apoptosis. The results in Fig. 6 show a correlation between the increase of annexin V-positive cells and a simultaneous reduction of CD14 expression on monocytes. Heidenreich et al. [20], investigating the apoptosis induction by IL-4 in monocytes in vitro, showed an association between down-regulation of CD14 molecule and consecutive apoptosis. Schmidt et al. [12], studying apoptosis induced by glucocorticoids, and Carracedo et al. [21], evaluating the association between low CD14 expression and apoptosis, have obtained similar results. Experiments to clarify CD14 signal transduction pathways regarding regulation of monocyte apoptosis induced by myriadenolide is currently being carried out in our laboratory. There are several reports demonstrating apoptosis in monocytes induced by immunosuppressive agents and anti-inflammatory cytokines [22 –24]. In all of them, the CD14 receptor was reported as the main molecule involved in monocyte senescence regulation. Apoptosis, as evoked in monocytes by steroids or anti-inflammatory cytokines, may therefore be an important mechanism in the treatment of immunomediated diseases in addition to the well-known monocyte-deactivating effects of these mediators [12]. In this context, the selective effect of myriadenolide on monocyte population may become a useful tool to study further the role of these cells in the immune system and inflammatory processes. Furthermore, as these cells are also host infectious agents, such as HIV, and to protozoan parasites, such as Leishmania and Trypanosoma, the myriadenolide may serve as a model for developing new drugs that could alter the course of infections caused by these agents.

Acknowledgements The authors thank Fiocruz, CNPq and PRONEX for their financial support and fellowships.


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