HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Boyadjieva, N. Articles by Sarkar, D. K. Search for Related Content PubMed PubMed Citation Articles by Boyadjieva, N. Articles by Sarkar, D. K.

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. Sarkar^1,*

^* 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

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Natural killer cells have been implicated in immune surveillance against tumors and bacterial and viral infections (1, 2, 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, 6, 7, 8, 9, 10). A few studies conducted using humans also show reduction in NK cell functions following alcohol administration (11, 12, 13). The mechanism by which ethanol alters NK cell function is not known.

NK cells quickly respond to immune activation signals (14, 15, 16). NK cell cytotoxic activity is increased by the lymphokine IFN-, which has a number of opioid-like effects (17). Additionally, IFN-mediated NK cell cytolytic activity is blocked by the opioid receptor antagonist (18). The opioid peptide -endorphin (-EP)³ is produced in the hypothalamus, in the pituitary, and in other peripheral glands (19, 20, 21). -EP enhances splenic lymphocyte proliferation in response to concanavalin A and increases the killing activity of NK cells (22, 23, 24). Opioid receptors are present on lymphocytes (25). Because the function of endogenous -EP is altered in alcoholic patients and alcohol-treated animals (26, 27, 28, 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

Top Abstract Introduction Materials and Methods Results Discussion References

 
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.

Intraparaventricular administration of -EP or naltrexone

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 ethanol-fed 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/µl. 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 µl/h in each PVN for a period of 16 h. Control animals received an additional 0.5 µl 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.

Flow cytometric analysis

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 µl 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 µg/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 µl 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.

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 ⁵¹Cr 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.

Assay for blood levels of ethanol

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.

Radioimmunoassay (RIA) of IR--EP

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 µl 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

Top Abstract Introduction Materials and Methods Results Discussion References

 
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).

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.

View larger version (41K): [in this window] [in a new window]   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. *, p < 0.001 significantly different from the ad libitum-fed and pair-fed groups at the same time period.

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).

View larger version (23K): [in this window] [in a new window]   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. *, p < 0.001 significantly different from the ad libitum-fed and pair-fed groups at the same time period.

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.

View larger version (24K): [in this window] [in a new window]   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. *, p < 0.001 significantly different from the ad libitum-fed and pair-fed groups at the same time period.

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 concentration-dependent 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.

View larger version (18K): [in this window] [in a new window]   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. *, 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 alone-treated group.

View larger version (25K): [in this window] [in a new window]   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.

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, 34, 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.

View larger version (27K): [in this window] [in a new window]   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 µg/18 µl) or vehicle (18 µl) 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.

View larger version (29K): [in this window] [in a new window]   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 µg/5 µl) or vehicle (5 µl) 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.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The action of ethanol on NK cells in rats has not been well studied. To our knowledge, this is the first study in which time-dependent 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 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 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, 27, 28, 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, 47, 48, 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, 52, 53, 54, 55). However, there are contradictory evidence from studies on enkephaline and morphine on NK cell functions. The treatment with morphine decreased 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, 23, 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 µ 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, 61, 62). Although the inhibitory influence of glucocorticoids on NK cell function remains to be elucidated in humans (63, 64, 65), studies in rats suggest that corticotropin-releasing hormone activates the sympathetic nervous system and releases norepinephrine to inhibit NK cell function (66, 67, 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 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.

	Footnotes

 
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: sarkar{at}aesop.rutgers.edu

³ 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.

Received for publication January 26, 2001. Accepted for publication September 18, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Herberman, R. B., J. R. Ortaldo. 1981. Natural-killer cells: their role in defense against disease. Science 214:24.[Abstract/Free Full Text]
2. Blank, S. E., D. A. Duncan, G. G. Meadows. 1991. Suppression of natural killer cell activity by ethanol consumption and food restriction. Alcohol. Clin. Exp. Res. 15:16.[Medline]
3. Hercend, T., R. E. Schmidt. 1988. Characteristics and uses of natural killer cells. Immunol. Today 9:291.[Medline]
4. Lewis, C. E., J. O’ D. McGee. 1992. The Natural Killer Cell IRL Press, Oxford.
5. Meadows, G. G., S. E. Blank, D. D. Duncan. 1989. Influence of ethanol consumption on natural killer cell activity in mice. Alcohol. Clin. Exp. Res. 13:476.[Medline]
6. Gallucci, R. M., L. J. Pfister, G. G. Meadows. 1994. Effects of ethanol consumption on enriched natural killer cells from C57BL/6 mice. Alcohol. Clin. Exp. Res. 18:625.[Medline]
7. Abdallah, R. M., J. R. Starkey, G. G. Meadows. 1982. Ethanol and related dietary effects on mouse natural killer cell activity. Immunology 50:131.
8. Wu, W. J., 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.[Abstract/Free Full Text]
9. Carson, E., 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., 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.[Abstract/Free Full Text]
11. Bounds, W., K. W. Betzing, R. M. Steward, 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, J. C. Gillin. 1990. Major depressive disorder, alcoholism, and reduced natural killer cell cytotoxicity. Arch. Gen. Psychiatry 47:713.[Abstract]
13. Cook, R. T., F. Li, D. Vandersteen, Z. K. Ballas, B. L. Cook, D. R. LaBrecque. 1997. Ethanol and natural killer cells: activity and immunophenotype in alcoholic humans. Alcohol. Clin. Exp. Res. 21:974.[Medline]
14. Otraldo, J. R., J. P. Gerard, L. E. Henderson, R. H. Neubauer, H. Rabin. 1983. Responsiveness of purified natural killer cells to pure interleukin-2 (IL-2). J. J. Oppenheim, and H. Rabin, eds. Interleukin Lymphokines and Cytokines 63. Academic Press, New York.
15. Santoli, D., N. Koprowski. 1979. Mechanisms of activation of human natural killer cells against tumor and virus-infected cells. Immunol. Rev. 44:125.[Medline]
16. Ahmed, M. S., J. Llanos, 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.[Medline]
18. Kay, N. E., J. Allen, J. E. Morley. 1984. Endorphins stimulate normal human peripheral blood lymphocyte natural killer activity. Life Sci. 35:53.[Medline]
19. Rossier, J., T. M. Vargo, S. Minick, N. Ling, F. E. Bloom, 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, 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, J. L. Roberts. 1987. The hypothalamic-pituitary-adrenal axis revisited. Ann. NY Acad. Sci. 512:176.[Abstract]
22. Mathews, P. M., C. J. Froelich, Jr W. L. Sibbit, A. D. Bankhurst. 1983. Enhancement of natural cytotoxicity by -endorphin. J. Immunol. 130:1658.[Abstract]
23. Mandler, R. N., W. Z. E. Biddison, R. Mandler, S. A. Serrate. 1986. -Endorphin augments the cytolytic activity and interferon production of NK cells. J. Immunol. 136:934.[Abstract]
24. Froehlich, C. J., A. D. Bankhurst. 1984. The effect of -endorphin on natural cytotoxicity and antibody-dependent cellular cytotoxicity. Life Sci. 35:261.[Medline]
25. Shahabi, N. A., K. M. Linner, B. M. Sharp. 1990. Murine splenocytes express a naloxone-insensitive binding site for -endorphin. Endocrinology 126:1442.[Abstract]
26. Froehlich, J. C.. 1993. Interaction between alcohol and the endogenous opioid system. S. Zakhari, ed. Alcohol and the Endocrine System, Monograph 23 21. National Institute on Alcohol Abuse and Alcoholism Research Monograph Series, U.S. Government Printing Office, Washington, D.C..
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.[Medline]
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, M. Passeri. 1992. Plasma -endorphin but not met-enkephalin levels are abnormal in chronic alcoholics. Alcohol Alcohol. 27:471.[Abstract/Free Full Text]
30. De, A., N. Iv. Boyadjieva, M. Pastorcic, 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, 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.[Medline]
32. Sarkar, D. K., 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.[Abstract]
33. Mezey, E., J. Kiss, T. O’Donohue, R. Eskay, 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, T. O’Donohue. 1986. Localization of proopiomelanocortin mRNA in functional subsets of neurons defined by their axonal projections. J. Neurosci. Res. 16:89.[Medline]
35. O’Donohue, T. L., D. M. Dorsa. 1982. The opiomelanotropinergic neuronal and endocrine systems. Peptides 3:353.[Medline]
36. Ferson, M., A. Edwards, A. Lind, G. W. Milton, P. Hersey. 1979. Low natural killer-cell activity and immunoglobulin levels associated with smoking in human subjects. Int. J. Cancer 23:603.[Medline]
37. Klimas, N. G., R. Morgan, N. T. Blaney, D. Chitwood, J. B. Page, K. Milles, 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.[Medline]
38. Shaw, S., C. S. Lieber. 1978. Plasma amino acid abnormalities in the alcoholic: respective role of alcohol, nutrition and liver injury. Gastroenterology 74:677.[Medline]
39. Vierling, J. M., D. L. Nelson, W. Strober, 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, R. Wright. 1981. Natural killer activity in patients with biopsy-proven liver disease. Clin. Exp. Immunol. 45:361.[Medline]
41. Yew, M. S., S. Moore, 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, R. E. Olson. 1968. Nutrition status of alcoholics. Am. J. Clin. Nutr. 21:1329.[Abstract]
43. Charpentier, B., D. Franco, L. Paci, M. Charra, B. Martin, D. Vuitton, D. Fries. 1984. Deficient natural killer cell activity in alcoholic cirrhosis. Clin. Exp. Immunol. 58:107.[Medline]
44. Saxena, Q. B., E. Mezey, 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.[Medline]
45. Kronfol, Z., M. Nair, E. Hill, P. Kroll, K. Brower, J. Greden. 1993. Immune function in alcoholism: a controlled study. Alcoholism 17:279.
46. Sarkar, D. K., S. Minami. 1990. Effect of acute ethanol on -endorphin secretion from rat fetal hypothalamic neurons in cultures. Life Sci. 47:PL31.[Medline]
47. Boyadjieva, N., 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.[Medline]
48. Reddy, B. V., N. Boyadjieva, 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.[Medline]
49. Pastorcic, M., N. Boyadjieva, 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, D. K. Sarkar. 1997. Forskolin delays the ethanol-induced desensitization of hypothalamic -endorphin neurons in primary cultures. Alcohol. Clin. Exp. Res. 21:477.[Medline]
51. Oleson, D. R., D. R. Johnson. 1988. Regulation of human natural cytotoxicity by enkephalins and selective opiate agonists. Brain Behav. Immun. 2:171.[Medline]
52. Gabriovac, J., M. Antica, 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.[Medline]
53. Shavit, Y., J. W. Lewis, G. W. Terman, R. P. Gale, J. C. Liebeskind. 1984. Opioid peptides mediate the suppressive effect of stress on natural killer cell cytotoxicity. Science 223:188.[Abstract/Free Full Text]
54. Freier, D. O., B. A. Fuchs. 1994. A mechanism of action for morphine-induced immunosuppression: corticosterone mediates morphine-induced suppression of natural killer cell activity. J. Pharmacol. Exp. Ther. 270:1127.[Abstract/Free Full Text]
55. Weber, R. J., A. Pert. 1989. The periaqueductal gray matter mediate opiate-induced immunosuppression. Science 245:188.[Abstract/Free Full Text]
56. Smith, E. M., J. E. Blalock. 1981. Human lymphocyte production of corticotropin and endorphin-like substances: association with leukocyte interferon. Proc. Natl. Acad. Sci. USA 78:7530.[Abstract/Free Full Text]
57. Endo, Y., T. Sakato, S. Watanabe. 1985. Identification of proopiomelanocortin-producing cells in the rat pyloric antrum and duodenum by in situ mRNA-cDNA hybridization. Biomed. Res. 6:253.
58. Westly, H. J., A. J. Kleiss, K. W. Kelly, P. K. Y. Wong, P.-H. Yuen. 1986. Newcastle disease virus-infected splenocytes express the proopiomelanocortin gene. J. Exp. Med. 163:1589.[Abstract/Free Full Text]
59. Hazum, E., K.-J. Chang, P. Cuatrecases. 1979. Specific nonopiate receptors for -endorphin. Science 205:1033.[Abstract/Free Full Text]
60. Rivier, C.. 1995. Interaction between stress and immune signals on the hypothalamic-pituitary-adrenal axis of the rat: influence of drugs. D. K. Sarkar, and C. D. Barnes, eds. The Reproductive Neuroendocrinology of Aging and Drug Abuse 169. CRC Press, Boca Raton, FL.
61. Rivier, C., S. Lee. 1996. Acute alcohol administration stimulates the activity of hypothalamic neurons that express corticotropin-releasing factor and vasopressin. Brain Res. 726:1.[Medline]
62. Rasmussen, D. D., C. A. Bryant, B. M. Boldt, E. A. Colasurdo, N. Levin, C. W. Wilkinson. 1998. Acute alcohol effects on opiomelanocortinergic regulation. Alcohol. Clin. Exp. Res. 22:789.[Medline]
63. Bodner, G., A. Ho, M. J. Kreek. 1998. Effect of endogenous cortisol levels on natural killer cell activity in healthy humans. Brain Behav. Immun. 12:285.[Medline]
64. Gatti, G., R. G. Masera, L. Pallavicini, M. L. Sartori, A. Staurengi, F. Orlandi, 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.[Medline]
65. Holbrook, N. J., W. I. Cox, 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.[Abstract/Free Full Text]
66. Irwin, M.. 1994. Stress-induced immune suppression: role of brain corticotropin-releasing hormone and autonomic nervous system mechanisms. Adv. Neuroimmunol. 4:29.[Medline]
67. Irwin, M., R. Hauger, 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.[Abstract]
68. Nistico, G., M. C. Caroleo, M. Arbitrio, L. Pulvirenti. 1994. Corticotropin-releasing factor microinfused into locus coeruleus produces electro-cortical desynchronization and immunosuppression. Neuroimmunomodulation 1:135.[Medline]

This article has been cited by other articles:

				H. Zhang and G. G. Meadows Chronic alcohol consumption in mice increases the proportion of peripheral memory T cells by homeostatic proliferation J. Leukoc. Biol., November 1, 2005; 78(5): 1070 - 1080. [Abstract] [Full Text] [PDF]

				A. Arjona and D. K. Sarkar Circadian Oscillations of Clock Genes, Cytolytic Factors, and Cytokines in Rat NK Cells J. Immunol., June 15, 2005; 174(12): 7618 - 7624. [Abstract] [Full Text] [PDF]

				I. Tegeder and G. Geisslinger Opioids As Modulators of Cell Death and Survival--Unraveling Mechanisms and Revealing New Indications Pharmacol. Rev., September 1, 2004; 56(3): 351 - 369. [Abstract] [Full Text] [PDF]

				N. I. Boyadjieva, K. Chaturvedi, M. M. Poplawski, and D. K. Sarkar Opioid Antagonist Naltrexone Disrupts Feedback Interaction between {micro} and {delta} Opioid Receptors in Splenocytes to Prevent Alcohol Inhibition of NK Cell Function J. Immunol., July 1, 2004; 173(1): 42 - 49. [Abstract] [Full Text] [PDF]

				A. Arjona, N. Boyadjieva, and D. K. Sarkar Circadian Rhythms of Granzyme B, Perforin, IFN-{gamma}, and NK Cell Cytolytic Activity in the Spleen: Effects of Chronic Ethanol J. Immunol., March 1, 2004; 172(5): 2811 - 2817. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Boyadjieva, N. Articles by Sarkar, D. K. Search for Related Content PubMed PubMed Citation Articles by Boyadjieva, N. Articles by Sarkar, D. K.