0145-6008/96/2005-0929$03.00/0 ALCOHOLISM: CLINICAL AND EXPERIMENTAL RESEARCH

Vol. 20, No. 5 August 1996

Serum and Ascitic Levels of Soluble Intercellular Adhesion Molecule-1 in Patients with Alcoholic Liver

Cirrhosis: Relation to Biochemical Markers of Disease Activity and Alcohol Intake

lstvan Nagy and Yvette MAndi

The overexpression of intercellular adhesion molecule-1 (ICAM-1) has been shown to be involved in the pathogenesis of various necro- inflammatory diseases, including alcoholic hepatitis. Shedding of this molecule from cell surfaces results in a circulating form, soluble CAM-1 (SICAM-1). In this work, the serum and ascitic concentra- tions of SICAM-1 were studied in relation to clinical and laboratory data in patients with alcoholic liver cirrhosis of different disease ac- tivities. Elevated circulating concentrations of this adhesion mole- cule were found in all cirrhotic patients, the highest in those with superimposed sewere alcoholic hepatitis, and the levels in regularly drinking cirrhotics without severe alcoholic hepatitis were likewise significantly higher than in those who had stopped drinking. The serum SICAM-1 concentration was best related to the serum AST activity, and also exhibited significant correlations with the pro- thrombin activity, serum bilirubin, albumin, peripheral leukocyte count, Maddrey’s discriminant function value, Child grading, and an- tecedent alcohol consumption. Multivariate regression analysis re- vealed that the serum AST and prothrombin activities were indepen- dent predictors of the circulating SICAM-1 concentration. The concentration of SICAM-1 in the uninfected ascitic fluid of cirrhotics was about seven times lower than that in the serum; the ratio of its ascitic and serum levels was lower than that of the ascitic and serum total protein concentrations. These data contradict a significant in- traperitoneal production of the molecule. It is concluded that the serum SICAM-1 level may be useful as a marker for the current dis- ease activity (the severity of underlying acute necroinflammatory re- actions) in alcoholic liver cirrhosis.

Key Words: Intercellular Adhesion Molecule-1, Liver Cirrhosis, Al- coholic Hepatitis, Ascites.

HE RECRUITMENT of leukocytes from the blood T and their localization to a site of inflammation is me- diated through interactions of different adhesion mole- cules.’.2 Intercellular adhesion molecule-1 (ICAM-1) is a cytokine-inducible cell surface glycoprotein of the immu- noglobulin supergene family that functions as a counterre- ceptor for P,-integrin-type adhesion proteins with distinct binding sites for lymphocyte function-associated antigen-1

From the 1st Department of Medicine (I.N.) and Institute of Microbiology

Received for publication December 14, 1995; accepted March 11, I995 This work was supported by Hungarian Research Grants ETT-MI4 and

ETT-M16. Some parts of this work were presented at the Scientific Sessions of the Digestive Disease Week, May 14-17, 1995, San Diego, CA.

Reprint requests: Istvan Nagy, M.D., 1st Department of Medicine, A. Szent-Gyowi Medical University, P.O. Box 469, H-6701 Szeged% Hungary.

Copyright 0 1996 by The Research Society on Alcoholism.

(Y M.), A. Szent-Gyo;rgyi Medical University, Szeged, Hungary.

Alcohol Clin Erp Res, Vol20, No 5, 1996: pp 929-933

and macrophage antigen-1 (MAC-I), expressed on leuko- cytes.”2 An increased tissular expression of ICAM-1 has been demonstrated in a variety of necroinflammatory disorders, including liver di~eases.3,~ In alcoholic hepatitis, a strong ex- pression of ICAM-1 on hepatocytes, correlating well with the histological degree of hepatocellular damage, parenchymal inflammation, and p,-integrin expression on infiltrating leu- kocytes, has recently been and a firm involvement of the ICAM-l/&-integrin pathway in the pathogenesis of leu- kocyte-mediated hepatic injury has been pr~posed.’.~

The shedding of ICAM-1 from cell surfaces results in a soluble form of this molecule (SICAM-l), containing its five extracellular domains; it can also be detected in the circulation and may thereby prove useful in the investigation, diagnosis, and therapeutic monitoring of inflammatory disorders associ- ated with the overexpression of ICAh4-1.8 There are only a few and partly contradictory data in the literature as concerns serum SICAM-1 in alcoholic liver disease^.^*^.'^ In contrast with a recently published paper,7 in a pilot study we found that serum concentrations of SICAM-1 in alcoholic liver cirrhotic patients with superimposed acute alcoholic hepatitis were distinctly higher than those in cirrhotics without alcoholic hepatitis,” similarly as in two other previous st~dies,~.’’ sug- gesting that the circulating levels of SICAM-1 may reflect the “disease activity” (i.e., the severity of underlying acute necro- inflammatory reactions) in alcoholic liver cirrhosis. To test this hypothesis, in this study, the serum levels of SICAM-1 were examined in comparison with comprehensive clinical and lab- oratory data on patients with alcohol-induced liver cirrhosis of clinically different disease activities, ranging from inactive cirrhosis to a superimposed severe alcoholic hepatitis (SAH). The concentrations of SICAM-1 in the ascitic fluid were also measured to assess whether intraperitoneal production of the molecule occurred, similarly, for example, as for inter- leukin-6.12

METHODS

Subjects

A total of 52 subjects were studied; all had given their informed consent.

Normal controls (n = 20; 13 males and 7 females; age range = 36 to 59 years): These were healthy nonalcoholic volunteers (blood donors), who

929

930

3 3000 - . M a j 2500-

8 2000- Y 2; g 1000 -

;;I 1500 -

NAGY AND MANDl

had no known recent or chronic illnesses, and who gave normal routine laboratory test results.

Akoholic liver cirrhotics (n = 32; 25 males and 7 females; age range = 40 to 70 years): These were patients who had consumed at least 80 g of ethanol/day for >10 years and either who had histologically proven mi- cronodular cirrhosis (27 of 32) or whose clinical evaluation (history, typical findings of physical, laboratory, endoscopic and sonographic ex- aminations; exclusion of other liver diseases) indicated the presence of alcoholic liver cirrhosis (5 of 32); 19 of them had ascites.

The 32 cirrhotic patients were divided into three clinically predefined subgroups: 10 of them had given up alcohol consumption >1 month before the study (subgroup 1: abstainers with “inactive cirrhosis”). The other 22 patients were regular drinkers and were studied within a few (1 to 5) days after their last drink. Subgroup 2 consisted of 11 regularly drinking patients with “active cirrhosis” without clinical evidence of su- perimposed SAH, as indicated by a Maddrey’s discriminant function score 14.6 x prothrombin time (sec) + serum bilirubin (mgldl); Ref. 131 of <93. Eleven of the 22 regularly drinking cirrhotics yielded clinical and labora- tory evidence of superimposed S A H (subgroup 3), with a Maddrey’s score of >93,” with jaundice, leucocytosis, and a history of recent, heavy (>150 g/day) ethanol ingestion. From ethical considerations, concurrent liver biopsies, sampled within a few days of blood sampling for SICAM-1, were taken only from patients whose liver cirrhosis had not been previously proven by histology, and who had acceptable coagulation parameters and no ascites (7 of 32). In the present study, therefore, a comparison of slCAM-1 data with liver histology was not possible. Five of the 11 patients with clinically diagnosed S A H died after admission; histology at autopsy confirmed the presence of SAH.

The severity of the liver disease was assessed according to the Child- Pugh criteria.I4 At the time of the investigation, none of the patients were receiving steroids or immunomodulatory therapy, and none of them yielded any evidence of bacterial infection, malignancy, or gastrointestinal bleeding. All subjects tested negative for hepatitis B surface antigen and antibodies to hepatitis C virus and human immunodeficiency virus.

Assays

Venous blood was collected in sterile vacuum blood collection tubes and allowed to clot at room temperature for 30 min. Ascitic fluid was obtained from 15 of the 19 patients with ascites (through diagnostic or therapeutic paracentesis under sterile conditions), immediately cooled, and centrifuged. Serum and cell-free ascites samples were stored at -20°C until assayed for slCAM-1 by an enzyme-linked immunosorbent assay (Bender & Co. GmbH, Vienna, Austria), as previously described.” Rou- tine blood and ascitic fluid analyses were performed by standard clinical chemistry methods in our clinical laboratory.

Starisrical Analysis

Data were analyzed for distribution normality by the Kolmogorov- Smirnov test. Normal distribution data are given as means 2 SE; other- wise, the median and the range of values are presented. For comparison between groups, one-way analysis of variance, the Kruskal-Wallis test, the Mann Whitney U test, the Student r test, or the x 2 test was used, as appropriate. Correlation coefficients were calculated by the Spearman rank correlation procedure. To assess the independent effects of predictor variables on the slCAM-1 concentration (dependent variable), a multi- variate linear regression modeling technique with forward stepwise addi- tion of independent variables was used. Only p < 0.05 was considered significant.

RESULTS

Circulating SICAM- 1

The serum SICAM-1 concentrations in the healthy con- trols displayed a normal distribution, ranging from 150 to

4000

3500 1

500 1

** :: . .

0.. . *.* .*

o ! I Abstainer Regular Regular cirrhotics drinkers drinkers (n=10) without with

SAH SAH ( n = l 1 ) (n=11)

Fig. I. Individual levels of serum SICAM-1 in subgroups of patients with alcoholic liver cinhosis. Dashed line denotes upper limit of normal value (375 pg/ml). Statistical analysis: Kruskal-Wallis test followed by Dunn’s test.

Table 1. Clinical Characteristics and Blwd Chemistry Parameters in Subgroups of Patients with Alcoholic Liver Cirrhosis

Regular drinkers with Abstainers with inactive cirrhosis Active cirrhosis Cirrhosis + SAH

Characteristics (n = 10) (n = 11) (n = 1 1 )

Age (Yr) Alcohol consumption

Child’s class (A/B/C) AST (unitdliter) ALT (unitditer) y G T (unitditer) Total bilirubin (pmol/liter) Prothrombin activity (%) Albumin (ghiter) Maddrey’s df value$ SICAM-1 (pg/ml)

(g/day)

56.5 t 3.1 -

7/3/0 34.2 t 3.8 31.1 2 3.8

18.0 2 0.8 83.0 2 4.1

70.9 2 2.2 582 2 38

87.1 f 14.5

38.2 t 0.9

50.2 ? 2.8 125 t 9.3

61312 90.5 2 6.8t 46.5 2 4.0t 571 t 115t 38.6 t 7.1 74.8 -t 4.6 34.1 2 2.1

1244 2 83t 79.8 2 3.6

48.1 f 1.5 229 2 lo’

01111 a’.t 143 2 7.4’7

51.5 2 3.9t 152 (84-81 1) 285 2 5W.t 37.3 2 4.l’,t 25.9 2 1.5t 152 2 17’,t

2613 ? 174’.t

Values are means t SE. except that the y-GT of patients with SAH is pre- sented as median and range. Normal values: AST and ALT, <40 unitsfliter; y-GT, 4 0 unitsfliter; total bilirubin, <17.1 pmol/liter; prothrombin. >65%; albumin, >35 g/liter; and SICAM-1, <375 pg/ml.

’ p < 0.05 between the regular drinkers with active cinhosis and those with SAH (Kruskal-Wallis tests followed by Dunn’s test or one-way analysis of variance followed by the Bonferroni t test or x 2 tests (for Child’s class).

t p < 0.05 vs. the subgroup “abstainers.” $ Maddreys’s discriminant function value 14.6 x prothrombin time (sec) +

bilirubin (mg/dl)].

375 pg/ml, with a mean 5 SE of 254 t 14 pg/ml. There was no difference between the sexes. The upper limit of the normal value, calculated as mean + 2 SD, was 375 pg/ml. The serum SICAM-1 concentration for all cirrhotic patients was above the normal range (median: 1250; range: 390 to 3600 pg/ml). Division of the cirrhotic patients into three subgroups (abstainers, regular drinkers without SAH, and regular drinkers with S A H ) improved the distribution nor- mality of serum SICAM-1 and liver function test values (Fig. 1, Table 1). The highest SICAM-1 concentrations (about 10 times those of the controls), clearly exceeding those in the other cirrhotic subgroups, were found in the sera of patients with SAH, and the levels of the regularly

SOLUBLE ICAM-1 IN ALCOHOLIC CIRRHOSIS 93 1

Table 2. Correlation of Serum SICAM-1 Concentration with Laboratory Test Parameters and Alcohol Intake in Patients with Alcohdic Liver Cirrhosis

Variable n r D value

AST ALT ASTIALT Total bilirubin Prothrombin (Quick Om) Maddrey's df value' White blood cell count Total protein Albumin Alkaline phosphatase r G T Creatinine Age Child score Alcohol intake Time since last drink

32 32 32 32 32 32 32 32 32 32 32 32 32 32 22 10

0.915 0.637 0.704 0.855

-0.831 0.864 0.708

-0.336 -0.658 0.156 0.300

-0.077 -0.337 0.745 0.835

-0.853

<0.0001 0.0004 0.0001

<0.0001 <0.0001 <O.OOol 0.0001 NS 0.0002 NS NS NS NS

~0.0001 0.0001 0.0105

n, number of patients: r, Spearman rank correlation coefficient; NS. not sig-

* Maddrey's discriminant function value [4.6 x prothrombin time (sec) + nificant.

bilirubin (mg/dl)].

drinking cirrhotics without SAH were also significantly higher than those of the abstainers. Levels of SICAM-1 were not different between survivors and nonsurvivors in patients with SAH. As concerns the other laboratory and clinical parameters on the cirrhotic patients, the serum SICAM-1 concentration correlated best with the serum AST activity, but also significantly with total bilirubin, pro- thrombin, and ALT activity; the AST/ALT ratio; serum albumin; peripheral leukocyte count; Maddrey's discrimi- nant function score; and the Child score. It did not corre- late significantly with the age or with the serum levels of alkaline phosphatase, y-glutamyltranspeptidase ( y-GT), or creatinine (Table 2).

Multivariate stepwise regression analysis with serum sI- CAM-1 as dependent, and laboratory test data as indepen- dent variables proved that the serum AST ( p = 0.0001) and prothrombin activity ( p = 0.0021) were independently as- sociated with the circulating SICAM-1 concentration and were the best predictors of the latter. Serum ALT, bilirubin and albumin, AST/ALT ratio, peripheral leukocyte count, and the Child score did not exhibit such a significant inde- pendent association.

As indicated in Table 1, the patients with SAH displayed relatively low serum y-GT activities. Exclusion of these 11 patients from the evaluation improved the correlation be- tween SICAM-1 and y-GT (r = 0 . 7 6 7 , ~ = 0.0006, n = 21).

In the regular drinkers, the level of circulating SICAM-1 revealed a significant positive correlation with the average daily alcohol intake, whereas in the abstainer cirrhotics, it gave a significant negative correlation with the duration of abstinence (Table 2).

Ascitic Levels of SICAM-1 The concentration of SICAM-1 in the ascitic fluid of

cirrhotic patients (n = 15) ranged from 125 to 260 pglml (187 2 12 pg/ml; mean 2 SE) and, in each patient, it was

much lower than the corresponding serum concentration (median: 1450; range: 650 to 3000 pglml). The level of SICAM-1 in the ascites correlated positively with that in the serum ( r = 0 . 7 7 2 , ~ = 0.001 by Spearman rank correlation test). The ratio of ascitic to serum SICAM-1 concentration (0.131 2 0.016) was significantly lower than the ratio of ascitic to serum total protein concentration (0.212 f 0.017; p < 0.05 by Student's t test).

DISCUSSION

In the present study, the serum concentrations of sI- CAM-1 were found to be elevated in patients with alcoholic liver cirrhosis and to be strongly correlated with the bio- chemical indices of disease activity, and with the parame- ters of antecedent alcohol consumption.

Theoretically, an increase in circulating sICAM-1 in liver diseases might be attributed to a diminished hepatic clear- ance or to increased release of SICAM-1 from the liver. Certain data on patients with inactive liver c i r rhosi~ '~ or cholestatic diseasesI6 suggest a possible hepatic elimination of the molecule; to date, however, there is no direct exper- imental evidence to support this mechanism. On the other hand, it has been evidenced that the liver tissue might be an active source of SICAM-1. Histological studies have dem- onstrated an overexpression of ICAM-1 in hepatic tissue and also on hepatocytes in liver i n f l a m m a t i ~ n . ~ - ~ " ~ In ad- dition, proinflammatory cytokines (interleukin-1, tumor ne- crosis-a, and interferon- y) may induce cell surface ICAM-1 expression and secretion shedding of the molecule by cul- tured human hepa to~y tes . ' ~~ '~ In clinical studies, elevated serum SICAM-1 levels relating to disease severity and se- rum levels of biochemical indices of hepatic injury (primar- ily serum transaminase activities) have recently been found in chronic liver diseases of immunopathological and viral

The significance of immunopathological processes has likewise been well recognized in the pathogenesis of alco- holic liver disease^.^^-^^ The harmful effects of alcohol may entail the modulation of immune responses, the distur- bances of cytokine homeostasis, and the induction of new antigenic structures (e.g., Mallory's hyalin, acetaldehyde- protein adducts) that can initiate destructive immune re- sponses targeting alcohol-exposed hepatocytes.25 Pro- longed heavy drinking leads to hepatocellular damage and inflammatory infiltration of varying degrees of severity; these events are most pronounced in alcoholic hepatitis. The cellular infiltrate consists predominantly of neutro- phils. The existence of a neutrophil-induced cytotoxicity, mediated by toxic-free radicals and cytolytic proteases, in the pathogenesis of alcoholic liver damage, is gaining ac- c e p t a n ~ e . ~ ~ ~ ~ ~ This putative mechanism is supported by numerous findings. The transendothelial migration of leu- kocytes and the exertion of their cytotoxicity require mul- tiple steps of cell adhesion regulated by the interaction of cell surface adhesion molecules, the expression of which is

origin.lS,16,19-22

932 NAGY AND MANDl

induced by proinflammatory cyt~kines. ' -~ An overproduc- tion of proinflammatory cytokines (tumor necrosis fac- tor-&, interleukin-1, interleukin-6, and interleukin-8), some of which are capable of inducing ICAM-1 expression on h e p a t o c y t e ~ ' ~ ~ ' ~ and activating leukocytes,' has commonly been observed in alcoholic liver diseases, especially in al- coholic he pa ti ti^.'^.^^ Furthermore, by means of immuno- histology, a codistribution of tumor necrosis factor-a with ICAM-1 expressed on hepatocytes and with the occurrence of 0,-integrin-positive polymorphonuclear leukocytes, es- pecially in areas of hepatocellular necrobiosis, has also been demonstrated in acute alcoholic hepatitk6 The up- regulation of both ICAM-1 on target cells and MAC-1 on neutrophils is necessary for neutrophil-induced cytotoxicity to O C C U ~ ~ ~ . ~ ~ ; chronic alcohol intoxication has been shown to upregulate the expression of P,-integrins on neutro- phils," and blocking ICAM-1 by an antibody or interfering with ICAM-1 induction has been found to attenuate exper- imental neutrophil-induced liver injury.28

Our findings on circulating SICAM-1 in alcoholic cirrhot- ics fit in well with previous histological demonstrat- ing a severity-related overexpression of ICAM-1 in alco- holic hepatitis, and support the hypothetic pathomecha- nism outlined herein: (1) the most striking increases in the serum concentration of sICAM-1 were observed in patients with superimposed S A H ; and (2) stepwise multiple regres- sion analysis revealed that the circulating levels of AST and prothrombin, two well-accepted biochemical markers of acute hepatocellular damage in alcoholic were the best independent predictors of the circulating level of SICAM-1. Not surprisingly, the serum level of sICAM-1 also correlated strongly with the parameters re- lating to antecedent alcohol consumption, with ethanol being the toxic causative agent in alcoholic hepatitis.

In accordance with these findings, Shimada et al.' and Ishii et a1." detected severity-related increases in the serum level of sICAM-1 when they compared patients with alco- holic hepatitis with those with inactive alcoholic cirrhosis. In contrast, Adams et al.7 found no significant difference in circulating sICAM-1 levels between patients with alcoholic hepatitis and those with simple cirrhosis. In that study, however, the patients were grouped purely on the basis of histological criteria, and thereby the conventional biochem- ical indices of hepatocellular damage also failed to show differences between these two patient groups; furthermore, their alcoholic hepatitis group included not only patients with SAH, but also others with almost normal liver function test values. Nevertheless, they too demonstrated significant correlations with liver biochemistry values, including serum AST, bilirubin, and prothrombin, and, similarly to us, found no correlation with serum ~rea t in ine .~ This latter argues against a significant role of the renal function in determin- ing the circulating level of sICAM-1.

The ascitic fluid of uninfected cirrhotic patients contains high amounts of bioactive interleukin-6, produced locally by the peritoneal macrophages, but only trace amounts of

the known ICAM-inducer cytokines interleukin-1 and tu- mor necrosis f a~ to r -a . ' ~ .~ ' Consistently with this, we found much lower sICAM-1 concentrations in the ascites than in the serum, and the ratio of the ascitic to the serum con- centration of sICAM-1 was significantly lower than that for the total protein. Similarly, as recently pre~ented,~ ' the concentration of sICAM-1 in the ascitic fluid was found to correlate well with that in the serum. Thus, a local intra- peritoneal production of this adhesion molecule in uncom- plicated liver cirrhosis seems to be very unlikely, and the results suggest a circulatory origin of ascitic SICAM-1. However, according to a recent study, the concentration of sICAM-1 may be higher in the ascitic fluid of patients with peritoneal cancer and spontaneous bacterial p e r i t ~ n i t i s . ~ ~

In conclusion, the highly significant associations found between the serum concentration of SICAM-1 and the levels of laboratory markers of acute hepatocellular injury, the amount of alcohol consumed, and the presence of superimposed S A H strongly indicate that the circulating SICAM-1 concentration-although not specific for liver diseases-might serve as a useful noninvasive test for as- sessment of the current disease activity (intensity of acute necroinflammatory reactions) in patients with alcoholic liver cirrhosis. We are aware of the limitations of this study; the question remains open as to how the serum level of SICAM-1 is related to the histologically determined sever- ity of alcoholic hepatitis. Much more experience is needed to estimate the specificity and sensitivity of this test (e.g., in establishing the diagnosis and prognosis of alcoholic hepa- titis). On the basis of these results, however, it seems reasonable to plan more extended, longitudinal studies involving the comparative evaluation of concurrent liver biopsies to establish the real value of this marker in the diagnostic work-up and therapeutic monitoring of patients with alcoholic liver diseases.

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