Cell Biology International (2006) 30, 114-121 - Jin Xu and others - Involvement of caspase-3 pathway in anti-apoptotic action of methionine enkephalin on CEMx174 cells in prolonged infection with simian immunodeficiency virus in vitro

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Cell Biology International (2006) 30, 114–121 (Printed in Great Britain)

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Involvement of caspase-3 pathway in anti-apoptotic action of methionine enkephalin on CEM×174 cells in prolonged infection with simian immunodeficiency virus in vitro

Jin Xu^a, Shuqin Xin^b, Hui Li^a, Lin Liu^b, Weiyi Xia^a, Pingfeng Li^a, Xinhua Liu^a and Gang Li^a^*

^aDepartment of Biochemistry and Molecular Biology, Peking University Health Science Center, Beijing 100083, China
^bMedicine and Health Analytical Center, Peking University Health Science Center, Beijing 100083, China

Abstract

The roles of methionine enkephalin, as an immunomodulator, on immunodeficiency virus-induced apoptosis of lymphocytes during prolonged infection are still unclear. In the present study, we evaluated the effects of methionine enkephalin on the viability, the profile of cell cycle and apoptosis, as well as the expression of apoptosis-related genes in CEM×174 cells infected with simian immunodeficiency virus for 72h. Our data demonstrated that methionine enkephalin maintains the viability of cells during the period of prolonged infection. Following co-incubation with the virus, CEM×174 cells were arrested at S phase, with increased mortality as a result of apoptosis. Methionine enkephalin could abolish virus-induced over-expression of caspase-3. Taken together all findings, we conclude that methionine enkephalin may maintain the viability of SIV-infected cells via suppressing the expression of caspase-3, which may have clinical implications in opioid peptide therapy for AIDS.

Keywords: Methionine enkephalin, Simian immunodeficiency virus, Lymphocytes, Apoptosis, Opiate, Receptor, AIDS.

^*Corresponding author. Department of Biochemistry and Molecular Biology, Peking University Health Science Center, 38 Xueyuan Road, Haidian District, Beijing 100083, China. Tel./fax: +86 10 82802891.

1 Introduction

Acquired immunodeficiency syndrome (AIDS) is a kind of serious epidemic which can be spread by repetitive use of syringes among drug users. Thus, it has been evident that AIDS is closely linked with opioids abuse. Exogenous opioids such as cocaine and heroine could aggravate the progress of AIDS (Thorpe et al., 2004; Terra et al., 2004; Tashkin, 2004). The importance of opiate addiction in the AIDS epidemic meant that gaining a better understanding of the mechanisms of opioids-induced immunoregulation was of more than academic interest (Peterson et al., 1998). Nevertheless, previous laboratorial studies indicate that administration of endogenous opiate peptides, such as methionine enkephalin, may improve immune functions in the case of opportunistic infection. Besides rheumatoid arthritis and malaria as reported, the effects of methionine enkephalin have also been applied for clinical therapy of AIDS (Singh and Singh, 2001; Takeba et al., 2001; Specter et al., 1994). There is increasing evidence that methionine enkephalin has widespread, receptor-mediated roles as growth regulators in non-neuronal cells and tissues. It has been proved to be synthesized and secreted from adrenal medulla, and functionally bridge the immune and central nervous system. Besides indirect interactions via central nervous system, methionine enkephalin could regulate immune function by direct interactions with immune cells. Recently, the studies from several laboratories have indicated that methionine enkephalin could operate as a communication signal of the immune system (Liu et al., 2003). All of the major properties of cytokines were shared by methionine enkephalin such as production by immune cells with paracrine, autocrine, and endocrine sites of action, functional redundancy, pleiotropy and effect that were both dose- and time-dependent (Peterson et al., 1998). Furthermore, several laboratories found that methionine enkephalin might enhance T cell-mediated immune responses and natural killer-cell activity in vitro, or promote host resistance (Sharp, 2003). Injections of methionine enkephalin increased both natural killer cytotoxicity and proliferation of mitogen-stimulated B and T cells; these effects were reversed by naloxone pretreatment (Faith et al., 1984, 1987a,b; Kowalski, 1998). Iglesias observed that in mouse primary cortical neurons, DAMGO (D-Ala2, NMe-Phe4, Gly-o15-enkephalin) reduced the percentage of apoptosis after 6, 12, 24 and 48h of serum withdrawal (Iglesias et al., 2003). Hayashi found that DADLE (D-Ala2, D-Leu5-enkephalin) at femtomolar to picomolar concentrations was anti-apoptotic in PC12 cells (Hayashi et al., 2002). This pentapeptide functions to up-regulate, or enhance, immune function in the majority of donor samples.

The potential uses of methionine enkephalin on the therapy of AIDS patients have recently been documented (Li et al., 2004b; Plotnikff et al., 1997; Gabrilovac and Marotti, 2000). It has been realized that the exhaustion of CD4+ T cells was the major cause of the deficiency in the function of the AIDS patients' immune system (Ameisen and Capron, 1991; Gougeon and Montagnier, 1993). Several studies suggest that apoptosis of CD4+ T cells could significantly contribute to AIDS pathogenesis although the mechanisms of the deletion of CD4+ T cells have not been fully elucidated. Methionine enkephalin is an endogenous opioid with preference for delta-opioid receptors (DOR). Evoking the DOR expression by activated CD4+ T cells significantly suppressed the expression of HIV-1 (Sharp et al., 2001). In DOR-transfected Jurkat cells, deltorphin and SNC-80 (delta opioids) concentration-dependently inhibited the production of p24 antigen, an index of HIV-1 expression (Sharp et al., 1998). Furthermore, Chao observed that methionine enkephalin dose-dependently suppressed interleukin (IL-6)-induced upregulation of HIV-1 expression (Chao et al., 1995). Although a great deal of compelling evidence has been accumulated on the effect of methionine enkephalin in the case of HIV infection, little is known about the molecular mechanisms on action of methionine enkephalin in the alleviation of pathological progression of AIDS. On the other hand, our recent studies indicated that methionine enkephalin could maintain survival of SIV-infected cells in the early stages of viral infection by the involvement of multiple molecular pathways (Li et al., 2004b). However, the precise mechanism of methionine enkephalin in promoting the survival of SIV-infected cells remains unclear. The aim of this work was to study the effects of methionine enkephalin on the expression of apoptosis-related proteins in lymphocytes infected with simian immunodeficiency virus (SIV) and hereby to explore the molecular mechanisms of prolonged survival of lymphocytes.

2 Materials and methods

2.1 Infection of CEM×174 cells with SIVmac239

The CEM×174 cell line, a hybrid of human T and B cells, was a gift from Institute of Experimental Animals in Chinese Academy of Medical Sciences and the cells were maintained in RPMI-1640 medium (Gibco USA) supplemented with 10% fetal calf serum at 37°C in a humidified atmosphere with 5% CO2. In SIVmac239 (a gift from Guangzhou University of Chinese Traditional Medicine) infected group, CEM×174 cells, at a density of 1.0×10⁵ cells/ml in 25-cm² flasks, were treated with 1/10 volume of SIVmac239 free-cells supernatant (1×10³ TCID50/mL). During culture, SIV-treated cells were gradually syncytized and depleted from the culture. To detect the effects of methionine enkephalin on prolonged infection of cultured CEM×174 cells, all assays were performed at 72h (and 96h in MTT assay) after treatment.

2.2 Determination of viability

To test the viability of cells, MTT colorimetric assay was performed as described previously (Hao et al., 2003). Briefly, 1×10⁴ CEM×174 cells were pipetted into a well of the 96-well microplate and loaded with SIV as described above. Concurrently, this cell and virus mixture was treated with 1μmol/L of methionine enkephalin. Hereafter, methionine enkephalin was continuously administrated into culture every 12h until harvest. Cells in all groups were cultured for various period of time (0, 24, 48, 72 and 96h). For antagonistic group, naloxone (10μmol/L) was added into culture half an hour in advance of treatment of methionine enkephalin. Methionine enkephalin (1μmol/L) was used for all assays in this study, which has been proved to be an optimal dose according to the dose–response curves in our previous experiments (Li et al., 2004b). Each sample was tested in parallel in sextuple to ninefold measurement. Four hours prior to measuring the absorbed values, 20μl of the 5mg/ml MTT stock solution was added. The reaction was ended by adding 150μl of DMSO. The crystallized MTT was dissolved, and the absorbance was measured on a Universal Microplate Reader (EL×800) at 570nm wavelengths.

2.3 Cell cycle analysis

The quantitative measurement of cell cycle was performed by flow cytometry analysis of nuclear DNA contents following propidium iodide staining as previously described (Li et al., 2004b). Briefly, CEM×174 cells were cultured in serum-free medium for 12h to arrest the cell cycle. The supernatant was then replaced by fresh medium containing 10% FCS and the cells were transferred into 6-well plates (3×10⁵ cells/well). For the SIV-infection group, the medium was supplemented with 1/10 SIVmac239 cultured supernatant. Then the cells were treated with methionine enkephalin (1μmol/L) every 12h for the maintenance of concentration. Cells were harvested following rinsing and resuspending into 1ml of 1× PBS (0.15μmol/L, pH 7.4) after 72h incubation. Cell suspension was then fixed by the addition of 2.8ml 100% ethanol, followed by 18h incubation in ice. Cells were rinsed once with 1× PBS, incubated for 30min at room temperature in a solution containing RNase A (20μl, 10mg/ml), and then stained by the addition of 10mg/ml of propidium iodide (PI) at a final concentration of 50μg/ml. DNA content was finally analyzed by FACScan-420 flow cytometer (Becton-Dickinso) as described previously (Li et al., 2002). The distribution of cells in different cell cycle stages was determined according to DNA content.

2.4 Annexin V affinity assay

The bivariate (Annexin V-FITC, Bosi Co)/propidium iodide (PI) analysis was performed to determine the cell apoptosis under the condition of treatment with SIV and methionine enkephalin. The procedure has been described previously (Hao et al., 2003). Briefly, after the cultured cells were treated with SIV, along with methionine enkephalin (1μmol/L) every 12h or/and naloxone (10μmol/L) for 72h, the cells were rinsed twice with cold PBS (free Ca²⁺ and Mg²⁺) and resuspended in 200μl of 1× binding buffer (10mg/L HEPES–NaOH, pH 7.4; 140mmol/L NaCl; 2.5mmol/L CaCl2). After the addition of 10μl of Annexin V-FITC and 5μl of PI/10⁵ cells, the cells were incubated at room temperature for 15min. The cells were analyzed on flow cytometry using CELLQuest software and phased into four regions based on the double-labeling of Annexin V and PI. The different labeling patterns in the assay identified the different cell fraction. The populations of vital cells, apoptotic cells, dead cells and damaged cells were separately shown in panels LL (lower left region, PI-negative/Annexin V-negative), LR (lower right region, PI-negative/Annexin V-positive), UR (upper right region, PI-positive/Annexin V-positive) and UL (upper left region, PI-positive/Annexin V-negative), respectively.

2.5 Immunoblotting assays

After treatment with SIV and methionine enkephalin as described above, the cell lysate (40μg) from each sample was subjected to 12% SDS-PAGE. Transfer of proteins from gels onto nitrocellulose membrane (Amersham, UK) was electrophoretically conducted in transblotting cell. Membranes were blocked by immersing in 5% nonfat milk (w/v)/PBS for 1h, and then incubated with anti-mAb (Santa Cruz, USA for bcl-2, bax and actin; Oncogene, USA for p53; Cell signaling Tech, USA for caspase-3) at room temperature for 2h. After rinsing with PBS/0.1% Tween-20, membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse (Zhongshan Boil Tech Co, Beijing) IgG secondary Ab. Immunocomplexes were visualized by incubation of the filters with the enhanced chemiluminescence (Zhongshan Boil Tech Co, Beijing) and exposure on an X-ray film. Quantitative analysis of bands was performed by an automated digitizing system (Image Master VDS) and the pix total scanned for each band was expressed as relative density.

2.6 RT-PCR assay

Reverse-transcriptase PCR (RT-PCR) was used to detect the expression of TNF-β mRNA in infected CEM×174 cells. Cells were treated with SIV and methionine enkephalin as described above and then lysed to extract the total RNA using Trizol reagents (GIBCO, USA). One microgram of total RNA was converted to cDNA by reverse transcriptase at 37°C for 1h using random primer. PCR amplification was performed using primers (5′-AAACCCTGCTGCTCACCTCATT-3′ and 5′-TGGATACACCATCTTCTGGG-3′) specific to TNF-β gene. The TNF-β specific primers should generate a 286-bp fragment. The primers used for amplification of actin, used for standardizing the results of amplification, were 5′-ATGTTTGAGACCTTCAACAC-3′ (sense) and 5′-CACGTCACACTTCATGATGG-3′ (antisense) (product 494bp). PCR amplification was performed using the following profile: denaturation at 94°C for 1min, followed by 30 cycles of denaturation at 94°C for 1min; annealing at 56°C for 45s, and primer extension at 72°C for 1min. The amplified DNA fragment was analyzed by 1.5% agarose gel electrophoresis and was visualized by ethidium bromide staining.

2.7 Statistical analyses

All the experiments were performed in triplicate and repeated at least three times. All data were expressed as x±sd and statistically analyzed by one-way ANOVA with the software SPSS 11.5.

3 Results

3.1 Effect of methionine enkephalin on the survival of CEM×174 cells infected with SIV

CEM×174 is susceptible to infection by SIVmac239 and usually used as a model of AIDS in the investigation in vitro. A typical syncytium formation in cultured cells could be observed from incubation day 2 and SIV-infected cells were gradually depleted from culture. MTT assay showed that methionine enkephalin could enhance the proliferation of normal cells and maintain the viability in infected cells (Fig. 1). The promoting effect of methionine enkephalin on the proliferative potentials of lymphocytes was observed at the incubation time points 72 and 96h. After administration of methionine enkephalin, the viabilities of normal and infected cells in the culture at 72h were slightly increased (7% and 11% compared to untreated groups, respectively). When the culture was prolonged to 96h, the total alive cells in infected group were decreased due to the viral infection. However, the methionine enkephalin treated cells still showed more survival than untreated groups. The elevated effect of methionine enkephalin on the viability of cells was more obvious in viral infected group (112%) than that in normal group (13%). The differences between methionine enkephalin-treated and untreated groups in both infected or normal groups were statistically significant (P<0.01 analyzed by ANOVA).

Fig. 1

Effects of methionine enkephalin on the viability of CEM×174 cells infected with SIVmac239 determined by MTT assay. Cells were treated with SIVmac239 and methionine enkephalin (1μmol/L) in 96-well plates for 96h as described under Section 2. The viability of cells at different time point was expressed as the absorbent value at 570nm. C: normal cells (control); CE: normal cells treated with methionine enkephalin; S: SIV-infected cells; SE: SIV-infected cells treated with methionine enkephalin. *P<0.05 and **P<0.01 vs corresponding to untreated group. The difference between normal and SIV-infected group in either treated or untreated group was statistically significant analyzed by ANOVA (#P<0.05, ##P<0.01). Data were representative of an experiment that was repeated three times presented as mean±sd of six to nine samples.

3.2 Effect of methionine enkephalin on cell cycle

The flow cytometric analysis of the cell cycle was summarized in Table 1, and showed that cells were arrested at S phase after 72h infection and the percentage of cells at G0–G1 phase was decreased in all infected groups (P<0.05) (Table 1). Compared to control, the populations of cells in methionine enkephalin-treated groups were only slightly changed in normal and infected groups at G0–G1 and S phases, respectively. The differences were not statistically significant, indicating that cells were greatly impaired at the prolonged stage of infection and failed to react with methionine enkephalin.

Table 1.

Effects of methionine enkephalin on the cell cycle of normal and SIV-infected CEM × 174 cells

Groups	G0–G1%		S%		G2–M%
	C	S	C	S	C	S
Control	67.18 ± 1.26	56.59 ± 0.45*	23.97 ± 2.12	34.49 ± 2.67*	8.85 ± 0.86	8.93 ± 2.23
E	71.1 ± 7.78	55.70 ± 1.42*	21.48 ± 5.18	36.29 ± 2.86*	7.43 ± 2.21	8.02 ± 1.44
N	65.82 ± 1.25	56.76 ± 1.50*	26.14 ± 0.77	34.06 ± 1.63*	8.05 ± 0.47	9.19 ± 0.13
E + N	65.21 ± 0.00	55.37 ± 0.21*	23.19 ± 1.81	34.37 ± 1.99*	10.54 ± 0.31	10.28 ± 1.79

3.3 Analysis of apoptosis

Annexin V/PI reagents were used widely to measure apoptosis. Fluorescence marked Annexin V can bind to the phosphatidyl serine (PS) in the outer membrane of apoptotic cells, and thus the cells can be detected through FCM. The results from Annexin V binding assay showed that more Annexin V/PI-negative vital cells were observed in all groups after an incubation period of 72h, but a lower proportion in SIV-infected cells. Compared to normal cells, the proportion of vital cells in SIV-infected group at LL panel was significantly reduced. In contrast, the population of apoptotic cells in SIV-infected group at UL and UR panels was greatly increased, indicating that more cells progress to apoptosis after viral infection (Fig. 2). ANOVA analysis showed the differences between normal and SIV-infected groups were statistically significant (P<0.05). Addition of methionine enkephalin or/and naloxone did not show demonstrable alteration on the profile of cells in any group, indicating a lack of response of cells to drugs at the late stage of infection.

Fig. 2

Effects of methionine enkephalin on the apoptosis of normal and SIV-infected CEM×174 cells. Ten thousand cells from each group was stained with PI and FITC-Annexin V and analyzed by flow cytometry. C: normal cells; CE: methionine enkephalin (1μmol/L) treated normal cells; CN: naloxone (10μmol/L) treated normal cells; CEN: methionine enkephalin (1μmol/L) plus naloxone (10μmol/L) treated cells; S: SIV-infected cells; SE: SIV-infected cells treated with methionine enkephalin (1μmol/L); SN: SIV-infected cells treated with naloxone (10μmol/L); SEN: SIV-infected cells treated with methionine enkephalin (1μmol/L) and naloxone (10μmol/L). Panels LL, LR, UR and UL represent, respectively, the populations of vital cells, apoptotic cells, dead cells and damaged cells. *P<0.05 and **P<0.01 vs corresponding to normal group analyzed by ANOVA. Data presented as mean±sd of three samples.

3.4 Analysis of apoptosis associated proteins by immunoblotting assays

As shown in Fig. 3, SIV infection could slightly elevate the expression of cellular p53. Methionine enkephalin showed a slight influence on the elevated expression. After equilibration with actin, the ratio of bax to bcl-2 of methionine enkephalin-treated normal cells was approximately decreased 10%, and that of infected cells approximately 35% at 72h infection, whereas the content of caspase-3 balanced with actin in SIV-infected cells was up-regulated. The elevated level of caspase-3 protein, but not p53, bcl-2 or bax, could be abolished by the administration of methionine enkephalin. Additionally, naloxone with methionine enkephalin had an apparent additive rather than antagonistic effect on the expression of caspase-3, indicating the involvement of the atypical opioid receptor.

Fig. 3

Effects of methionine enkephalin on the expression of p53, bcl-2, bax and caspase-3 in CEM×174 cells infected with SIV at 72h. SIV-infected CEM×174 were treated with methionine enkephalin (1μmol/L) or/and naloxone (10μmol/L) and collected at culture 72h. Cells lysates were prepared and subjected to SDS-PAGE followed by immunoblotting of proteins onto nitrocellulose. Western blotting analysis was undertaken according to the description under Section 2. Intensities of bands corresponding to actin were shown. C: normal cells; CE: methionine enkephalin (1μmol/L) treated normal cells; CN: naloxone (10μmol/L) treated normal cells; CEN: methionine enkephalin (1μmol/L) plus naloxone (10μmol/L) treated cells; S: SIV-infected cells; SE: SIV-infected cells treated with methionine enkephalin (1μmol/L); SN: SIV-infected cells treated with naloxone (10μmol/L); SEN: SIV-infected cells treated with methionine enkephalin (1μmol/L) and naloxone (10μmol/L). The immunoblots were representative of an experiment that was repeated three times.

3.5 Effects of methionine enkephalin on the expression of TNF β

At culture time 72h, the expression of TNF-β mRNA was moderately down-regulated by methionine enkephalin in the viral infected lymphocytes. The change was calculated up to 21.3% by density analysis compared to untreated group. Similar to the data in analysis of apoptosis, no obvious abolishment by naloxone on the expression of TNF-β was detectable. Interestingly, methionine enkephalin alone did not affect the expression of TNF-β in normal cells. An apparent additive of naloxone was still observed (Fig. 4).

Fig. 4

Effects of methionine enkephalin on expression of TNF β in normal and infected CEM×174 cells cultured for 72h by RT-PCR. Treatment groups: C: normal cells; CE: methionine enkephalin (1μmol/L) treated normal cells; CN: naloxone (10μmol/L) treated normal cells: CEN: methionine enkephalin (1μmol/L) plus naloxone (10μmol/L) treated normal cells; S: SIV-infected cells; SE: SIV-infected cells treated with methionine enkephalin (1μmol/L); SN: SIV-infected cells treated with naloxone (10μmol/L); SEN: SIV-infected cells treated with methionine enkephalin (1μmol/L) and naloxone (10μmol/L). The results were representative of an experiment that was repeated three times.

4 Discussion

Early in vitro functional studies demonstrated that opioid peptides could enhance the immune capacity and protect body against infections by exerting their action primarily in activating their cell membrane receptors on the surface of lymphocytes (Faith et al., 1984, 1987a,b). The observation that opioid peptides augmented cytolytic effector cell activity in vitro as well as parallel levels observed in vivo has led to studies evaluating the therapeutic potential of these naturally occurring peptides in individuals with viral infection. It has been reported that the continuous administration of methionine enkephalin to HIV-infected individuals improved some immunologic parameters including mitogen-stimulated blastogenesis, KN activity, and the percentages of T cell subset populations (Wybran and Plotnikoff, 1991). Coupled with these studies were the recent reports suggesting the reaction following the binding of opiates to its binding sites on the surface of cells was mediated in typical or atypical way, which in turn regulated downstream signaling pathways (Li et al., 2004b). However, molecular events underlying this differential regulation were not clearly understood. Although it has known that opioids affected the proliferation of cells, it was uncertain about the extent to which opioids directly affect the viability of SIV-infected lymphocytes. In the experiment of exogenous opioid alkaloid, the treatment of CEM×174 cells with morphine for 72h might result in the suppressive effect in SIV-infected group, indicating that lymphocytes impaired by virus still exhibited the sensitivity to the inhibitory role of morphine (Xu et al., 2004). Dissimilarly, continuous incubation of methionine enkephalin with SIV-infected lymphocytes could benefit the growth of the cells as expected. In agreement with previous reports, the effects of methionine enkephalin, termed as opioid growth factor, were confirmed. In the early stage of infection with SIV (<8h), decreased level of cAMP and activity of PKA, and as a result, associated with a significant decreased on phosphorylation of histone were one of the major reasons leading to alleviation of the apoptosis (Li et al., 2004b). However, a different mechanism in the prolonged viral infection stage was probably involved.

In view of the lack of data on the role of opioid peptides in the cellular behavior, particularly during prolonged infection, we inspected the influence of methionine enkephalin on the cell cycle. The results indicted that cell cycle was arrested at S phase by SIV-loading for 72h, though no obvious effect of methionine enkephalin on the profile of cell cycle was observed. Previous studies in our lab, as well as other laboratories, showed that immunodeficiency virus could induce the cells to arrest at G0–G1 phase (Li et al., 2004a; Zagon et al., 2000). Other study reported that proteins encoded by HIV and SIV vpr accessory genes might induce cell cycle arrest at the G2 phase of infected cells (Bouzar et al., 2003). The present data differed from previous data obtained in studies with special viral protein, with respect to the involvement of different mechanism. Although not statistically significant (P>0.05), a slight change in methionine enkephalin-treated normal, but not SIV-infected, cells could be observed, raising the possibility of an enervated response of cells to methionine enkephalin in the prolonged viral infection; further experiments are required to clarify this.

The measurements of apoptosis by FCM displayed that the population of vital cells in LL panel was obviously decreased and apoptotic cells as well as the fragments of dead cells were obviously increased when cells were infected with SIV for 72h. This indicates that infection of SIV could result in an accelerated progression of apoptosis. Like the results of cell cycle assay, methionine enkephalin failed to change the profile of the viability in all infected groups. We have noticed that methionine enkephalin could alleviate the apoptosis of cells in the early stage of infection, but no obvious effect was observed during the prolonged infection (Li et al., 2004b). It was therefore postulated that the impaired cells showed a lower susceptibility to the addition of methionine enkephalin.

In spite of its inability to alter the profile of cell cycle and distribution in Annexin V assay due to serious impairment by virus, methionine enkephalin had potently influenced intracellular expression of apoptosis-related proteins, leading to the relievable trend of apoptosis in the late stage of infection. Elevated level of p53 observed in this study, together with previously published data, was consistent with the fact that the over-expression of the apoptosis-related gene could be persistent in the whole process of apoptosis (Li and Kanneth, 2000). On the other hand, infection by immunodeficiency virus also often down-regulated the expression of the bcl-2 apoptosis-blocking gene in the cultured cells or AIDS patients (Rought et al., 1999; Richard et al., 2003; Regamey et al., 1999). Our present data showed that the decrement extent on the ratio of bax to bcl-2 in infected group was moderately changed compared with that in normal control. These virus-induced intracellular alterations were correlated with the accelerated cell death during the whole observation period compared to non-infected cells. Compared with the early finding on the expression of bcl-2, we postulate that anti-apoptosis effect of methionine enkephalin did not involve the bcl-2 and p53 mechanism in the late stage of infection. This observation was in agreement with data from our previous study (Xu et al., 2004). In the present study, the intracellular level of caspase-3 in CEM×174 cells infected with SIV was up-regulated, indicating the reason of accelerated death of lymphocytes. The abolishment of caspase-3 expression by the administration with methionine enkephalin into infected cells might serve to explain the increased survivability of lymphocytes. The pharmacological effects of methionine enkephalin on the abolishment of caspase-3 in SIV-infected lymphocytes have not been reported before.

Direct cytopathic effects could not explain the massive CD4+ T cell depletion in AIDS patients and several indirect mechanisms might be involved. Lymphocytes from HIV-1 individuals displayed increased levels of spontaneous apoptosis. Previous studies indicated that the progression of apoptosis might be ascribed to the enhanced level of TNF (Dianzani et al., 2003; Pascal et al., 2000). However, the difference on the decreased lever of TNF-β mRNA, based on our results, was limited. Thus, it is not convincing to account for the effect of methionine enkephalin on the alleviation of apoptosis induced by HIV was due to the depression of TNF β expression, at least not in the prolonged infection.

It was remarkable that a cooperative, rather than blocking, property of naloxone to methionine enkephalin was detectable in the present study. It was true for normal cells that the different effects of naloxone have been reported, which depends on the involvement of typical or atypical opioid receptor mechanism. For instance, the effect of morphine being unable to be abolished by naloxone has been documented (Tegeder et al., 2003). The additive effect of naloxone with opiate has also been observed in the investigation of the modulation of infection and type 1 cytokine expression parameters by opiate during in vitro co-infection with human T-cell leukemia virus type 1 and HIV-1 (Nyland et al., 2003). In the present study, naloxone was not able to reverse the effects of methionine enkephalin in SIV-infected cells, raising the possibility of a non-opioid receptor action. Moreover, the cells were seriously harmed by SIV in this study, which might also contribute to the abnormal cellular behavior. How the impaired cells respond to co-administration with methionine enkephalin and naloxone remains to be clarified.

In summary, all these findings led us therefore to conclude that caspase-3 played a key role in the apoptotic process of lymphocytes in the prolonged infection. Taking into account that the enhanced effects of methionine enkephalin on the viability of early-infected lymphocytes, we reasoned that administration of methionine enkephalin to AIDS patients in the early stage of infection should be a more valuable approach to improve the viability of impaired lymphocytes though it was also effective during prolonged infection. Accordingly, association of methionine enkephalin with other anti-virus medicines might open novel perspectives for the clinical therapy of AIDS.

Acknowledgments

This work is supported by the National Natural Science Foundation of China (No. 30060091 and 30271174) and the Foundation of National Education Ministry for graduate program (No. 20030001028).

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Received 19 July 2005/1 August 2005; accepted 22 August 2005

doi:10.1016/j.cellbi.2005.08.011

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