Effect of adrenalectomy on ethanol-associated immunosuppression

Int. Z lmmunopharmac., Vol. 12, No. 4, pp. 4 3 5 - 4 4 2 , 1990. Printed in Great Britain.

0192-0561/90 $3.00 + .00 International Society for Immunopharmacology.

E F F E C T OF A D R E N A L E C T O M Y ON E T H A N O L - A S S O C I A T E D IMMUNOSUPPRESSION THOMAS R. JERRELLS,*t CHERYL A. MARIETTA,* FORREST F. WEIGHT,* and MICHAEL J. ECKARDT§ *Department of Pathology, University of Texas Medical Branch, Galveston, TX 77550; *Laboratory of Physiologic and Pharmacologic Studies, National Institute on Alcohol Abuse and Alcoholism, Rockville, MD 20853; ~Laboratory of Clinical Studies, National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD 20892, U.S.A.

(Received 15 April 1989 and in final form 10 January 1990)

-The alterations in lymphoid cell numbers and lymphocyte function due to administration of ethanol was found to be associated with high levels of circulating corticosteroids. The role of corticosteroids in the ethanol-induced alterations in the immune system was studied by administering ethanol to adrenalectomized rats. The results of these experiments showed that the ethanol-induced loss of cells from the thymus was not completely prevented by adrenalectomy and the ethanol-induced loss of cells from the spleen was not affected by adrenalectomy. Likewise the ethanol-induced decrease in antibody production to the T-cell-dependent antigen sheep erythrocytes were not affected by adrenalectomy. The ability of animals to produce antibodies of the T-cell-independent antigen, TNP-Ficoll, was not affected by ethanol regardless of whether the animals had adrenal glands or not. These data indicate that adrenal corticosteroids are responsible for some but not all of the thymic involution due to ethanol intoxication. Also, adrenalectomized rats did not show as much impairment in lymphocyte proliferation as sham adrenalectomized animals after ethanol administration. However, this loss of cells from peripheral lymphoid organs such as the spleen and the decreased ability to respond to T-cell-dependent antigens is not influenced by adrenalectomy indicating mechanisms other than corticosteroids mediate these effects of ethanol.

Abstract

A number of observations have led to the suggestion that prolonged consumption of large amounts of ethanol (ETOH) results in alterations in host defense mechanisms leading to increased incidence of infections, certain cancers, and perhaps autoimmune diseases (Eckardt, Harford, Kaelber, Parker, Rosenthal, Ryback, Salmoiraghi, Vanderveen, & Warren, 1981; MacGregor, 1986; Lauria, 1963). Administration of E T O H to experimental animals as well as studies of human alcoholics have shown that E T O H consumption is associated with a number of immunological abnormalities including changes in lymphoid organs (Jerrells, Marietta, Echardt, Majchrowicz & Weight, 1986), and a loss of circulating leukocytes (Brayton, Stokes, Schwartz & Louria, 1970; Jerrells et al., 1986; MacGregor, Gluckman & Senior, 1978; Smith, Van Thiel, Whiteside, Janson, Magovern, Puet & Rabin, 1980; Spagnuolo & MacGregor, 1975; Tennenbaum, Ruppert, St. Pierre & Greenberg, 1969). Defects in cell-mediated immunity have been

shown and include delayed-type hypersensitivity (Berenyi, Straus & Cruz, 1974; Gluckman, Dvorak, & MacGregor, 1977; Tennenbaum et al., 1969) and lymphocyte proliferative responses to mitogens in vivo (Brayton et al., 1970; Jerrells et al., 1986; Roselle & Mendenhall, 1984; Young, Van der Weyden, Rose & Dudley, 1970). The possibility that some of the effects of ETOH are mediated by corticosteroids was suggested by our own work showing that the most profound changes in the immune system were evident at a time when animals had withdrawn from E T O H and blood E T O H levels were low or absent (Jerrells et al., 1986) and the finding that animals maintained at a constant blood ETOH level by administering ETOH in an inhalation chamber (Marietta, Jerrells, Meagher, Karanian, Weight & Echardt, 1988) did not show the alterations of lymphocyte function previously seen in other models. This suggestion is supported by the observations that ETOH administration affects corticosteroid metabolism

t To whom correspondence should be addressed. 435

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T. R. JERRELLSet al.

(Jenkins & Connolly, 1968; Kissin, Schenker & Schenker, 1960; Tabakoff, Jaffe & Ritzmann, 1978) and by findings that changes in corticosteroid levels produce immunological alterations similar to those observed in the discussed models of ETOH effects (Cupps & Fauci, 1982; Del Rey, Besedovsky & Sorkin, 1984; Kelso & Munck, 1984; Paciotti, Skwerer & Tamarkin, 1971; Warren & Vogel, 1985). The effects of corticosteroids have been studied by a variety of methods both in vitro and in vivo. It is clear from these studies that interactions of lymphoid cells and corticosteroids following administration of corticosteroids to intact animals or to cultures of lymphoid cells results in dramatic changes in cell viability and function. Further, a number of investigators have shown that corticosteroids are produced in response to a number of immunological and other stimuli and that this response generally leads to a down regulation of the immune response (Esterling & Rabin, 1987; Dennis & Mond, 1986). In many studies the effects of corticosteroids were appreciated by studying animals that were incapable of producing corticosteroids because of surgical removal of the adrenal glands (Bertini, Bianchi & Ghezzi, 1988; C h a n & Cheers, 1982; Ito, Ama, Hyodo, Shigeta & Ito, 1985; Kaiser, Hecht, Roth & Cooperband, 1985; Keller, Weiss, Schleifer, Miller & Stein, 1983; Van Zon, Eling, Schetters & Hermsen, 1985). It is interesting to note that adrenalectomized (ADX) animals were shown not to be immunosuppressed by removal of the adrenal glands and the only remarkable immunological change was exaggerated response to both mitogens and antigens in vitro (Calvano, Mark, Good & Fernandes, 1982). The present study was designed to investigate directly the possible role of corticosteroids in ETOHinduced immunological changes. To accomplish this we have studied the effect of ETOH on parameters of immunocompetence in ADX rats. We report here that the ETOH-induced loss of thymocytes is partially reversed by ADX, however the loss of cells from the spleen and the antibody response to a T-cell dependent antigen was affected by ETOH regardless of the level of corticosteroids. The antibody response to the T-cell independent antigen T N P - f i c o l l , was not affected by ETOH and in fact this response was enhanced by both ADX and ETOH. EXPERIMENTAL PROCEDURES

Animals and ethanol administration In initial experiments male S p r a g u e - Dawley rats (Charles Rivers) were treated with 8 - 11 g E t O H / k g body weight/day as described by Majchrowicz

(1975), the dose shown by us to produce a reproducible immunosuppression (Jerrells et al., 1986). In follow-up experiments adrenalectomized and sham adrenalectomized male S p r a g u e - D a w l e y rats were treated surgically by the supplier (Charles Rivers) and treated with 6 - 9 g E T O H / k g body weight/day essentially as described. Briefly, ETOH (20% w/v) in Sustacal (Mead Johnson and Co., Evansville, IN) was administered by oral intubation in fractional doses throughout the day to maintain intoxication. Control animals were intubated at the same time and administered the caloric equivalent of Sustacal only. Animals were intubated through day 4 and evaluated on day 5 for the characteristic signs of and responses to ETOH intoxication and withdrawal (Majchrowics, 1975). Beginning on day 5, all animals were maintained on Purina rat chow and water. At designated intervals during the course of ETOH administration, as well as during the subsequent abstinence period, animals were anesthetized with halothane and sacrificed by exsanguination accomplished by cardiac puncture. A sample of blood from each animal was analyzed for blood ETOH content. Corticosterone levels At various times during ETOH administration (day 1 - 4 ) , or after ETOH administration was discontinued, animals from the ETOH-treated and control diet groups were bled by cardiac puncture after halothane anesthesia. Blood was obtained at the same time on each day. Corticosterone levels were determined using a commercially available ~H RIA kit (Radioassay Systems Laboratories, Inc., Carson, CA). Each plasma sample was assayed in duplicate. Isolation and counts o f lymphocytes from thymus and spleen After sacrifice, the spleen and thymus were removed aseptically from each animal and rinsed in Hank's balanced salt solution (HBSS, Microbiological Associates, Walkersville, MD); single cell suspensions were prepared by passing individual organs through stainless steel sieves. Debris was allowed to settle out of suspension for 5 min at room temperature, and an aliquot of each cell suspension was removed for nucleated cell counts. Peripheral blood obtained in preservative-free heparin (50 U/ml of blood; Gibco Laboratories, Grand Island, NY) was evaluated for total leukocyte and erythrocyte count using an automated electronic cell counter (Model ZBI, Coulter Electronics, Hialeah, FL), and leukocyte differential was evaluated using Giemsa-

437

Ethanol-induced Steroid Immunosuppression stained thin smears. The proportion of each leukocyte type was determined by counting 200 cells on each slide, and duplicate slides were counted for each animal. In some experiments, viable cell numbers from each cell source were determined using trypan blue exclusion as the criterion for viability.

Lymphocyte proliferation to mitogens Lymphocytes, obtained from spleens as described above, were evaluated for ability to proliferate in response to T- and B-cell mitogens. Spleen cells were adjusted to 5 x 106 cells/ml in RPMI-1640 supplemented with 25 mM Hepes buffer, 1% glutamine, 5 0 g g / m l gentamicin, and 5% heatinactivated fetal bovine serum (Microbiological Associates). These cells were cultured in 0.1-ml (5 x 105 cells/well) aliquots in flat-bottom, 96-well microliter plates (Costar Plastics, Boston, MA). Mononuclear cells isolated from the peripheral blood were adjusted to 1 x 106 cells/ml in RPMI-1640 supplemented as described, and 0.1-ml aliquots were added to wells (1 x 105 cells/well) of a roundbottom, 96-well microliter plate (Linbro, Flow Laboratories, Hamden, CT). To quadruplicate wells 0.1 ml of media, or 0.1 ml of various concentrations of concanavalin A (con A; Cal Biochem, LaJolla, CA), phytohemagglutinin (PHA; Difco, Detroit, MI), or the rat B-cell mitogen STM (Ribi Biochemicals, Hamilton, MO), were added. Cultures were incubated at 37°C in a 5% CO2 atmosphere for 72 h. On the last day of culture, 1 gCi of tritiated thymidine (3HTdR, specific activity 5 Ci/mM; Amersham, Chicago, IL) was added to each well for the last 6 h of culture. Cultures were harvested onto filter strips using an automatic sample harvester (PhD, Cell Harvester, Cambridge Technology, Inc., Cambridge, MA), and incorporated radioactivity was determined by liquid scintillation spectroscopy. The responses to mitogens were expressed as net counts per minute (nCPM), calculated by subtracting the CPM of cultures receiving media only from the CPM of cultures receiving mitogen. Differences between responses of ETOH-treated animals and various control groups in terms of nCPM were determined using Student's t-test. In the majority of studies the control group consisted of adrenalectomized animals that received control diet by intubation. Quantitation o f antibody-producing cells At various times before and during ETOH administration, groups of ETOH-treated and control animals were given intraperitoneally 1.0 ml of a 10°70

Table 1. Corticosterone levels in ethanol-treated or control diet-treated rats Treatment* Day 1

2 3 4 5 9

Ethanol

Control

72 _+3' 123_+ 31 46 + 7 98-+-18 160_+33 19_+3

27 _+3 68_+26 12 _+ 18 7_+6 40_+ 3 23_+3

*Male Sprague-Dawley rats were intubated with liquid diet containing ethanol or isocaloric control diet for the indicated number of days. Ethanol was given for 4 days and thereafter animals were fed lab chow and water ad libitum. *Each point is the mean corticosterone serum level as ng/ml _+one standard deviation of 6 animals in each treatment group on each day. Untreated animals (n = 10) had corticosterone levels ranging from 10 to 42 ng/ml suspension of washed sheep erythrocytes (SRBC). Four days after immunization, animals were sacrificed, and the spleens removed. Single cell suspensions were prepared in HBSS, and antibodyproducing cell numbers were determined using a modification of the Jerne plaque assay (Cunningham & Szenberg, 1986). Responses to the T-cellindependent antigen T N P - f i c o l l were determined following immunization with 10/~g TNP - ficoll by intraperitoneal injection, and plaque-forming cells quantitated using TNP-treated SRBC as described (Rittenberg & Pratt, 1969). Data were expressed as mean plaque-forming cells per 10 6 nucleated spleen cells and as mean plaque-forming cells per spleen.

RESULTS

Previous studies by us demonstrated that administration of 8 - 11 g ETOH/kg body weight/ day to rats using oral intubation results in a loss of cells from the thymus, spleen and peripheral blood, as well as a dose dependent loss of lymphocyte function in vitro and in vivo. Maximal cell loss occurred when ETOH was no longer administered and signs associated with the withdrawal syndrome were evident (Jerrells et al., 1986). To determine if the immunological changes that result from administration of ETOH are associated with increased levels of corticosteroids, we measured serum levels of corticosterone at various times of ETOH administration. In accordance with other reports we found that animals administered ETOH produced significantly higher levels of corticosterone than animals intubated with control diet (Table 1).

T. R. JERREI_LS et al.

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Table 2. Effect of adrenalectomy in depletion of lymphoid cells after administration of ETOH

Day

Control/Control diet

Spleen

1 3 5 9

41 _+ 2 t 38_+4 37 +_ 3 42 _+ 2

Thymus

1 3 5 9

137 125 130 141

_+ 12 +_ 16 __ 18 _+ 12

Animal group and diet* ADX/Control diet ADX/ETOH 43 41 45 40

+_ 3 _+ 3 _+ 5 _+ 3

129 133 121 127

_+ 8 _+ 12 _+ 9 _+ 13

36 _+ 4 (84) + 19_+ 2(46) t 28 +_+_1.5 (62) 8 38 _+ 2 (95) 126 87 79 112

_+ 17 (98) _+ 12 (65) 8 _+ 9 (65) 8 _+ 17 (88)

Sham A D X / E T O H 38 23 22 31

± 3 (88) +_2(56) 3 _+ 15 (49) 8 _+ 0.5 (78)

131 _+_9 (102) 64 _+ 7 (48) 3 59 -4- 8 (49) 8 96 _+ 11 (76)

*Male Sprague-Dawley rats untreated (Control) adrenalectomized (ADX) or Sham adrenalectomized, were intubated with liquid diet containing ETOH or an isocaloric control diet for the indicated days. ETOH was given for 4 days and thereafter animals were fed lab chow and water ad lib#urn. Evidence of withdrawal was evident at day 5 in Sham A D X / ETOH group. tMean nucleated cell counts obtained from six animals in each group _+ one standard deviation. +Values in parentheses are percentage of the value obtained for ADX rats intubated with control diet. 3Significantly different from value obtained at each time point for ADX rats intubated with control diet. Table 3. Effects of adrenalectomy on proliferative responses of spleen lymphocytes from ETOH-treated rats Source of cells*

StimuluU Media

Untreated ADX-Control diet Sham ADX-ETOH ADX-ETOH

1.33 1.97 1.70 2.50

_+ 0.5 + + 0.2 _+ 0.3 _+ 0.2

Con A

196.0 223.7 92.4 256.6

_+ 21.0 _+ 14.3 ± 15.08 _+ 31.2

PHA

47.3 49.7 3.90 125.0

+_ 5.0 _+ 6.2 _+ 1~ + 14.28

LPS

46.0 43.3 17.9 38.4

_+ 6.1 +_ 5.8 _+ 2.3 ~ +_ 4.2

*Spleen cells were obtained from ADX or sham-ADX rats treated for 4 days with ETOH or control diet, and after a further 18 h after treatment was discontinued. tLymphocytes were cultured in the presence of 100~1 of complete medium, concanavalin A at 5/ag/ml, phytohemagglutinin at 10 t~g/ml, or bacterial lipopolysaccharide (STM) at 10 tag/ml. +Each data point is the mean counts per minute _+ standard deviation of quadruplicate cultures x 10 -3. 8Significantly different from ADX-control diet results at P~<0.05. I m p o r t a n t l y , we f o u n d t h a t the h i g h e s t level o f c o r t i c o s t e r o n e o c c u r r e d in a n i m a l s t h a t were withdrawing from ETOH ( 1 0 0 - 160 n g / m l vs 40 n g / m l in c o n t r o l a n i m a l s ) . Because this t i m e was a s s o c i a t e d w i t h the m o s t p r o f o u n d c h a n g e s in the i m m u n e s y s t e m s f u r t h e r e x p e r i m e n t s were designed to assess t h e role o f c o r t i c o s t e r o i d s in E T O H i n d u c e d i m m u n o s u p p r e s s i o n . T o this e n d we studied the r e s p o n s e o f A D X rats to d e t e r m i n e t h e possible role o f a d r e n a l c o r t i c o s t e r o i d s in the o b s e r v e d effects o f E T O H o n the i m m u n e s y s t e m . A d r e n a l e c t o m i z e d ( A D X ) a n i m a l s did n o t tolerate t h e dose o f 8 - 11 g E T O H / k g b o d y w e i g h t / d a y ; the dose was a d j u s t e d to 6 - 9 g E T O H / k g b o d y w e i g h t / day, a n d was well tolerated. Similar c h a n g e s in c o r t i c o s t e r o n e levels i n d u c e d by h i g h doses o f E T O H (8 - 11 g E T O H / k g b o d y w e i g h t / d a y ) were n o t e d in a d r e n a l g l a n d - b e a r i n g rats t h a t received the lower dose or E T O H (120 ___ 8 n g / m l o n d a y 5) a n d it was

verified t h a t n o detectable s e r u m levels o f corticosterone were p r e s e n t d u r i n g E T O H a d m i n i s t r a t i o n a n d s u b s e q u e n t w i t h d r a w a l (data n o t s h o w n ) . Cell c o u n t s o f t h y m u s , spleen, a n d peripheral b l o o d f r o m a d r e n a l g l a n d - b e a r i n g rats given 6 - 9 g E T O H / k g b o d y w e i g h t / d a y resulted in a significant loss o f l y m p h o i d cells in these s h a m - A D X rats over t i m e (Table 2); as previously r e p o r t e d (Jerrells et al., 1986), this loss was greatest at the t i m e w i t h d r a w a l signs were evident. A l t h o u g h the cell loss was n o t as d r a m a t i c in A D X rats given E T O H , a c o n s i s t e n t loss o f cells f r o m the t h y m u s a n d spleen o c c u r r e d a n d was evident in repeated e x p e r i m e n t s . In these e x p e r i m e n t s significant loss o f l y m p h o c y t e s f r o m the peripheral blood was n o t o b s e r v e d , a l t h o u g h we n o t e d a c o n s i s t e n t lower n u m b e r o f circulating l y m p h o c y t e s (data n o t s h o w n ) . T h e s e results m a y h a v e b e e n i n f l u e n c e d by the smaller doses a d m i n i s t e r e d per day, necessitated b e c a u s e A D X rats

Ethanol-induced Steroid Immunosuppression

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Fig. 1. Responses of ETOH-treated, ADX, or sham-ADX rats to SRBC (panel a), or TNP - ficoll (panel b). ADX (121)and sham-ADX (IN) rats were treated with ETOH for 4 days or ADX rats were treated with Sustacal only (VI). Each point represents the mean plaque-forming cell numbers of spleens from three rats in each group. Control rats maintained on lab chow and water were immunized at the same time and plaque assay results showed 17 × 103 ___3 plaque forming cells/ spleen to sheep erythrocytes and 27 _+ 103 _+ 5 plaque forming cells/spleen to TNP.

could not tolerate 8 - 11 g E T O H / k g body weight/ day. A possible mediating role for E T O H in the observed effects in the A D X group was supported further by the failure to observe differences in the lymphoid cell counts for A D X rats incubated with vehicle only and untreated litter mates examined at the same time (Table 2). Both groups were studied at identical time points as E T O H - t r e a t e d animals. Although cell counts varied from animal to animal, any stress associated with intubation was apparently adjusted to and had little measurable effect on the immune system of these animals. The functional capabilities of spleen and peripheral blood lymphocytes were investigated using proliferation assays to the T- and B-cell mitogens con A, P H A , and lipopolysaccharide (LPS) (Table 3). When spleen cells were examined during the withdrawal period, there was a marked increase in proliferation to P H A , and lesser increases in responses to con A and LPS, in A D X rats administered E T O H when compared to the responses of cells in A D X rats intubated with vehicle, or s h a m - A D X rats intubated with E T O H . The increased proliferation of spleen cells from A D X animals as compared to s h a m - A D X animals also was

evident over the course of E T O H administration and to most doses of mitogen used. Similar data were obtained using mononuclear cells isolated from the peripheral blood (data not shown). As in our previous studies (Jerrells et al., 1986) a complete dose range of each mitogen was used and similar results were noted regardless of the dose used. The most obvious suppression of proliferation was noted when suboptimal conditions were used (48 h of incubation and suboptimal amounts of mitogen) and this was essentially reversed in A D X rats as shown in Table 3 (data not shown). The effects of corticosteroids on the ability of ETOH-treated rats to m o u n t a primary immune response was investigated using plaque-forming cell responses to SRBC or T N P - f i c o l l . Regardless of whether the rats were A D X or sham A D X , a marked suppression of the primary antibody response to the T-cell-dependent SRBC was noted in rats administered E T O H (Fig. 1). In contrast, E T O H administration to s h a m - A D X or A D X rats did not markedly inhibit the immune response to the T-cellindependent antigen T N P - ficoll; in fact, A D X rats given E T O H produced an elevated response to this antigen. The response of control rats to SRBC was

440

T. R. JERRELLSet al.

not significantly different than ADX rats given control diet (not shown). Expression of these data as Pfu/106 spleen cells did not alter the conclusions.

DISCUSSION Corticosteroids have been shown to have multiple effects on the immune system including thymic atrophy, increase in circulating neutrophils, and impairments in lymphocyte proliferative responses as well as function of cells playing an accessory role in the immune response (Cupps & Fauci, 1982; Del Rey et al., 1984; Kelso & Munck, 1984; Warren & Vogel, 1985). Alcohol abuse in humans and the administration of ETOH to experimental animals have been shown to alter corticosteroid metabolism (Jenkins & Connolly, 1968; Kissin et al., 1960; Tabakoff et al., 1978) and prompted the experiments presented above. It was first demonstrated that administration of ETOH to rats resulted in circulating levels of corticosterone above those levels produced by intubation of vehicle only, and that increased levels of corticosterone accompanied the ETOH withdrawal syndrome. Concurrent loss of lymphoid cells and of immunological functioning was observed. As steroid levels have clearly been associated with immunosuppression in other systems (Cupps & Fauci, 1982), rats were ADX to eliminate the major source of steroids. It would be nai've to assume that the only effect of ADX is to eliminate the production of adrenal corticosteroids. Our own data clearly show that the removal of the adrenal glands has an effect on the animals ability to tolerate relatively high doses ( 8 - 11 g/kg) of ETOH. This effect may be due to the effects of ADX on metabolic processes or electrolyte and water balances. The relatively profound endocrine changes resulting from ADX notwithstanding it has been shown in this study as well as by others (Calvano et al., 1982), that ADX alone does not profoundly affect the immune responses examined in this study except to augment these responses. It is likely that this is due to the elimination of the blunting effects on lymphocyte function of the normal levels of corticosteroids. Previous studies by this laboratory as well as other workers have demonstrated that administration of ETOH results in a loss of thymocytes and lymphocytes from the peripheral blood (Brayton et al., 1970; Jerrells et al., 1986, MacGregor et al., 1978; Slone, Smith & Van Thiel, 1977; Smith et al., 1980; Spagnuolo & MacGregor, 1975; Tennenbaum et al., 1969; Young et al., 1970). The present study

has shown that adrenalectomy partially reverses the loss of thymocytes, and lessens the loss of peripheral blood lymphocytes. With respect to cell changes, these data suggest that ETOH mediates a loss of thymocytes by both corticosteroid-dependent and -independent mechanisms. Previously described changes in cell composition of the peripheral blood of ETOH-treated rats (Jerrells et al., 1986) (increase in polymorphonuclear cells and decrease in lymphocytes) were reversed in ADX rats although some lymphoid cell loss was noted in peripheral blood. Corticosteroids have been shown to be lympholytic and affect the bone marrow and marginal pool of polymorphonuclear neutrophils (Cupps & Fauci, 1982; Del Ray et al., 1984; Kelso & Munck, 1984), and it is likely that the effects on peripheral blood leukocytes described in ETOHtreated rats are due to increased steroid production. It also has been shown that corticosteroids will alter normal lymphocyte recirculation patterns and it is possible that changes in the composition of the peripheral blood result from this. It was consistently observed that ETOH administration to ADX rats resulted in a loss of thymocytes and spleen cells, with spleen cell loss being essentially the same as that seen in animals with intact adrenal glands. The mechanism of this cell loss is unclear, however this has been also described by others (Ito et al., 1985). Ingestion of ETOH by adrenal-gland-bearing mice has been shown by us to result in similar changes in thymus and spleen cellularity (Jerrells et al., 1990). Flow cytometry analysis have shown that both Tand B-cells are lost from the spleen and predominantly immature (CD4 ÷ CD8 ~) cells are lost from the thymus (manuscript in preparation). In preliminary studies, ADX did not alter the cell types depleted from the spleen and thymus by ETOH only the magnitude of depletion. Administration of dexamethasone (15 mg/kg) subcutaneously to ADX rats resulted in a loss of thymocytes similar to that noted in ETOH-treated sham ADX rats (data not shown) further suggesting the role of corticosteroids in this system. Although the more definitive experiment using corticosterone pellets to restore physiological levels in ADX animals has not been done in this system, it is clear that some aspects of the effects of ETOH on the immune system are due to the stress of withdrawal. Defects in lymphocyte proliferation to nonspecifc mitogenic stimulation are at least partially prevented by adrenalectomy; and administration of ETOH to ADX rats usually resulted in a markedly better response to mitogenic stimulation at higher doses, and also in antibody response to T-cell-independent

Ethanol-induced Steroid Immunosuppression antigens, t h a n in controls. This is consistent with o u r u n p u b l i s h e d d a t a t h a t a d d i t i o n o f E T O H to cultures o f lymphocytes f r o m u n t r e a t e d rats to a t t a i n levels in culture consistent with b l o o d E T O H levels results in a n e n h a n c e m e n t in p r o l i f e r a t i o n . As it is generally accepted t h a t T-cell m i t o g e n s a n d T-cell-independent antigens directly b i n d to receptors o n the cell a n d it is possible t h a t the observed e n h a n c e m e n t o f cell p r o l i f e r a t i o n a n d T - i n d e p e n d e n t antigen response is due to cell m e m b r a n e changes induced directly by E T O H . Such changes m i g h t include e n h a n c e d receptor m o b i l i z a t i o n or internalization. F u r t h e r studies are required to answer this question. It is also possible t h a t E T O H s o m e h o w alters l y m p h o k i n e influences o n the B-cell s u b p o p u l a t i o n t h a t is responsive to T - i n d e p e n d e n t antigens, however, n o d a t a exists to s u p p o r t this idea.

441

In s u m m a r y , the d a t a generated in this study indicate t h a t some p a r a m e t e r s o f i m m u n o c o m p e t e n c e are indirectly suppressed by E T O H a p p a r e n t l y t h r o u g h the action o f adrenal corticosteroids. This conclusion is based o n the reversal o f some aspects o f i m m u n o s u p p r e s s i o n by A D X . Currently, efforts are u n d e r w a y to test the effects o f corticosteroid a n t a g o n i s t s in this system b u t to date we have been u n a b l e to identify a suitable specific antagonist. M o r e i m p o r t a n t l y , o u r d a t a also indicate t h a t some aspects o f E T O H - i n d u c e d changes in the i m m u n e system, n o t a b l y the loss o f spleen cells a n d the p r i m a r y a n t i b o d y response to sheep erythrocytes, are likely to be m e d i a t e d by m e c h a n i s m s other t h a n corticosteroid p r o d u c t i o n .

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