T-cell activation, expression of adhesion molecules and response to ethanol in alcoholic cirrhosis

ELSEVIER

Immunology

Letters

50 (1996) 179- 183

T-cell activation, expression of adhesion molecules and response to ethanol in alcoholic cirrhosis Juan Luis Santos-Perez”, Antonio Diez-Ruizb, Luis Luna-Casadob, Jose Antonio Helmut Wachter”, Dietmar FuchsCT*, Francisco Gutierrez-Gea”

Soto-Mash,

“Department of Biochemistry, University Hospital of’ Granada. Granada, Spain bDepartment of Medicine, University Hospital of Granada, Granada, Spain ‘Institute of Medical Chemistry and Biochemistry, University of Innsbruck, Fritz Pregl StraJe 3, A-6020 Innsbruck. Austria Received

23 February

1996; accepted

26 March

1996

Abstract Abnormal immune function is a well-recognized feature in patients with alcoholic cirrhosis. It may contribute to the pathogenesis of the disease and to the clinical consequences. Nevertheless, a potential role of ethanol to elicit immune disturbances in patients is still unclear. To further examine the immune mechanisms which potentially are involved in alcoholic cirrhosis and the relationship to ethanol, we have determined the expression of surface antigens CD4, CDS, and of adhesion molecules CD25 LFA-1, ICAM- and LFA-3 in patients and in response to stimulation with OKT-3, IL-2 and with ethanol in vitro. In addition, we quantified the production of IL-2, TNF-a and IFN-11 by lymphocytes of alcoholic cirrhosis patients compared to controls. Lymphocytes from patients showed increased basal and stimulated expression of CD4, CD25, LFA-1, ICAM- and LFA-3 molecules and increased TNF-(r production in comparison to controls. When lymphocytes from patients were co-cultured with ethanol, the overexpression of activation markers and TNF-c( production was similar to that obtained with mitogens. In contrast, a predominant suppressive effect of ethanol was observed in lymphocytes from controls. Our study underlines the importance of a chronic state of immune activation in alcoholic cirrhosis. The data further suggest a role of ethanol to stimulate immune response and to be directly involved in the development of disease. Keywords:

Alcoholic cirrhosis; Ethanol; T cell activation;

Adhesion molecules; Cytokines

1. Introduction Alcoholic liver disease is accompanied by a wide range of immunological disturbances that combines findings of immunodeficiency and of immune activation as well [l-3]. Moreover, it is unclear whether the immunological changes in patients with alcoholic liver disease are the cause of hepatic damage and progression to cirrhosis or consequences of the established disease in relation to impaired protein synthesis and diminished antigen removal capacity by the liver [4,5]. The first step to address this question might be to investigate a potential immunogenic capacity of ethanol in patients. Thus far, immunological studies regarding the role of ethanol in alcoholic liver disease are incon* Corresponding 5072865. 0165-2478/96/$12.00

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elusive [1,2]. Discrepant findings are reported on a possible ethanol-mediated immunopathogenesis of alcoholic liver disease and some aspects of immune function in patients [2,6]. The present status of research is often complicated by the fact that, frequently, different methods and quite heterogenous patient populations such as hepatic steatosis, alcoholic hepatitis or liver cirrhosis have been studied [l]. In the present work we examined the expression of CD25, a surface marker of cell activation, and of adhesion molecules and the synthesis of specific cytokines in lymphocyte cultures of patients with established alcoholic cirrhosis. In parallel, changes of surface expression and cytokine synthesis were monitored in cell cultures in response to mitogens. Finally, we investi-, gated the in vitro response of lymphocytes cultures to ethanol.

180

J.L. Santos-Perez

et al. 1 Immunology Letters 50 (1996) I79- 183

2. Materials and methods

2. I. Patients Twenty patients (17 males, 3 females; mean age 58 f 9.5 years) admitted to the Internal Medicine and Digestive Departments of the University Hospital of Granada were included in the study. All the patients were diagnosed with alcoholic cirrhosis on the basis of a history of daily alcohol consumption of at least 80 g for more than 10 years and on clinicoanalytical criteria and biopsy with histological features according to internationally accepted criteria [7]. Alcoholic cirrhosis was defined as established cirrhosis without evidence of acute alcoholic hepatitis. According to the Child-Pugh’s score for severity of liver disease [8], 4 patients were in grade A, 3 in grade B and 13 were in grade C. Other diseases like acute infections or treatments, by, e.g. corticosteroids, that might have affected the immune status of the patients have been ruled out. At the time of recruitment, cirrhosis was stable and no patient had consumed alcohol during at least 1 month prior to the study. The control group consisted of 20 sex- and age-matched healthy blood donors who were identified as non-drinkers. The study was carried out in accordance with the Declaration of Helsinki, and approved by the Hospital ethics authorities. Ten milliliters of venous blood was collected into sterile endotoxin-free, EDTA-K3 anticoagulated blood collection tubes. Blood was immediately diluted in phospate buffered saline (PBS) in a 1: 1 proportion and processed in sterile conditions under laminar flow. 2.2. Cell cultures Peripheral blood mononuclear cells from alcoholic cirrhosis patients and controls were isolated by FicollHypaque density gradient centrifugation (Pharmacia, Uppsala, Sweden), then suspended in PBS and after repeated centrifugation, resuspended in RPMI- 1640 medium (Sigma, St. Louis, MO, USA) with penicillin (100 IU/ml), gentamicin (0.05 mg/ml) and fungizone (2.5 g/ml). The final cell concentration was adjusted to lo7 cells/ml. These cells were immediately frozen in cryotubes at 70°C and, after 24 h introduced in liquid nitrogen at - 120°C until use. All the assays were performed at the same time and within 6 months from sample collection. A pilot study was performed to determine the effect of different ethanol concentrations on cell survival. We used lymphocytes from control subjects, cultured during 3 days and with increasing concentrations of ethanol (2.5 x 10e3, 5 x 10e3, 10 x lo-’ and 20 x 10 - 3 g/l). The assays were performed in quadruplicate and the estimation of cellular viability by the exclusion with ethidium bromide and flow cytometry

count. The maximum ethanol concentration not decreasing the proportion of viable cells compared to basal cultures was found as 5 x 10 ~ 3 g/l. This concentration is also compatible with serum levels achieved by the ingestion of a moderate quantity of alcohol. Two hundred milliliters of peripheral blood mononuclear cells suspension was cultured in triplicate in complete medium in 96-well round-bottomed microwell plates (Nunc, Roskilde, Denmark). As a control, cell proliferation was tested without mitogen and with OKT3 monoclonal antibody (Ortho Diagnostic Systems, Raritan, NJ, USA; concentration: 2.5 x 10 -’ g/l), interleukin-2 (Sigma; 5000 IUjl), and ethanol (Merck, Darmstadt, Germany; in the mentioned concentration of 5 x 10 - 3 g/l), The plates were incubated in an atmosphere of 5% CO,. After centrifugation the supernatants were harvested and frozen at - 70°C until analysis and the cells were resuspended in PBS. 2.3. Flow cytometric measurement of antigen expression

One hundred milliliter aliquots of cell suspensions were incubated with the various antibodies at 4°C in the dark for 30 min. The monoclonal antibodies used were 0KT3 (identifying CD3 antigen), 0KT4 (CD4). OKT8 (CD8) and OKT26a (CD25) from Ortho and CD45RA (CD45RA antigen), LFA-1 (CD1 la), ICAM1 (CD54) and LFA-3 (CD58) from Immunotech (Marseille, France). Samples were run immediately after staining. Flow cytometry analysis (Cytoron, Ortho Diagnostic Systems) with an argon laser at a wavelength of 488 nm was performed to determine the percentage of fluorescent cells. Non-specific IgG-FITC (OK-Control, Ortho Diagnostic Systems) was used as a negative control in order to set negative regions in the fluorescence histogram. Cells were analyzed by gating them on lymphocytes region on the forward/right scatter cytogram. A total number of 1000 cells in the triggered region was counted, and the number of positive cells was established by considering the percentage of cells in the positive region in the fluorescence histogram. 2.4. Cytokines measurements in culture supernatants From ten random alcoholic cirrhosis patients and ten controls we harvested culture supernatants and determined the concentrations of interleukin-2 (IL-2), tumour necrosis factor-a (TNF-or) and interferon-l) (IFN-y) by immunoenzymatic methods. IL-2 and TNF(r were measured with the Biokine kits (T Cell Diagnostics Inc., Cambridge, MA, USA) and IFN-1, by means of the IFN-y-EASIA kit (Medgenix Diagnostics, Fleurs, Belgium) according to the manufacturers’ instructions.

J.L. Santos-Perezet al.llmmurdogy

2.5. Statistical analysis

All the results in the tables are given as mean f SD; in the figures mean f SEM. is provided. Two-way analysis of variance was employed to compare antigen expression between groups and before and after stimulation. When statistical significance existed (P < 0.05), a multiple analysis was done with the Newman-Keuls method. Correlations between variables were tested for by the Spearman’s rank regression coefficient.

3. Results In basal cultures, an increased expression of CD25 antigen on the surface of lymphocytes from cirrhotics was found when compared with controls (Table 1). The adhesion molecules LFA-1, ICAM-I and LFA-3 were more expressed in cirrhotics than in controls, and higher concentrations of TNF-a were detected in the supematants of cirrhotic patients. There existed significant correlations of ICAM- with CD4’(rs = 0.52, P < 0.05), CD25 (TS = 0.81, P < 0.01) LFA-1 (YS = 0.76, P < 0.01) and LFA-3 (rs = 0.56, P < 0.05). CD25 was related, besides, to LFA-1 (rs = 0.77, P < 0.01) and LFA-3 (YS = 0.63, P < 0.01). No significant correlations were found between these variables and the Child-Pugh groups of patients. Culture of lymphocytes with 0KT3 led to an increased expression of CD4, CD25 and LFA-3 molecules on the surface of lymphocytes of alcoholic cirrhosis patients compared to controls, and there was a decreased expression of CDS (Table 2). Also an increased production of TNF-(w was observed of lymphocytes from alcoholic cirrhosis patients upon stimulation with OKT3. Exposure of cultured lymphocytes to exogenous IL-2 induced a diminished expression of CD4 and CD8 antigens, diminished production of IL-2 and elevated Table I Mean 5 SD values of the expression of surface antigens and adhesion molecules (percentages of total cell counts) IL-2, TNF-I (pglml) and IFN-11 (W/ml) concentrations in supematants of basal lymphocyte cultures from controls and cirrhotic patients (ns. = not significant) Controls CD4 CD8 CD25 LFA-I ICAM-I LFA-3 IL-2 TNF-a IFN-y

53.10 33.60 9.10 50 19.40 6.30 68.60 5.60 2.66

+ k k F & + f i +

7.51 5.62 2.60 10.16 2.50 2.54 18.47 3.85 0.28

60.70 30.10 25.20 57 41.50 52.60 48.20 41.50 2.30

& * + f * + + & 2

Table 2 Expression of surface antigens and adhesion molecules (percentages of total cell counts, .Y + SD.), IL-2 (pg/ml), TNF-G( (pg/ml) and IFN-;a (IUlml) concentrations in supernatants of OKT3-stimulated lymphocytes cultures from controls and cirrhotic patients (ns. = not significant)

CD4 CD8 CD25 LFA-I LCAM- I LFA-3 IL-2 TNF-r IFN-;s

14.10 9.39 5.65 9.60 II.08 10.01 15.97 68.06 0.49

n.s. n.s. < 0.01 < 0.01 < 0.01 c 0.01 ns. < 0.01 n.s.

72.8 43.00 52.2 73.6 63.6 70.1 90.1 7.4 3.2

k f + $ _+ rf; k * +

Significance (P)

Cirrhotics

Controls 4.83 7.61 13.44 1.71 4.92 6.17 40.24 3.99 0.64

18.2 33.00 68.3 12.5 66.2 76.4 66.10 45.4 3.00

I_ & i k i k _+ k *

13.5 8.89 14.73 14.33 II.39 3.28 54.05 46.99 0.31

< 0.01 < 0.01 < 0.01 n s. n.s. i 0.01 n.s. < 0.01 n-s.

LFA-I, ICAM-I and LFA-3 expression in cirrhotic patients compared to controls (Table 3). No significant differences were found in TNF-a and IFN-y production. When lymphocytes were cultured with ethanol, an almost two-fold higher expression of CD25 in cirrhotics compared to basal values was seen (Fig. 1). A stimulatory effect of ethanol was also indicated by the expression of ICAM-1, LFA-1 and LFA-3 in cirrhotics. In contrast, only ICAM- expression was increased, but LFA-1, LFA-3 and CD4 expression and IL-Z production decreased in control cells after stimulation with ethanol (Fig. 1). When comparing the effects induced by ethanol between controls and patients, a significantly increased expression of CD4, CD25 LFA-1, ICAM- and LFA-3 and an enhanced production of TNF-a was observed in cirrhotic patients (all P < O.Ol)(Table 4).

Table 3 Expression of surface antigens and adhesion molecules (percentages of total cell counts. s + S.D.), IL-2 (pg/ml), TNF-a (pg/ml) and IFN-7 (IU/ml) in supematants of IL-2-stimulated lymphocytes cultures from controls and cirrhotic patients (us. = not significant).

Significance (P)

Cirrhotics

I81

Letters50 (1996) 179-183

CD4 CD8 CD25 LFA-1 [CAM-I LFA-3 IL-2 TNF-3 IFN-;

Controls

Cirrhotics

84.8 36.8 71.00 18.00 13.00 55.1 134.6 6.00 3.2

79.3 32.6 76.8 83.6 16.8 76.7 10.3 17.6 2.90

i 6.88 k 1.53 + 7.62 + 10.99 ;t 5.58 * 10.31 + 42.4 + 4.09 +_ 0.64

+ k + + k f i_ & +

14.68 Il.97 15.89 9.94 12.5 1.63 29.83 20.02 0.48

Significance (P) < 0.01

< 0.01 n.s. < 0.01
182

J.L. San&s-Perez

et al. /Immunology

Letters 50 (1996) 179-183

SO_

CD25

CD4 CD6

ICAM-I IL-2 IFN-8 LFA-1 LFA-3 TNF-Or

CD6

LFA-1

LFA-3

TNF-w

Fig. 1. Expression of surface antigens and adhesion molecules (percentages of total cell counts, x + S.E.M.), and of IL-2 (pg/ml), TNF-cr (pg/ml) and IFN-y (IUjml) in supematants of basal (open bars) and ethanol-stimulated (hatched bars) lymphocyte cultures from controls (left) and cirrhotics (right) (P values are shown on the top, n.s. = not significant).

4. Discussion

There was a significantly increased expression of CD25 in basal cultures of lymphocytes from alcoholic cirrhosis patients which is also found in the proliferative assays upon stimulation with OKT3. The CD25 antigen, the receptor for IL-2, is a 55-kDa molecule expressed by activated T lymphocytes, which is a reliable and a specific indicator of early cell activation [9]. The appearance of CD25 antigen only requires protein synthesis but is independent from DNA synthesis or cell division [lo]. Thus, increased expression of CD25 in patients reflects T cell activation in vivo. Similar to this finding, in the basal culture of lymphocytes, the cell surface expression of adhesion molecules ICAM- 1, LFA-1 and LFA-3 is higher in alcoholic cirrhosis patients than in controls. ICAM- and LFA-3 are adhesion molecules and members of the immunoglobulin superfamily that are essential for the interaction of T cells with other immune and/or target cells by mediating adhesion [l 11. LFA-1 is an integrin adhesion molecule, which is present on most leukocytes and it is the natural ligand for ICAM-1. The affinity between LFA-1 and ICAM- is enhanced within a few minutes Table 4 Expression of surface antigens and adhesion molecules (percentages of total cell counts, x + SD.), IL-2 (pg/ml), TNF-a (pg/ml) and IFN-7 (W/ml) in supematants of ethanol-stimulated lymphocytes cultures from controls and cirrhotic patients (n.s. = not significant).

CD4 CD8 CD25 LFA-I ICAMLFA-3 IL-2 TNF-x IFN-~1

39.3 30.5 10.6 40.7 25.4 40.8 56.00 4.4 3.3

+ + & k + * f * k

Significance (P)

Cirrhotics

Controls 5.69 8.59 3.43 13.25 3.02 3.35 13.26 3.4 0.92

64.8 31.1 49.70 63.3 52.5 58.6 66.4 37.9 3.1

& 1 + & i + i f i

16.12 7.17 11.99 9.63 11.29 6.76 39.32 73.15 1.08

< 0.01 n.s. (0.01 -z 0.01 < 0.01
after T cell receptor stimulation [ 121. The relative increase of ICAMwas much greater than that of LFA-I or LFA-3 which would agree with the fact that ICAM- is clearly upregulated upon activation whereas LFA-1 and LFA-3 rather undergo conformational changes which increases their affinity. The overexpression of the adhesion phenomena in lymphocytes cultures from alcoholic cirrhosis patients appears to reflect a persistent activation of the immune response. These findings are in line with the increase of adhesion molecules expressed in the liver [ 131, on peripheral blood mononuclear cells (Luna-Casado, unpublished) and of the shedded serum soluble forms in alcoholic cirrhosis patients [14]. In the present study we found increased baseline production of TNF-a by lymphocytes of alcoholic cirrhosis patients which agrees with a high in vitro synthesis and increased serum levels of the pro-inflammatory cytokines IL-I, IL-6 and TNF-cr, as well as soluble TNF receptors in alcoholic cirrhosis [15- 171. No significant differences in IFN-y were found. Inflammatory cytokines are associated with the clinical and biochemical manifestations of acute-phase response [ 181. TNF-c( is able to enhance ICAM-l-mediated adhesion of neutrophils to vascular endothelium as part of the inflammatory process [19]. By this way, it is likely that increased concentrations of inflammatory cytokines and expression of adhesion molecules could mediate the liver damage in alcoholic cirrhosis patients. A decreased production of IL-2 in response to IL-2 exposure was found in lymphocyte cultures of cirrhotic patients compared to controls. These data are consistent with previous studies [1,20], but contrast with the increased expression of the IL-2 receptor. However, with the exception of TNF-K production in OKT-3 stimulated cultures, the differences of antigen expression levels between stimulated and unstimulated cultures were much smaller than the differences observed at baseline; the data indicate that the in vivo situation is more conclusive. In fact, in alcoholic cirrhosis high

J.L. Santos-Perez et al. / Immunology Letters 50 (1996) 179-183

concentrations of serum neopterin and sTNF-Rs also confirm a chronic activation of the lymphocyte-monocyte/macrophage system [ 171. Interestingly, reduced Tcell proliferative response and diminished production of IL-2 and IFN-7 in vitro was found to parallel increased serum IFN-y and/or neopterin and soluble IL-2 receptor [21,22] in other chronic immune activation states such as HIV infection or cancer. Ethanol co-culture of lymphocytes from patients with alcoholic cirrhosis induces a quite dramatic overexpression of the activation marker CD25, and the adhesion molecules, ICAM-1, LFA-1 and LFA3 were moderately increased. Compared to controls, the cells also released increased amounts of TNF-a in supernatants. Interestingly, there were no such changes observed in ethanol-cultured lymphocytes from controls, with the exception of a moderate increase of ICAM- expression. Rather, suppressive effects of ethanol with respect to basal values were observed, indicating that the stimulatory capacity of ethanol on the T lymphocytes is obtained only in cirrhotic patients. In the absence of any racial or geographic differences between patients and controls, the results imply that chronic exposure of lymphocytes to ethanol in patients may alter their way of response. The originally inhibitory effect of ethanol on lymphocytes may be even reversed and become stimulatory in the situation of chronic excessive alcohol intake. Ethanol might act as a hapten by binding to other structures [23]. In fact, there is evidence of antibodies against ethanol-modified cells [24], and ethanol was found to alter TNF-production and apoptosis in vitro earlier [25]. Therefore, our findings strongly suggest an immunomodulatory effect of ethanol on the lymphocytes of alcoholic cirrhosis patients and argues in favour of an immune pathogenesis in alcoholic liver disease. Further studies are needed to better define the diverse effects of ethanol on patients’ and controls’ lymphocytes. In conclusion, patients with alcoholic cirrhosis present with a chronic immune activation state as shown by increased expression of T-cell activation markers, adhesion molecules and TNF-a production by cultured lymphocytes. Ethanol enhances the expression of these activation parameters suggesting a direct role in immune mechanisms of alcoholic cirrhosis.

183

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