Beta-endorphin enhances the replication of neurotropic human immunodeficiency virus in fetal perivascular microglia

Journal of Ncuroimmunology Journal of Neuroimmunology

ELSEVIER

61 (lY95) 97-104

Beta-endorphin enhances the replication of neurotropic human immunodeficiency virus in fetal perivascular microglia Kamaraju S. Sundar a,*, Lakshmi S. Kamaraju a, James Dingfelder b, James McMahon ‘, Sastry Gollapudi d, William H. Wilson a, Ling-yuan Kong ‘, John S. Hong ‘, Jay M. Weiss f, James E. Lee a a Laboratory of Neuroimmunology,

’ Laboratory

Department of Psychiatry, Box 3552, Duke Unioersity Medical Center, Durham, NC 27710, USA b East Towne OBGYN, Chapel Hill, Chapel Hill, NC, USA ’ Eue Surgical Centers, National Boulecard, Los Angeles, CA, USA ’ Department of Medicine, University of California at Irvine, Irvine, CA, USA of Moleatlar Neuroscience, National Institute of Encironmental Health Sciences, Research Triangle Park, NC, USA ’ Department of Psychiatry, Emory University Medical School, Atlanta, GA, USA Received 17 January

1995; revised 17 May 1995; accepted

17 May 1995

Abstract The effect of an endogenous opiate, /3-endorphin, on the replication of HIV was investigated in brain perivascular microglia. Beta-endorphin enhanced the synthesis of p-24 antigen and transactivation of HIV promoter. Dialysed culture supematants of endorphin-treated microglia re-activated latent HIV infection. These culture supernatants showed elevated levels of interleukin-lfi, IL-6

and tumor necrosis factor c~. Sub-optimal concentration of /3-endorphin potentiated GP-120-induced synthesis of these cytokines. Nalaxone reversed P-endorphin-induced, but not GP-120-induced, cytokine production and enhanced HIV replication. These results suggest that endogenous opiates may contribute to the progression of AIDS dementia complex. Keywords:

AIDS; Cytokines;

Microglia;

Opiate: HIV replication

1. Introduction Several investigators reported a variety of behavioral and cognitive dysfunctions in patients with AIDS (Petit0 et al., 1985; Navia et al., 1986a,b; Price et al., 1988,199O; Price and Brew, 1988; Johnson et al., 1988), which are collectively described as AlDS dementia complex (ADC). Observations of isolation of HIV from seronegative subjects and synthesis of HIV-specific antibodies in the brain (Ho et al., 1985; Resnick et al., 1985a,b; Amadori et al., 1988; Chiodi et al., 1988) suggest that infection of the brain with HIV may occur very early in the course of AIDS and perhaps even in the absence of clinical abnormalities. In multinucleated encephalitis, seen in ADC, there appears to be a direct correlation between the concentration of HIV and severity of the disease. Further evidence for the involvement of increased HIV replication

* Corresponding

author. Phone ,:919) 684 6205; Fax (919) 684 3071

0165-5728/95/$09.50 8 1995 Elsevier Science B.V. All rights reserved SSDI 0165-5728(95)00089-2

in ADC is provided by the transient amelioration of some of these symptoms by anti-HIV treatment with azidothymidine (AZT) (Pizza et al., 1988; Schmitt et al., 1988; Yarchoan et al., 1988; Hollweg et al., 1991; Tozzi et al., 1993). In patients with AIDS, the extent of viral replication and cytokine production in HIV-infected peripheral lymphoid cells appear to depend upon co-factors, such as opportunistic infections. However, opportunistic infections in the brain, in the early stages of AIDS, are uncommon and the role of host factors in contributing to the progression of ADC are not clearly known. Exogenous opiate, morphine, enhances the replication of HIV and SIV in lymphocytes (Peterson et al., 1990,1991,1992; Chuang et al., 1993). Opiates appear to mediate these effects by inducing the synthesis of cytokines (IL-1 and IL-41 in macrophages and lymphocytes (Apte et al., 1989,199O; Heijnen et al., 1991; Van den Bergh et al., 1993). Exogenous opiate, morphine, was recently shown to induce the production of tumor necrosis factor (Y in human fetal and

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murine microglia (Chao et al., 1993; Peterson et al., 1994). In this study, the effect of P-endorphin on the synthesis of cytokines and on the replication of HIV in fetal brain-derived macrophage were examined. The results presented here demonstrate that /3-endorphin enhances the replication of HIV as well as enhances the transactivation of HIV promoter region in fetal perivascular microglia. In addition, dialysed culture supematant of these microglia which were pretreated with P-endorphin re-activated latent infection of HIV in a monocytic cell line. Beta-endorphin appears to mediate these effects by inducing the synthesis of pro-inflammatory cytokines in perivascular microglia. These effects are reversible by naloxone which is an antagonist for opiate receptors.

2. Materials and methods

2.1. Reagents Beta-endorphin was purchased from Peninsula Laboratories. ELISA kits for the detection of cytokines were obtained from Cistron Biotechnology. Ul cells, which are latently infected with HIV, recombinant, glycosylated GP120 and neurotropic HIV-l,,_r, were obtained from the AIDS Research Resource Program, Division of AIDS, NINAID, NIH. 2.2. Collection of brain tissue The protocol to collect and utilize fetal tissue has been approved by the Institutional Review Board of the Duke University Medical Center. The tissues were collected from legally terminated pregnancies (16-24 weeks of gestation). Fetal tissue collected from clinics in the Triangle area were derived from dilatation and curettage. The physician generally removes the cranium (before it passes into the collection jar) and inverts the cranial contents into the transport medium. Eve Surgical Centers in Los Angeles, CA, pioneered the intact dilatation and evacuation (IDE) in the early 1980s. The paracentesis capitis is performed to reduce traction to the mother. However, it incidentally affords an advantage to scientists interested in fetal research. The IDE provides advantages in that fetal demise is central nervous system based, as opposed to other techniques which cause cardiac asystole. This provides tissue that is being perfused right until the moment of collection. These tissues were transported in transport medium containing DMEM, 10% fetal calf serum and heparin (100 units/ml). None of these subjects received prostaglandin. 2.3. Isolation of brain perivascular

microglia

Culture of brain-derived perivascular microglia was carried out immediately, essentially as described by Hayes et al. (1988). The brain tissue was minced with sterile scalpels

in dissociation buffer (0.137 M NaCl, 0.005 M KCl, 0.0007 M Na,HPO,, 0.015 M HEPES and 0.01 M glucose, pH 7.5) containing 0.02% collagenase and DNAse (200 U/ml) and then incubated at 37°C for 15 min. The tissue was gently passed through sterile pipettes and agitated every 5 min. Clumps of brain tissue were allowed to settle down and the dissociated cells were aspirated. The remaining brain fragments were further digested several times with above-mentioned enzymes till tissue was completely dissociated. The enzymatic digestion was stopped by the addition of fetal bovine serum (final concentration 10%). Subsequent handling of the cells was carried out at 4°C and the cells were passed through 130 and 80 t.Lrn Nylon mesh (Tetco). These cells were incubated at (4°C) with sheep red blood cells (SRBC) which were coated with sub-agglutinating concentration of antibody to SRBC (hemolysin). Brain microglia, which binds to the Fc portion of hemolysin, form erythrocyte rosettes (EA). These EA cells were subjected to Ficoll-Hypaque density gradient (1.072 g/ml) centrifugation at 450 X g for 10 min at 4°C. The interface containing EA cells were collected, and washed. Sheep red blood cells were lysed with cold hypotonic shock (Hayes et al., 1988). The cells were numerated by Trypan blue viability test and then distributed into culture plates as desired in DMEM + 5% fetal bovine serum (low endotoxin, and heat-inactivated) or human Al3 serum. The cells were examined by flow cytometry using FITC-labelled CDllb monoclonal antibody (IgGl, Immunotech). As negative control, FITC-labelled isotypic antibody controls were used. These cells were examined in a FACScan (BectonDickinson). The presence of CD4 antigen was determined by direct immunofluorescence test using FITC-labelled CD4 monoclonal antibody and isotypic antibody controls (Immunotech). The number of CDCpositive cells were enumerated in a Leitz fluorescent microscope. The functional capacity of these cells was evaluated by the phagocytosis of hemolysin-coated sheep red blood cells. The cultures were incubated at 37°C in a humidified atmosphere containing 5% CO,. 2.4. Effect of rotropic HN-1

@endorphin

on the replication

of neu-

was grown in PHA-stimulated peripheral HIV-l,,_,, blood mononuclear cells. The medium was supplemented periodically with the culture supernatant of PHA-stimulated monocytes. In this experiment, fetal brain perivascular microglia were cultured in DMEM containing 10% heat-inactivated human AB serum. They were pre-treated with 10 pg polybrene for 1 h. Then these cells were infected with a neurotropic HIV-l,,_,r (containing 3 ng of p24 antigen/O.5 X lo6 cells). Two hours later, the cells were rinsed with medium. Cells were then incubated with medium alone or /3-endorphin (10m7 M). On day 4, one half of the medium was replaced with fresh medium containing twice the concentration of P-endorphin to

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maintain a final concentration of low7 M. In order to determine the effect of nalaxone, HIV-l,,_,,-infected cells were pre-treated with naloxone (10e8 M) for 1 h and /3-endorphin (10m7 M) was added. Every time p-endorphin was added to naloxone-treated cultures, these cells were pre-treated with naloxone as described above. On day 7 and 14 the cells were lysed by three cycles of freezing and thawing and the cell lysate was analysed for the presence of p-24 antigen using ELISA kits from Incstar. 2.5. Transactivation of HIV long terminal repeat (HIVLTR) The entire promoter region of the HIV-l IIIB, which is linked to the reporter gene chloramphenicol acetyl transferase (CAT), was obtained from Dr. Warner Greene. Brain-derived perivascular microglia, cultured as described above from fetal brain tissue, were added into 6-well tissue culture plates (1.0 X 106/well) and were incubated for 24 h. Transfection of brain mi’croglia was carried out essentially as described by McCutchan and Pagan0 (1968) using DEAE Dextran. The cells were rinsed with PBS and were then incubated for 60 min .with HIV-LTR-CAT construct in Tris-buffered saline containing DEAE dextran (10 mg/ml) and dextrose. The cells were rinsed with warm Tris buffer and were treated1 with 0.1 mM chloroquine for 30 min. Additional cells were transfected with SV40-RSVCAT expression vector as negative control. The cells were incubated at 37°C in Dulbecco’s modified medium supplemented with 10% heat-inactivated fetal bovine serum. 24 h later, transfected cells were treated with different concentrations of /3-endorphin (10.m7-10-9 M). Trypsinized cells were collected in Eppendorf tubes and were lysed by three cycles of freezing and thawing. Cell extract was incubated in a water bath at 70°C to inactivate heat-labile endogenous acetylase enzymes. The expression of the reporter gene chloramphenicol acetyl transferase was determined as described by Neumann et al. (1987) using tritiated acetylCoA enzyme (0.1 &i) in a fluor diffusion assay. 2.6. Reactivation of latent HN infection by the culture supernatant of P-endorphin-treated brain perivascular microglia Two-million fetal brain perivascular microglia were treated with medium alone or /3-endorphin (10m7 M). In order to determine the effect of nalaxone, cells were pretreated with naloxone as described above and the P-endorphin was then added. Culture supematants were dialysed and the supernatant was examined for the presence of cytokines which are known to reactivate latent HIV infection in U-l cells. In this experiment, 500000 Ul cells were treated with the above culture supematants. 48 h later, reactivation of latent HIV infection was evaluated by determining the synthesis of p-24 antigen by ELISA test (Incstar, Co).

99

2.7. Effect of P-endorphin on cytokine synthesis Fetal brain-derived perivascular microglia (2 X lo6 cells), isolated from four different brains, were cultured in 24-well plates. The cells were treated with different concentrations of P-endorphin (10-7-10-10 M). Cells were also treated with GP-120 and sub-optimal concentrations of P-endorphin (10e9 M). GP-120 used in these experiments was glycosylated, recombinant wild-type (HIV-l,,,) and consisted of > 90% full-length polypeptide as assayed by SDS-PAGE electrophoresis. The entire length GP-120 gene was cloned, signal sequences removed and substituted with that of human t-PA and the protein was expressed in CHO cells. (This reagent was obtained through the AIDS Research Resource Program, Division of AIDS, NINAID, NIH:GP-120 from HIV-,,, from Dr. Nancy Ha&wood of Chiron corporation). Cultures were also treated with heat-inactivated GP-120 (BO”C/lO min) as additional control. 24 h later, culture supernatant was evaluated for the presence of IL-26 and TNFa! by ELISA (Cistron, Biotechnology). To determine IL-l/3, the cells were lysed by three cycles of freezing and thawing and the clarified supernatant was examined by ELISA (Cistron Biotechnology). 2.8. Statistical analysis Preliminary analysis indicated that the variances changed with the increase of means scores; therefore, data were converted to log,, form for analysis. Results of effects of /3-endorphin was compared to appropriate controls and were the analysis were examined with paired t-tests. Since each experiment had several t-tests, Bonferroni correction of the alpha probability was employed by dividing the 0.05 level by the number of t-tests to be done. This correction tends to be very conservative. In one experiment, in addition to Bonferroni correction, the results were also analysed by Rank order analysis as suggested by Conover and Iman (1981). The effect of p-endorphin on HIV replication was analysed by ANOVA.

3. Results

3.1. Characterization of brain perivascular microglia As reported by Hayes et al. (1988), from each gram of brain tissue OS-l.0 X lo6 cells were obtained. Depending upon the gestation period, it was possible to isolate between 8 and 15 X lo6 cells from the brain tissue derived from the 16-24 weeks gestation period. Shipping brain tissue from Los Angeles did not adversely affect the survival of brain macrophages. Isolated cells were characterized by flow cytometry using surface markers for CDllb, as well as by the phagocytosis of hemolysin coated sheep red blood cells. As seen in Fig. 1, these cells

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25000 z :

20000

h 5 ._

15000

zi 2

10000

0 5000

o\

0

Fluorescence intensity Fig. 1. Purified fetal brain-derived perivascular microglia, isolated as described in Materials and methods, were examined by flow cytometry using fluoresceine-labelled CDllb monoclonal antibody in Beckman FACScan. Over 95% of these CDllb-positive cells also engulfed hemolysin-coated sheep red blood cells and 18-26% were positive for CD4 antigen (data not shown).

exhibited CDllb marker. Over 95% of these cells also exhibited phagocytosis of hemolysin-coated sheep red blood cells. Number of CD4-positive cells in these preparations ranged from 18 to 26% (data not shown).

=

40

E

q HIV-control

P

HIV+Beta

E

30 ._cn

HIV+Beta

E+N

: c E

> I

0

HIV-LTR

10 +

beta

9

8

endorphin

7 (-Log

10

M)

Fig. 3. Beta-endorphin enhances the transactivation of HIV promoter in fetal perivascular microglia. These cells were transfected with HIV-LTRCAT as described in Materials and methods. The cultures were treated with different concentrations of p-endorphin and 24 h later, the CAT gene expression was determined by fluor diffusion assay. Data are means&- S.E. from perivascular microglia cultures derived from five different brains. Beta-endorphin has no effect on the expression of CAT in cells which were transfected with RSV promoter.

3.2. Beta-endorphin enhances the replication fetal brain perivascular microglia

of HIV in

When 0.5 X lo6 perivascular microglia were infected with neurotropic HIV-l,,,,, replication of HIV was seen at low level as evidenced by the synthesis of intracellular p-24 at 7 and 14 days post infection. ANOVA analysis indicated that treatment of HIV-infected cells with p-endorphin (10e7 M) resulted in a significant increase in the

Cells+Naloxo 20

,7.5 ._ 3 s E s 5 u)

z z h

o&

10

0 7 days

Incubation

14 days period

Fig. 2. Beta-endorphin enhances the replication of neurotrophic HIV1 ,a+,_. Fetal brain perivascular microglia were isolated from four different fetal brains and were distributed into wells at a concentration of 0.5 X 106. They were treated with polybrene (10 pg/ml). The cells in each well were infected with 3 ng of HIV-l,.,,. In addition to the infected cell controls, additional cells were treated with P-endorphin (Beta E, 10e7 M) or P-endorphin (lo-’ M) plus naloxone (N, lo-’ M). Cells were lysed at 7 and 14 days post infection and the lysate was examined for p-24 by ELISA. Data are means + S.E. of brain perivascular microglial cultures derived from four different brains.

z Q2.5 z I Control

Beta E

Fig. 4. Culture supematant of brain perivascular microglia re-activates latent HIV infection in Ul cells. Five-hundred Ul cells were incubated with the culture supematant of brain perivascular microglia which were treated with medium alone, P_endorphin or naloxone plus pendorphin. 48 h later, the culture supernatant was analyzed for the presence of p-24 antigen by ELISA. Data are meansfS.E. of cultures derived from four different brains.

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synthesis of p-24 antigen both at 7 and 14 days (P I 0.003). Although there was a significant day effect (P I 0.018), there was no significant interaction effect. Pre-treatment of HIV-infected cells with naloxone abolished enhanced replication of HIV due to P-endorphin (Fig. 2). Beta-endorphin significantly enhanced the replication of the virus at a dose of lo-* M (data not shown). 3.3. Beta-endorphin LTR

Control

1 (3

a

9

7

Concentration of beta endorphin (-log 10 M)

ff?J IL1

Beta

E

T

GP-120

fl

0

7

Beta E + GP-120

the transactiuation

of HIV-

Expression of chloramphenicol acetyl transferase was compared between those cells treated with /I-endorphin 10e7, 10m8, 1O-9 and lo-” M and medium and LTRtransfected controls. Beta-endorphin at concentrations of 10e7 and lo-’ M significantly differed from each of the two control conditions (the Bonferroni 0.05 probability was set at P I 0.007). At lower concentrations, p-endorphin did not enhance the transactivation of HIV promoter (Fig. 3). Beta-endorphin has no effect on the expression of CAT when the cells were transfected with RSV promoter (data not shown). 3.4. Reactivation

TNF

enhances

of HIV infection in Ul cells

The culture supematant of control brain perivascular microglia did not show significant ability to reactivate latent HIV infection in Ul cells (p-24 antigen 230 pg/ml). In sharp contrast, treatment of Ul cells with the culture supernatant of brain perivascular microglia which were incubated with Pendorphin (10m7 M) resulted in a significant synthesis of p-24 antigen (n = 4, 7700 pg/ml; paired t-test P I 0.001; Fig. 4). Culture supematant of perivascular microglia, which were pre-treated with naloxone followed by /3-endorphin, did not reactivate latent HIV infection in Ul cells (Fig. 4).

Medium

a

Concentration of beta endorphin (-log 10 M)

Fig. 5. (A) Induction of cytokines by B-endorphin and GP-120. Fetal brain-derived perivascular microglia were cultured with different concentrations of S-endorphin. Culture supematant or lysate were examined by ELISA for IL-6, TNFa and ILIP, respectively. The results presented are means+S.E. from four different brain perivascular microglial cell cultures. (Bl In order to determine the effect of Pendorpbin on pre-stimulated brain perivascular microglia, cells were treated with P-endorphin (low9 M), GP-120 (500 ng/ml) or a combination of the two and cytokine synthesis was determined by ELISA as described in (A). Data are means *SE. from cell cultures from four different brains. (Cl Naloxone reversed the effects of /3-endorphin. In order to determine whether P-endorphin is mediating through classical opiate receptor, brain perivascular microglia were pre-treated with naloxone (10-s Ml for 1 h followed by exposure to Pendorphin or GP-120 or a combination of the two. Cytokines were examined by ELISA as described in (A). The results indicate that &endorphin mediates these effects by interacting with classical opiate receptor. Naloxone did not inhibit TNFa induced by GP-120 (GP-120 = 5825 f 1374 pg/ml vs. GP-120 + naloxone = 7130 f 1484 pg/ml, respectively). Data are means f S.E. from perivascular microglial cultures of four different brains.

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3.5. induction of pro-inflammatory cytokines by p-endorphin in untreated and GP-I20-treated brain perivascular microglia Brain-derived perivascular microglia, rapidly isolated in these experiments, constitutively produced IL-1 /3, IL-6 and TNFcv (122 f 23, 46 + 15 and 205 + 72 pg/ml, respectively). Beta-endorphin at a concentration of lop7 and lo-* M enhanced the constitutive expression of IL-l/3, IL-6 and TNFa. A lo-’ M dose of /3-endorphin had a minimal, but statistically significant, effect on the synthesis of these cytokines (Bonferroni 0.05 probability was set at P LS0.007; Fig. 5A). GP-120 induced the synthesis of these cytokines in bfiin perivascular microglia significantly. In order to rule out the possibility of endotoxin contamination of GP-120 preparations with lipopolysaccharide, GP-120 preparations were heat-inactivated (SO’C for 30 min). Heat-inactivated GP-120 failed to induce any cytokines, indicating that the cytokine induction observed in these experiments is due to the glycosylated GP-120. When these cells were treated with a combination of heat-inactivated GP-120 and sub-optimal concentrations of Pendorphin (10e9 M), the levels of cytokines observed did not significantly differ from the results of the cells incubated with P-endorphin (lop9 M) alone. GP-120-induced cytokine synthesis was augmented by sub-optimal concentrations of /3-endorphin (10e9 MI (Rank order analysis and Bonferroni 0.05 probability was set at P I 0.006; Fig. 5B). In order to determine whether P-endorphin mediates these effects by binding to classical opiate receptor, brain perivascular microglia were pretreated with naloxone (lo-’ M) for 1 h and then incubated with /3-endorphin (10p7, lo-’ Ml, GP-120 (500 ng/ml) or a combination of GP-120 and P-endorphin (10e9 M). The culture supematants were examined for the presence of TNFa! by ELISA test. Nalaxone blocked the synthesis of TNFa induced by p-endorphin. However, naloxone failed to inhibit the constitutive expression as well as GP-120 induced TNFa synthesis in fetal brain perivascular microglia (Fig. SC>. There is a significant difference in ranks (by Bonferroni criteria; P I 0.006).

4. Discussion The results presented here demonstrate that p-endorphin influences different stages of the replication of HIV, namely: synthesis of the virus, transactivation of the promoter region and reactivation of the latent infection. Beta-endorphin appears to mediate these effects by inducing the synthesis of pro-inflammatory cytokines in fetal brain-derived perivascular microglia. Nalaxone, a classical opiate receptor antagonist, abolished the potentiating effects of /3-endorphin on the replication of neurotropic HIV and on the synthesis of cytokines. The cells isolated from

fetal brains expressed CD4 marker (18-24%). CD4-positive cells were considered to be perivascular microglia (Peudenier et al., 1991) and expression of CD4 in these cells can be regulated by stimuli (Perry and Gordon, 1987; Sawada et al., 1992). In addition, infection of microglia with HIV was shown to be dependent on the expression of CD4 antigen (Jordan et al., 1991). Therefore, these cells were referred to as perivascular microglia. Pro-inflammatory cytokines are shown to enhance the replication of HIV and are postulated to contribute to the progression of AIDS and AIDS dementia complex (Rosenberg and Fauci, 1990; Poli and Fauci, 1992a,b; Poli et al., 1992; Fauci, 1993a,b; Vitkovic et al., 1994). These immune activation of HIV by cytokines occur either at transcriptional as well as translational events (Poli and Fauci, 1992a). These cytokines enhance the transactivation of HIV-LTR which involves the induction of and binding of NFkB transcription factor to the LTR region (Rosenberg and Fauci, 1990). Therefore, the ability of P-endorphin to enhance the replication of HIV may be directly related to the induction of IL-lp, IL-6 and TNFa in fetal microglia. This is supported by the observation that naloxone inhibited the synthesis of TNFcv and the replication of HIV induced by P-endorphin. Beta-endorphin may also directly influence the transactivation of HIV promoter by elevating levels of second messengers and the pathways of the protein phosphorylation, such as protein kinase C (Guitart and Nestler, 1993). Cytokines induced by P-endorphin may amplify these signals to enhance the transactivation of HIV-LTR as well as HIV replication. Our results are in agreement with others investigators, who demonstrated that P-endorphin induces the synthesis of IL1 in macrophages (Apte et al., 1989,199O; Heijnen et al., 1991; Chao et al., 1993). Peterson et al. (19941 similarly demonstrated that morphine potentiated TNF-cr synthesis in LPS stimulated murine and human fetal microglia and these culture supematants re-activated latent HIV infection in U-l cells. These effects were also blocked by nalaxone. Opiates have been postulated to play an important role in infectious diseases, including AIDS (Rouveix, 1992; Donahoe, 1993). The results presented here extend these observations by demonstrating that cytokines released by endogenous opiates enhance the transactivation of HIV-LTR as wells enhance the replication of HIV. Although stress has been postulated to contribute to the progression of AIDS (Glaser and Kiecolt-Glaser, 1987), the role of stress in patients who progress rapidly has not been systematically investigated. This is particularly important in the light of these observations, since p-endorphin is one of the major hormones released during stress. Stress, which is also was shown to release IL-10 in the brains (Saperstein et al., 1992), and endogenous or exogenous opiates are likely to contribute to the progression of AIDS dementia complex. Although P-endorphin enhanced the replication of HIV at 10T7 and 10e8 M concentrations, these doses appear to be pharmacological.

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The concentrations of P-endorphin at a cellular level may be significantly higher than those seen in the cerebrospinal fluid. Stress, therefore, may be a contributing factor, particularly in the early stages of AIDS, where low levels of viral replication may be synergistically augmented by pendorphin.

Acknowledgements

This work is supported by a grant from the National Institutes of Mental Health MH51524.

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