Int. J. lmmunopharmac., Vol. 18, No. 5, pp. 305-309, 1996 Copyright Š 1996InternationalSocietyfor Immunopharmacology Publishedby ElsevierScienceLtd. Printedin Great Britain 0192~)561/96$15.00+ .00
Pergamon
PII: S0192--0561 (96)00033-1
A N T I - R E T R O V I R A L A C T I V I T Y OF M E T H I O N I N E E N K E P H A L I N A N D AZT IN A M U R I N E CELL C U L T U R E JOENG-IM SIN,* NICHOLAS PLOTNIKOFFt and STEVEN SPECTER*:~ *Department of Medical Microbiology and Immunology, University of South Florida College of Medicine, 12901 N. Bruce B. Downs Blvd, Tampa, FL 33612, U.S.A. "~Department of Pharmacodynamics, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, U.S.A. (Received 7 March 1996; revised 10 M a y 1996)
Abstract--Previously, this laboratory has demonstrated that azidothymidine used in combination with methionine enkephalin, an opioid pentapeptide, was more effectivethan AZT alone in inhibiting diseaseprogression due to murine retrovirus infections. In order to study the mechanism(s) by which Met-ENK mediates-antiviral effects, when used in combination with AZT in Friend leukemia virus infected mice, an in vitro focus forming assay was used. AZT at 1 ng/ml inhibited FLV replication by 30-50% in the susceptible Mus dunni cell line. By contrast, the immunostimulatory neuropeptide, Met-ENK, displayed no direct inhibition of viral replication. This suggests that Met-ENK does not have any direct anti-retroviral activity. Subsequent testing of Met-ENK in the presence of AZT showed no ability of this peptide to promote inhibition of viral replication due to AZT. By contrast, in the presence of mouse spleencells,as a source of lymphocytes,in vitro combination treatments using AZT and Met-ENK reduced FLV replication by 67%, compared to 47% using AZT alone. The inhibition due to Met-ENK was abrogated when spleen cells were pretreated with naloxone, an opioid antagonist. Therefore, we conclude that Met-ENK effects are mediated via opioid receptors on spleen cells and that the observed anti-FLV activity is dependent on the use of Met-ENK stimulated spleen cells in combination with AZT. Copyright Š 1996 International Society for Immunopharmacology Keywords: methionine enkepbalin, AZT, retrovirus, Friend leukemia virus.
The need for effective therapies for the human immunodeficiency virus (HIV) and other human retroviruses has led to the examination of both antiviral and immunomodulatory drugs. While 3'-azido-3'deoxythymidine (AZT) has been demonstrated to provide some benefit to HIV infected patients it has done little to prevent the progression of the acquired immunodeficiency syndrome (AIDS). Thus, newer approaches to therapy have sought to combine therapies for treatment of HIV infection and AIDS. In this regard murine retroviruses have been studied as AIDS models to evaluate the efficacy of therapy. Several studies have indicated that AZT could provide some protection against the murine retroviurus, Friend leukemia virus (FLV), and when combined with immunostimulants, including interleukin 2 and interferon, was more effective (Ciolli et al., 1991; Johnson et al., 1990; Morrey et al., 1990; Portnoi et al., 1990; Simard and Jolicoeur, 1991). Nevertheless, these approaches, using the same immunostimulantsas well as others, when applied to HIV infected individuals
have yet to demonstrate significant benefit (Johnson and Hirsch, 1990; Specter and Hadden, 1992). We have demonstrated more recently that the combination of an immunostimulatory neuropeptide, methionine enkephalin (Met-ENK), and AZT is more effective than either substance alone in reducing the morbidity and mortality due to the murine retroviruses FLV and the BM5 virus complex (Specter et al., 1994). This effect appeared to be the result of an antiviral effect of AZT and an immunostimulatory effect of Met-ENK, since AZT alone provided some protection while Met-ENK had no antiviral effect when used alone. Met-ENK has been shown to be an immunostimulator both in mice and humans, increasing CD4 lymphocyte counts in patients with AIDS or AIDS related complex (Wybran et al., 1987). Met-ENK has also been demonstrated to boost natural killer (NK) cell activity (Ghanta et al., 1991; Hsueh et al., 1992; Puente et al., 1992), cytokine levels and cytokine receptor expression (Marotti et al., 1994; Singh et al.,
~Author to whom correspondence should be addressed. 305
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1994; Wybran et al., 1987). Thus, there is a strong suggestion that the effects that we have observed could be the result of a boost of immune function. More recently, Met-ENK treatment was reported to enhance N K cell cytotoxicity and virus specific cytotoxic T lymphocyte activity in mice infected with influenza A virus (Burger et al., 1995). Thus, it is important to understand the mechanism(s) by which Met-ENK is contributing to the abrogation of morbidity and mortality due to murine retrovirus. In this regard mouse spleen cells were used to determine if Met-ENK effects were mediated by a mixed lymphocyte population. The present study examined the effects of AZT and Met-ENK in vitro using FLV and the susceptible Mus dunni (Dunni) cell line to measure antiviral therapy. Studies revealed that AZT could reduce viral replication but that Met-ENK did not and the neuropeptide did not boost AZT activity. However, mouse spleen cells that were stimulated with Met-ENK when added to AZT were capable of decreasing virus infectivity as measured by a focus forming assay on the Dunni cells.
METHODS
Mice--BALB/c mice were purchased from Jackson Laboratories (Bar Harbor, ME) at 6--8 weeks of age. The mice were housed in groups of 10 per plastic cage and fed water and mouse food pellets ad libitum. Friend leukemia virus--FLV used in these studies was the N/B tropic polycythemia inducing strain that contains the entire FLV complex, composed of a helper lymphatic leukemia virus and a defective spleen focus forming virus obtained from Dr B. Chesebro (Hamilton, MT). Virus was prepared as a 10% suspension (w/v) of spleen homogenate from FLV infected mice in phosphate buffered saline (PBS). Stock preparations contained approximately 5000 infectious doses per ml and were stored at - 7 0 째 C until used. Azidothymidine--The drug was provided in powder form as a gift from Burroughs-Wellcome Co. (Research Triangle Park, NC) and is soluble in water. Methionine enkephalin and naloxone, an opioid receptor antagonist Met-ENK, a pentapeptide consisting of Met-Phe-Gly-Gly-Tyr (MW--574 d), was provided as a powder by TNI Pharmaceuticals (Tulsa, OK). Naloxone hydrochloride (MW--389 d) was obtained as a white solid from Research Biochemicals International (RBI) (Natick, MA). Naloxone was selected as a general antagonist that utilizes multiple opioid receptors and its activity is indicative of an
opioid receptor mediated activity. Each drug was suspended in PBS solution for tissue culture. Naloxone was used at two, five and ten times the concentration of Met-ENK. Focus forming assay--Virus titers were determined by measuring focus forming units (FFU) on Mus dunni cells kindly provided by Dr B. Chesebro (Hamilton, MT). Dunni cells were grown to confluence at 37째C in 5% CO2, 95% air, in 24 well cluster plates (Costar, Cambridge, MA) containing 2 ml "complete medium"/well. This consisted of RPMI 1640 supplemented with 10% FBS, 1% L-glutamate, 100 ~tg penicillin and streptomycin/ml and 0.25% 1 M HEPES buffer solution. To enhance FLV infectivity, 8 I~g polybrene/ml was added to the medium. Subsequently, a confluent monolayer of cells was infected with approximately 50 F F U FLV. Thereafter, treatment of Dunni cells with drugs and/or spleen cells was initiated 2 hr post viral infection. After 3 days incubation, infected cells and mock infected (supernatant fluid from non-infected spleens) controls were stained with monoclonal anti-FLV gp70 antibody (supplied by B. Chesebro), followed by goat antimouse IgG labelled with horseradish peroxidase. Foci were then developed by addition of substrate and yellowish-brown stained areas were counted. Each treatment regimen group was tested in four replicates. All experiments were repeated at least three times. Spleen cell preparation--Spleens were aseptically removed from BALB/c mice and a single cell suspension was made by mincing the spleen with a sterile scissors and forceps in a plastic dish containing complete medium. Suspensions of cells were passed through a 22 gauge needle to break up clumps and then the cell debris was removed by pipetting. After allowing the larger pieces to settle down for 5 rain at room temperature, the splenocyte suspension was harvested. Suspended cells were examined for viability using trypan blue dye exclusion and adjusted to 1 x 108 cells/ml. For the focus forming assay, 10 6 spleen cells were added to each well 2 h post infection of the Dunni cells with FLV. Statistical analysis--Statistical analysis was done using the paired Student's t-test. Experimental values were compared with the untreated control and AZT treated control values, p values < 0.05 were considered significant. RESULTS
Effects o f A Z T and methionine enkephalin on Friend leukemia virus replication in Mus dunni cells AZT was tested for its ability to inhibit FLV replication over a dose range from 50 pg/ml to 100 lag/ml
Anti-retroviral Activity of Methionine Enkephalin and AZT (Table 1). All data presented here are a representative experiment that has been repeated two additional times with like results. The three experiments were not averaged because the number of F F U in control cultures varied too greatly. While the lowest A Z T concentrations tested had no effect, 1 ng A Z T / m l and greater significantly inhibited virus replication. Inhibition by 1 ng/ml A Z T in three separate experiments (data not shown) was 35% (Table 1), 39%, 44%, respectively. A Z T at 10 ng/ml or higher completely inhibited viral replication. In order to determine whether M e t - E N K could contribute further to the anti-viral effect of A Z T all future experiments were performed using 1 ng A Z T / m l medium. M e t - E N K was tested over a concentration range of 10 ng/ml to 100 pg/ml. N o direct antiviral effect of M e t - E N K was seen on F L V replication in Dunni cells (Table 1). Subsequent experiments examined the combination of A Z T (1 ng/ml) and M e t - E N K (1-100 p.g/ml) for the ability to inhibit F L V replication. As noted in Table 2, A Z T inhibited F L V replication by approximately 40% but the addition of M e t - E N K did not further reduce infectivity of the virus. Toxicity studies, to insure that A Z T or M e t - E N K effects alone or in combination did not damage cells, indicated no toxicity, as measured by cell growth, at the concentrations tested (data not shown). Table 1. Effects of AZT or methionine enkephalin on Friend leukemia virus replication in Mus dunni cells Group Control c AZT 50 pg/ml 100 pg/ml 500 pg/ml 1 ng/ml 10 ng/ml 100 ng/ml Methionine enkephalin 10 ng/ml 1 }tg/ml 10 ~tg/ml 50 ~tg/ml 100 pg/ml
# Focus forming units + S.D. a
Inhibition (%)b
32.2 + 1.9 32.3 _ 2.5 28.8 ___1.0 26.3 + 4.2 20.8___2.1" 0.0" 0.0"
0 11 18 35 100 100
26.4 + 4.7 30.0 +__7.9 31.0+3.5 29.3+ 1.5 32.3 + 1.5
18 7 4 9 0
a Mus dunni cells were grown to confluence in 24 well cluster plates. Two hours post FLV infection, drug treatment was initiated. After 3 days at 37째C in 5% CO2, 95% air the cultures were stained with immunoperoxidase labelled antigp70 monoclonal antibody and then loci were counted. Data represent a typical result in one of three experiments. b Inhibition (%) calculated as: [FFU c o n t r o l - F F U drug treatments]/FFU control x 100. Four replicates were used for each group. * Statistically significant at p < 0.05 using the Student's t-test compared to control.
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Table 2. Effect of AZT and methionine-enkephalin combined for treating FLV in cell culture Group Control c AZT 1 ng/ml AZT + Met-ENK AZT + Met-ENK AZT + Met-ENK AZT + Met-ENK
# Focus forming Inhibition units + S.D. a (%)b
1 ~tg/ml 10 Bg/ml 50 Bg/ml 100 ixg/ml
59.5 _ 3.5 36.5 4-5.3* 36.8___3.2* 35.3_ 3.4* 35.8+2.6* 39.5 __+5.2*
-39 38 41 40 34
Mus dunni cells were grown to confluence in 24 well cluster plates. Two hours post FLV infection, drug treatment was initiated. After 3 days at 37째C in 5% CO2, 95% air the cultures were stained with immunoperoxidase labelled antigp70 monoclonal antibody and foci were counted. b Inhibition (%) calculated as: [FFU control - FFU drug treatments]/FFU control x 100. c Four repficates were used for each group. * Statistically significant at p < 0.05 using the Student's t-test compared to control.
Ability o f mouse spleen cells stimulated with Met-ENK to further decrease FL V replication Because M e t - E N K has been demonstrated to boost immune functions, and had inhibitory effects on F L V infection in vivo, we next tested this opioid peptide for the induction of antiviral activity in mouse spleen cells. One million spleen cells were added with MetE N K to Dunni cells with or without AZT. As seen in Table 3, the M e t - E N K stimulated spleen cells had no effect on replication of FLV. However, when spleen cells were added to Dunni cells in the presence of A Z T , the opioid further reduced F L V replication as compared to A Z T alone. This effect of M e t - E N K was dose related.
Reversal o f the effects of Met-ENK by the opioid antayonist naloxone Naloxone hydrochloride is a non-selective antagonist for opiates that binds to the various opioid receptors. Thus, this c o m p o u n d was used to determine if the spleen cell stimulatory effects o f M e t - E N K were mediated by specific binding to receptors. D a t a in Table 4 show that naloxone was capable of abrogating the ability of M e t - E N K to stimulate spleen cells to inhibit F L V replication when in the presence of A Z T . Naloxone did not show any statistically significant F L V inhibitory effects at the concentrations shown. However, at a higher concentration (870 BM) naloxone had a metabolic inhibitory effect that caused the Dunni cells to detach from the plastic and die (data not shown).
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Table 3. Effect of AZT and methionine enkephahn stimulated spleen cells combined for treating FLV in cell culture Group Control b AZT 1 ng/ml Met-ENK 1 #g/ml Met-ENK 10 vg/ml Met-ENK 50/tg/ml Met-ENK 100 ktg/ml A Z T + M e t - E N K 1 ktg/ml AZT+Met-ENK 10 lag/ml AZT+Met-ENK 50 lxg/ml AZT+Met-ENK 100 #g/ml
# Focus forming units _+ S.D. a 39.0_ 2.5 20.8 +4.9* 40.3 + 5.7 38.0+__2.9 39.5 _ 4.2 38.0__+2.2 19.0__ 1.8" 16.8 _ 1.9' 14.3 +2.1",** 13.0_+1.0",**
Inhibition (%) .... 47c 0
3 0 3 51c 57 63 67
_ ---
--9d 19o 31d 38a
Mus dunni cells were grown to confluence in 24 well cluster plates. Two hours post FLV infection, drug treatment was initiated. In addition to drugs, 10 6 spleen cells were added to Dunni cells. After 3 days at 37째C in 5% CO2, 95% air the cultures were stained with immunoperoxidase labelled antigp70 monoclonal antibody and then foci were counted. b Four replicates were used for each group. c Inhibition (%) calculated as: [FFU control - FFU drug treatments]/FFU control x 100. d Inhibition (%) calculated as: [FFU AZT alone - FFU drug combination]/FFU AZT alone x 100. * Statistically significant at p < 0.05 using the Student's t-test compared to control. **Statistically significant at p<0.05 using the Student's ttest compared to AZT alone. Table 4. Blockage of the anti-FLV effects of Met-ENK stimulated spleen cells by naloxone Group Control b AZT 1 ng/ml AZT + Nal (174 t~M) AZT + Nal (435 aM) A Z T + Met-ENK 50 ktg/ml (87 pM) AZT + Met-ENK + Nal (174 ~tM) AZT + Met-ENK + Nal (435/~M)
# Focus forming units + S.D. a
Inhibition (%)
50.8 _+ 1.7 32.7+2.1" 29.5 + 1.3" 29.0 _ 3.6* 23.7+ 1.2",**
36c 42 43 53c
10 11 28d
32.3 __+4.0*
36
1
32.5 + 2.5"
36
1
Mus dunni cells were grown to confluence in 24 well cluster plates. Two hours post FLV infection, naloxone was added to spleen cells 10 min before Met-ENK. After 3 days at 37째C in 5% CO2, 95% air the cultures were stained with immunoperoxidase labelled anti-gp70 monoclonal antibody and then foci were counted. b Four replicates were used for each group. c Inhibition (%) calculated as: [FFU control - FFU drug treatments]/FFU control x 100. d Inhibition (%) calculated as: [FFU AZT alone - FFU drug combination]/FFU AZT alone x 100. * Statistically significant at p < 0.05 using the Student's t-test compared to control. **Statistically significant at p<0.05 using the Student's ttest compared to AZT alone.
DISCUSSION The ability of M e t - E N K to c o m b i n e with A Z T to inhibit the replication o f F L V was d e m o n s t r a t e d to be d e p e n d e n t on stimulation of spleen cells by the opioid peptide. This is consistent with results seen in B A L B / c mice infected with F L V a n d then treated with A Z T a n d M e t - E N K (Specter et al., 1994). In those studies M e t - E N K h a d no antiviral activity unless it was used in c o m b i n a t i o n with A Z T . The in vitro studies reported here indicate t h a t M e t - E N K h a d n o direct antiviral effect against FLV, since M e t - E N K only boosted the antiviral effect of A Z T w h e n spleen cells stimulated with the opioid were present in infected cell cultures. These data further s u p p o r t o u r earlier suggestion t h a t the M e t - E N K effect was a n i m m u n o s t i m u l a t o r y effect r a t h e r t h a n a direct antiviral activity. The general opioid antagonists, n a l o x o n e or naltrexone block i m m u n o s t i m u l a t o r y effects o f MetE N K (Hsueh et al., 1992; W y b r a n et al., 1979; Zaitsev e t al., 1991). F u r t h e r m o r e , mu, delta a n d k a p p a opioid receptors of different binding properties are present o n m u r i n e splenocytes ( T a u b et al., 1991). Studies using opioid receptor agonists a n d antagonists showed t h a t a delta specific receptor is involved in i m m u n o s t i m u l a t i o n , whereas a m u specific receptor is involved in i m m u n o s u p p r e s s i o n ( M a z u m d e r et al., 1993). Linner a n d co-workers also report t h a t stimulation of lymphoid cells by M e t - E N K is via a delta receptor, specifically delta 2 receptor b u t n o t delta 1 (Linner et al., 1995). In o u r system, p r e t r e a t m e n t o f spleen cells with n a l o x o n e blocked M e t - E N K effects on reduction o f F F U f o r m a t i o n (Table 4). This suggests t h a t M e t - E N K is interacting with an opioid receptor on m o u s e spleen cells for c o m b i n a t i o n effects with A Z T . F u r t h e r studies have been initiated to determine the specific opioid receptor involved in the antiviral effect associated with M e t - E N K stimulation o f m o u s e splenocytes. Preliminary data indicate t h a t s u p e r n a t a n t fluid from M e t - E N K or C o n A stimulated spleen cells is capable o f c o m b i n i n g with A Z T to further inhibit F L V replication. This observation suggests t h a t MetE N K stimulates some soluble p r o d u c t that has antiviral activity. It is possible t h a t M e t - E N K is stim u l a t i n g one or m o r e cytokine(s) t h a t c a n c o n t r i b u t e to the inhibition of F L V replication. Previous reports indicate t h a t M e t - E N K is capable o f stimulating the p r o d u c t i o n o f interleukin (IL)-1, IL-2, IL-4, a n d interferon (IFN)-7 as well as greater expression o f IL-2 receptors ( M a r o t t i et al., 1994; Singh et al., 1994; W y b r a n et al., 1987). Thus, there is a n indication t h a t the i m m u n e stimulatory effect o f M e t - E N K is via one or more o f these p r o d u c t s which can limit F L V infec-
Anti-retroviral Activity of Methiouine Enkephalin and AZT tion, both in vivo and in vitro, in the presence of an antiviral drug such as AZT. Two possible mechanisms that may be involved in the M e t - E N K mediated effect are: the production of cytokines of a helper T lymphocyte type 1 response may be promoting a host protective effect; and the
309
induction of interferons may contribute by a direct inhibition of virus replication. This latter possibility seems less likely since spleen cells stimulated with MetE N K when used alone did not inhibit FLV. Investigation of these mechanisms is in progress.
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