Chronic Ethanol Inhibits NK Cell Cytolytic
Activity: Role of Opioid Peptide β-Endorphin
This information is current as
of June 14, 2017.
Subscription
Permissions
Email Alerts
J Immunol 2001; 167:5645-5652; ;
doi: 10.4049/jimmunol.167.10.5645
http://www.jimmunol.org/content/167/10/5645
This article cites 64 articles, 15 of which you can access for free at:
http://www.jimmunol.org/content/167/10/5645.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2001 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
References
Nadka Boyadjieva, Madhavi Dokur, Juan P. Advis, Gary G.
Meadows and Dipak K. Sarkar
Chronic Ethanol Inhibits NK Cell Cytolytic Activity: Role of
Opioid Peptide ␤-Endorphin1
Nadka Boyadjieva,* Madhavi Dokur,* Juan P. Advis,* Gary G. Meadows,† and
Dipak K. Sarkar1*
N
atural killer cells have been implicated in immune surveillance against tumors and bacterial and viral infections (1–3). Activated NK cells circulate through blood
and lymph and accumulate at sites of injury, infections, and tumors
(4). Although an increased incidence of certain forms of cancers
and infections has been documented in chronic alcoholics, very
few studies have been conducted on the NK cell functions. Although some inconsistencies in findings exist, most studies indicate that NK cell cytotoxicity is reduced in ethanol-treated animals
(5–10). A few studies conducted using humans also show reduction in NK cell functions following alcohol administration (11–
13). The mechanism by which ethanol alters NK cell function is
not known.
NK cells quickly respond to immune activation signals (14 –16).
NK cell cytotoxic activity is increased by the lymphokine IFN-␥,
which has a number of opioid-like effects (17). Additionally, IFNmediated NK cell cytolytic activity is blocked by the opioid receptor antagonist (18). The opioid peptide ␤-endorphin (␤-EP)3
*Department of Animal Sciences, Rutgers, The State University of New Jersey, New
Brunswick, NJ 08901; and †Department of Pharmaceutical Sciences, Cancer Prevention and Research Center, Washington State University, Pullman, WA 99164
Received for publication January 26, 2001. Accepted for publication September
18, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants AA-08757 and
AA-00220 (to D.K.S.).
2
Address correspondence and reprint requests to Dr. Dipak K. Sarkar, Department of
Animal Sciences, Rutgers, The State University of New Jersey, New Brunswick, NJ
08901-8525. E-mail address: [email protected]
Abbreviations used in this paper: ␤-EP, ␤-endorphin; PVN, paraventricular nucleus;
CSF, cerebrospinal fluid; IR-␤-EP, immunoreactive ␤-EP; YAC, yeast artificial chromosome; RIA, radioimmunoassay; POMC, proopiomelanocortin.
3
Copyright © 2001 by The American Association of Immunologists
is produced in the hypothalamus, in the pituitary, and in other
peripheral glands (19 –21). ␤-EP enhances splenic lymphocyte
proliferation in response to concanavalin A and increases the killing activity of NK cells (22–24). Opioid receptors are present on
lymphocytes (25). Because the function of endogenous ␤-EP is
altered in alcoholic patients and alcohol-treated animals (26 –29),
questions arise as to whether the abnormality in immune function,
particularly the changes in NK cell activity in the alcoholic, is
secondary to altered ␤-EP function. The present study is conducted
to investigate the role of ␤-EP in ethanol-modulated NK cell functions using the Fischer-344 male rat as an animal model. In this
report, we provide evidence that ethanol-induced inhibition of NK
cell cytolytic activity may be partly due to reduced ␤-EP-regulated
NK cell function.
Materials and Methods
Animals and feeding design
Male Fischer-344 rats of 150 –175 g body weight were maintained on a
12-h light/dark cycle (lights on 7:00 a.m. and lights off at 7:00 p.m.) and
were either ad libitum-fed rodent chow, pair-fed an isocaloric liquid diet,
or fed an ethanol-containing liquid diet as described by us previously (30).
The liquid diet consisted of vitamins (0.31%, w/v), minerals (0.50%, w/v),
sucrose (11.36%, w/v; pair-fed group), or ethanol (8.7%, v/v; ethanol-fed
group) and Sustacal (83.3%, v/v; Mead Johnson, Evansville, IN). Ethanol
substitution for sucrose provided ⬃37% ethanol-derived calories to the
ethanol-fed rats. Graduated ball-barrel cylinders containing the freshly prepared diet were placed in the animals’ cages 1 h before lights off (6:00
p.m.) for 1, 2, 3, or 4 wk. Animals were given free access to water. The
body weights of the animals were recorded before sacrifice by decapitation.
At the end of experiments, animals were weighed and decapitated. Spleen
tissues were immediately removed and used for NK cell cytolytic activity.
The hypothalamic tissues and trunk blood samples were also taken for
analysis of tissue and plasma immunoreactive (IR)-␤-EP levels. Animal
surgery and care were in accordance with institutional guidelines and complied with National Institutes of Health policy.
0022-1767/01/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
The role of ␤-endorphin (␤-EP) in ethanol-altered NK cell cytolytic activity is studied using male Fischer-344 rats as an animal
model. Ethanol was administered for 1, 2, 3, or 4 wk in a liquid diet containing 8.7% ethanol (v/v), which means that 37% of the
total calories were derived from ethanol. Rats treated with ethanol for 1 wk showed an increase in hypothalamic and plasma levels
of immunoreactive (IR)-␤-EP, but displayed no significant effect on NK cell activity determined by 51Cr release assay, as compared
with those in pair-fed and ad libitum-fed animals. However, animals treated with ethanol for 2, 3, or 4 wk showed decreased
hypothalamic and plasma levels of IR-␤-EP and decreased splenic NK cell activity. No significant decrease in the number of
splenocytes and NK cells or in the percentage of NK cells was seen until after 3 and 4 wk of ethanol treatment. Exposure in vitro
of splenic lymphocytes obtained from control animals to various concentrations of ␤-EP increased NK cell activity. The opiate
antagonist naltrexone blocked the ␤-EP-stimulated effect. The in vitro NK cell response to ␤-EP was reduced in the splenocytes
obtained from animals treated with ethanol for 2 wk, but not in those obtained from animals treated with ethanol for 1 wk as
compared with those in control animals. Additionally, ␤-EP administration into the paraventricular nucleus of the hypothalamus
stimulated NK cell cytolytic activity, whereas the opiate blocker administration reduced NK cell activity. The NK cell responses
to paraventricular nucleus ␤-EP were reduced in the animals treated with ethanol for 2 wk. These data provide evidence for the
first time that ethanol inhibits NK cell cytolytic activity, possibly by reducing ␤-EP-regulated splenic NK cell function. The
Journal of Immunology, 2001, 167: 5645–5652.
␤-EP MEDIATION OF ETHANOL EFFECT ON NK CELLS
5646
Intraparaventricular administration of ␤-EP or naltrexone
Assay for blood levels of ethanol
Male Fischer rats weighing 150 –175 g body weight were anesthetized with
sodium pentobarbital (33 mg/kg body weight) and were stereotaxically
implanted with a bilateral guide cannula (Plastics One, Ronkonkoma, NY)
using a Kopf stereotaxic apparatus. In each rat, bilateral guide cannulae
were positioned 3 mm apart, immediately lateral to each paraventricular
nucleus (PVN; 1.8 mm behind bregma, 1.5 mm lateral to the midline, and
7.7 mm below the skull surface) and were closed by a dummy infusion
cannula. Seven days after stereotaxic surgery, rats were pair-fed or ethanolfed for an additional 2 wk. At the end of this period, ␤-EP, naltrexone, or
artificial cerebrospinal fluid (CSF) was infused into each PVN. The ␤-EP
and naltrexone solutions were made using artificial CSF, and the concentration of these solutions was 200 ng/␮l. Five microliters of ␤-EP or naltrexone solution or CSF were infused through each PVN cannula during a
5-min period. The animals that received ␤-EP were additionally infused
with 100 ng of ␤-EP/0.5 ␮l/h in each PVN for a period of 16 h. Control
animals received an additional 0.5 ␮l CSF/h in each PVN for a period of
16 h. ␤-EP or CSF was continuously perfused using an Alzet mini pump
(model 2002; ALZA, Palo Alto, CA). The osmotic pump was implanted
s.c. and was connected with two infusion cannulae using a Y-connector.
Each infusion cannula reached the PVN through the permanently implanted bilateral guide cannula. Rats infused with naltrexone were sacrificed 4 h after treatment, whereas ␤-EP- or CSF-treated animals were sacrificed after completion of the 16-h infusion. Immediately after sacrifice,
spleen tissues and brains were removed. Spleens were used for NK cell
cytolytic activity. Brains were frozen on dry ice until brain sequential coronal sections were performed to verify the exact position of the infusion site.
Ethanol levels in plasma samples were determined using a commercially
available enzymatic assay (Sigma-Aldrich, St. Louis, MO), where ethanol
concentrations were determined from the absorbency at 340 nm. The minimum amount of alcohol level detected by this assay was 10 mg/dl.
Flow cytometric analysis was used to determine the percentage of NK cells
in spleen cell preparations. Splenic lymphocytes were isolated as described
above in medium containing RPMI 1640, 1% penicillin and streptomycin,
2% glutamine, and 10% untreated FBS and were pelleted by centrifugation.
Cells were resuspended in flow cytometry buffer (PBS with 0.1% BSA and
0.1% sodium azide, pH 7.4) at a concentration of 106 cells/100 ␮l in a
96-well U-bottom culture plate and were pelleted again by centrifugation.
Cells were stained according to the following procedures. The Ab was
titered and used at an optimum concentration of 1 ␮g/well. The rat monoclonal Ab 2.4G2 was added initially to all wells to block Ab binding via F
receptors. Irrelevant Ab-stained cells were used as controls. The plates
were kept on ice during the labeling and washing steps. NK cells were
identified using FITC-labeled anti-CD161 Ab. Cells were incubated with
this Ab for 30 min, washed with flow cytometry buffer twice, and finally
suspended in 200 ␮l of flow cytometry buffer for analysis. Flow cytometric
analysis was performed on a FACScan with Lysis 2 data analysis software
(BD Biosciences, Mountain View, CA). Lymphocytes were gated according to forward and side scatter properties. Data were accumulated for 104
gated cells.
The IR-␤-EP levels in plasma and the hypothalamus were measured by a
RIA as described by us previously (32). Hypothalamic tissues were extracted with 0.1 N HCl, and 10 ␮l of extract of each sample was used in
duplicate in the RIA. Plasma samples from blood were obtained and used
directly for measurement of IR-␤-EP levels. The RIA system used a ␤-EP
antiserum Y10 (S. S. C. Yen, University of California, San Diego, CA),
which cross-reacted with ␤-EP (100%) and with ␤-lipotropin (15–20%) on
a molar basis. The minimum amount of ␤-EP detectable was 3 pg/tube at
1/28,000 antiserum dilution. IR-␤-EP characterized by gel chromatography
from hypothalamic tissue extracts and from plasma extracts showed a major ␤-EP component and a small ␤-lipotropin component (32), suggesting
that the values obtained by this RIA represented mostly ␤-EP peptides. The
protein content in hypothalamic extracts was determined using the
bicinchroninic acid (Pierce, Rockford, IL) protein assay reagents. Protein
values were used to calculate the amount of IR-␤-EP per microgram of
protein in the tissue samples.
Statistics
The mean and SE of the data were determined and are presented in the text
and figures. Data were analyzed using one-way ANOVA. The differences
between groups were determined using the Student-Newmann-Keuls test.
A value of p ⬍ 0.05 was considered to be a significant difference.
Results
Blood alcohol levels and body weights
As expected from our previous findings (30), the alcohol treatment
regimen significantly elevated the levels of blood alcohol (n ⫽ 5;
milligrams per decaliter; day 7, 93.7 ⫾ 16.0; day 14, 131.4 ⫾ 21.0;
day 21, 127.1 ⫾ 9.0; and day 28, 119.0 ⫾ 7.6). The liquid diet
regimen used for alcohol administration did not produce a nutritional deficit because body weights of pair-fed and ad libitum-fed
rats were not significantly different. However, alcohol-fed rats
showed significantly (p ⬍ 0.01) reduced body weight gain as compared with pair-fed and ad libitum-fed rats after 4 wk of treatment
(Table I).
Basal NK cell cytolytic activity
NK cell activity
NK cell cytolytic activity of splenic lymphocytes was determined against
yeast artificial chromosome (YAC)-1 lymphoma cells by a standard 4-h
51
Cr release cytolytic assay as described by us previously (2). Splenocytes
were obtained from whole spleens by pressing tissue through stainless steel
wire-mesh screens. Erythrocytes were removed by a 5-s hypotonic shock
with sterile distilled water. The percentages of cytolytic activity at E:T
ratios of 200:1, 100:1, 50:1, and 25:1 were converted to lytic units per 106
effector cells according to Pross et al. (31). Each assay was conducted in
quadruplicate.
The effect of ethanol consumption on splenic NK cell activity was
determined in rats given an ethanol-containing diet for 1, 2, 3, and
4 wk. As shown in Fig. 1, ethanol intake for 2, 3, and 4 wk reduced
NK cell activity as compared with the activity of pair-fed or ad
libitum-fed animals. Ethanol intake for 1 wk did not have any
significant effect on NK cytolytic activity. The cytolytic activities
of 1-wk ethanol-treated animals were similar to those of pair-fed or
ad libitum-fed animals.
Table I. Changes in body weight at different time intervals in animals fed with a rodent chow ad libitum,
fed with a liquid ethanol diet (alcohol-fed), or pair-fed a liquid diet with sucrose substituted for ethanol
(pair-fed) for 4 wka
Body Weight (g)
Treatment
0 Day
5 Days
10 Days
21 Days
28 Days
Ad lib-fed
Pair-fed
Alcohol-fed
145 ⫾ 2
150 ⫾ 4
140 ⫾ 3
168 ⫾ 3
170 ⫾ 6
161 ⫾ 3
195 ⫾ 6
190 ⫾ 5
185 ⫾ 2
220 ⫾ 4
212 ⫾ 3
208 ⫾ 4
232 ⫾ 4
227 ⫾ 3
217 ⫾ 3*
a
Data are mean ⫾ SE of five animals.
ⴱ, p ⬍ 0.001 significantly different from the ad libitum-fed and pair-fed groups at the same time period.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
Flow cytometric analysis
Radioimmunoassay (RIA) of IR-␤-EP
The Journal of Immunology
5647
Immunoreactive ␤-EP levels in the plasma and hypothalamic
tissue samples
To measure whether the ethanol administration altered in vivo levels of ␤-EP in rats, we determined the concentrations of IR-␤-EP
in hypothalamic extract and in plasma. As shown in Fig. 2A, animals given an ethanol diet for 2, 3, or 4 wk had significantly
reduced levels of IR-␤-EP in the hypothalamic extracts as compared with those in pair-fed or ad libitum-fed animals. In contrast,
animals given the ethanol diet for 1 wk had significantly increased
levels of IR-␤-EP in the hypothalamic extracts. Similarly, the
plasma levels of IR-␤-EP were significantly higher following 1 wk
of ethanol consumption, but were significantly reduced after 2, 3,
or 4 wk of ethanol consumption (Fig. 2B).
FIGURE 2. Effects of ethanol on hypothalamic content (A) and plasma
levels (B) of IR-␤-EP. Mediobasal hypothalamus and plasma samples of ad
libitum-fed, pair-fed, and alcohol-fed male rats used in NK cell activity
determination (Fig. 1) were collected and used in determination of IR-␤-EP
levels by RIA. Data are mean ⫾ SE of six to nine observations. ⴱ, p ⬍
0.001 significantly different from the ad libitum-fed and pair-fed groups at
the same time period.
Effects of chronic ethanol on NK cell numbers in the spleen
Flow cytometric analysis was used to determine the percentage of
NK cells in spleen cell preparations. As shown in Fig. 3, a–c,
animals given an ethanol diet for 1 and 2 wk had no significant
change in splenocyte number, total NK cell number, or percentage
of NK cell population. However, animals given an ethanol diet for
3 and 4 wk had significantly reduced number of splenocytes, total
NK cell population, and percentage NK cell population.
NK cell cytolytic response to in vitro ␤-EP and opiate
antagonist naltrexone
The parallel changes in NK cell activity and tissue and plasma
levels of ␤-EP following alcohol treatment suggest the possibility
of an involvement of the opioid peptide in alcohol-regulated NK
cell function. To test this possibility, we determined the effects of
␤-EP and the opioid receptor blocker naltrexone on splenic NK
cell activity in vitro. As shown in Fig. 4A, ␤-EP increased the in
vitro NK cell cytolytic activity of splenocytes in a concentrationdependent manner. Naltrexone inhibited the ␤-EP-stimulated NK
cell activity (Fig. 4B). Naltrexone by itself did not have any effect
on in vitro NK cell activity. As shown in Fig. 5, 1 wk of ethanol
consumption did not affect the in vitro NK cell response to ␤-EP.
However, 2 wk of ethanol consumption significantly reduced the in
vitro NK cell cytolytic response to ␤-EP.
NK cell cytolytic response to in vivo ␤-EP and opiate antagonist
naltrexone
The role of ␤-EP in ethanol-modulated NK cell function was evaluated in vivo by determining the effects of hypothalamic administration of ␤-EP or naltrexone on splenic NK cell activity. The
opioid peptide and its blocker were infused into the PVN, as a
large number of ␤-EP terminals and ␤-EP-sensitive receptors were
localized in this part of the hypothalamus (19, 33–35). As shown
in Fig. 6, ␤-EP infusion into the PVN increased the NK cell cytolytic activity in ad libitum- or pair-fed control animals. The infusion of the opioid peptide into the PVN failed to increase NK
cell activity in animals fed with an alcohol diet for 2 wk. PVN
administration of the opiate blocker naltrexone in control pair-fed
animals significantly inhibited NK cell activity (Fig. 7). However,
naltrexone failed to significantly reduce NK cell activity in animals
fed with alcohol for 2 wk.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
FIGURE 1. Effects of ethanol on splenic NK cell activity in vivo. Male
rats were fed with rodent chow ad libitum (ad libitum-fed) or fed with a
liquid ethanol diet (alcohol fed) for 1, 2, 3, and 4 wk. A group of rats was
pair-fed a liquid diet with sucrose substituted for ethanol (pair-fed) for 1,
2, 3, or 4 wk. Spleens from these rats were used for NK cell cytolytic assay
using the YAC-1 lymphoma cells, and the cytolytic activity was determined in lytic units. The E:T ratio ranged from 200:1 to 25:1. Data are
mean ⫾ SE of six to nine observations. ⴱ, p ⬍ 0.001 significantly different
from the ad libitum-fed and pair-fed groups at the same time period.
5648
␤-EP MEDIATION OF ETHANOL EFFECT ON NK CELLS
Discussion
FIGURE 3. Changes in the number of splenocytes and NK (CD161⫹)
cells after ethanol treatment. Animals were fed with rodent chow ad libitum, or fed with a liquid ethanol diet (alcohol fed) or pair-fed a liquid diet
with sucrose substituted for ethanol (pair fed) for 1, 2, 3, or 4 wk. Spleens
from these rats were used to determine the number of total splenocytes (A)
and to identify by flow cytometric analysis the number of NK (CD161⫹)
cells (B) or the percentage of NK (CD161⫹) cells from the total splenocytes (C). The CD161 Ab, specific for the NK Ag expressed by NK cells,
was used to measure the percentage or number of NK (CD161⫹) cells
present in spleen cell suspensions. Data are mean ⫾ SE of five to seven
animals. ⴱ, p ⬍ 0.001 significantly different from the ad libitum-fed and
pair-fed groups at the same time period.
The data obtained from in vivo studies presented in this study
demonstrate that ethanol consumption for 2, 3, or 4 wk suppresses
NK cell activity and that ethanol’s inhibition of NK activity is
associated with decreased plasma and hypothalamic content of
␤-EP in male rats. In vivo and in vitro studies also provided evidence that central ␤-EP and peripheral ␤-EP increase NK cell activity and that an opioid receptor antagonist, naltrexone, blocks
these responses. Additionally, ethanol-treated animals showed a
reduced NK cell response to ␤-EP. Together, these data provide
evidence for the first time that chronic administration of alcohol
reduces NK cell cytolytic activity by altering ␤-EP-regulated NK
cell function.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
FIGURE 4. Effects of ␤-EP and naltrexone on splenic NK cell activity
in vitro. A, Lymphocytes were isolated from spleens of male rats and incubated with various concentrations of ␤-EP or no ␤-EP at 37°C for a
period of 2 h before NK cell cytolytic assay. B, Lymphocytes were isolated
from spleens of male rats and preincubated with different concentrations of
naltrexone at 37°C for 2 h. ␤-EP was then added to the cells at a concentration of 1 ng/ml. Lymphocytes were incubated at 37°C for an additional
2-h period before NK cell cytolytic assay. Controls received vehicle. NK
cell activity was measured in a 4-h cytolytic assay in the presence of drugs
using the YAC-1 lymphoma cell line. Data are mean ⫾ SE of lytic units
obtained from five to nine experiments. ⴱ, p ⬍ 0.001 significantly different
from the rest of the groups. a, p ⬍ 0.001 significantly different from the
control group. b, p ⬍ 0.001 significantly different from the ␤-EP alonetreated group.
The Journal of Immunology
The action of ethanol on NK cells in rats has not been well
studied. To our knowledge, this is the first study in which timedependent effects of ethanol on NK cells were determined. With
careful attention to nutritional status and ethanol intake, studies in
which mice are given high levels of ethanol (20%, w/v) in the
drinking water showed inhibition of NK cell cytolytic activity (2,
6, 7). Wu and Pruett (8, 10), in a binge alcohol model, also showed
inhibition of NK cell cytolytic activity and a decrease in cell percentage and number in mice. Data presented in this report indicate
that ethanol has an inhibitory effect on NK cell cytolytic activity
independent of NK cell loss at 2 wk. Thereafter, it is probably a
combination of loss and the independent effect on NK cytolytic
activity.
The relevance of rodent models of alcohol intake to NK cells in
humans modulated by alcohol is still largely unknown. Assessments of NK cell activity in humans is complicated by the fact that
many patients use tobacco and other drugs that are known to affect
NK cell activity (36, 37). Furthermore, alcoholics are frequently
malnourished (38). They may have underlying viral infections and
FIGURE 6. Effect of ethanol consumption on ␤-EP-stimulated NK cell
activity in vivo. Male rats were ad libitum-fed a chow diet, pair-fed a liquid
diet, or fed an alcohol-containing liquid diet for 1 wk (A) or 2 wk (B).
These rats were infused with ␤-EP (2.6 ␮g/18 ␮l) or vehicle (18 ␮l) into
each PVN for a period of 16 h and were then sacrificed. NK cell activity
was measured in a 4-h cytolytic assay in the presence of drugs using the
YAC-1 lymphoma cell line. The E:T ratio ranged from 200:1 to 25:1. Data
are mean ⫾ SE of lytic unit obtained from five to six experiments. a, p ⬍ 0.001
significantly different from ad libitum-fed and pair-fed animals. b, p ⬍ 0.001
significantly different from the vehicle-treated group fed similar diet.
may have varying degrees of liver damage, which also affects immune responses. Patients with chronic active hepatitis and cirrhosis have low NK activity (39, 40). However, it is unclear whether
the immunological abnormalities in human alcoholics result from
generalized malnutrition, malabsorption of specific nutrients, or
direct ethanol exposure (38, 41, 42). Charpentier et al. (43) found
that NK cell activity was impaired only in alcoholic subjects with
inactive cirrhosis. Additionally, the decrease was more pronounced in cirrhotic patients with severe malnutrition. In contrast,
Saxena et al. (44) observed enhanced NK cell activity in peripheral
blood lymphocytes from alcoholic subjects despite compounding
factors such as inadequate nutritional status, clinical illness, drug
addiction, and the fact that 70% of the subjects smoked. In other
studies, sober chronic alcoholics who were well nourished and free
of underlying diseases had no demonstrable abnormality in immune function, including NK cell activity (45). Additionally, NK
cell activity was decreased in depressed patients with alcoholism
(12). A recent publication by Cook et al. (13) showed a trend
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
FIGURE 5. Effect of ethanol consumption on ␤-EP-stimulated NK cell
activity in vitro. Lymphocytes were isolated from ad libitum-fed, pair-fed,
and alcohol-fed male rats treated for a period of 1 wk (A) or 2 wk (B).
Lymphocytes were cultured at 37°C and were treated with vehicle (controls) or ␤-EP at 1 ng/ml for 2 h before NK cell cytolytic assay. NK cell
activity was measured in a 4-h cytolytic assay in the presence of ␤-EP. Data
are mean ⫾ SE obtained from five rats. a, p ⬍ 0.001 significantly different
from ad libitum-fed and pair-fed animals. b, p ⬍ 0.001 significantly different
from the vehicle-treated group treated and fed a similar diet.
5649
5650
toward lower NK cell activity in alcoholics without liver disease
and a loss of NK cells in alcoholics with liver disease. Although
additional research is needed to clarify the role of alcohol in human alcoholics, a majority of these studies suggest a reduced NK
cell function.
The data on the varying effects of ethanol on hypothalamic ␤-EP
are in agreement with previously published studies in rats (26 –29).
It has been shown that acute treatment of ethanol stimulates hypothalamic content of ␤-EP and its precursor proopiomelanocortin
(POMC) mRNA, whereas chronic treatment with ethanol reduced
␤-EP and POMC mRNA levels in this tissue. Similar biphasic
actions of ethanol on ␤-EP and POMC mRNA levels were observed in hypothalamic cells in primary culture (46 – 49). It is believed that hypothalamic ␤-EP neurons, when exposed to a longer
duration of ethanol, develop desensitization due to adaptive
changes in cellular signaling mechanisms (50). The data presented
in this study suggest that the plasma ␤-EP exhibits a biphasic response to ethanol depending on the length of treatment. The source
of ␤-EP in plasma is believed to be primarily from the pituitary
gland (19). However, it is also possible that the other peripheral
tissues, including the adrenal gland and lymphocytes, contribute to
the circulatory levels of ␤-EP (20, 21). It could be concluded that
ethanol administration for a period of 2 wk or longer also reduces
␤-EP secretion from the peripheral organs that contribute to the
circulatory ␤-EP.
The results presented in this study demonstrate that the reduction of hypothalamic and plasma ␤-EP following chronic ethanol
intake correlates with the reduction of NK cell cytolytic activity.
The positive association between the changes of NK cell function
and hypothalamic ␤-EP levels in ethanol-treated animals provides
correlative evidence for an involvement of the opioid peptide in
controlling NK cell function. This view is further supported by
both in vivo and in vitro data that ␤-EP stimulates NK cell function, which is blocked by the opiate antagonist naltrexone. The
ability of morphine and enkephaline to affect various types of immune cells has been documented previously (51–55). However,
there are contradictory evidence from studies on enkephaline and
morphine on NK cell functions. The treatment with morphine de-
creased NK cell activity (53, 54), whereas the treatment with enkephaline increased NK cell activity or had no effect (51, 52). The
morphine inhibitory effect on NK cell appears not to be mediated
via hypothalamic opioid system, as the morphine effect is only
observed after direct administration into the periaqueductal gray
matter but not after administration into different areas of the hypothalamus (55).
The stimulatory action of ␤-EP on NK cell function could occur
by increasing the killing activity or by increasing the cytokine
secretion. The data presented in this study revealed the cytolytic
activity of NK cells. In a preliminary study, we determined the
plasma immunoreactive IFN-␥ levels in alcohol-treated rats and
pair-fed animals. We found no changes in the plasma levels of
IFN-␥ following 2 and 4 wk of alcohol administration (data not
shown). Although these data support the concept that ␤-EP increases the killing activity of NK cells, further studies determining
in vivo and in vitro levels of cytokines (e.g., IFN-␥), killing molecules (e.g., perforin and granzyme), and NK surface markers
(e.g., Fas ligand) are needed to establish the mechanism by which
the opioid peptide alters NK cell function.
Previously, it has been shown that the ␤-EP peptide enhances
splenic lymphocyte proliferation in response to concanavalin A
and increases the killing activity of NK cells (18, 22–24). IR-␤-EP
is also present in lymphocytes (56) and macrophages (20). POMC
mRNA transcript has been identified in splenic lymphocytes (57,
58). Both acetylated and nonacetylated ␤-EP are produced in
splenic lymphocytes (20). [125I]␤-EP can bind to splenocytes (26),
and the bound [125I]␤-EP can be displaced by N-acetylated ␤-EP
and C-terminal fragments of ␤-EP but not by naloxone (25). Recently, a high-affinity ⑀-like opioid receptor has been identified on
lymphocytes that is specific for ␤-EP (59). In addition to these
high-affinity opioid receptors, lymphocytes possess ⭸, ␬, and ␮
opioid receptors (59). It has been shown that endogenous opioids
up-regulate human and rat NK cell activity (18, 22). Hence, it
appears that ␤-EP is an important regulator of NK cell function.
Although a positive association between ␤-EP and NK cell activity following 2 wk of ethanol treatment was observed in this
study, the early action of ethanol on ␤-EP and NK cell function did
not correlate positively. Considering the stimulatory action of
␤-EP on NK cells, we anticipated that initial ethanol consumption
would stimulate NK cells because ethanol treatment stimulates hypothalamic and peripheral ␤-EP. However, 1 wk of ethanol treatment did not significantly change NK cell function. One explanation for this discrepancy is that during the early phase of action,
ethanol stimulation of ␤-EP-regulated NK cell function was overcome by NK cell inhibitory factors. It has been shown previously
that acute ethanol treatment elevates secretion of hormones from
the hypothalamic pituitary adrenal axis, leading to increased release of plasma corticosterone and hypothalamic corticotropin-releasing hormone (60 – 62). Although the inhibitory influence of
glucocorticoids on NK cell function remains to be elucidated in
humans (63– 65), studies in rats suggest that corticotropin-releasing hormone activates the sympathetic nervous system and releases norepinephrine to inhibit NK cell function (66 – 68). Hence,
it is possible that the stimulatory influence of ␤-EP during the early
ethanol administration is also compensated by the inhibitory influences of the hormones of the hypothalamic pituitary
adrenal axis.
In summary, the data presented in this study indicate that ␤-EP
stimulates NK cell cytolytic activity. Additionally, the lymphocytes appear to produce the receptors for the opioid peptide (65).
In this study, we have presented evidence that chronic administration of ethanol reduces the levels of both hypothalamic and peripheral ␤-EP. We have also provided evidence that the influence
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
FIGURE 7. Effect of ethanol consumption on naltrexone-inhibited NK
cell activity in vivo. Male rats were ad libitum-fed a chow diet, pair-fed a
liquid diet, or fed alcohol-containing liquid diet for 2 wk. These rats were
infused with a single bolus of naltrexone (1 ␮g/5 ␮l) or vehicle (5 ␮l) into
each PVN, and were sacrificed 4 h later. NK cell activity was measured in
a cytolytic assay using the YAC-1 lymphoma cell line. The E:T ratio
ranged from 200:1 to 25:1. Data are mean ⫾ SE of lytic unit obtained from
five to six experiments. a, p ⬍ 0.001 significantly different from pair-fed
and vehicle-treated animals.
␤-EP MEDIATION OF ETHANOL EFFECT ON NK CELLS
The Journal of Immunology
of hypothalamic and peripheral ␤-EP on NK cell cytolytic activity
is reduced by ethanol treatment. This addictive agent lowered the
response to PVN-administered ␤-EP, as well as the direct response
to ␤-EP in vitro. Hence, alcohol’s inhibitory action on NK cell
function may involve a reduction in both.
References
30. De, A., N. Iv. Boyadjieva, M. Pastorcic, and D. K. Sarkar. 1996. Potentiation of
estrogen’s mitogenic effect on the pituitary gland by alcohol consumption. Int.
J. Oncol. 7:643.
31. Pross, H. F., M. G. Baines, P. Rubin, P. Shragge, and M. S. Paterson. 1981.
Spontaneous human lymphocyte-mediated cytotoxicity against tumor target cell:
the quantitation of natural killer cell activity. J. Clin. Immunol. 1:51.
32. Sarkar, D. K., and S. S. C. Yen. 1985. Changes in ␤-endorphin-like immunoreactivity in pituitary portal blood during the estrous cycle and after ovariectomy in
rats. Endocrinology 116:2075.
33. Mezey, E., J. Kiss, T. O’Donohue, R. Eskay, and M. Palkovits. 1988. Distribution
of pro-opiomelanocortin derived peptides (ACTH, ␣-MSH, ␤-endorphin) in the
rat hypothalamus. Brain Res. 328:341.
34. Wilcox, J. N., J. L. Roberts, B. M. Chronwall, J. F. Bishop, and T. O’Donohue.
1986. Localization of proopiomelanocortin mRNA in functional subsets of neurons defined by their axonal projections. J. Neurosci. Res. 16:89.
35. O’Donohue, T. L., and D. M. Dorsa. 1982. The opiomelanotropinergic neuronal
and endocrine systems. Peptides 3:353.
36. Ferson, M., A. Edwards, A. Lind, G. W. Milton, and P. Hersey. 1979. Low
natural killer-cell activity and immunoglobulin levels associated with smoking in
human subjects. Int. J. Cancer 23:603.
37. Klimas, N. G., R. Morgan, N. T. Blaney, D. Chitwood, J. B. Page, K. Milles, and
M. A. Fletcher. 1990. Alcohol and immune function in HIV-1 seronegative,
HTLV- I/II seronegative and positive men on methadone. Prog. Clin. Biol. Res.
325:103.
38. Shaw, S., and C. S. Lieber. 1978. Plasma amino acid abnormalities in the alcoholic: respective role of alcohol, nutrition and liver injury. Gastroenterology 74:
677.
39. Vierling, J. M., D. L. Nelson, W. Strober, and B. M. Bundy. 1977. In vitro
cell-mediated cytotoxicity in primary biliary cirrhosis and chronic hepatitis.
J. Clin. Invest. 60:1116.
40. Serdengecti, S., D. B. Jones, G. Holdstock, and R. Wright. 1981. Natural killer
activity in patients with biopsy-proven liver disease. Clin. Exp. Immunol. 45:361.
41. Yew, M. S., S. Moore, and M. M. Biesele. 1981. Effects of chronic “moderate”
alcohol consumption on vitamins A and C status of male Sprague-Dawley rats.
Nutr. Rep. Int. 23:427.
42. Neville, J. N., J. A. Eagles, G. Samson, and R. E. Olson. 1968. Nutrition status
of alcoholics. Am. J. Clin. Nutr. 21:1329.
43. Charpentier, B., D. Franco, L. Paci, M. Charra, B. Martin, D. Vuitton, and
D. Fries. 1984. Deficient natural killer cell activity in alcoholic cirrhosis. Clin.
Exp. Immunol. 58:107.
44. Saxena, Q. B., E. Mezey, and W. H. Adler. 1980. Regulation of natural killer
activity in vivo: the effect of alcohol consumption on human peripheral blood
natural killer activity. Int. J. Cancer 26:413.
45. Kronfol, Z., M. Nair, E. Hill, P. Kroll, K. Brower, and J. Greden. 1993. Immune
function in alcoholism: a controlled study. Alcoholism 17:279.
46. Sarkar, D. K., and S. Minami. 1990. Effect of acute ethanol on ␤-endorphin
secretion from rat fetal hypothalamic neurons in cultures. Life Sci. 47:PL31.
47. Boyadjieva, N., and D. K. Sarkar. 1994. Effects of chronic ethanol on ␤-endorphin secretion from hypothalamic neurons in primary cultures: evidence for ethanol tolerance, withdrawal and sensitization responses. Alcohol. Clin. Exp. Res.
18:1497.
48. Reddy, B. V., N. Boyadjieva, and D. K. Sarkar. 1995. Effect of ethanol, propanol,
butanol and catalase enzyme blockers on ␤-endorphin secretion from primary
cultures of hypothalamic neurons: evidence for mediatory role of acetaldehyde in
ethanol stimulation of ␤-endorphin release. Alcohol. Clin. Exp. Res. 19:339.
49. Pastorcic, M., N. Boyadjieva, and D. K. Sarkar. 1994. Comparison of the effects
of ethanol and acetaldehyde on proopiomelanocortin mRNA expression and
␤-endorphin secretion from hypothalamic neurons in primary cultures. Cell. Mol.
Neurosci. 5:580.
50. Boyadjieva, N., B. Reddy, and D. K. Sarkar. 1997. Forskolin delays the ethanolinduced desensitization of hypothalamic ␤-endorphin neurons in primary cultures. Alcohol. Clin. Exp. Res. 21:477.
51. Oleson, D. R., and D. R. Johnson. 1988. Regulation of human natural cytotoxicity
by enkephalins and selective opiate agonists. Brain Behav. Immun. 2:171.
52. Gabriovac, J., M. Antica, and M. Osmak. 1992. In vivo bidirectional regulation
of mouse natural killer (NK) cell cytotoxic activities by leu-enkephalins: reversibility by naloxone. Life Sci. 50:29.
53. Shavit, Y., J. W. Lewis, G. W. Terman, R. P. Gale, and J. C. Liebeskind. 1984.
Opioid peptides mediate the suppressive effect of stress on natural killer cell
cytotoxicity. Science 223:188.
54. Freier, D. O., and B. A. Fuchs. 1994. A mechanism of action for morphineinduced immunosuppression: corticosterone mediates morphine-induced suppression of natural killer cell activity. J. Pharmacol. Exp. Ther. 270:1127.
55. Weber, R. J., and A. Pert. 1989. The periaqueductal gray matter mediate opiateinduced immunosuppression. Science 245:188.
56. Smith, E. M., and J. E. Blalock. 1981. Human lymphocyte production of corticotropin and endorphin-like substances: association with leukocyte interferon.
Proc. Natl. Acad. Sci. USA 78:7530.
57. Endo, Y., T. Sakato, and S. Watanabe. 1985. Identification of proopiomelanocortin-producing cells in the rat pyloric antrum and duodenum by in situ mRNAcDNA hybridization. Biomed. Res. 6:253.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
1. Herberman, R. B., and J. R. Ortaldo. 1981. Natural-killer cells: their role in
defense against disease. Science 214:24.
2. Blank, S. E., D. A. Duncan, and G. G. Meadows. 1991. Suppression of natural
killer cell activity by ethanol consumption and food restriction. Alcohol. Clin.
Exp. Res. 15:16.
3. Hercend, T., and R. E. Schmidt. 1988. Characteristics and uses of natural killer
cells. Immunol. Today 9:291.
4. Lewis, C. E., and J. O’ D. McGee. 1992. The Natural Killer Cell. IRL Press,
Oxford.
5. Meadows, G. G., S. E. Blank, and D. D. Duncan. 1989. Influence of ethanol
consumption on natural killer cell activity in mice. Alcohol. Clin. Exp. Res. 13:
476.
6. Gallucci, R. M., L. J. Pfister and G. G. Meadows. 1994. Effects of ethanol consumption on enriched natural killer cells from C57BL/6 mice. Alcohol. Clin. Exp.
Res. 18:625.
7. Abdallah, R. M., J. R. Starkey, and G. G. Meadows. 1982. Ethanol and related
dietary effects on mouse natural killer cell activity. Immunology 50:131.
8. Wu, W. J., and S. B. Pruett. 1994. Ethanol decreases the number and activity of
splenic natural killer cells in a mouse model for binge drinking. J. Pharmacol.
Exp. Ther. 271:722.
9. Carson, E., and S. B. Pruett. 1995. Development and characterization of a binge
drinking model in mice for evaluation of the immunological effects of ethanol.
Alcohol. Clin. Exp. Res. 20:132.
10. Wu, W. J., and S. B. Pruett. 1996. Suppression of splenic natural killer cell
activity in a mouse model for binge drinking: role of the neuroendocrine system.
J. Pharmacol. Exp. Ther. 278:1331.
11. Bounds, W., K. W. Betzing, R. M. Steward, and R. F. Holcombe. 1994. Social
drinking and the immune response: impairment of lymphokine-activated killer
activity. Am. J. Med. Sci. 11:85.
12. Irwin, M., C. Caldwell, T. L. Smith, S. Brown, M. A. Schuckit, and J. C. Gillin.
1990. Major depressive disorder, alcoholism, and reduced natural killer cell cytotoxicity. Arch. Gen. Psychiatry 47:713.
13. Cook, R. T., F. Li, D. Vandersteen, Z. K. Ballas, B. L. Cook, and
D. R. LaBrecque. 1997. Ethanol and natural killer cells: activity and immunophenotype in alcoholic humans. Alcohol. Clin. Exp. Res. 21:974.
14. Otraldo, J. R., J. P. Gerard, L. E. Henderson, R. H. Neubauer, and H. Rabin. 1983.
Responsiveness of purified natural killer cells to pure interleukin-2 (IL-2). In
Interleukin Lymphokines and Cytokines. J. J. Oppenheim and H. Rabin, eds.
Academic Press, New York, p. 63.
15. Santoli, D., and N. Koprowski. 1979. Mechanisms of activation of human natural
killer cells against tumor and virus-infected cells. Immunol. Rev. 44:125.
16. Ahmed, M. S., J. Llanos, and C. M. Blatties. 1984. Interleukin-1 interacts with
opioid binding sites. Proc. Soc. Neurosci. Abstr. 10:1109.
17. Dafny, N. 1983. Modification of morphine withdrawal by interferon. Life Sci.
32:303.
18. Kay, N. E., J. Allen, and J. E. Morley. 1984. Endorphins stimulate normal human
peripheral blood lymphocyte natural killer activity. Life Sci. 35:53.
19. Rossier, J., T. M. Vargo, S. Minick, N. Ling, F. E. Bloom, and R. Guillemin.
1977. Regional dissociation of ␤-endorphin and enkephalin contents in rat brain
and pituitary. Proc. Natl. Acad. Sci. USA 11:5162.
20. Lolait, S. J., J. A. Clements, A. J. Markwick, C. Cheng, M. McNally, A. I. Smith,
and J. W. Funder. 1986. Pro-opiomelanocortin messenger ribonucleic acid and
post-translational processing of ␤-endorphin in spleen macrophages. J. Clin. Invest. 77:1776.
21. Ganong, W. F., M. F. Dallman, and J. L. Roberts. 1987. The hypothalamicpituitary-adrenal axis revisited. Ann. NY Acad. Sci. 512:176.
22. Mathews, P. M., C. J. Froelich, W. L. Sibbit, Jr., and A. D. Bankhurst. 1983.
Enhancement of natural cytotoxicity by ␤-endorphin. J. Immunol. 130:1658.
23. Mandler, R. N., W. Z. E. Biddison, R. Mandler, and S. A. Serrate. 1986. ␤-Endorphin augments the cytolytic activity and interferon production of NK cells.
J. Immunol. 136:934.
24. Froehlich, C. J., and A. D. Bankhurst. 1984. The effect of ␤-endorphin on natural
cytotoxicity and antibody-dependent cellular cytotoxicity. Life Sci. 35:261.
25. Shahabi, N. A., K. M. Linner, and B. M. Sharp. 1990. Murine splenocytes express
a naloxone-insensitive binding site for ␤-endorphin. Endocrinology 126:1442.
26. Froehlich, J. C. 1993. Interaction between alcohol and the endogenous opioid
system. In Alcohol and the Endocrine System, Monograph 23. S. Zakhari, ed.
National Institute on Alcohol Abuse and Alcoholism Research Monograph Series, U.S. Government Printing Office, Washington, D.C., p. 21.
27. Gianoulakis, C. 1990. Characterization of the effects of acute ethanol administration on the release of ␤-endorphin peptides by the rat hypothalamus. Eur.
J. Pharmacol. 180:21.
28. Gianoulakis, C. 1996. Implications of endogenous opioids and dopamine in alcoholism: human and basic science studies. Alcohol Alcohol. 31(Suppl. 1):33.
29. Vescovi, P. P., V. Coiro, R. Volpi, A. Gianni, and M. Passeri. 1992. Plasma
␤-endorphin but not met-enkephalin levels are abnormal in chronic alcoholics.
Alcohol Alcohol. 27:471.
5651
5652
58. Westly, H. J., A. J. Kleiss, K. W. Kelly, P. K. Y. Wong, and P.-H. Yuen. 1986.
Newcastle disease virus-infected splenocytes express the proopiomelanocortin
gene. J. Exp. Med. 163:1589.
59. Hazum, E., K.-J. Chang, and P. Cuatrecases. 1979. Specific nonopiate receptors
for ␤-endorphin. Science 205:1033.
60. Rivier, C. 1995. Interaction between stress and immune signals on the hypothalamic-pituitary-adrenal axis of the rat: influence of drugs. In The Reproductive
Neuroendocrinology of Aging and Drug Abuse. D. K. Sarkar and C. D. Barnes,
eds. CRC Press, Boca Raton, FL, p. 169.
61. Rivier, C., and S. Lee. 1996. Acute alcohol administration stimulates the activity
of hypothalamic neurons that express corticotropin-releasing factor and vasopressin. Brain Res. 726:1.
62. Rasmussen, D. D., C. A. Bryant, B. M. Boldt, E. A. Colasurdo, N. Levin, and
C. W. Wilkinson. 1998. Acute alcohol effects on opiomelanocortinergic regulation. Alcohol. Clin. Exp. Res. 22:789.
63. Bodner, G., A. Ho, and M. J. Kreek. 1998. Effect of endogenous cortisol levels
on natural killer cell activity in healthy humans. Brain Behav. Immun. 12:285.
␤-EP MEDIATION OF ETHANOL EFFECT ON NK CELLS
64. Gatti, G., R. G. Masera, L. Pallavicini, M. L. Sartori, A. Staurengi, F. Orlandi,
and A. Angeli. 1993. Interplay in vitro between ACTH, ␤-endorphin and glucocorticosteroids in the modulation of spontaneous and lymphokine-inducible
human natural killer (NK) cell activity. Brain Behav. Immun. 7:16.
65. Holbrook, N. J., W. I. Cox, and H. C. Horner. 1983. Direct suppression of natural
killer activity in human peripheral blood leukocyte cultures by glucocorticoids
and its modulation by interferon. Cancer Res. 43:4019.
66. Irwin, M. 1994. Stress-induced immune suppression: role of brain corticotropin-releasing
hormone and autonomic nervous system mechanisms. Adv. Neuroimmunol. 4:29.
67. Irwin, M., R. Hauger, and M. Brown. 1992. Central corticotropin-releasing hormone activates the sympathetic nervous system and reduces immune function:
increased responsivity of the aged rat. Endocrinology 131:1047.
68. Nistico, G., M. C. Caroleo, M. Arbitrio, and Pulvirenti, L. 1994. Corticotropin-releasing factor microinfused into locus coeruleus produces electrocortical desynchronization and immunosuppression. Neuroimmunomodulation 1:135.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017