1965. 8. Donahoe,. R.M., and. Falek,. A. Neuroimmunomodulation by opiates and other drugs of abuse: relationship to HIV infection and. AIDS. In Psychological.
Functional
alterations
of swine
peripheral
blood
mononuclear
cells by methadone Thomas W. Molitor7 Michael and Phillip K. Peterson’ *Departments
of Large
of Minnesota,
St.
School,
Animal
Paul;
P. Murtaugh,’
Clinical
‘Department
Sciences
Robert
and
of Medicine,
Veterinary
Hennepin
Pathobiology,
County
an
ideal model for the suppression of immune 51: 124-128; 1992.
Words: natural cells
investigation cell functions.
methadone cytotoxicity
immunosuppression activity swine peripheral .
respiratory
.
blood
mono-
agents in humans [6, 8, 14, 21, 26, 27, 33]. The results of these studies have been difficult to interpret because of cornplicating factors that contribute in unpredictable ways to the impairment of immune function, such as malnutrition,
In vitro
(2, studies
occurrence 5, 26]. have
of polydrug
abuse
among
Journal
Medicine,
University
of Minnesota
Medical
MATERIALS
AND
Preparation
Healthy, crossbred castrated male swine, 6 to 12 weeks of age to 40 kg), were purchased from a local swine producer. Blood was collected by jugular venipuncture in the presence or absence of heparin. Swine sera were prepared from nonheparinized blood. Heparinized blood was diluted 1:1 in phosphate-buffered saline (PBS) and layered onto a FicollHypaque gradient (density 1.076 g/ml). After centrifugation for 30 mm at 800g the mononuclear cell layer was aspirated and the cells washed twice in PBS. The cells were counted with a hemocytometer and diluted to 1-2 x 106 cells/ml in RPMI 1640 containing either 2 to 10% autologous pig Serum or 5% fetal bovine serum (FBS). The purified PBMNCs were 10 to 15% monocytes and 85 to 90% lymphocytes as determined by nonspecific esterase staining [20, 37]. Cell viability was assessed by staining with ethidium bromide-acridine orange [23].
Intracellular
Protein
Synthesis the
that
immune
cell
of Leukocyte
Biology
Volume
function
5!,
and
of protein
and
incorporation
of
Abbreviations:
shown
METHODS
RNA RNA
Synthesis by
PBMNCs
L-[3H]leucine
was
and
assessed
[3H]uridine
by into
voluncan
be modulated by opiates [26, 27, 36]. Some activities that opiates inhibit include T cell rosetting [9, 35], expression of surface markers [9], lymphoproliferative mitogenic responses [12, 32], and interferon y production [24]. Chronic exposure of human peripheral blood mononuclear cells (PBMNCs) to morphine or methadone in vitro has also been
124
Veterinary
University
Chao,’
( 20
Abuse of addictive substances, especially opiate drugs, has been associated with an increased incidence and/or severity of infectious disease [1, 1!]. The recent appearance of acquired immunodeficiency syndrome (AIDS) and the importance of intravenous drug abusers as a high-risk group have prompted heightened interest in testing the hypothesis that drugs of abuse may act as cofactors to promote the spread of human immunodeficiency virus (HIV) and the progression of AIDS. Attemps have been made to ascertain the effects of the opiates methadone and morphine on the immune response and the ability to resist infectious disease
the
of
Center,
Chun
of
J.
INTRODUCTION
stress, and teer subjects
“College
Medical
Gekker,’
associated with diminished capacity of the monocytes in this mixed cell population to generate reactive oxygen intermediates, superoxide anion (02), and hydrogen peroxide [25, 26]. The modulating effects ofopiates on immune cells may act directly through opiate receptors, which immune cells are known to have on their surface [3, 4, 35]. The need for a clinically relevant animal model to avoid the confounding factors prompted us to investigate the effects ofmethadone on immune functions in swine. As a first step, we examined whether methadone would impair the function of swine PBMNCs in vitro. Our findings indicate that metadone in a dose-dependent manner suppressed two monocyte functions of PBMNCs, superoxide anion production and phagolysosome formation, as well as natural killer (NK) activity, a lymphocyte function.
Cell Key burst nuclear
Genya
Minneapolis
Abstract: In vitro exposure of the synthetic opiate drug methadone allowed evaluation of putative immunomodulatory activities of swine peripheral blood mononuclear cells. Respiratory burst, an index of microbicidal activity, was suppressed by methadone in a dosedependent manner following exposure for 48 h. The suppression was blocked by the opiate antagonist naloxone. Another macrophage function phagosome-lysosome fusion was impaired by exposure to methadone. A primary lymphocyte-mediated function natural killer cell activity was also affected. In contrast, the macrophage function antibody-mediated phagocytosis was not affected. Because the functions affected by methadone are critical to host defenses against pathogenic organisms, our findings suggest that opiate-mediated immunomodulation merits further study. Moreover, our studies suggest that swine may provide opiate-mediated Leukoc. Biol.
E. CIick
February
1992
bovine
PBMNC, line;
AIDS,
serum; PMA,
HIV,
peripheral
Reprint 225
Received
immunodeficiency
syndrome;
immunodeficiency mononuclear
myristate
virus; cell;
acetate;
PBS,
SRBC,
NK,
FBS, natural
fetal killer;
phosphate-buffered
sheep
red
blood
sacell;
ThA,
acid. requests:
Veterinary
Minnesota,
blood
phorbol
trichloroacetic
acquired human
Thomas Teaching
St. May
W.
Molitor,
Hospitals,
Paul,
MN
55108.
2,
1991;
accepted
Large 1365
July
Gortner 10,
1991.
Animal Avenue,
Clinical
Sciences, University
of
trichloroacetic acid (‘TCA)-precipitable materials [19]. cells to be labeled were incubated in 24-well plates, lively at a density of 1 x 106/ml in the presence of 0 M methadone in RPMI containing 10% autologous Twenty tCi of [3H]!eucine or 24 jCi of [3H]uridine
Briefly, respecto 106 sera. (Amer-
sham, Arlington Heights, IL) was added at the designated times for 4 h. Plates were then chilled on ice for 5 mm and 150 tl of 50% ICA was added to all wells. The fluid from each well was mixed thoroughly, transferred to a separate 12 x 75 mm tube, and sonicated at 30 watts for 20 s. The protein and RNA precipitates were then applied to a glass fiber filter that was under vacuum. The filters were washed twice with cold 5% TCA, twice with 70% ethanol, and once with 95% ethanol. Filters were dried under a heat lamp, placed in vials containing scintillation fluid, and counted in a liquid scintillation spectrophotometer.
Treatment The
of PBMNCs
National
Institute
with of
Methadone Drug
Abuse
and
Naloxone
(Bethesda,
MD)
kindly provided the methadone hydrochloride and naloxone hydrochloride. Methadone at concentrations ranging from 1018 to 106 M was dissolved in RPMI 1640 medium. Swine PBMNCs were cultured with various concentrations of methadone for 48 h prior to functional analyses as described [ 25, 26]. The volume of each well remained constant throughout incubation by the addition of RPM! 1640 medium. In selected experiments, naloxone at either 1012 or 106 M was incubated for 1 h prior to the addition of methadone. In all cases cell viability was determined both prior to and following incubation with methadone and/or naloxone.
Phagocytosis Antibody-mediated phagocytosis was measured using SiCr labeled opsonized sheep red blood cells (SRBCs) as described [15, 30]. Briefly, equal volumes ofa 5% SRBC suspension was mixed with a 1:10 (v/v) solution of hyperimmune rabbit anti-SRBC serum. The mixture was incubated at 37#{176}Cfor 1 h and then washed extensively with PBS. The SRBCs were radiolabeled with 100 zCi of Na25tCrO4 (New England Nuclear, Boston, MA; specific activity, 518 mCi/mg) at 37#{176}Cfor 1 h with gentle agitation. They were then washed twice and resuspended to 1% (v/v), and 0.3 ml was added to each 16-mm well containing PBMNCs. Phagocytosis after the cells tized SRBCs. by hypotonic for 40 5 and phagocytosed of 1% Triton a scintillation counter. All
was allowed to proceed for 1 h at 37#{176}C;therewere washed with PBS to remove nonphagocyResidual extracellular erythrocytes were lysed shock by the addition of 0.5 ml of 0.2% NaC1 then 0.5 ml of 1.6% NaCl. To determine radioactivity, cells were lysed by the addition X-100. Subtle contents were then transferred to vial and radioactivity was counted in a gamma experiments were performed in triplicate.
lysosomes granules.
revealed distinguishable The number of cells
200 cells formation
orange intracytoplasmic showing orange granules
in
was recorded. For detection of phagosome-lysosome only cell populations enriched for macrophages
2 h adherence
to
Respiratory
plastic
Burst
were
by
used.
Activity
The amount of O2 produced by PBMNCs was determined by calculating the superoxide dismutase-inhibitable reduction of ferricytochrome c as previously described [25, 281. Briefly, PBMNCs were cultured 48 h under various conditions in microtiter plates, centrifuged, and resuspended in medium alone or medium containing superoxide dismutase. Ferricytochrome C, with or without a respiratory burst stimulus (phorbol myristate acetate, PMA), was then added [26]. Plates were incubated at 37#{176}Cfor 60 mm and absorbance was read at 550 nm on a Multiskan MC spectrophotometer. The nanomoles of 02 released per 10 cells was calculated by multiplying the absorbance reading by 15.67 [28].
Natural Effector plates were h with beled three K562 period tion, each percent nations Lytic culated defined specific
Killer
Cell
Activity
PBMNCs were placed in 96-well round-bottom at 54 x 10k, 18 x 10k, and 6 x 10 cells/well. The cells incubated at 37#{176}Cin modified Clicks medium for 48 or without methadone. The K562 target cells were lawith Na25tCrO4 (Amersham) for 1 h at 37#{176}C,washed times in PBS, and counted. A total of 10 SiCrlabeled target cells was added to each well and the incubation continued for an additional 4 h [15, 37]. After incubathe plates were centrifuged and aliquots removed from well for counting in a gamma spectrophotometer. The specific lysis was calculated from triplicate determitaking into account total and spontaneous release. units were calculated from the specific lysis values calat various effector-target ratios. Lytic units were as the number of effector cells required to obtain lysis of the K562 target cells.
Statistical Where
Analyses appropriate,
data
were
expressed
as
mean
±
SE.
In
each experiment, n refers to the number of individual pigs used. For comparison of data for multiple groups, analyses of variance were performed using a standard computer program (Statistical Analysis Service Inc., Cary, NC). for cornparison of means of two groups, statistical significance was assessed by Student’s t-test; significance was defined as P < 0.05.
RESULTS
Phagosome-Lysosome
Formation
Phagosome-lysosome fusion was detected as described [10]. Acridine orange was added to cultures of adherent cell (1 x 106 cells/rn!) (1 rig/culture) 20 mm before the addition of heat-inactivated Candida albicans. Cells were cultured on 14-mm glass coverslips in 24-well microtiter plates for 48 h. The culture medium was then removed, 0.2 ml of 5 x 10 inactivated yeast was added, and the samples were incubated at 37#{176}Cfor 60 mm. After incubation, coverslips were washed twice and mounted on glass slides and cells were examined under ultraviolet light. Characteristics of fused phagosome-
PMA-Induced Swince
O2 Production
PBMNCs
cultured
for
48 h did
not
produce
detecta-
ble amounts of O2 ( < 5 nmol/106 cells/h. After stimulation with PMA for 60 mm, 31 ± 5.3 nmol 02/106 cells/h was obtamed from PBMNC cultures (Fig. 1). Pretreatment of PBMNCs with methadone for 48 h markedly suppressed 02 production. production blocked PBMNCs
Molzior
At a was at 106 M of all
ci aL
concentration of 10i2 M methadone, 02 suppressed 60%, and it was completely (Fig. 1). Similar results were obtained with animals tested (n = 10).
Methadone
Effects
on
Swine
PBMNCs
125
40
Phagocytosis
and
To determine phage functions antibody-mediated 30
formation of opsonized methadone lysosome
L)
0
r.D20
Phagolysosome
whether from
Formation
methadone PBMNCs, phagocytosis
modulated other macrothe effect of methadone on and phagosome-lysosome
was
examined. Antibody-mediated phagocytosis SRBCs was not influenced by exposure (data not shown). In contrast, phagosomeformation following ingestion of nonopsonized
yeast particles was significantly (P cells preincubated with methadone compared to control cells cultured done (Fig. 2).
C/) LU
to
< 0.05) compromised in at either !02 or 106 M in the absence of metha-
1 10
Natural
0 Control
10.18
iO-ls
Efl#{232}ctofmethadone
PBMNCs
from
sence
(control
done.
Cells
peroxide
five then
release
expressed
P
