OBJECTIVES:

Excessive alcohol consumption is associated with the generation of antibodies against neoantigens induced by ethanol metabolism. However, the associations between such immune responses, ethanol consumption, and liver injury remain unclear.

METHODS:

Eight-six male alcoholics with (n = 54) or without (n = 32) liver disease, and 20 male volunteers (6 abstainers, 14 moderate drinkers) underwent clinical, morphological, and biochemical assessments of liver status and ethanol consumption.

RESULTS:

Antiacetaldehyde adduct IgAs in both groups of alcoholics were significantly higher than those in the controls. Elevated IgGs occurred in patients with liver disease, whereas IgMs were high in the heavy drinkers without apparent liver disease. Liver disease patients had high levels of both proinflammatory (IL-2, IL-6, IL-8, TNF-) and antiinflammatory (IL-10) cytokines, whereas those without liver disease showed elevated IL-6, IL-8, and IL-10 only. Ethanol consumption correlated significantly with antiadduct IgA and IL-6 levels, which also showed parallel changes upon abstinence.

CONCLUSIONS:

Alcoholic liver disease is associated with the generation of IgAs and IgGs against acetaldehyde-derived antigens and enhanced levels of both pro- and antiinflammatory cytokines, whereas elevated IgA, IL-6, and IL-10 characterize alcoholics without liver disease. These data suggest that immunological mechanisms may play a role in the sequence of events leading to liver disease in some patients with excessive drinking.

INTRODUCTION

Alcoholic liver disease in humans has been previously found to be associated with elevated serum IgA and abnormal tissue deposition of IgA (^1,2). A growing body of evidence has further indicated that excessive alcohol consumption leads to the production of immune responses, which are specifically directed to sequential and conformational epitopes generated in covalent binding reactions between proteins and ethanol metabolites (^{3,4,5,6,7,8,9}). Studies have also suggested a role for such immunological reactions in the adverse effects of ethanol in tissues (^4,6,10). IgA antibodies against acetaldehyde-derived neo-antigens have been previously found in patients with alcoholic liver disease, the titers being significantly higher than those in patients with non-alcoholic liver disease, or non-drinking controls (⁷). However, the sequence of events in the sensitization to ethanol, generation of immune responses, and tissue injury has remained unclear.

The present studies were designed to investigate immunoglobulin isotype-specific responses against acetaldehyde-derived antigens in male alcoholics representing a wide range of alcohol consumption and severity of liver injury. The antibody titers were compared with the levels of proinflammatory (IL-2, IL-6, IL-8, TNF-) and antiinflammatory (IL-10, TGF-1) cytokines that are known to modulate immunological functions in vivo. The data indicate distinct differences in the antibody and cytokine responses, which could play a role in the development of liver injury in some alcoholic patients.

METHODS

Patients and Control Subjects

We studied 86 male alcoholic patients (mean age 48 12 yr) all of whom showed a well-documented history of excessive ethanol consumption. The main clinical and laboratory characteristics of the study population are summarized in Table 1. There were 54 patients with biopsy-proven liver disease, who had consumed ethanol in amounts exceeding 80 g/day either continuously or during repeated episodes of binge drinking. This sample included patients in whom liver histology ranged from mild fibrosis and fatty change to cirrhosis with a wide distribution of morphological abnormalities related to alcoholic hepatitis, as assessed according to previously established combined morphological index (CMI) of liver disease severity (^11,12). In addition, the study population included 32 heavy drinkers, who had been admitted for detoxification, but were devoid of clinical and laboratory evidence of significant liver disease. Because of ethical considerations, these patients were not biopsied. They showed a history of continuous ethanol consumption or binge drinking, which had consisted of a mean of 130 g/day during the period of 4 wk prior to sampling. The documentation of ethanol intake was based on detailed personal interviews using a time-line follow-back technique. The patients were asked how many alcohol-containing drinks (standard drink = 12 g of ethyl alcohol corresponding to one beer, one glass of table wine, or three centiliters of 40% proof spirit) they had consumed during (1) 24 h, (2) 1 wk, and (3) 4 wk preceding admission. The mean duration of abstinence prior to sampling was 2 2 days. A follow-up with supervised abstinence and repeated sampling during hospitalization over a period of 8 2 days was carried out from 17 patients. Blood alcohol concentrations during this time were controlled by repeated ethanol analyses from breath air. All patients were negative for hepatitis B virus antigen or hepatitis C serology.

Table 1 - Main Clinical and Laboratory Characteristics of the Study Subjects.

Full table

The reference population consisted of 20 apparently healthy male volunteers (mean age 48 17 yr) who were either abstainers (n = 6) or moderate drinkers (n = 14) whose mean daily ethanol consumption was 20 g (range 1–60 g). In the latter group, the mean duration of abstinence was 3 days and the amount of alcohol had not exceeded two drinks during the 24 h preceding blood sampling.

All serum samples were stored at -70°C until analysis. All participants of the study gave their informed consent and the study was carried out according to the provisions of the Declaration of Helsinki.

Preparation of Acetaldehyde-Modified Antigens In Vitro

Human erythrocyte protein was prepared from EDTA-blood of a teetotaler. The erythrocytes were separated by centrifugation and washed thrice with an equal volume of phosphate-buffered saline (PBS: 7.9 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 137 mM NaCl, 2.7 mM KCl, pH 7.4), lysed with polyoxyethylene ether, 0.1% V/V in borate buffer (Hemolysis Reagent, DIAMAT™ Analyzer System, Bio-Rad), and incubated for 35 min at 37°C to remove unstable Schiff bases. The hemolysate was brought into a hemoglobin protein concentration of 12 mg/mL with PBS and stored frozen in aliquots at -70°C prior to use. Acetaldehyde diluted in PBS was added to aliquots of the freshly prepared hemoglobin, containing 12 mg protein/mL, to obtain a final acetaldehyde concentration of 10 mM. The mixture was allowed to react in a tightly sealed container at 4°C overnight. Protein adducts were reduced by the addition of sodium cyanoborohydride (10 mM) and mixing for 5 h at 4°C. All protein solutions were dialyzed twice against PBS at 4°C and stored in small aliquots for single use at -70°C. Samples representing unmodified protein were prepared and treated similarly to that of the modified protein except for the addition of acetaldehyde.

Measurements of the Antibody Titers

The microtiter plates (Nunc-Immuno Plate, Maxisorb™, InterMed, Denmark) were coated with acetaldehyde-modified hemoglobin, or corresponding unmodified proteins (background) in PBS (3 g protein in 100 l/well) and incubated for 1½ h at +37°C. Nonspecific binding was blocked by incubation with 0.2% gelatin in PBS (150 l/well) for 1 h at +37°C. The sample sera were diluted (1:40) in PBS, which contained 0.04% Tween-20 (PBS-Tween). The final volume of 50 l of each serum dilution were allowed to react with the coated proteins for 1 h at +37°C followed by extensive washing with PBS-Tween. Alkaline phosphatase-linked goat antihuman immunoglobulins IgA, IgG, or IgM (Jackson ImmunoResearch Laboratories, Inc., West Grove) were used to label antibody-antigen complexes (50 l/well). The immunoglobulins were diluted in PBS-Tween containing 8 mM MgCl₂ and a small amount of dithiothreitol (DTT). The plates were allowed to incubate at +4°C overnight. After washing, 100 L of p-nitrophenylphosphate-solution was added for color reaction substrate (Alkaline Phosphatase Substrate Kit, Bio-Rad Laboratories, Hercules, CA). Color reactions were stopped by adding 100 l of 0.4 M NaOH and the optical densities were read at 405 nm by Anthos HTII microplate reader (Anthos Labtec Instruments, Salzburg, Austria).

Other Methods

The concentrations of interleukins (IL-2, IL-6, IL-8, IL-10, TNF-, and TGF-1) in serum were determined using Quantikine high sensitivity ELISA kits according to the instructions of the manufacturer (R&D Systems, Abington Science Park, UK). The concentration of carbohydrate-deficient transferrin (CDT) was measured by a competitive radioimmunoassay after microcolumn separation (CDTect). Serum GT, aspartate aminotransferase (AST), albumin, total immunoglobulins, and bilirubin were measured by standard clinical chemical methods. All analyses were carried out in an accreditated laboratory (SFS-EN 45001, ISO/IEC Guide 25) of EP Central Hospital.

Calculations and Statistical Methods

The data are expressed as mean SD. In the antibody titer analyses, the immunoassay values (OD₄₀₅) obtained in a reaction with the sample and the unconjugated protein (background) were subtracted from the corresponding values measured from the reaction between the sample and the acetaldehyde-protein-conjugate to get a difference score for each sample. The data are expressed as units/liter (U/L) referring to the difference score (OD₄₀₅ 10³). The comparisons between the alcoholic and the non-alcoholic groups were carried out using Student's t-test or the Mann-Whitney test for parameters with skewed distributions of values. The differences between three or more groups were analyzed using ANOVA and the differences were considered statistically significant at p < 0.05. Square root transformation was used to yield non-skewed distributions before ANOVA analyses when necessary. Linear regression analysis was used to calculate correlations between the different variables. The analyses were carried out using GraphPadPrism statistical software (San Diego, CA).

RESULTS

Titers of IgA, IgG, and IgM antibodies against acetaldehyde-modified epitopes in alcoholics with or without liver disease, in moderate drinkers and in abstainers are shown in Figure 1. The patients with alcoholic liver disease showed elevated mean titers of IgA and IgG antibodies compared to abstainers. The IgA titers were also significantly higher in the alcoholics without liver disease than in moderate drinkers (p < 0.01) or in abstainers (p < 0.001), whereas IgG titers did not differ between these groups. IgM titers were most notably elevated in alcoholics without liver disease. Interestingly, the values in moderate drinkers were also higher than those in abstainers (p < 0.05). The levels of total unspecific IgA, IgG or IgM were not significantly different between any of these groups (data not shown).

Figure 1.

Titers of IgA, IgG, and IgM antibodies against acetaldehyde-modified epitopes in alcoholics, moderate drinkers, and abstainers. The patients with alcoholic liver disease showed the highest titers of IgA and IgG antibodies. The IgA titers were significantly higher in alcoholics with (321 217 U/L) (p < 0.001) or without (143 118 U/L) (p < 0.01) liver disease than those in moderate drinkers (56 82 U/L) or in abstainers (22 24 U/L). The highest IgG titers occurred in the ALD patients (364 138 U/L) (p < 0.05) whereas the groups of alcoholics without liver disease (293 168 U/l), moderate drinkers (256 114 U/L), and abstainers (167 130 U/L) were not significantly different. IgM titers were most notably elevated in alcoholics without liver disease (656 143 U/L) (p < 0.001). The values in moderate drinkers (593 78 U/L) were also higher than those in abstainers (393 241 U/L) (p < 0.05) (U/L = OD₄₀₅ 10³).

Full figure and legend (29K)

In order to examine the possible regulation and downregulation of the antibody responses, cytokine measurements were also carried out for each patient. The patients with ALD showed significantly elevated values of proinflammatory cytokines (IL-2, IL-8, and TNF-), whereas in patients without liver disease these cytokines, except for IL-8, remained at the levels of healthy controls (Figs. 2A–C, respectively). The levels of IL-6 (Fig. 2D) were high both in the groups of alcoholics with and without liver disease, as compared to the groups of moderate drinkers or abstainers (p < 0.001 for both comparisons). The mean levels of the antiinflammatory cytokine IL-10 were higher in all the alcohol consuming groups, including moderate drinkers, than that of the abstainers (p < 0.001) (Fig. 2E). In contrast, the mean levels of TGF-1 did not differ significantly in these comparisons (Fig. 2F). Interestingly, relatively low values of this cytokine were observed in patients suffering from liver disease. The CMI of liver disease severity showed a significant correlation with the proinflammatory cytokines, IL-6 (r_s= 0.59, p < 0.001) and IL-8 (r_s= 0.63, p < 0.001) whereas not with the other cytokines or with the antibody levels.

Figure 2.

The levels of pro- and antiinflammatory cytokines in alcoholics, moderate drinkers, and abstainers. Proinflammatory cytokines IL-2 (A) and TNF- (C) were elevated only in alcoholic liver disease patients such that the levels in this group were significantly higher than those in any of the other groups (p < 0.001). IL-8 (B) was also high in ALD patients (p < 0.001) in comparison with all the other groups. The patients without apparent liver disease also showed higher values of IL-8 than those in the moderate drinkers (p < 0.01) or abstainers (p < 0.01). IL-6 (D) in both groups of alcoholics was significantly higher than those in the control groups (p < 0.001 for both comparisons). The levels of the antiinflammatory IL-10 cytokine (E) were also higher in the alcoholic groups as compared to the abstainers (p < 0.001). Some moderate drinkers also showed elevations in IL-10 levels, which, however, did not reach significance when compared to the levels of abstainers (p= 0.06). The levels of TGF-1 were found to be relatively low in ALD patients, although not significantly different from those in the group of abstainers (F).

Full figure and legend (28K)

Figure 3 shows the mean (SD) titers of IgA, IgG, and IgM antibodies against the acetaldehyde-derived antigens in 17 alcoholics without apparent liver disease at the time of admission and after supervised abstinence, as compared to the levels obtained from moderate drinkers and abstainers. IgA levels were significantly lower in the samples taken after 8 2 days of supervised abstinence (p < 0.05), whereas IgG or IgM levels did not change significantly. After this period of abstinence, significant changes were also noted in the levels of IL-6 (-47%), IL-10 (-82%), and TNF- (-41%), whereas the levels of IL-2 ( 0%), IL-8 (-26%), and TGF-1 (+3%) remained relatively stable. However, the IL-6 levels after abstinence remained higher than those in the reference groups (p < 0.05 for moderate drinkers; p < 0.001 for abstainers). The mean daily ethanol consumption from the period of 1 month (mean 172 g, range 68–470 g) preceding admission showed a highly significant correlation with the anti-adduct IgA levels (r= 0.77, p < 0.001) and IL-6 levels (r= 0.83, p < 0.001).

Figure 3.

Mean (SD) titers of IgA, IgG, and IgM against the acetaldehyde-derived antigens in 17 alcoholics at the time of admission and after supervised abstinence, as compared to the values obtained from moderate drinkers and abstainers. IgA levels were significantly different between the samples taken at admission as compared to those taken after 8 2 days of supervised abstinence (p < 0.05), whereas IgG or IgM levels did not differ significantly between these groups U/L = OD₄₀₅ 10³.

Full figure and legend (22K)

DISCUSSION

The present studies show that the generation of immune responses against acetaldehyde-modified epitopes and enhanced IL-6 and IL-10 cytokine responses occur early in the sequence of events leading from excessive alcohol consumption to clinical signs of alcoholic liver injury. IgM isotype antibodies, the predominant component of early immune responses, and IgA antibodies were both higher in the alcohol consumers without apparent liver disease than in the abstainers. In turn, IgG antibodies and excessive amounts of both pro- and antiinflammatory cytokines parallel the evolution of liver injury suggesting that a disturbed balance in the immune regulation may play a role in the pathogenesis of liver disease (ALD) in some alcoholic patients.

Previous studies in patients with ALD have shown a generalized increase in circulating IgA concentrations together with increased tissue IgA deposition, which have been suggested to result from decreased IgA catabolism or from decreased IgA excretion (^2,4,13,14). The present findings on different immunoglobulin isotypes indicate that the generation of excess IgAs in alcoholics is antigen-driven. It is possible that IgA antibodies result from intestinally induced B-cell responses against ethanol-derived neoantigens since the epithelial tissues in alcohol abusers are continuously exposed to ethanol and mucosal immunity has been shown to be highly adaptable to the antigenic load of the environment (¹⁵). In line with this view, the IgA responses also show a strong correlation with the actual amounts of recent ethanol ingestion. The gastrointestinal tract is rich in enzymes capable of metabolizing ethanol to acetaldehyde (^16,17,18). Studies in experimental animals have also suggested an association between intestinal ethanol metabolism and the production of liver damage (^19,20). Previously, endotoxemia has been shown to be common in ALD patients, and such patients may also have IgA antibodies to components of endotoxin (²¹) and to human gut luminal aspirates (²²). Thus, there may be intestinally induced B lymphocyte responses against several enteric antigens. IgAs against endotoxin-derived components have previously been reported in patients with ALD although not in alcohol consumers without liver injury (^17,21). It is possible that in ALD patients a continuous antigen load together with high endotoxin levels could enhance the mucosal response to gut-derived antigens at the same time when IgA absorption is increased as a result of mucosal injury. Endotoxin can also stimulate immune induction by inhibiting T regulatory cells through a T cell-like receptor/interleukin 6 dependent pathway (²³).

It is currently not known whether the immune responses occurring in the alcoholic patients represent protective or harmful mechanisms for the liver. The present observations on the associations between ethanol consumption, antibody profiles, IL-6 levels, and IL-10 levels may indicate that the early responses, which precede liver disease, could reflect regulation of tissue damage and immune protection mechanisms. This view is also supported by the differences in the immune parameters between abstainers and apparently healthy moderate drinkers. IgA antibodies may contribute to exclusion and neutralization of antigens resulting from acetaldehyde modification of proteins and cellular constituents (^4,5,6). In line with this view, IgA antibodies and IL-6 levels show parallel changes in the follow-up of alcoholics with abstinence. Previously, IL-6 levels have also been shown to readily respond to therapeutic interventions with lifestyle modifications and vitamin E in non-alcoholic steatohepatitis (NASH) (²⁴). IL-6 release into bile occurs with the same time course as that for transcellular transport of IgA in the liver (²⁵). A relatively high concentration of IgA after ethanol consumption may also be found in Kupffer cells. Under conditions where Kupffer cell phagocytosis is impaired, there may be increased production of IgG immunoglobulins (²⁶). Upon excessive antigenic stimulation, the generation of IgA immune complexes may, however, be damaging since these can cause monocytes to release mediators of tissue damage. Such immune complexes could also play an initiating role in the development of renal lesions in alcoholics (¹). IgG antibodies, which were observed in some patients with liver disease, are known as mediators of several immunopathogenic consequences including complement activation and induction of cytotoxic reactions. In patients with advanced ALD, recent studies have also described the occurrence of specific T-cell responses to adducted proteins (¹⁰).

The present findings may further indicate that a disturbed balance between ethanol-derived antigen loading and immunological protection mechanisms could also play a role in the progression of liver disease in alcoholics. In ALD patients, there appears to be a skewed balance in the ratios of pro- and antiinflammatory cytokines. The IL-6 and IL-10 cytokines were found to show early changes also reflecting the current status of drinking. IL-6 is a multifunctional cytokine that has been previously implicated in the hepatic acute phase response (²⁷) and control of immunoglobulin production (²⁸). Recent evidence has shown that IL-6 is also required for normal liver regeneration after injury or partial hepatectomy and may thereby provide hepatoprotection by conferring resistance to injury (^29,30). IL-6 has been previously shown to inhibit TNF- production and reduce antigen presentation (^31,32). IL-10 has been recently implicated as having antifibrogenic effects since IL-10 deficiency in IL-10-/- mice potentiates liver fibrogenesis as a result of enhanced inflammation or direct effects on matrix regulation (³³). In mice, a mechanical tissue injury causes a rapid induction of IL-10 mRNA and promotion of Th2 responses to antigens (³⁴). IL-6 and IL-10 are both Th2 IgA stimulating cytokines (³⁵). Interestingly, although TGF-1 expression has been previously associated with the development of fibrosis in alcoholics (³⁶), the present data show no significant differences in circulating TGF-1 in patients with ALD as compared to abstainers. The proinflammatory cytokines IL-8, IL-2, and TNF- were found to be markedly more dominant in the cytokine profiles of the ALD patients than in those without liver disease. Of these, IL-8 is able to attract neutrophils and its enhanced expression also coincides with decreased hepatocyte survival in cultured human hepatocytes (³⁷). The levels of IL-2 (influencing the growth and differentiation of Th1-cells) and TNF- (inducing activation of inflammatory cells, expression of adhesion molecules, and platelet activation) were especially pronounced in ALD patients. Excessive release of such cytokines can also increase the production of reactive oxygen species from hepatocytes and induce apoptosis (³⁸). Several recent studies have emphasized the importance of increased TNF- production and Kupffer cell activation as key responses in hepatic inflammation (^38,39). Studies in ethanol-exposed rats (³⁹) and in ob/ob mice, a model of non-alcoholic fatty liver disease (⁴⁰), have shown that treatment with inhibitors of TNF- may reduce hepatic inflammation and necrosis.

In this work, we chose to study male patients only because immunological responses in vivo may show significant gender dependence (⁴¹). Men and women are known to have markedly different incidences of a variety of autoimmune disorders. Sex steroid hormones have been suggested to play a role in the regulation of immune responses, although the specific mechanisms have remained obscure. For instance, estradiol inhibits the suppressive activity of a subset of T-lymphocytes bearing Fc-receptors for immunoglobulin G (^41,42). However, a substantial interindividual variation was noted in several study parameters. It is possible that different degrees of liver injury, different amounts and patterns of ethanol intake prior to sampling, and different genetic susceptibilities may all play a role in explaining such variation.

In conclusion, the present data showing characteristic immune responses and cytokine profiles in the expression of ALD indicate that such mechanisms may play a role in the sequence of events leading from excessive ethanol consumption to tissue injury. Future studies appear warranted to examine the possibility whether modulation of the immunopathogenesis could provide new approaches into the treatment of the alcoholic patients and whether the immune parameters could also be used as biological staging markers of ethanol-induced liver disease.

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Acknowledgements

The studies were supported in part by a grant from the Finnish Foundation for Alcohol Studies.