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Cytokines in detoxified patients with chronic alcoholism without liver disease: increased monocytic cytokine production
Cytokines in Detoxified Patients with Chronic Alcoholism without Liver Disease: Increased Monocytic Cytokine Production Cai Song, Aihua Lin, Raf De Jong, Erik Vandoolaeghe, Gunter Kenis, Eugene Bosmans, Alex Whelan, Simon Scharpe, and Michael Maes Background: The aim of the present study was to examine the production of interferon-g, tumor necrosis factor-a (TNF-a), granulocyte–macrophage colony stimulating factor (GM-CSF), interleukin-6 (IL-6), IL-10, IL-4, IL-5, IL-1 receptor antagonist (IL-1RA), and prostaglandin E2 in relation to the number of leukocytes in the blood of detoxified, chronic alcoholic patients without apparent liver disease (AWLD). Methods: Phytohemagglutinin 1 lipopolysaccharide-induced production of the above variables as well as the number of white blood cells and differentials were determined in detoxified AWLD patients and normal volunteers. Results: Detoxified AWLD patients have a significantly higher production of IL-6, IL-10, TNF-a, GM-CSF, and IL-1RA and significantly increased numbers of leukocytes and neutrophils compared to normal volunteers. Conclusions: Detoxified AWLD patients show an increased production of proinflammatory cytokines, i.e., IL-6, TNF-a, and GM-CSF, as well as negative immunoregulatory proteins, such as IL-10 and IL-1RA. Biol Psychiatry 1999;45:1212–1216 © 1999 Society of Biological Psychiatry Key Words: Cytokines, alcoholism, depression, interleukin-6, interleukin-10, tumor necrosis factor-a, leukocytes
Introduction
A
cute administration of alcohol and chronic alcoholism have been implicated in the onset of a variety of immune defects in vivo and in vitro, including various signs of immunosuppression (Nair et al 1990; Mili et al 1992; Szabo et al 1995), such as abnormal interleukin-2
From the Clinical Research Center for Mental Health, Antwerp, Belgium (CS, AL, EV, MM); Department of Medical Biochemistry, University of Antwerp, Antwerp, Belgium (AL, SS); Department of Psychology, Carleton University, Ottawa, Canada (CS); Pathology Center, St James Hospital, Dublin, Ireland (AW); and Eurogenetics, Transportstraat, Belgium (RDJ, GK, EB). Address reprint requests to Michael Maes, MD, PhD, Clinical Research Center for Mental Health, University Department of Psychiatry, AZ Stuivenberg, 267 Lange Beeldekensstraat, 2060 Antwerp, Belgium. Received August 22, 1997; revised February 5, 1998; accepted February 12, 1998.
© 1999 Society of Biological Psychiatry
(IL-2)-supported and mitogen-induced responsiveness of lymphocytes, and suppression of tumor necrosis factor-a (TNF-a) and IL-2 production (Cook et al 1991; Verma et al 1993). Alcohol suppresses lipopolysaccharide (LPS)induced production of proinflammatory cytokines (Nelson et al 1989; Szabo et al 1995). Chronic alcohol dependent patients without liver disease show also increased numbers of leukocytes, neutrophils, or monocytes (Khoruts et al 1990; Kanwar and Tikoo 1992). Alcoholic liver disease is characterized by activation of the monocyte–macrophage system, with increased production of TNF-a, IL-1, and IL-6 (Martinez et al 1992; Rodriguez-Rodriguez et al 1995; Ruiz et al 1993; Khoruts et al 1990). One study performed in abstinent (detoxified) chronic alcoholic patients without overt liver disease (AWLD), however, detected signs of T-cell activation (Cook et al 1991). Prostaglandin E2 (PGE2), a proinflammatory peptide, was found to be elevated in acute-alcohol treated monocytes and stomach tissue of alcoholic patients (Smith et al 1991; Szabo et al 1992). The main sources of PGE2 during immune responses are monocyte cells (Sergeeva et al 1998). PGE2 has a biphasic control of lymphocyte proliferation and, therefore, plays an important role in immune cell function regulation (Sergeeva et al 1998); however, it has remained elusive whether detoxified AWLD patients exhibit any changes in cytokine or PGE2 production. Based on the above literature and the recent findings that AWLD patients have signs of T-cell activation (Cook et al 1991), we have chosen to measure T-cell and monocyte-derived cytokines, since these are main regulators of immune homeostasis (Cavaillon 1996). Two polar categories of T helper cells (Th) have been described in humans, i.e., Th1-like cells, which secrete interferon-g (INF-g), and Th2-like cells, which secrete IL-4 and IL-5 (Mosmann and Sad 1996). Several other cytokines are produced by both Th1- and Th2-like cells and/or cells of the monocyte–macrophage lineage, e.g., IL-6, IL-10, TNF-a, and granulocyte–macrophage stimulating factor (GM-CSF). The aims of the present study were to examine i) PGE2, Th1-like (IFN-g) and Th2-like (IL-4 and IL-5) 0006-3223/99/$20.00 PII S0006-3223(98)00083-3
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cytokines, the proinflammatory cytokines, IL-6, TNF-a, and GM-CSF, and the negative immunoregulatory proteins, IL-10, and the IL-1 receptor antagonist (RA) in detoxified AWLD patients; and ii) the number of leukocytes and differentials in relation to the above cytokines or monocytic products.
Methods and Materials Subjects Twenty-four subjects participated in this study, i.e., 12 normal volunteers and 12 chronic alcohol-dependent (303.9) inpatients. The patients were classified according to DSM-III-R criteria on the basis of the Structured Interview for DSM-III-R Diagnosis (SCID) (Spitzer et al 1990), administered by one of the authors (EV). The normal volunteers were excluded for a current and past history of psychiatric disorders, using the SCID, and a positive family history of psychiatric disorders in first-degree relatives, using a semistructured interview. The healthy volunteers were nondrinkers and were free of any medication during at least 1 month previous to blood sampling. None had ever been taking psychotropic drugs. The alcohol-dependent patients had an alcohol consumption of more than 300 mL pure alcohol/day for at least 2 years. We excluded the following alcoholic patients: i) patients with overt liver disease, such as hepatitis or liver cirrhosis, and patients with pulmonary or urinary tract infections or hepatitis B surface antigen (HbsAG) and HIV positive patients; and ii) patients with a recent or past history of other axis-I diagnoses (beside alcohol dependence), such as schizophrenic, paranoid, organic mental, and affective disorders. A positive family history of psychiatric disorders in the AWLD patients was not considered as an inclusion or exclusion criterion. Inclusion criteria for normal volunteers and alcoholic patients included mean cell volume (MCV), g-glutamyl transpeptidase (g-GT), and cholinesterase (CHE) values within the normal laboratory limits and a normal physical examination. A semistructured interview was employed to exclude subjects i) with any acute or chronic medical illness, such as autoimmune disorders, inflammatory bowel disease, rheumatoid arthritis, and endocrine disorders; and ii) who suffered from acute infectious or allergic reactions for at least 2 weeks prior to the study.
Methods After an overnight fast, blood samples were taken at 7:30 AM (630 min). In AWLD patients blood was collected 32–34 days after admission. Immediately after admission into hospital, alcoholic patients received diazepam (20 – 40 mg/day), which was tapered off during a 2-week period. Consequently, patients were drug-free for 18 –20 days before blood samplings were carried out. None of the patients suffered from withdrawal phenomena. Five subjects were taking disulfiramum, 200 – 400 mg/day, which was started 21 days after admission into hospital. No other medications were administered to the patients. All patients remained hospitalized during the study.
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Total number of leukocytes and white blood cell (WBC) differentiation, and red blood cell (RBC) parameters, i.e., RBC count, MCV, mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), hematocrit (Ht), and hemoglobin (Hb), were measured with a Technicon H2 (Bayer, Brussels, Belgium) fully automated blood cell counter. The analytical interassay coefficient of variation (CV) values obtained in our laboratory were as follows: number of leukocytes 1.5%, neutrophils 0.9%, lymphocytes 3.1%, monocytes 7.2%, and RBC 1.3%; Hb 1.0%, Ht 1.3%, MCV 1.3%, MCH 1.5%, and MCHC 1.5%. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), g-GT, lactate dehydrogenase (LDH), amylase, and creatinine kinase (CK), amylase, serum ureum and creatinine, and CHE (E.C.3.1.1.8) were measured with the dry chemistry method (Ortho Clinical Diagnostics). Cytokine and PGE2 concentrations were studied by stimulating whole blood with phytohemagglutinin (PHA) 1 lipopolysaccharide (LPS) and analyzing culture supernatant (De Groote et al 1992). Heparin (100 IU/mL) blood samples were diluted 1:10 in RPMI-1640 medium (Life Technologies, Belgium) containing 1% of penicillin (100 IU/mL) (Sigma), PHA (Murex, Belgium), 5 mg/mL, and LPS (Sigma), 20 mg/mL (De Groote et al 1992). For a blank, the blood from each sample was diluted with RPMI-1640 containing 1% penicillin. Samples (400 mL) were pipetted into 24 well-plates prefilled with medium (1200 mL) and incubated for 48 hours or 72 hours in a humidified atmosphere at 37°C, 5% CO2. After incubation, the plates were centrifuged at 1500 rpm for 10 min. Supernatants were taken off carefully under sterile conditions, divided into Eppendorf tubes, and frozen immediately at 270°C until thawed for assay of PGE2 (48 hours) and IFN-g, TNF-a, IL-6, IL-10, IL-1RA, GM-CSF, IL-4, and IL-5 (72 hours). All immune–inflammatory variables were quantified by means of enzyme-linked immunosorbent assay methods (Eurogenetics, Tessenderlo, Belgium) based on appropriate and validated sets of monoclonal antibodies. PGE2 concentrations in culture supernatant were measured by means of an enzyme immunoassay (Cayman Chemical Company, Ann Arbor, MI). The optimal dilution for the supernatant has been determined and, consequently, we employed a 15-fold dilution in this assay. All samples from normal volunteers and detoxified AWLD patients were assayed at the same time, in a single run with a single lot number of reagents and consumables employed by a single operator (CS or AL). The intraassay CV values for all variables were less than 8%.
Statistical Analysis The independence of classification systems was ascertained by means of analysis of contingence (x2 test). Group mean differences were examined by means of analysis of variance (ANOVA) or analysis of covariance (ANCOVA). Relationships between variables were checked by means of Pearson’s product– moment correlation coefficients. Transformations (square root) were used to normalize the distribution of the variables or to adjust for heterogeneity of variance between study groups.
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Table 1. Demographic Data and Erythron Characteristics in Detoxified AWLD Patients and Normal Volunteers (NV) Variables Age (years) Men/women RBC (106/mL) Ht (%) Hb (g/dL) MCV (fL) MCH (pg) MCHC (g/dL)
NV
AWLD
F/x2
df
p value
36.6 (9.5) 10/2 5.04 (0.51) 45.2 (3.5) 15.1 (1.1) 89.9 (4.3) 30.1 (1.6) 33.5 (0.8)
41.0 (8.9) 11/1 4.73 (0.37) 43.1 (2.1) 14.4 (0.7) 91.2 (5.7) 30.6 (2.3) 33.5 (0.9)
1.4 0.4 2.8 3.0 3.3 0.4 0.3 0.02
1/22 1 1/20 1/20 1/20 1/20 1/20 1/20
.2 .5 .1 .09 .08 .5 .6 .9
Results are shown as mean (SD).
Results Table 1 shows that there were no significant differences in age or gender between detoxified AWLD patients and normal controls. The erythron variables were not significantly different between detoxified AWLD patients and normal controls. Table 2 shows that, in detoxified AWLD patients, mean blood concentrations of ureum, LDH, g-GT, AST, ALT, CHE, amylase, and creatinine were all in the normal laboratory range. There was only 1 subject with slightly elevated LDH (823 U/L) and AST (106 U/L) values. There were no significant differences either in serum g-GT (F 5 1.3, df 5 1/22, p 5 .2; mean in normal controls: 35.2 6 15.2 U/mL) or CHE (F 5 0.3, df 5 1/22, p 5 .6; mean in normal controls: 8468 6 2119 U/mL) between normal controls and detoxified AWLD patients. In AWLD patients, there were no significant correlations between the above variables and the immune variables. There were positive correlations between IFN-g and IL-6 (r 5 .43, p , .05), TNF-a (r 5 .75, p , .001), and IL-1RA (r 5 .53, p , .01); between IL-6 and TNF-a (r 5 .55, p , .01), IL-10 (r 5 .51, p , .05), and IL-1RA (p , .01); between TNF-a and IL-10 (r 5 .45, p , .05); between IL-1RA and TNF-a (r 5 .73, p , .001) and IL-10 (r 5 .60, p , .01); between GM-CSF and IL-1RA (r 5 .48, p , .05); and between IL-4 and IL-5 (r 5 .64, p , Table 2. Comparison of Enzyme Concentrations between Detoxified AWLD Patients and the Normal Laboratory Ranges Variables Ureum LDH g-GT AST ALT Alcalic phosphatase Amylase CHE Creatinine
AWLD
Normal ranges
Units
21.3 (9.2) 484 (152) 35.2 (15.2) 37.5 (26.9) 36.8 (16.8) 61.0 (15.3) 58.9 (21.6) 7982 (1756) 0.93 (0.17)
19 – 43 316 – 618 8 –78 17–59 21–72 38 –126 30 –110 4650 –10440 0.5–1.2
mg/dL U/L U/L U/L U/L U/L U/L U/L mg/dL
All results are expressed as mean (SD).
.01). There were significant and positive correlations between the number of leukocytes and IL-6 (r 5 .49, p , .05), TNF-a (r 5 .49, p , .05), IL-10 (r 5 .46, p , .05), IL-1RA (r 5 .76, p , .001), and GM-CSF (r 5 .69, p , .001). There were significant and positive correlations between the number of neutrophils and IL-6 (r 5 .48, p , .05), IL-1RA (r 5 .56, p , .01), and GM-CSF (r 5 .61, p , .01). There was a significant and positive correlation between number of monocytes and IL-1RA (r 5 .54, p , .01). Due to the multiple intercorrelations between the variables measured, we did not use a Bonferroni p correction, since this could induce type II errors of inference. Table 3 shows that the number of leukocytes and neutrophils was significantly higher in detoxified AWLD patients than in normal volunteers. There was a trend toward a significant increase in the number of monocytes in alcoholic patients. The production of TNF-a, IL-6, IL-10, IL-1RA, and GM-CSF was significantly higher in detoxified AWLD patients than in normal volunteers. Covarying for age and sex did not change any of these results. There were no significant differences in any of the cytokine concentrations, except IL-10, between patients with (n 5 5) or without (n 5 7) use of disulfiramum, i.e., IL-10 was significantly lower (F 5 7.1, df 5 1/10, p 5 .02) in detoxified AWLD patients with (788 6 418 pg/mL) than without (1509 6 581 pg/mL) use of disulfiramum. IL-6 (F 5 15.9, p 5 .001), TNF-a (F 5 19.8, p 5 .0006), IL-1RA (F 5 10.5, p 5 .005), IL-10 (F 5 19.8, p 5 .0006), and GM-CSF (F 5 9.8, p 5 .006) (all df 5 1/16) were significantly higher in detoxified AWLD patients without use of disulfiramum than in the normal volunteers.
Discussion The major finding of this study is that detoxified AWLD patients have a significantly increased production of cytokines and monocytic products, such as IL-6, TNF-a, GM-CSF, IL-10, and IL-1RA. Since the stimulated pro-
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Table 3. Measurements of Immune Variables in Detoxified AWLD Patients and Normal Volunteers (NV) Variables Leukocytes (AN) Neutrophils (%) Lymphocytes (%) Monocytes (%) Neutrophils (AN) Lymphocytes (AN) Monocytes (AN) PGE2 (pg/mL) IFN-g (IU/mL) TNF-a (pg/mL) IL-6 (pg/mL) IL-10 (pg/mL) IL-1RA (ng/mL) GM-CSF (pg/mL) IL-4 (pg/mL) IL-5 (pg/mL)
NV
AWLD
F
df
p
6.53 (1.32) 53.1 (9.5) 33.0 (7.8) 8.21 (3.22) 3.47 (1.02) 3.67 (0.47) 0.52 (0.16) 149 (117) 1171 (633) 614 (222) 27,181 (5582) 736 (223) 5.35 (1.72) 606 (413) 49 (41) 65 (67)
9.35 (1.66) 57.2 (7.7) 29.1 (7.7) 6.74 (1.0) 5.39 (1.39) 2.67 (0.63) 0.63 (0.12) 147 (84) 1718 (977) 1535 (1418) 48,583 (25,543) 1208 (621) 9.84 (4.65) 1546 (668) 42 (26) 77 (59)
18.9 1.2 1.4 2.2 13.1 0.5 3.2 0.0 2.6 5.0 7.0 5.5 10.2 16.0 0.2 0.3
1/20 1/20 1/20 1/20 1/20 1/20 1/20 1/18 1/21 1/21 1/21 1/21 1/21 1/21 1/20 1/21
.0005 .3 .2 .1 .002 .5 .08 .96 .1 .03 .01 .03 .004 .0009 .6 .6
Results are expressed as mean (SD). The data were processed after square-root transformation. %, percentages in total white blood cell count; AN, absolute number of leukocytes (in 10E9/L).
duction of INF-g, a Th1-like cytokine, and IL-4 and IL-5, two Th2-like cytokines, was not altered in detoxified AWLD patients, it can be concluded that no Th1-like or Th2-like response had occurred. The significant increased production of IL-1RA, IL-6, TNF-a, and GM-CSF suggests that detoxified AWLD patients show an exaggerated response of monocytic cells. Moreover, the cytokine profile obtained in detoxified AWLD patients suggests increased production of proinflammatory cytokines, i.e., IL-6, TNF-a, and GM-CSF, as well as negative immunoregulatory proteins, i.e., IL-10 and IL-1RA. Previous research found an increased production of proinflammatory cytokines from mononuclear cells in those alcohol-dependent patients who suffered from liver diseases, such as cirrhosis or hepatitis (Martinez et al 1992, 1993; Rodriguez-Rodriguez et al 1995; Ruiz et al 1993; Khoruts et al 1990). In the present study, however, detoxified alcohol-dependent patients were selected to participate if they had normal values of MCV, g-GT, and cholinesterase, were HBsAG negative, and had negative physical examination. Moreover, no significant alterations in the several erythron variables or serum liver enzymes were detected in the detoxified AWLD patients. No significant correlations were detected between the immune–inflammatory variables and serum liver enzyme concentrations. Thus, the increased production of cytokines in detoxified AWLD patients does not seem to result from liver diseases. The mechanisms by which chronic use of alcohol may have increased the production of several cytokines some weeks after detoxification remains unclear. Damage to tissues other than the liver by chronic alcohol abuse may be involved. Another possibility is that chronic alcohol intake could have primed cells of the
monocyte–macrophage lineage. In this respect, Zuiable et al (1992) showed that chronic, but not acute, incubation of monocytes/macrophages with alcohol significantly enhanced the activities of this cell line. In detoxified AWLD patients no significant alterations in PGE2 release could be found. Results from previous studies, which examined the effects of acute and chronic treatment with alcohol, are not consistent, showing increased, decreased, or unaltered PGE2 production (Bode et al 1988; Smith et al 1991; Szabo et al 1992). Another finding is that detoxified AWLD patients have significantly increased numbers of leukocytes and neutrophils. These findings extent those of Khoruts et al (1990), who found that leukocytes and monocytes were both increased in chronic alcohol-dependent patients without liver disease. A leukopenia and increased numbers of neutrophils and monocytes were reported by other investigators (Homann and Hasselbalch 1992; Kanwar and Tikoo 1992). We found that the leukocytosis and neutrophilia in detoxified AWLD patients was positively related to the production of hematopoietic cytokines, such as GM-CSF, IL-6, and TNF-a. Since the latter have a stimulatory effect on hematopoiesis, and therefore may increase the number of leukocytes, monocytes, and neutrophils in the blood (Cavaillon 1996), increased production of these cytokines may explain leukocytosis and neutrophilia in detoxified AWLD patients. There is some evidence that an increased production of proinflammatory cytokines may be related to the etiopathology of major depression and depression due to a general medical condition (Maes et al 1993a, 1993b; Maes 1997; Yirmiya 1997). In this respect, prominent depressive symptoms, sufficient to meet the criteria for depres-
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sion, are present in 50% of the alcoholic patients the first weeks of abstinence (Schukit 1983). Therefore, it may be hypothesized that the increased production of proinflammatory cytokines observed in newly detoxified alcoholic patients may play a role in the depressive syndrome that frequently occurs in that condition. The research reported was supported in part by the Staglin (NARSAD) Investigator Award to Dr. M. Maes, USA; and the Clinical Research Center for the Mental Health (CRC-MH), Antwerp, Belgium.
References Bode C, Ito T, Rollenhagen A, Bode JC (1988): Effect of acute and chronic alcohol feeding on prostaglandin E2 biosynthesis in rat stomach. Dig Dis Sci 33:814 – 818. Cavaillon J-M (1996): Les Cytokines. Paris: Masson. Cook RT, Garvey MJ, Booth BM, Goeken JA, Stewart B, Noel M (1991): Activated CD-8 cells and HLA DR expression in alcoholics without overt liver disease. J Clin Immunol 11: 246 –253. De Groote D, Zangerle PF, Gevaert Y, Fassotte MF, Belgium Y, Noizat-Pirenne F, et al (1992): Direct stimulation of cytokines (IL-1b, TNF-a, IL-6, IL-2, IFN-g and GM-CSF) in whole blood. I. Comparison with isolated PBMC stimulation. Cytokine 4:239 –248. Homann C, Hasselbalch HC (1992): Hematological abnormalities in alcoholism. Ugeskrift Laeger 154:2184 –2187. Kanwar KC, Tikoo A (1992): Hematological lesion in rat following heavy alcohol ingestion. J Environ Pathol Toxicol Oncol 11:241–245. Khoruts A, Stahnke L, McClain CJ, Logan G, Allen J (1990): Circulating tumour necrosis factor, interleukin-1 and interleukin-6 concentrations in chronic alcoholic patients. Hepatology 13:267–275. Maes M (1997): The immune pathophysiology of major depression. In: Honig A, van Praag HM, editors. Depression: Neurobiological, Psychopathogical and Therapeutic Advances. London: Wiley, pp 197–215. Maes M, Bosmans E, Meltzer HY, Scharpe´ S, Suy E (1993a): Interleukin-1b: A putative mediator of HPA-axis hyperactivity in major depression? Am J Psychiatry 150:1189 –1193. Maes M, Scharpe S, Meltzer HY, Bosmans E, Suy E, Calabrese J, et al (1993b): Relationships between interleukin-6 activity, acute phase proteins, and function of the hypothalamicpituitary-adrenal axis in severe depression. Psychiatry Res 49:11–27. Martinez F, Abril ER, Earnest DL, Watson RR (1992): Ethanol and cytokine secretion. Alcohol 9:455– 458. Martinez F, Thomas NM, Darban H, Cox TJ, Wood S, Watson RR (1993): Interleukin-6 and interleukin-8 production by mononuclear cells of chronic alcoholics during treatment. Alcohol Clin Exp Res 17:1193–1197.
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Mili F, Flanders WD, Boring JR, Annest JL, DeStefano F (1992): The associations of alcohol drinking and drinking cessation to measures of the immune system in middle-aged men. Alcohol Clin Exp Res 16:688 – 694. Mosmann TR, Sad S (1996): The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol Today 17:138 –146. Nair MP, Kronfol ZA, Schwartz SA (1990): Effects of alcohol and nicotine on cytotoxic functions of human lymphocytes. Clin Immunol Immunopathol 54:395– 409. Nelson S, Bagby G, Summer WR (1989): Alcohol suppresses lipopolysaccharide-induced tumor necrosis factor activity in serum and lung. Life Sci 44:673– 676. Rodriguez-Rodriguez E, Gonzales-Reimers E, Santolaria-Fernandez F, Milena-Abril A, Rodriguez-Moreno F, OramasRodriguez J, et al (1995): Cytokine levels in acute alcoholic hepatitis: A sequential study. Drug Alcohol Depend 39:23– 27. Ruiz AD, Perez JLS, Martinez GL, Calvin JG, Extremera BG, Gea FG (1993): Tumour necrosis factor, interleukin-1 and interleukin-6 in alcoholic cirrhosis. Alcohol Alcohol 28:319 – 323. Schukit M (1983): Alcoholic patients with secondary depression. Am J Psychiatry 140:711–714. Sergeeva MG, Gonchar MV, Mevkh AT, Varfolomeyev SD (1998): Prostaglandin E-2 biphasic control of lymphocyte proliferation: Inhibition by picomolar concentrations. FEBS Lett 418:235–238. Smith GS, Myers SI, Bartula LL, Miller TA (1991): Adaptive cytoprotection against alcohol injury in the rat stomach is not due to increased prostanoid synthesis. Prostaglandins 41: 207–223. Spitzer RL, Williams JBW, Gibbon M, First MB (1990): Structured Clinical Interview According to DSM-III-R. New York: American Press. Szabo G, Verma BK, Fogarasi M, Catalano DE (1992): Induction of transforming growth factor-beta and prostaglandin E2 production by ethanol in human monocytes. J Leukoc Biol 52:602– 610. Szabo G, Mandrekar P, Catalano D (1995): Inhibition of superantigen-induced T cell proliferation and monocyte Il-1 beta, TF-alpha, and IL-6 production by acute ethanol treatment. J Leukoc Biol 58:342–345. Verma BK, Fogarasi M, Szabo G (1993): Down-regulation of tumor necrosis factor a activity by acute alcohol treatment in human peripheral blood monocytes. J Clin Immunol 13:8 –22. Yirmiya R (1997): Behavioral and psychological effects of immune activation: Implications for depression due to a general medical condition. Curr Opin Psychiatry 10:470 – 476. Zuiable A, Wiener E, Wickramasinghe SN (1992): In vitro effects of ethanol on the phagocytic and microbial killing activities of normal monocytes and monocyte-derived macrophages. Clin Lab Haematol 14:137–147.
Introduction
A
cute administration of alcohol and chronic alcoholism have been implicated in the onset of a variety of immune defects in vivo and in vitro, including various signs of immunosuppression (Nair et al 1990; Mili et al 1992; Szabo et al 1995), such as abnormal interleukin-2
From the Clinical Research Center for Mental Health, Antwerp, Belgium (CS, AL, EV, MM); Department of Medical Biochemistry, University of Antwerp, Antwerp, Belgium (AL, SS); Department of Psychology, Carleton University, Ottawa, Canada (CS); Pathology Center, St James Hospital, Dublin, Ireland (AW); and Eurogenetics, Transportstraat, Belgium (RDJ, GK, EB). Address reprint requests to Michael Maes, MD, PhD, Clinical Research Center for Mental Health, University Department of Psychiatry, AZ Stuivenberg, 267 Lange Beeldekensstraat, 2060 Antwerp, Belgium. Received August 22, 1997; revised February 5, 1998; accepted February 12, 1998.
© 1999 Society of Biological Psychiatry
(IL-2)-supported and mitogen-induced responsiveness of lymphocytes, and suppression of tumor necrosis factor-a (TNF-a) and IL-2 production (Cook et al 1991; Verma et al 1993). Alcohol suppresses lipopolysaccharide (LPS)induced production of proinflammatory cytokines (Nelson et al 1989; Szabo et al 1995). Chronic alcohol dependent patients without liver disease show also increased numbers of leukocytes, neutrophils, or monocytes (Khoruts et al 1990; Kanwar and Tikoo 1992). Alcoholic liver disease is characterized by activation of the monocyte–macrophage system, with increased production of TNF-a, IL-1, and IL-6 (Martinez et al 1992; Rodriguez-Rodriguez et al 1995; Ruiz et al 1993; Khoruts et al 1990). One study performed in abstinent (detoxified) chronic alcoholic patients without overt liver disease (AWLD), however, detected signs of T-cell activation (Cook et al 1991). Prostaglandin E2 (PGE2), a proinflammatory peptide, was found to be elevated in acute-alcohol treated monocytes and stomach tissue of alcoholic patients (Smith et al 1991; Szabo et al 1992). The main sources of PGE2 during immune responses are monocyte cells (Sergeeva et al 1998). PGE2 has a biphasic control of lymphocyte proliferation and, therefore, plays an important role in immune cell function regulation (Sergeeva et al 1998); however, it has remained elusive whether detoxified AWLD patients exhibit any changes in cytokine or PGE2 production. Based on the above literature and the recent findings that AWLD patients have signs of T-cell activation (Cook et al 1991), we have chosen to measure T-cell and monocyte-derived cytokines, since these are main regulators of immune homeostasis (Cavaillon 1996). Two polar categories of T helper cells (Th) have been described in humans, i.e., Th1-like cells, which secrete interferon-g (INF-g), and Th2-like cells, which secrete IL-4 and IL-5 (Mosmann and Sad 1996). Several other cytokines are produced by both Th1- and Th2-like cells and/or cells of the monocyte–macrophage lineage, e.g., IL-6, IL-10, TNF-a, and granulocyte–macrophage stimulating factor (GM-CSF). The aims of the present study were to examine i) PGE2, Th1-like (IFN-g) and Th2-like (IL-4 and IL-5) 0006-3223/99/$20.00 PII S0006-3223(98)00083-3
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cytokines, the proinflammatory cytokines, IL-6, TNF-a, and GM-CSF, and the negative immunoregulatory proteins, IL-10, and the IL-1 receptor antagonist (RA) in detoxified AWLD patients; and ii) the number of leukocytes and differentials in relation to the above cytokines or monocytic products.
Methods and Materials Subjects Twenty-four subjects participated in this study, i.e., 12 normal volunteers and 12 chronic alcohol-dependent (303.9) inpatients. The patients were classified according to DSM-III-R criteria on the basis of the Structured Interview for DSM-III-R Diagnosis (SCID) (Spitzer et al 1990), administered by one of the authors (EV). The normal volunteers were excluded for a current and past history of psychiatric disorders, using the SCID, and a positive family history of psychiatric disorders in first-degree relatives, using a semistructured interview. The healthy volunteers were nondrinkers and were free of any medication during at least 1 month previous to blood sampling. None had ever been taking psychotropic drugs. The alcohol-dependent patients had an alcohol consumption of more than 300 mL pure alcohol/day for at least 2 years. We excluded the following alcoholic patients: i) patients with overt liver disease, such as hepatitis or liver cirrhosis, and patients with pulmonary or urinary tract infections or hepatitis B surface antigen (HbsAG) and HIV positive patients; and ii) patients with a recent or past history of other axis-I diagnoses (beside alcohol dependence), such as schizophrenic, paranoid, organic mental, and affective disorders. A positive family history of psychiatric disorders in the AWLD patients was not considered as an inclusion or exclusion criterion. Inclusion criteria for normal volunteers and alcoholic patients included mean cell volume (MCV), g-glutamyl transpeptidase (g-GT), and cholinesterase (CHE) values within the normal laboratory limits and a normal physical examination. A semistructured interview was employed to exclude subjects i) with any acute or chronic medical illness, such as autoimmune disorders, inflammatory bowel disease, rheumatoid arthritis, and endocrine disorders; and ii) who suffered from acute infectious or allergic reactions for at least 2 weeks prior to the study.
Methods After an overnight fast, blood samples were taken at 7:30 AM (630 min). In AWLD patients blood was collected 32–34 days after admission. Immediately after admission into hospital, alcoholic patients received diazepam (20 – 40 mg/day), which was tapered off during a 2-week period. Consequently, patients were drug-free for 18 –20 days before blood samplings were carried out. None of the patients suffered from withdrawal phenomena. Five subjects were taking disulfiramum, 200 – 400 mg/day, which was started 21 days after admission into hospital. No other medications were administered to the patients. All patients remained hospitalized during the study.
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Total number of leukocytes and white blood cell (WBC) differentiation, and red blood cell (RBC) parameters, i.e., RBC count, MCV, mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), hematocrit (Ht), and hemoglobin (Hb), were measured with a Technicon H2 (Bayer, Brussels, Belgium) fully automated blood cell counter. The analytical interassay coefficient of variation (CV) values obtained in our laboratory were as follows: number of leukocytes 1.5%, neutrophils 0.9%, lymphocytes 3.1%, monocytes 7.2%, and RBC 1.3%; Hb 1.0%, Ht 1.3%, MCV 1.3%, MCH 1.5%, and MCHC 1.5%. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), g-GT, lactate dehydrogenase (LDH), amylase, and creatinine kinase (CK), amylase, serum ureum and creatinine, and CHE (E.C.3.1.1.8) were measured with the dry chemistry method (Ortho Clinical Diagnostics). Cytokine and PGE2 concentrations were studied by stimulating whole blood with phytohemagglutinin (PHA) 1 lipopolysaccharide (LPS) and analyzing culture supernatant (De Groote et al 1992). Heparin (100 IU/mL) blood samples were diluted 1:10 in RPMI-1640 medium (Life Technologies, Belgium) containing 1% of penicillin (100 IU/mL) (Sigma), PHA (Murex, Belgium), 5 mg/mL, and LPS (Sigma), 20 mg/mL (De Groote et al 1992). For a blank, the blood from each sample was diluted with RPMI-1640 containing 1% penicillin. Samples (400 mL) were pipetted into 24 well-plates prefilled with medium (1200 mL) and incubated for 48 hours or 72 hours in a humidified atmosphere at 37°C, 5% CO2. After incubation, the plates were centrifuged at 1500 rpm for 10 min. Supernatants were taken off carefully under sterile conditions, divided into Eppendorf tubes, and frozen immediately at 270°C until thawed for assay of PGE2 (48 hours) and IFN-g, TNF-a, IL-6, IL-10, IL-1RA, GM-CSF, IL-4, and IL-5 (72 hours). All immune–inflammatory variables were quantified by means of enzyme-linked immunosorbent assay methods (Eurogenetics, Tessenderlo, Belgium) based on appropriate and validated sets of monoclonal antibodies. PGE2 concentrations in culture supernatant were measured by means of an enzyme immunoassay (Cayman Chemical Company, Ann Arbor, MI). The optimal dilution for the supernatant has been determined and, consequently, we employed a 15-fold dilution in this assay. All samples from normal volunteers and detoxified AWLD patients were assayed at the same time, in a single run with a single lot number of reagents and consumables employed by a single operator (CS or AL). The intraassay CV values for all variables were less than 8%.
Statistical Analysis The independence of classification systems was ascertained by means of analysis of contingence (x2 test). Group mean differences were examined by means of analysis of variance (ANOVA) or analysis of covariance (ANCOVA). Relationships between variables were checked by means of Pearson’s product– moment correlation coefficients. Transformations (square root) were used to normalize the distribution of the variables or to adjust for heterogeneity of variance between study groups.
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Table 1. Demographic Data and Erythron Characteristics in Detoxified AWLD Patients and Normal Volunteers (NV) Variables Age (years) Men/women RBC (106/mL) Ht (%) Hb (g/dL) MCV (fL) MCH (pg) MCHC (g/dL)
NV
AWLD
F/x2
df
p value
36.6 (9.5) 10/2 5.04 (0.51) 45.2 (3.5) 15.1 (1.1) 89.9 (4.3) 30.1 (1.6) 33.5 (0.8)
41.0 (8.9) 11/1 4.73 (0.37) 43.1 (2.1) 14.4 (0.7) 91.2 (5.7) 30.6 (2.3) 33.5 (0.9)
1.4 0.4 2.8 3.0 3.3 0.4 0.3 0.02
1/22 1 1/20 1/20 1/20 1/20 1/20 1/20
.2 .5 .1 .09 .08 .5 .6 .9
Results are shown as mean (SD).
Results Table 1 shows that there were no significant differences in age or gender between detoxified AWLD patients and normal controls. The erythron variables were not significantly different between detoxified AWLD patients and normal controls. Table 2 shows that, in detoxified AWLD patients, mean blood concentrations of ureum, LDH, g-GT, AST, ALT, CHE, amylase, and creatinine were all in the normal laboratory range. There was only 1 subject with slightly elevated LDH (823 U/L) and AST (106 U/L) values. There were no significant differences either in serum g-GT (F 5 1.3, df 5 1/22, p 5 .2; mean in normal controls: 35.2 6 15.2 U/mL) or CHE (F 5 0.3, df 5 1/22, p 5 .6; mean in normal controls: 8468 6 2119 U/mL) between normal controls and detoxified AWLD patients. In AWLD patients, there were no significant correlations between the above variables and the immune variables. There were positive correlations between IFN-g and IL-6 (r 5 .43, p , .05), TNF-a (r 5 .75, p , .001), and IL-1RA (r 5 .53, p , .01); between IL-6 and TNF-a (r 5 .55, p , .01), IL-10 (r 5 .51, p , .05), and IL-1RA (p , .01); between TNF-a and IL-10 (r 5 .45, p , .05); between IL-1RA and TNF-a (r 5 .73, p , .001) and IL-10 (r 5 .60, p , .01); between GM-CSF and IL-1RA (r 5 .48, p , .05); and between IL-4 and IL-5 (r 5 .64, p , Table 2. Comparison of Enzyme Concentrations between Detoxified AWLD Patients and the Normal Laboratory Ranges Variables Ureum LDH g-GT AST ALT Alcalic phosphatase Amylase CHE Creatinine
AWLD
Normal ranges
Units
21.3 (9.2) 484 (152) 35.2 (15.2) 37.5 (26.9) 36.8 (16.8) 61.0 (15.3) 58.9 (21.6) 7982 (1756) 0.93 (0.17)
19 – 43 316 – 618 8 –78 17–59 21–72 38 –126 30 –110 4650 –10440 0.5–1.2
mg/dL U/L U/L U/L U/L U/L U/L U/L mg/dL
All results are expressed as mean (SD).
.01). There were significant and positive correlations between the number of leukocytes and IL-6 (r 5 .49, p , .05), TNF-a (r 5 .49, p , .05), IL-10 (r 5 .46, p , .05), IL-1RA (r 5 .76, p , .001), and GM-CSF (r 5 .69, p , .001). There were significant and positive correlations between the number of neutrophils and IL-6 (r 5 .48, p , .05), IL-1RA (r 5 .56, p , .01), and GM-CSF (r 5 .61, p , .01). There was a significant and positive correlation between number of monocytes and IL-1RA (r 5 .54, p , .01). Due to the multiple intercorrelations between the variables measured, we did not use a Bonferroni p correction, since this could induce type II errors of inference. Table 3 shows that the number of leukocytes and neutrophils was significantly higher in detoxified AWLD patients than in normal volunteers. There was a trend toward a significant increase in the number of monocytes in alcoholic patients. The production of TNF-a, IL-6, IL-10, IL-1RA, and GM-CSF was significantly higher in detoxified AWLD patients than in normal volunteers. Covarying for age and sex did not change any of these results. There were no significant differences in any of the cytokine concentrations, except IL-10, between patients with (n 5 5) or without (n 5 7) use of disulfiramum, i.e., IL-10 was significantly lower (F 5 7.1, df 5 1/10, p 5 .02) in detoxified AWLD patients with (788 6 418 pg/mL) than without (1509 6 581 pg/mL) use of disulfiramum. IL-6 (F 5 15.9, p 5 .001), TNF-a (F 5 19.8, p 5 .0006), IL-1RA (F 5 10.5, p 5 .005), IL-10 (F 5 19.8, p 5 .0006), and GM-CSF (F 5 9.8, p 5 .006) (all df 5 1/16) were significantly higher in detoxified AWLD patients without use of disulfiramum than in the normal volunteers.
Discussion The major finding of this study is that detoxified AWLD patients have a significantly increased production of cytokines and monocytic products, such as IL-6, TNF-a, GM-CSF, IL-10, and IL-1RA. Since the stimulated pro-
Cytokines and Alcoholism
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Table 3. Measurements of Immune Variables in Detoxified AWLD Patients and Normal Volunteers (NV) Variables Leukocytes (AN) Neutrophils (%) Lymphocytes (%) Monocytes (%) Neutrophils (AN) Lymphocytes (AN) Monocytes (AN) PGE2 (pg/mL) IFN-g (IU/mL) TNF-a (pg/mL) IL-6 (pg/mL) IL-10 (pg/mL) IL-1RA (ng/mL) GM-CSF (pg/mL) IL-4 (pg/mL) IL-5 (pg/mL)
NV
AWLD
F
df
p
6.53 (1.32) 53.1 (9.5) 33.0 (7.8) 8.21 (3.22) 3.47 (1.02) 3.67 (0.47) 0.52 (0.16) 149 (117) 1171 (633) 614 (222) 27,181 (5582) 736 (223) 5.35 (1.72) 606 (413) 49 (41) 65 (67)
9.35 (1.66) 57.2 (7.7) 29.1 (7.7) 6.74 (1.0) 5.39 (1.39) 2.67 (0.63) 0.63 (0.12) 147 (84) 1718 (977) 1535 (1418) 48,583 (25,543) 1208 (621) 9.84 (4.65) 1546 (668) 42 (26) 77 (59)
18.9 1.2 1.4 2.2 13.1 0.5 3.2 0.0 2.6 5.0 7.0 5.5 10.2 16.0 0.2 0.3
1/20 1/20 1/20 1/20 1/20 1/20 1/20 1/18 1/21 1/21 1/21 1/21 1/21 1/21 1/20 1/21
.0005 .3 .2 .1 .002 .5 .08 .96 .1 .03 .01 .03 .004 .0009 .6 .6
Results are expressed as mean (SD). The data were processed after square-root transformation. %, percentages in total white blood cell count; AN, absolute number of leukocytes (in 10E9/L).
duction of INF-g, a Th1-like cytokine, and IL-4 and IL-5, two Th2-like cytokines, was not altered in detoxified AWLD patients, it can be concluded that no Th1-like or Th2-like response had occurred. The significant increased production of IL-1RA, IL-6, TNF-a, and GM-CSF suggests that detoxified AWLD patients show an exaggerated response of monocytic cells. Moreover, the cytokine profile obtained in detoxified AWLD patients suggests increased production of proinflammatory cytokines, i.e., IL-6, TNF-a, and GM-CSF, as well as negative immunoregulatory proteins, i.e., IL-10 and IL-1RA. Previous research found an increased production of proinflammatory cytokines from mononuclear cells in those alcohol-dependent patients who suffered from liver diseases, such as cirrhosis or hepatitis (Martinez et al 1992, 1993; Rodriguez-Rodriguez et al 1995; Ruiz et al 1993; Khoruts et al 1990). In the present study, however, detoxified alcohol-dependent patients were selected to participate if they had normal values of MCV, g-GT, and cholinesterase, were HBsAG negative, and had negative physical examination. Moreover, no significant alterations in the several erythron variables or serum liver enzymes were detected in the detoxified AWLD patients. No significant correlations were detected between the immune–inflammatory variables and serum liver enzyme concentrations. Thus, the increased production of cytokines in detoxified AWLD patients does not seem to result from liver diseases. The mechanisms by which chronic use of alcohol may have increased the production of several cytokines some weeks after detoxification remains unclear. Damage to tissues other than the liver by chronic alcohol abuse may be involved. Another possibility is that chronic alcohol intake could have primed cells of the
monocyte–macrophage lineage. In this respect, Zuiable et al (1992) showed that chronic, but not acute, incubation of monocytes/macrophages with alcohol significantly enhanced the activities of this cell line. In detoxified AWLD patients no significant alterations in PGE2 release could be found. Results from previous studies, which examined the effects of acute and chronic treatment with alcohol, are not consistent, showing increased, decreased, or unaltered PGE2 production (Bode et al 1988; Smith et al 1991; Szabo et al 1992). Another finding is that detoxified AWLD patients have significantly increased numbers of leukocytes and neutrophils. These findings extent those of Khoruts et al (1990), who found that leukocytes and monocytes were both increased in chronic alcohol-dependent patients without liver disease. A leukopenia and increased numbers of neutrophils and monocytes were reported by other investigators (Homann and Hasselbalch 1992; Kanwar and Tikoo 1992). We found that the leukocytosis and neutrophilia in detoxified AWLD patients was positively related to the production of hematopoietic cytokines, such as GM-CSF, IL-6, and TNF-a. Since the latter have a stimulatory effect on hematopoiesis, and therefore may increase the number of leukocytes, monocytes, and neutrophils in the blood (Cavaillon 1996), increased production of these cytokines may explain leukocytosis and neutrophilia in detoxified AWLD patients. There is some evidence that an increased production of proinflammatory cytokines may be related to the etiopathology of major depression and depression due to a general medical condition (Maes et al 1993a, 1993b; Maes 1997; Yirmiya 1997). In this respect, prominent depressive symptoms, sufficient to meet the criteria for depres-
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sion, are present in 50% of the alcoholic patients the first weeks of abstinence (Schukit 1983). Therefore, it may be hypothesized that the increased production of proinflammatory cytokines observed in newly detoxified alcoholic patients may play a role in the depressive syndrome that frequently occurs in that condition. The research reported was supported in part by the Staglin (NARSAD) Investigator Award to Dr. M. Maes, USA; and the Clinical Research Center for the Mental Health (CRC-MH), Antwerp, Belgium.
References Bode C, Ito T, Rollenhagen A, Bode JC (1988): Effect of acute and chronic alcohol feeding on prostaglandin E2 biosynthesis in rat stomach. Dig Dis Sci 33:814 – 818. Cavaillon J-M (1996): Les Cytokines. Paris: Masson. Cook RT, Garvey MJ, Booth BM, Goeken JA, Stewart B, Noel M (1991): Activated CD-8 cells and HLA DR expression in alcoholics without overt liver disease. J Clin Immunol 11: 246 –253. De Groote D, Zangerle PF, Gevaert Y, Fassotte MF, Belgium Y, Noizat-Pirenne F, et al (1992): Direct stimulation of cytokines (IL-1b, TNF-a, IL-6, IL-2, IFN-g and GM-CSF) in whole blood. I. Comparison with isolated PBMC stimulation. Cytokine 4:239 –248. Homann C, Hasselbalch HC (1992): Hematological abnormalities in alcoholism. Ugeskrift Laeger 154:2184 –2187. Kanwar KC, Tikoo A (1992): Hematological lesion in rat following heavy alcohol ingestion. J Environ Pathol Toxicol Oncol 11:241–245. Khoruts A, Stahnke L, McClain CJ, Logan G, Allen J (1990): Circulating tumour necrosis factor, interleukin-1 and interleukin-6 concentrations in chronic alcoholic patients. Hepatology 13:267–275. Maes M (1997): The immune pathophysiology of major depression. In: Honig A, van Praag HM, editors. Depression: Neurobiological, Psychopathogical and Therapeutic Advances. London: Wiley, pp 197–215. Maes M, Bosmans E, Meltzer HY, Scharpe´ S, Suy E (1993a): Interleukin-1b: A putative mediator of HPA-axis hyperactivity in major depression? Am J Psychiatry 150:1189 –1193. Maes M, Scharpe S, Meltzer HY, Bosmans E, Suy E, Calabrese J, et al (1993b): Relationships between interleukin-6 activity, acute phase proteins, and function of the hypothalamicpituitary-adrenal axis in severe depression. Psychiatry Res 49:11–27. Martinez F, Abril ER, Earnest DL, Watson RR (1992): Ethanol and cytokine secretion. Alcohol 9:455– 458. Martinez F, Thomas NM, Darban H, Cox TJ, Wood S, Watson RR (1993): Interleukin-6 and interleukin-8 production by mononuclear cells of chronic alcoholics during treatment. Alcohol Clin Exp Res 17:1193–1197.
C. Song et al
Mili F, Flanders WD, Boring JR, Annest JL, DeStefano F (1992): The associations of alcohol drinking and drinking cessation to measures of the immune system in middle-aged men. Alcohol Clin Exp Res 16:688 – 694. Mosmann TR, Sad S (1996): The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol Today 17:138 –146. Nair MP, Kronfol ZA, Schwartz SA (1990): Effects of alcohol and nicotine on cytotoxic functions of human lymphocytes. Clin Immunol Immunopathol 54:395– 409. Nelson S, Bagby G, Summer WR (1989): Alcohol suppresses lipopolysaccharide-induced tumor necrosis factor activity in serum and lung. Life Sci 44:673– 676. Rodriguez-Rodriguez E, Gonzales-Reimers E, Santolaria-Fernandez F, Milena-Abril A, Rodriguez-Moreno F, OramasRodriguez J, et al (1995): Cytokine levels in acute alcoholic hepatitis: A sequential study. Drug Alcohol Depend 39:23– 27. Ruiz AD, Perez JLS, Martinez GL, Calvin JG, Extremera BG, Gea FG (1993): Tumour necrosis factor, interleukin-1 and interleukin-6 in alcoholic cirrhosis. Alcohol Alcohol 28:319 – 323. Schukit M (1983): Alcoholic patients with secondary depression. Am J Psychiatry 140:711–714. Sergeeva MG, Gonchar MV, Mevkh AT, Varfolomeyev SD (1998): Prostaglandin E-2 biphasic control of lymphocyte proliferation: Inhibition by picomolar concentrations. FEBS Lett 418:235–238. Smith GS, Myers SI, Bartula LL, Miller TA (1991): Adaptive cytoprotection against alcohol injury in the rat stomach is not due to increased prostanoid synthesis. Prostaglandins 41: 207–223. Spitzer RL, Williams JBW, Gibbon M, First MB (1990): Structured Clinical Interview According to DSM-III-R. New York: American Press. Szabo G, Verma BK, Fogarasi M, Catalano DE (1992): Induction of transforming growth factor-beta and prostaglandin E2 production by ethanol in human monocytes. J Leukoc Biol 52:602– 610. Szabo G, Mandrekar P, Catalano D (1995): Inhibition of superantigen-induced T cell proliferation and monocyte Il-1 beta, TF-alpha, and IL-6 production by acute ethanol treatment. J Leukoc Biol 58:342–345. Verma BK, Fogarasi M, Szabo G (1993): Down-regulation of tumor necrosis factor a activity by acute alcohol treatment in human peripheral blood monocytes. J Clin Immunol 13:8 –22. Yirmiya R (1997): Behavioral and psychological effects of immune activation: Implications for depression due to a general medical condition. Curr Opin Psychiatry 10:470 – 476. Zuiable A, Wiener E, Wickramasinghe SN (1992): In vitro effects of ethanol on the phagocytic and microbial killing activities of normal monocytes and monocyte-derived macrophages. Clin Lab Haematol 14:137–147.