Download anti-retroviral activity of methionine enkephalin and azt in a murine

Int. J. lmmunopharmac., Vol. 18, No. 5, pp. 305-309, 1996
Copyright © 1996InternationalSocietyfor Immunopharmacology
Publishedby ElsevierScienceLtd. Printedin Great Britain
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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 © 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
306
J.-I. SIN et al.
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 ° 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
37°C 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 37°C 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.
307
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 37°C 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).
308
J.-l. SIN et al.
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 37°C 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
37°C 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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