Immunology 1991 73 140-146
ADONIS 001928059100128J
T-lymphocyte activation pathways in alcoholic liver disease F. SPINOZZI, A. BERTOTTO,* F. RONDONIJt R. GERLI, F. SCALISE* & F. GRIGNANI Departments of Internal Medicine and *Paediatrics, Perugia University and tDivision of Internal Medicine, Assisi General Hospital, Italy
Accepted
ftr
publication
11
February 1991
SUMMARY Immune system derangement in cirrhotic patients with evidence of malnutrition is a well-recognized characteristic of chronic alcohol abuse. However, in vitro studies on cellular immune function performed with lectin mitogens have produced conflicting results. The recent development of more accurate immunological techniques for studying lymphocyte transformation, that use monoclonal antibodies directed against surface structures (CD3 and CD2) involved in antigen recognition, as well in adesion functions, prompted us to study discrete in vitro T-cell hypo-responsiveness in a series of alcoholic liver disease (ALD) patients with no evidence of malnutrition or hepatic cirrhosis. The results indicated that the CD2 pathway is markedly defective in ALD T lymphocytes, accompanied by reduced interleukin-2 (IL-2) receptor expression upon in vitro activation. This defect cannot be reversed by the addition of recombinant IL-2 (rIL-2) or rIL- 1. Faulty intracellular signal transduction by protein kinase C (PKC) and defective intracellular Ca2' mobilization may be responsible for the CD2 pathway impairment. The addition of small amounts of phorbol 12myristate, 13-acetate, but not Ca2+ ionophore A23187, is able to overcome the defect, thereby suggesting a direct PKC involvement. The hypothesis of a direct ethanol effect on transmembrane signal transduction systems is suggested by the demonstration of an expansion of circulating virgin (naive) T cells (CD3' /UCHLI '`7) that binds tyrosine phosphatase (CD45RA antigen) on their surface.
INTRODUCTION Alcohol significantly inhibits cell-mediated immunity. Clinical evidence includes a high incidence of both tuberculosis and head and neck cancers among heavy smokers.' I It has been reported that the ability of circulating lymphocytes to undergo blast transformation under mitogenic activation is impaired in alcoholic liver disease (ALD),j'5 especially when malnutrition or chronic drinking have already produced a degree of liver damage.67 In addition, a serum factor that blocks mitogeninduced lymphocyte transformation in vitro has been described in alcoholic cirrhosis.8 However, a number of studies have indicated a normal in vitro response to phytohaemagglutinin (PHA)9"' with a relative increase in both absolute number and function of helper T cells, suggesting that the immune system may be partially activated rather than depressed in ALD patients."'' Despite the fact that the clinically well-recognized defective host defence is partially attributable to the direct effect of ethanol on neutrophil and mononuclear phagocyte functions,'6"7 it is unlikely that opsonization and phagocytosis play a decisive role in the host's immune response to infections caused by intracellular micro-organisms, such as viruses. Although this suggests that there may also be a defect in cell-mediated Correspondence: Dr F. Spinozzi, Clinica Medica 1, Policlinico Monteluce, 06100 Perugia, Italy.
immunity, a linear correlation between alcohol abuse and functional impairment of T cells has proved difficult to substantiate (reviewed by ref. 2). A number of different experimental systems have been proposed for studying T-cell activation signals. Antigen activation is mediated by the T-cell receptor (TcR) antigen complex. which in man, consists of a disulphide-linked clonotypic heterodimer antigen recognition unit (Ti) non-covalently bound to the CD3 antigen on the cell membrane." Normal human T cells can be activated by anti-CD3 monoclonal antibody (mAb) in an interleukin-2 (IL-2)-dependent autocrine fashion in the presence of accessory cells (AC)." Furthermore, the CD2 structure, the receptor for sheep red blood cells (SRBC) on human T cells, has been reported to serve as an alternative T-cell activation pathway that is independent of both antigen and, to a lesser extent, AC.21' This pathway can be activated in vitro by appropriate pairs of anti-CD2 mAb which recognize distinct conformational epitopes on the CD2 molecule.2' It has been suggested that the CD2 antigen and the TcR complex are structurally and functionally linked.'2 In particular, antigenmediated T-cell activation appears to depend on the interaction between TcR and CD2 structures that results from the simultaneous binding of the TcR with antigen and of the CD2 molecule with its natural ligand, lymphocyte function-associated antigen 3,45 which is also expressed on the surface of AC.
140
T-lymphocyte activation in alcoholic liver disease Since these in vitro models induce both IL-2 receptor (IL-2R) expression and IL-2 production26 and promote the proliferation of naive (CD3+/CD45RA+/UCHLI -low) and memory (CD3+/ CDw29+/UCHLl - high) T cells differently, i.e. by interaction with monocytes20.27 or by different expression of adhesion molecules,28'29 they offer a better physiological stimulus for studying T-lymphocyte functions than other mitogens.30'3' Since the poor proliferation of naive cells to doses of agonist mAb that induce strong responses from memory T cells reflects their decreased capacity to respond to specific antigens in vivo, and since the irreversible shift from naive to a memory T-cell state leads to the acquisition of the ability to help B-cell differentiation and develop a secondary response to antigens,28 we investigated the proliferative response of peripheral blood lymphocytes (PBL) from ALD patients, with no evidence of malnutrition, to anti-CD3 and anti-CD2 mAb in an attempt to better understand the effects chronic ethanol intoxication exerts on T-lymphocyte functions. MATERIALS AND METHODS
Patients Fifteen patients with ALD were studied. The diagnosis was made according to well-defined criteria,32 consisting of increased echogenicity of liver parenchyma, which was further histologically confirmed as steatosis. The ages ranged from 24 to 63 years, eight were male and seven female. All showed multiple florid physical signs of ALD with spider angiomata, hepatosplenomegaly (but no hypersplenism) and flapping tremors. Patients with major medical illnesses, such as inflammatory bowel disease, diabetes, delirium tremens, alcoholic hepatitis, intestinal haemorrhage, pancreatic or hepatic insufficiency or concomitant infections, were excluded from the study. To avoid enrolling patients with malnutrition and/or poor caloric intake, a nutritional assessment based on dietary evaluation, height, weight and triceps skinfold thickness measurement was performed. Protein serum levels, creatinine excretion and creatinine-height-index were also determined and only non-malnutrished subjects were entered in the study protocol. Liver enzymes, such as serum aspartate transaminase (SGOT), alanine transaminase (SGPT), alkaline phosphatase and y-glutamil-transpeptidase, were raised in all subjects studied. Daily alcohol intake was from 100 to 300 g for 2-5 years. There were no reductions in platelet, total leucocyte or absolute lymphocyte COUitS.
Cell preparation PBL from ALD patients and 13 control subjects were isolated
by Ficoll-Hypaque (Lymphoprep, Nycomed AS, Oslo, Norway) density gradient centrifugation, resuspended in RPMI1640 supplemented with 10% foetal calf serum, 4 mM, Lglutamine, 100 U/ml penicillin and 100 ,g/ml streptomycin (complete medium; Gibco, Grand Island, NY) and separated into SRBC rosette-enriched (E+) and rosette-depleted (E-) subsets, as described previously.33 The E- cells suspensions were then irradiated at 3000 rads and used as source of AC. E+ cell populations were passed through nylon-wool columns and treated with the OKMI mAb (Ortho, Raritan, NJ) plus rabbit complement (Cedarlane, Ornby, Ontario, Canada) to eliminate residual contaminating monocytes. On the basis of their reactivity with the anti-CD3 mAb (OKT3, Ortho) (> 98'% with
141
immunofluorescence analysis), these mononuclear cells were considered highly purified T-cell subsets. Membrane immunofluorescence studies T cells from both ALD and control PBL samples were identified by OKT3, anti-TI I1 and TI 12 (a generous gift from Dr S. F. Schlossman, Harvard Medical School, Boston, MA) mAb in an indirect immunofluorescence assay. Briefly, mononuclear cells (1 x 106/ml) were suspended in complete medium supplemented with 0-01% sodium azide and incubated with an optimal dilution of each mAb for 30 min in an ice bath. After two washings, the cell pellet was incubated with a fluoresceinconjugated goat anti-mouse IgG (Kallestad, Chaska, MA) for 30 min in an ice bath. After a further three washings, at least 300 cells were counted under a Leitz Dialux microscope, equipped with an epi-illumination mercury arc lamp and phase contrast, to determine the percentage of positive cells. The IL-2R was identified on both resting and stimulated T cells by direct immunofluorescence staining with a fluoresceinated anti-Tac mAb (Becton-Dickinson, Mountain View, CA). Memory and naive T cells were evaluated in both ALD and control samples by two-colour cytofluorimetric analysis (FACScan; BectonDickinson). The first-step mAb used in these experiments were anti-CD3 (OKT3) (Ortho), UCHL1 (Dakopatts, Glostrup, Denmark) and CD45RA (2H4) (Coulter Immunology, Hialeah, FL). The cells were then counterstained with isotype-specific goat anti-mouse IgG conjugated with either fluorescein isothiocyanate or phycoerithrin (Southern Biotechnology Associated, Inc., Birmingham, AL). Isotype-matched mAb which do not react with human leucocytes served as controls. Cell proliferation assay PBL or T cells (5 x 104/well) were cultured in triplicate in complete medium in 96-well round-bottomed microwell plates (Nunc, Roskilde, Denmark). Cell proliferation was triggered by adding PHA (Gibco), the OKT3 mAb, or a combination of the anti-T I 12 and anti-T I 13 mAb (the latter also donated by Dr S. F. Schlossman). Phorbol 12-myristate, 13-acetate (PMA; Sigma, St Louis, MO), human recombinant IL-2 (rIL-2; Janssen, Beerse, Belgium; specific activity 90 x 106 U/mg protein), human recombinant IL-I (rIL-1; Janssen; specific activity 1-3 x 107 U/mg protein) and calcium ionophore A23187 (Ca2+ A23 187; Sigma) were added in some culture experiments. Stock solutions of each mitogen or reagent were diluted in complete medium and used at the indicated concentrations. The plates were incubated in a 5% CO2 humidified atmosphere at 37 for 72 hr. Cell proliferation was assessed by measuring [3H] thymidine ([3H]TdR) incorporation (0 5 pCi; specific activity 25 Ci/mmol; Amersham Int., Amersham, Bucks, U.K.) during the last 6 hr of culture and counting in a liquid scintillation counter. Results are expressed as net counts per minute (c.p.m.) [3H]TdR incorporation and reflect absolute c.p.m. [3H]TdR uptake minus background c.p.m. (c.p.m. incorporated in the absence of mitogen). Statistical anali sis Due to the non-normal distribution of samples, the MannWhitney U-test was applied for statistical evaluation. Values of P < 0-05 were considered as significant.
F. Spinozzi et al.
142
(b)
(a)
100
.00
,ArTIT
VIVTV
0
III
0
10
5000
5
2
110
0
4
0o
10 1
V
IVIIf IT
l03 102
Fluorescence Intensity (loglo) Figure 1. Expression of UCHL1 antigen on CD3+ lymphocytes in controls (a) and ALD patients (b). The cytofluorimetric analysis was done on gated CD3 + cells. In normal T cells, the antigen distribution is characterized by a small percentage of low-positive (naive) and a peak of high-positive cells (memory), whereas in ALD patients there is an evident peak of low UCHL 1-positive 'naive' T lymphocytes.
Table 1. Expression of CD45RA and UCHL1 antigens on T lymphocytes of normal controls and ALD patients
CD3+CD45RA+ (range) CD3 + UCHLlIow (range) CD3+UCHLIhigh (range) *
% controls
% ALD
31 (26-35) 35 (29-38) 17 (15-19)
55 (50-57)* 62 (58-67)* 19 (17-21)
P
ADONIS 001928059100128J
T-lymphocyte activation pathways in alcoholic liver disease F. SPINOZZI, A. BERTOTTO,* F. RONDONIJt R. GERLI, F. SCALISE* & F. GRIGNANI Departments of Internal Medicine and *Paediatrics, Perugia University and tDivision of Internal Medicine, Assisi General Hospital, Italy
Accepted
ftr
publication
11
February 1991
SUMMARY Immune system derangement in cirrhotic patients with evidence of malnutrition is a well-recognized characteristic of chronic alcohol abuse. However, in vitro studies on cellular immune function performed with lectin mitogens have produced conflicting results. The recent development of more accurate immunological techniques for studying lymphocyte transformation, that use monoclonal antibodies directed against surface structures (CD3 and CD2) involved in antigen recognition, as well in adesion functions, prompted us to study discrete in vitro T-cell hypo-responsiveness in a series of alcoholic liver disease (ALD) patients with no evidence of malnutrition or hepatic cirrhosis. The results indicated that the CD2 pathway is markedly defective in ALD T lymphocytes, accompanied by reduced interleukin-2 (IL-2) receptor expression upon in vitro activation. This defect cannot be reversed by the addition of recombinant IL-2 (rIL-2) or rIL- 1. Faulty intracellular signal transduction by protein kinase C (PKC) and defective intracellular Ca2' mobilization may be responsible for the CD2 pathway impairment. The addition of small amounts of phorbol 12myristate, 13-acetate, but not Ca2+ ionophore A23187, is able to overcome the defect, thereby suggesting a direct PKC involvement. The hypothesis of a direct ethanol effect on transmembrane signal transduction systems is suggested by the demonstration of an expansion of circulating virgin (naive) T cells (CD3' /UCHLI '`7) that binds tyrosine phosphatase (CD45RA antigen) on their surface.
INTRODUCTION Alcohol significantly inhibits cell-mediated immunity. Clinical evidence includes a high incidence of both tuberculosis and head and neck cancers among heavy smokers.' I It has been reported that the ability of circulating lymphocytes to undergo blast transformation under mitogenic activation is impaired in alcoholic liver disease (ALD),j'5 especially when malnutrition or chronic drinking have already produced a degree of liver damage.67 In addition, a serum factor that blocks mitogeninduced lymphocyte transformation in vitro has been described in alcoholic cirrhosis.8 However, a number of studies have indicated a normal in vitro response to phytohaemagglutinin (PHA)9"' with a relative increase in both absolute number and function of helper T cells, suggesting that the immune system may be partially activated rather than depressed in ALD patients."'' Despite the fact that the clinically well-recognized defective host defence is partially attributable to the direct effect of ethanol on neutrophil and mononuclear phagocyte functions,'6"7 it is unlikely that opsonization and phagocytosis play a decisive role in the host's immune response to infections caused by intracellular micro-organisms, such as viruses. Although this suggests that there may also be a defect in cell-mediated Correspondence: Dr F. Spinozzi, Clinica Medica 1, Policlinico Monteluce, 06100 Perugia, Italy.
immunity, a linear correlation between alcohol abuse and functional impairment of T cells has proved difficult to substantiate (reviewed by ref. 2). A number of different experimental systems have been proposed for studying T-cell activation signals. Antigen activation is mediated by the T-cell receptor (TcR) antigen complex. which in man, consists of a disulphide-linked clonotypic heterodimer antigen recognition unit (Ti) non-covalently bound to the CD3 antigen on the cell membrane." Normal human T cells can be activated by anti-CD3 monoclonal antibody (mAb) in an interleukin-2 (IL-2)-dependent autocrine fashion in the presence of accessory cells (AC)." Furthermore, the CD2 structure, the receptor for sheep red blood cells (SRBC) on human T cells, has been reported to serve as an alternative T-cell activation pathway that is independent of both antigen and, to a lesser extent, AC.21' This pathway can be activated in vitro by appropriate pairs of anti-CD2 mAb which recognize distinct conformational epitopes on the CD2 molecule.2' It has been suggested that the CD2 antigen and the TcR complex are structurally and functionally linked.'2 In particular, antigenmediated T-cell activation appears to depend on the interaction between TcR and CD2 structures that results from the simultaneous binding of the TcR with antigen and of the CD2 molecule with its natural ligand, lymphocyte function-associated antigen 3,45 which is also expressed on the surface of AC.
140
T-lymphocyte activation in alcoholic liver disease Since these in vitro models induce both IL-2 receptor (IL-2R) expression and IL-2 production26 and promote the proliferation of naive (CD3+/CD45RA+/UCHLI -low) and memory (CD3+/ CDw29+/UCHLl - high) T cells differently, i.e. by interaction with monocytes20.27 or by different expression of adhesion molecules,28'29 they offer a better physiological stimulus for studying T-lymphocyte functions than other mitogens.30'3' Since the poor proliferation of naive cells to doses of agonist mAb that induce strong responses from memory T cells reflects their decreased capacity to respond to specific antigens in vivo, and since the irreversible shift from naive to a memory T-cell state leads to the acquisition of the ability to help B-cell differentiation and develop a secondary response to antigens,28 we investigated the proliferative response of peripheral blood lymphocytes (PBL) from ALD patients, with no evidence of malnutrition, to anti-CD3 and anti-CD2 mAb in an attempt to better understand the effects chronic ethanol intoxication exerts on T-lymphocyte functions. MATERIALS AND METHODS
Patients Fifteen patients with ALD were studied. The diagnosis was made according to well-defined criteria,32 consisting of increased echogenicity of liver parenchyma, which was further histologically confirmed as steatosis. The ages ranged from 24 to 63 years, eight were male and seven female. All showed multiple florid physical signs of ALD with spider angiomata, hepatosplenomegaly (but no hypersplenism) and flapping tremors. Patients with major medical illnesses, such as inflammatory bowel disease, diabetes, delirium tremens, alcoholic hepatitis, intestinal haemorrhage, pancreatic or hepatic insufficiency or concomitant infections, were excluded from the study. To avoid enrolling patients with malnutrition and/or poor caloric intake, a nutritional assessment based on dietary evaluation, height, weight and triceps skinfold thickness measurement was performed. Protein serum levels, creatinine excretion and creatinine-height-index were also determined and only non-malnutrished subjects were entered in the study protocol. Liver enzymes, such as serum aspartate transaminase (SGOT), alanine transaminase (SGPT), alkaline phosphatase and y-glutamil-transpeptidase, were raised in all subjects studied. Daily alcohol intake was from 100 to 300 g for 2-5 years. There were no reductions in platelet, total leucocyte or absolute lymphocyte COUitS.
Cell preparation PBL from ALD patients and 13 control subjects were isolated
by Ficoll-Hypaque (Lymphoprep, Nycomed AS, Oslo, Norway) density gradient centrifugation, resuspended in RPMI1640 supplemented with 10% foetal calf serum, 4 mM, Lglutamine, 100 U/ml penicillin and 100 ,g/ml streptomycin (complete medium; Gibco, Grand Island, NY) and separated into SRBC rosette-enriched (E+) and rosette-depleted (E-) subsets, as described previously.33 The E- cells suspensions were then irradiated at 3000 rads and used as source of AC. E+ cell populations were passed through nylon-wool columns and treated with the OKMI mAb (Ortho, Raritan, NJ) plus rabbit complement (Cedarlane, Ornby, Ontario, Canada) to eliminate residual contaminating monocytes. On the basis of their reactivity with the anti-CD3 mAb (OKT3, Ortho) (> 98'% with
141
immunofluorescence analysis), these mononuclear cells were considered highly purified T-cell subsets. Membrane immunofluorescence studies T cells from both ALD and control PBL samples were identified by OKT3, anti-TI I1 and TI 12 (a generous gift from Dr S. F. Schlossman, Harvard Medical School, Boston, MA) mAb in an indirect immunofluorescence assay. Briefly, mononuclear cells (1 x 106/ml) were suspended in complete medium supplemented with 0-01% sodium azide and incubated with an optimal dilution of each mAb for 30 min in an ice bath. After two washings, the cell pellet was incubated with a fluoresceinconjugated goat anti-mouse IgG (Kallestad, Chaska, MA) for 30 min in an ice bath. After a further three washings, at least 300 cells were counted under a Leitz Dialux microscope, equipped with an epi-illumination mercury arc lamp and phase contrast, to determine the percentage of positive cells. The IL-2R was identified on both resting and stimulated T cells by direct immunofluorescence staining with a fluoresceinated anti-Tac mAb (Becton-Dickinson, Mountain View, CA). Memory and naive T cells were evaluated in both ALD and control samples by two-colour cytofluorimetric analysis (FACScan; BectonDickinson). The first-step mAb used in these experiments were anti-CD3 (OKT3) (Ortho), UCHL1 (Dakopatts, Glostrup, Denmark) and CD45RA (2H4) (Coulter Immunology, Hialeah, FL). The cells were then counterstained with isotype-specific goat anti-mouse IgG conjugated with either fluorescein isothiocyanate or phycoerithrin (Southern Biotechnology Associated, Inc., Birmingham, AL). Isotype-matched mAb which do not react with human leucocytes served as controls. Cell proliferation assay PBL or T cells (5 x 104/well) were cultured in triplicate in complete medium in 96-well round-bottomed microwell plates (Nunc, Roskilde, Denmark). Cell proliferation was triggered by adding PHA (Gibco), the OKT3 mAb, or a combination of the anti-T I 12 and anti-T I 13 mAb (the latter also donated by Dr S. F. Schlossman). Phorbol 12-myristate, 13-acetate (PMA; Sigma, St Louis, MO), human recombinant IL-2 (rIL-2; Janssen, Beerse, Belgium; specific activity 90 x 106 U/mg protein), human recombinant IL-I (rIL-1; Janssen; specific activity 1-3 x 107 U/mg protein) and calcium ionophore A23187 (Ca2+ A23 187; Sigma) were added in some culture experiments. Stock solutions of each mitogen or reagent were diluted in complete medium and used at the indicated concentrations. The plates were incubated in a 5% CO2 humidified atmosphere at 37 for 72 hr. Cell proliferation was assessed by measuring [3H] thymidine ([3H]TdR) incorporation (0 5 pCi; specific activity 25 Ci/mmol; Amersham Int., Amersham, Bucks, U.K.) during the last 6 hr of culture and counting in a liquid scintillation counter. Results are expressed as net counts per minute (c.p.m.) [3H]TdR incorporation and reflect absolute c.p.m. [3H]TdR uptake minus background c.p.m. (c.p.m. incorporated in the absence of mitogen). Statistical anali sis Due to the non-normal distribution of samples, the MannWhitney U-test was applied for statistical evaluation. Values of P < 0-05 were considered as significant.
F. Spinozzi et al.
142
(b)
(a)
100
.00
,ArTIT
VIVTV
0
III
0
10
5000
5
2
110
0
4
0o
10 1
V
IVIIf IT
l03 102
Fluorescence Intensity (loglo) Figure 1. Expression of UCHL1 antigen on CD3+ lymphocytes in controls (a) and ALD patients (b). The cytofluorimetric analysis was done on gated CD3 + cells. In normal T cells, the antigen distribution is characterized by a small percentage of low-positive (naive) and a peak of high-positive cells (memory), whereas in ALD patients there is an evident peak of low UCHL 1-positive 'naive' T lymphocytes.
Table 1. Expression of CD45RA and UCHL1 antigens on T lymphocytes of normal controls and ALD patients
CD3+CD45RA+ (range) CD3 + UCHLlIow (range) CD3+UCHLIhigh (range) *
% controls
% ALD
31 (26-35) 35 (29-38) 17 (15-19)
55 (50-57)* 62 (58-67)* 19 (17-21)
P
