Influence of ethanol on CD2 receptors oflymphocytes in alcohol dependentinpatients and healthy individualsLAUR TOOMASPOEG, OLEV TOOMLA, MA8 RT SAARMA, VEIKO VASAR

Toomaspoeg L, Toomla O, Saarma M, Vasar V. Influence of ethanol on CD2 receptors oflymphocytes in alcohol dependent inpatients and healthy individuals. Nord J Psychiatry2000;54:287–291. Oslo. ISSN 0803-9488.

The objective of this research was to assess the alcohol effect on E-rosette formation in 6itro.The results reveal that the intoxicating dose of alcohol (0.2‰–5.0‰) in healthy volunteersand alcoholics’ blood can differently affect the E-rosette formation: in the healthy volunteersit suppressed while in the alcoholics it stimulated the E-rosette formation. Both the suppres-sion and stimulation were closely dependent on the alcohol consumption. Though the bloodof alcoholics contained fewer E-rosette forming cells than that of the healthy volunteers, theinfusion of 2.0‰ alcohol in 6itro to the alcoholics’ blood raised their E-rosette formation tothe level of healthy volunteers.• Alcoholism, Ethanol, E-rosettes.

L. Toomaspoeg, Department of Psychiatry, University of Tartu, Raja Street 31, 50417 Tartu,Estonia; Accepted: 14 April 2000.

The research on alcoholics and laboratory animals in6i6o has provided evidence which enables us to

confirm that alcohol has a toxic effect on the immunesystem. Still, we think that the result of an in 6i6oexperiment is difficult to interpret because it is presentedas a kind of earmark that can be influenced by thefollowing factors: 1) the breakdown of alcohol and themetabolites formed; 2) the impact of alcohol and themetabolites on the digestive system; 3) the impact ofalcohol and the metabolites on the brain and through thebrain, the rest of the body; 4) the breakdown of alcoholand the metabolites on the immune system.

Our earlier study (1) on 19 patients revealed that theintoxicating amount (0.2–5.0‰) of alcohol in vitro in thehealthy volunteers’ and the alcoholics’ blood differentlyaffected the E-rosette formation: unlike in the healthyvolunteers, alcohol stimulated the E-rosette formation inthe alcoholics. To determine the alcoholic effect moreprecisely, we decided to increase the number of patients.

For a review of the literature about the immunepathology of alcoholics and the alcohol effect on E-rosette formation in 6itro, please see our previous article(1).

Materials and MethodsThe series80 male alcoholics and 101 healthy male volunteerswere included in the study. The experimental popula-

tion included only patients without any clinical and/orlaboratory evidence of hepatitis, cirrhosis, pancreatitisor other serious somatic diseases.

The average ages of the alcoholics and the healthyvolunteers were 39.7 and 35.4 years, respectively.

All the alcoholics met the diagnostic criteria foralcohol dependence according to ICD-10 researchcriteria.

E-rosette forming reactionThe E-rosette forming reaction of lymphocytes in ahuman organism is based on the lymphocytes’ power tobind CD2 receptors on their membrane with sheeperythrocytes, so that the rosette-like cellular clusters areformed (Fig. 1). E-rosettes will be referred to in thefinal part of the discussion.

The patients’ blood samples were collected at theiradmission into the hospital (11:30–12:30) before ameal. Mononuclear cells were separated from the bloodby Boyum (2). E-rosette forming reaction was per-formed by Han et al. (3) and Brohee et al. (4). To eachspecimen 0.2‰, 0.35‰, 0.5‰, 0.65‰, 1.0‰, 2.0‰,3.0‰, 4.0‰ and 5.0‰ of ethanol were respectivelyadded to obtain final concentration. To the controltube a physiological solution was added. The tubeswere incubated at +4°C for 24 hours. Thereafter, lightmicroscopy was used to count the number of E-rosettes, originating from the specimens, per 200lymphocytes.

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Fig. 1. E-rosette forming cell.

neurological and mental disorders, continuous con-sumption of alcohol in small amounts due to decreasedtolerance and degradation of personality.

ResultsResults of ethanol influence on the E-rosette formingreaction on a whole group are given in Table 1. Asshown in Table 1, any amount of alcohol inhibits theE-rosette formation in the healthy volunteers, while itstimulates the E-rosette formation in the alcoholics.Both in the healthy volunteers and in the alcoholics thequantity of E-rosette formation is varying significantly.The standard deviations are given in Table 1.

Effect of alcohol on the E-rosette forming reaction ofthe lymphocytes of the healthy volunteers, dependingon the amount of alcohol used, is given in Table 2 andFig. 2. It is demonstrated that inhibition of the E-rosette formation of the healthy volunteers depends onthe drinking habit: the less alcohol a person consumes,the bigger the inhibiting effect of alcohol on E-rosetteformation.

Age, as a rule, has its impact on all functions of thehuman organism, including that of the immune system.Effect of alcohol on the E-rosette forming reaction ofthe lymphocytes of the healthy volunteers depends onage as demonstrated in Table 3. A correlation betweenthe patients’ age, the amount of alcohol consumptionand the immunologic condition is demonstrated inTable 4.

In order to search a relationship between age, alcoholconsumption, and E-rosette formation in the healthyvolunteers, a factor analysis was made. The results arepresented in Table 5.

Results of a similar analysis of alcoholics are pre-sented in Tables 6 and 7.

To compare the effect of alcohol on E-rosette form-ing reaction in different persons, we used indicator(IM—index of modulation) which takes into consider-ation individual differences of forming E-rosettes anddirection of alcohol action that is to say whether alco-hol stimulates or inhibits E-rosette forming. Thereforewe used the following formula:

In the course of statistical analysis we divided healthyvolunteers according to their self-estimation into 3groups depending on use of amount of alcohol: totalabstainers, mild consumers, and moderate consumers.

According to their clinical symptoms, the alcoholicswere divided into three stages. The first stage werecharacterized by excessive consumption of alcohol, withepisodic drinking bouts lasting many days, loss ofcontrol about the amount of alcohol and an increasedtolerance to alcohol. The second stage were character-ized by the withdrawal syndrome with autonomic disor-ders, high tolerance to alcohol, regular consumption ofalcohol and changes of personality. The third stagewere characterized by regular withdrawal states with

IM (Index of modulation)=Count of E-rosettes with ethanol−Count of E-rosettes without ethanol

Count of E-rosettes without ethanol�100

Table 1. E-rosettes in blood, expressed at different alcohol concentrations.

Healthy volunteers (n=101) Alcohol dependency (n=80)Concentration ofP (Healthy volunteers vs alcoholics)Mean9SDethanol Mean9SD

73.894.30‰ 63.696.6 \0.0010.2‰ 69.395.7* 7098.2* NS

71.798.4*69.895.2*0.35‰ NS0.5‰ NS71.395.9* 70.498.7*

72.298.1*0.65‰ NS71.095.8*72.398.6*1.0‰ B0.0569.795.4*

B0.00172.698.7*69.195.0*2.0‰70.295.6* 72.198.4*3.0‰ NS

4.0‰ 68.695.2* 72.398.7* B0.0015.0‰ 67.795.4* 70.399.0* B0.05

Remarks: Significantly different from ethanol concentration 0 per mills (*PB0.001).

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Table 2. Indexes of E-rosette modulation of the healthy volunteers, depending on their alcohol consumption.

Total abstainers Mild consumers Moderate consumers(n=29)(n=30) (n=42)Concentration of

A B Cethanol P(AB) P(BC)P(AC)

0.2‰ −5.7 −8.5 −2.5 B0.02 B0.05 B0.0010.35‰ −9.7 −7.9 3.1 NS B0.001 B0.0010.5‰ −10.0 −2.4 2.2 B0.001 B0.001 B0.0010.65‰ −5.6 −4.7 −0.5 NS B0.001 B0.0011.0‰ −8.2 −6.3 −1.3 NS B0.001 B0.0012.0‰ −7.7 −7.1 −3.9 NS B0.001 B0.013.0‰ −5.1 −6.0 −2.9 NS B0.01 B0.014.0‰ −9.7 −7.8 −2.9 NS B0.001 B0.0015.0‰ −9.4 −8.8 −5.8 NS B0.01 B0.05

Table 3. Indexes of E-rosette modulation of the healthy volunteers, depending on their age.

16–27 years 31–43 44–56(n=30) years (n=45) years (n=26)

Concentration of ethanol A B C P= (AB) P= (AC) P= (BC)

0.2‰ −7.5 −5.4 −5.2 NS NS NS0.35‰ −5.0 −5.5 −5.2 NS NS NS0.5‰ −5.7 −3.4 −0.4 NS B0.05 B0.010.65‰ −3.7 −4.9 −1.7 NS NS B0.011.0‰ −6.7 −4.5 −5.7 NS NS NS2.0‰ −5.0 −7.4 −6.2 B0.05 NS NS3.0‰ −4.1 −5.6 −4.4 NS NS NS4.0‰ −6.9 −7.5 −6.0 NS NS NS5.0‰ −10.1 −7.7 −6.6 NS B0.01 NS

Table 4. Correlation between alcohol consumption, age, and IM of E-rosettes in healthy volunteers.

Variable R of usage of alcohol R of age P of usage of alcohol P of age

Age 0.120 NSE-RFC 0‰ Alc −0.399 0.147 B0.001 NSIM 0.2‰ Alc 0.190 0.204 NS B0.05IM 0.35‰ Alc 0.687 0.046 B0.001 NSIM 0.5‰ Alc 0.730 0.401 B0.001 B0.001IM 0.65‰ Alc 0.385 0.199 B0.001 B0.05IM 1.0‰ Alc 0.533 0.215 B0.001 B0.05IM 2.0‰ Alc 0.343 −0.057 B0.001 NSIM 3.0‰ Alc 0.187 0.081 NS NSIM 4.0‰ Alc 0.557 0.117 B0.001 NSIM 5.0‰ Alc 0.262 0.228 B0.01 B0.05

DiscussionUnlike the blood of healthy volunteers, the blood ofalcoholics contains considerably fewer E-rosettes. Otherresearchers have received similar results (5). Therefore,we avoid further discussion of the low amount ofE-rosettes in the alcoholics’ blood. Instead, it is interest-ing to discuss the effect of ethanol in 6itro on thelymphocyte E-rosette formation in alcoholics and healthyvolunteers. Several researchers have stated the inhibitoryeffect of alcohol on the lymphocytes’ E-rosette formation

in 6itro (6, 7). In those studies, no correlation has beendemonstrated between the alcohol effect in 6i6o and thealcohol effect on in 6itro E-rosette formation, i.e. alcoholconsumption pattern has been discarded. The results ofour study confirm that alcohol consumption determinesthe intensity of the E-rosette formation on exposure toalcohol in 6itro. Thus, the less a person consumes alcohol,the more inhibiting effect the alcohol will have; and themore a person consumes alcohol, the higher the stimulat-ing effect of alcohol will be on E-rosette formation.

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Fig. 2. Indexes of modulation of E-RFC of healthy volun-teers and alcoholic patients.

Table 6. Correlation between alcohol consumption, age, andIM of E-rosettes in alcoholic patients.

P of UsageUsage ofof alcoholVariable Agealcohol P of age

B0.0010.713AgeE-RFC 0‰ Alc 0.199 0.176 NS NSIM 0.2‰ Alc −0.210 −0.266 NS B0.05IM 0.35‰ Alc 0.010 0.044 NS NSIM 0.5‰ Alc 0.007 −0.027 NS NSIM 0.65‰ Alc 0.250 0.094 B0.05 NS

NSB0.010.1730.330IM 1.0‰ AlcNS0.0750.210IM 2.0‰ Alc NS

NSNS−0.113IM 3.0‰ Alc 0.010−0.168 −0.156 NSIM 4.0‰ Alc NS

−0.181 NS NSIM 5.0‰ Alc −0.217

According to the anamnesis, the duration of thealcohol consumption had its significant impact on theE-rosette formation: the IM of healthy volunteers had astronger correlation with the quantity of the alcoholconsumed than with the consumers’ age. Also, thefactor analysis of the data received from healthy volun-teers reveals that the IM has a stronger correlation withthe quantity of alcohol consumed than with the con-sumers’ age. In the healthy volunteers, the alcoholinhibits the E-rosette formation (except in the highconsumers of alcohol who were not classified as alco-holics), while in the alcoholic patients the alcohol al-ready stimulates E-rosette formation. It became evidentthat the stronger the patient’s addiction to alcohol is,the bigger the stimulative effect of alcohol on E-rosetteformation in 6itro.

Unlike in the healthy volunteers, the statistics in thealcoholic patients is difficult to interpret. The reasonmay be that the alcoholic patients have lost the verystrong correlation between the IM and the mode ofalcohol consumption. On the one hand, it may be dueto the overwhelming effect that alcohol has on a humanorganism, including the function of lymphocytes. On

Table 7. Results of factor analysis of data about alcoholicpatients.

2 3Variable 1

0.909 −0.033Usage of alcohol 0.1530.019Age 0.876 0.005

% of E-RFC 0.333−0.194 0.828−0.3460.605 0.394IM 0.2‰ Alc

IM 0.35‰ Alc 0.584 0.015 0.0770.570 0.488IM 0.5‰ Alc −0.047

IM 0.65‰ Alc 0.921 0.128 0.095IM 1.0‰ Alc 0.793 0.294 0.124IM 2.0‰ Alc 0.735 0.064 0.213IM 3.0‰ Alc 0.630 −0.156 0.323IM 4.0‰ Alc 0.721 −0.246 0.443IM 5.0‰ Alc 0.301 −0.330 0.726

the other hand, the exact determination of the mode ofalcohol consumption of alcoholics is quite difficult.While the responses from healthy volunteers about theiralcohol consumption are plausible, the responses fromalcoholic patients almost never are.

The analyses have revealed that the sheep erythro-cytes are bound by CD2 receptors, which are glyco-proteins that consist of 360 aminoacids (8) and occuron 99% of T-lymphocytes. It has been confirmed thatthe CD2 receptor is an essential link in the activation ofthe T-lymphocytes (9).

The T11 sheep erythrocyte binding glycoprotein isexpressed throughout human T-lymphocyte ontogenyand appears to play an important physiological role inT-cell activation. Thus, the treatment of T-cells withcertain monoclonal anti-T11 antibodies results in anti-gen-independent polyclonal T-cell activation as assessedby proliferation and lymphokine secretion (10). Mono-clonal antibodies blocking antibody-dependent cytotox-icity, pokewed mitogen induced interleukin-2 release, orConcanavalin-A and pokewed mitogen inducedlymphocyte proliferation were found among anti-CD2

Table 5. Results of factor analysis of data about healthyvolunteers.

Variable 1 2 3 4

Usage of alcohol 0.226 0.839 0.276 0.098Age −0.107 −0.069 0.879 0.157% of E-RFC 0.261 −0.761 0.323 −0.134

0.096IM 0.2‰ Alc 0.123 0.7820.0340.443 0.4590.067IM 0.35‰ Alc 0.683

IM 0.5‰ Alc 0.371 0.614 0.609 0.110IM 0.65‰ Alc 0.734 0.160 0.355 0.227IM 1.0‰ Alc 0.451 0.334 0.322 0.603

0.826 0.089IM 2.0‰ Alc −0.0780.238IM 3.0‰ Alc 0.873 −0.052 0.084 0.125IM 4.0‰ Alc 0.628 0.361 0.139 0.400

0.014IM 5.0‰ Alc 0.207 0.7910.098

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reagents. Inhibition of lectin-dependent cellular cyto-toxicity was found as an exclusive effect of anti-CD2antibodies. Several anti-CD2s blocked natural killeractivity and/or pokewed mitogen induced interferonproduction (11).

Unlike humoral immune responses, which are medi-ated through antibodies and complement and can betransferred in serum to unimmunized subjects, cellularimmune responses require the direct participation ofeffector cells such as T lymphocytes. The functionalactivities of both helper T lymphocytes and cytotoxic Tlymphocytes are initiated by the binding of specificantigen presented in association with the major histo-compatibility complex (MHC) on the target cell toT-cell-antigen receptors. The interaction begins with thebinding of antigen in conjunction with an MHCmolecule on the target cell by the T-cell-antigen recep-tor. Accessory molecules, such as lymphocyte functionantigens (LFA) 1 and 3, intercellular adhesion molecule1, CD2, CD28, and B7 (T-cell costimulatory factor),are involved in this interaction through their enhance-ment of cell-cell adhesion or transduction of additionalcell-activation signals (12).

Lymphocyte function-associated antigen 3 (LFA-3) isa widely distributed cell surface glycoprotein that bindsto the T lymphocyte CD2 surface glycoprotein. Thisinteraction mediates cytolytic T lymphocyte-target cellconjugate formation and adhesion of thymocytes tothymic epithelial cells. CD2 is also the E rosette recep-tor of T lymphocytes and mediates rosetting with au-tologous E by binding to LFA-3 (13).

That sheep erythrocytes are used to bind T-cell CD2receptors may be due to their higher LFA-3 antigen,compared to human erythrocytes (14).

The CD2 receptors of alcoholic patients can be lessexpressive due to the lower activity of their T-lymphocytes, which in turn inhibits the E-rosette for-mation by CD2. There is evidence that FHA-stimulatedreactivity of T-lymphocytes in alcoholic patients hasremarkably declined, compared to the healthy volun-teers (15). The decline in reactivity of lymphocytes ofalcoholic patients may be due to the insufficiency ofalcohol in their organism, which may be due to theirgrowing addiction to alcohol. Adding alcohol to E-rosette specimens improves the function of lymphocytesso that they can form E-rosettes equivalent to those inhealthy volunteers (please look at Table 1. E-rosettes in

sober healthy volunteers—73.8% and in alcoholic pa-tients with 2.0‰ alcohol—72.6%). If the results areconfirmed, it is likely that the immune system plays asignificant role in the pathogenesis of alcohol addiction.

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10. Siliciano RF, Pratt JC, Schmidt RE, Ritz J, Reinherz EL.Activation of cytolytic T lymphocyte and natural killer cellfunction through the T11 sheep erythrocyte binding protein.Nature 1985;317:428–30.

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13. Selvaraj P, Dustin ML, Silber R, Low MG, Springer TA. Defi-ciency of lymphocyte function-associated antigen 3 (LFA-3) inparoxysmal nocturnal hemoglobinuria. Functional correlatesand evidence for a phosphatidylinositol membrane anchor. JExp Med 1987;166:1011–25.

14. Selvaraj P, Dustin ML, Mitnacht R, Hunig T, Springer TA,Plunkett ML. Rosetting of human T lymphocytes with sheepand human erythrocytes. Comparison of human and sheep lig-and binding using purified E receptor. J Immunol1987;139:2690–5.

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Laur Toomaspoeg, M.D., Ph.D., Olev Toomla, M.D., Ph.D.,Mart Saarma, M.D., Ph.D., Veiko Vasar, M.D., Ph.D.,Department of Psychiatry, University of Tartu, Raja Street 31,50417 Tartu, Estonia

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