Blood Cell Responses Following Heavy Alcohol Consumption Coincide with Changes in Acute Phase Reactants of Inflammation, Indices of Hemolysis and Immune Responses to Ethanol Metabolites

Abstract

Aberrations in blood cells are common among heavy alcohol drinkers. In order to shed further light on such responses, we compared blood cell status with markers of hemolysis, mediators of inflammation and immune responses to ethanol metabolites in alcohol-dependent patients at the time of admission for detoxification and after abstinence. Blood cell counts, indices of hemolysis (LDH, haptoglobin, bilirubin), calprotectin (a marker of neutrophil activation), suPAR, CD163, pro- and anti-inflammatory cytokines and autoantibodies against protein adducts with acetaldehyde, the first metabolite of ethanol, were measured from alcohol-dependent patients (73 men, 26 women, mean age 43.8 ± 10.4 years) at baseline and after 8 ± 1 days of abstinence. The assessments also included information on the quantities of alcohol drinking and assays for biomarkers of alcohol consumption (CDT), liver function (AST, ALT, ALP, GGT) and acute phase reactants of inflammation. At baseline, the patients showed elevated values of CDT and biomarkers of liver status, which decreased significantly during abstinence. A significant decrease also occurred in LDH, bilirubin, CD163 and IgA and IgM antibodies against acetaldehyde adducts, whereas a significant increase was noted in blood leukocytes, platelets, MCV and suPAR levels. The changes in blood leukocytes correlated with those in serum calprotectin (p < 0.001), haptoglobin (p < 0.001), IL-6 (p < 0.02) and suPAR (p < 0.02). The changes in MCV correlated with those in LDH (p < 0.02), MCH (p < 0.01), bilirubin (p < 0.001) and anti-adduct IgG (p < 0.01). The data indicates that ethanol-induced changes in blood leukocytes are related with acute phase reactants of inflammation and release of neutrophil calprotectin. The studies also highlight the role of hemolysis and immune responses to ethanol metabolites underlying erythrocyte abnormalities in alcohol abusers.

Patterns of IgA Autoantibody Generation, Inflammatory Responses and Extracellular Matrix Metabolism in Patients with Alcohol Use Disorder

“…Adduct formation can also induce IgA antibodies, which have been previously observed in a high percentage of patients with AUD [11,12,14,55,56]. Such immune responses may be required for the exclusion and neutralization of the neoantigens although an overwhelming antigenic stimulation could also cause a release of inflammatory mediators and aggravate tissue damage [14,57,58].…”

“…The results are expressed as units/milliliter (U/mL). The analyses for the IgA antibodies against the acetaldehyde-derived epitopes in proteins (HbAch-IgA) were carried out as previously described [37,58]. In essence, acetaldehyde-modified antigen was prepared in vitro using human hemoglobin as a carrier protein.…”

“…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, which has been previously shown to effectively lead to the generation of immunogenic epitopes in acetaldehyde-protein condensates [11,37,58]. Protein adducts were reduced by addition of sodium cyanoborohydride (10 mM) and mixing for 5 h at +4 • C to create stable condensates.…”

“…Protein adducts were reduced by addition of sodium cyanoborohydride (10 mM) and mixing for 5 h at +4 • C to create stable condensates. For measuring the antibody titers, microtiter plates (Nunc-Immuno Plate, Maxisorb TM , Thermo Scientific, Waltham MA, USA, Roskilde, Denmark) were coated with acetaldehyde-modified hemoglobin or corresponding unmodified proteins (background) in PBS (3 µg protein in 100 µL/well) [37,58]. Optical densities of color reactions were read at 405 nm with Anthos HTII microplate reader (Anthos Labtec Instruments, Salzburg, Austria).…”