Journal of Hepatology, 1992; 14: 146- 150 6 1992Elsevier Science Publishers B.V. All rights reserved. 0168-8278/92/$05.00
146 HEPAT 00995
Nitric oxide production by monocytes in alcoholic liver disease
N.C.A. Hunt and R.D. Goldin Department of Histopathology, St. Mary’s Hospital Medical School, London, United Kingdom
(Received 7 January 1990)
Nitric oxide, initially described as an endothelial-derived relaxing factor, has recently been recognised as a mediator of macrophage function. We have studied the production of nitric oxide by peripheral blood monocytes from both normal volunteers and alcoholics. This was measured indirectly by assessing nitrite formation. Normal monocytes were found to produce a basal level of nitrite, which could be stimulated more than 6-fold using endotoxin. This effect was abrogated by the addition of nitric oxide synthesis inhibitor, L-n-monomethyl-arginine. A striking difference was observed in the monocytes obtained from alcoholics with and without evidence of alcoholic hepatitis. Whereas the latter behaved in a similar manner to the controls, the former had markedly increased basal levels. In the hepatitis group there was also substantial inhibition of production by t_-n-monomethyl-arginine. We believe that these results indicate that nitric oxide derived from monocytes may play a role in the pathogenesis of alcoholic liver disease, especially alcoholic hepatitis.
Much work has been carried out on the role of various cells, including hepatocytes, neutrophils and lymphocytes, in the pathogenesis of alcoholic liver disease (ALD) (1). There is increasing evidence to suggest that cells of the mononuclear phagocyte family are involved, as effector cells, in this process (2). Of particular interest are the hepatic sinusoidal macrophages (Kupffer cells) and, more recently, peripheral blood monocytes; both, when stimulated, are known to produce a variety of mediators including interleukin-1, and tumour necrosis factor (cachectin) (3). Early reports suggested a decrease in the number of hepatic sinusoidal macrophages, as assessed by measuring the number of lysozyme-positive cells, in patients with increasingly severe ALD (4). Thus, the decrease was greatest in patients with cirrhosis and least in those with steatosis alone. Later work supported this decrease in lysozyme-positive cells. However, using a number of other antibodies it was also shown that certain classes of hepatic sinusoidal macrophages were in fact more abundant and overall there is an increase in num-
bers of hepatic sinusoidal macrophages, even in the earliest stages of ALD (5). It has been suggested that functional impairment of hepatic sinusoidal macrophages may be of greater importance in ALD than the decrease in cell number. In support of this it has been shown that alcohol-exposed macrophages produce decreased amounts of lysozymes (6). Decreased macrophage function in conjunction with portosystemic shunting, in certain groups of ALD patients, is believed to contribute to the endotoxaemia seen in these people (6,7). Endotoxin is itself a known activator of monocytes and macrophages (8). The fact that endotoxin stimulates the production of nitric oxide by macrophages, including hepatic sinusoidal macrophages has been well documented (9). It has been shown that endotoxin can stimulate monocytes to produce increased amounts of tumour necrosis factor and, moreover, the monocytes of alcoholics show an increased basal production of this mediator (10). The object of the present study was to investigate the production of nitric oxide
Correspondence: R.D. Goldin, Department of Histopathology, St. Mary’s Hospital Medical School, London W2, U.K.
MONOCYTJZS, NITRIC OXIDE
AND ALCOHOLICS
147
by monocytes in patients with ALD and to see to what extent they, and normal monocytes, responded to endotoxin.
NMMA: Sigma). All samples were incubated for 24 h at 37 “C, 5% CO,/95% air, before the supematants were removed for subsequent nitrite assay
Materiais and Methods
Nitrite assay Nitric oxide production was measured indirectly using a quantitative, calorimetric assay based on the Griess reaction, a sensitive assay for nitrite ions (11). Nitrite and nitrate ions are the stable, anionic, products formed in a molar ratio of 3:2 from nitric oxide as a result of its decompos8ttion (12). In the present study triplicate 100~,ulaliquots of supernatants were added to an equal volume of freshly prepared Griess reagent. In this system nitrite ions react with 1% sulphanilamide in 5% ortho-phosphoric acid/O. 1% N-l-naphthylethylenediamine dihydrochloride to yield an azo-chromophore, the absorbance of which was measured at 550 nm using a Titretek 96well plate reader. All samples were assayed against a blank comprising RPMI-1640 medium incubated for 24 h in the same plates as the samples but in the absence of cells.
Subjects .4 total of 54 subjects were recruited for the study. The control group consisted of 15 normal volunteers (mean age = 37.7 years, age range = 19-61, eight males and seven females). The alcoholic hepatitic group consisted of 22 patients, all with histories of excessive alcohol intake (mean age = 42.8 years, age range = 18-65,14 males and eight females). All were out-patients with histological diagnosis of alcoholic hepatitis with varying degrees of fibrosis. In addition, a third group of 17 alcohol abusers with non-hepatic liver disease were studied (mean age = 39.0 years, age range = 23-57, ten males and seven females). These patients ail had liver biopsies showing fatty change with varying degrees tif fibrosis but no evidence of alcoholic hepatitis. In each case serological markers of viral hepatitis, including hepatitis C virus, were negative. All biopsies were reviewed prior to inclusion in the study in order to confirm the histological diagnosis (RDG.) Monocyte extraction and culture Monocytes were extracted from peripheral blood on a density gradient materiai (IEstopaque, Sigma). This and all subsequent procedures required specific precautions in order to minimise contamination by LPS. Such measures included baking of all laboratory glassware, use of pyrogen-free disposables and endotoxin-free culture media. Briefly, peripheral blood mononuclear cells were removed from the density gradient, washed twice in endotoxin-free Hanks’ Balanced Salts Solution, and finally resuspended in RPMI-1640 (RPM]-1640, endotoxin-free. Phenol red-free; Sigma) supplemented with 10% decomplemented fetal calf serum (low endotoxin; Flow, U.K.) and 2 mmol t_-glutamine (Sigma). Cells were then plated out at a concentration of 2-4-l@ per ml in individual 16mm wells of tissue cuiture plates (24 well; Nunclon). A Trypan blue exclusion analysis was performed at this stage and showed a mean viability of 95% with a range of 92-97%. Monocyte enrichment was achieved by allowing the cells to adhere to the plates for 2 h at 37 “C, after which time the medium was removed by aspiration after vigorous washing to remove any non-adherent cells. For the final incubation cells were cultured in either RPMI-1640 (supplemented as above) alone, medium with the addition of 1 &ml endotoxin (Escherichia co/i lipopolysaccharide, L4516; Sigma), or medium with 1 ,@ml endotoxin plus 300 ,uM t-n-monomethyl-arginine (L-
Protein assay Briefly, PBMCs were removed from the density gradient, washed twice in endotoxin-free Hanks’ Balanced Salts Solution, and finally resuspended in RMPI-1640 medium (RMPI-1640, low endotoxin, Phenol red-free; Sigma Chemicals) supplemented with 10% decomplemented fetal bovine serttm (low endotoxin; Flow, U.K.). Cells iyere then plated out at a concentration of plates (Nunclan, 24 well). A Trypan blue exclusion analysis was performed at this stage and showed a mean viability of 95% with a range of 92-97%. Monocyte enrichment was achieved by allowing the cells to adhere to the plates for 2 h at 37 “C, after which time the medium was removed by aspiration after vigorous washing to displace any non-adherent cells. For the final incubation cells were cultured in either RPM&1640 alone, RPMI-1640 with the addition of 1 ,ug/ml endotoxin (addition of 1 pg/ml endotoxin, E. coli, L4516; Sigma) or RPMI-1640 with the addition of endotoxin (as before), plus 3OOpM L-NMMA (Sigma). All samples were incubated for 24 h at 37 “C, 5% C02/95% air before the supematants were removed for subsequent nitrite (NO,-) assay. The concentration of endotoxin used, 1 @ml, stimulates maximal nitrite production. Concentrations of endotoxin in the range O.l-lOO&ml were also studied, but produced no increase in nitrite production (results not shown). Statktical analysis To assess the significance of the differences means a modified Student’s t-test was used.
between
N.C.A. HUNT AND R.D. GOLDIN
148 Results Normal monocytes The monocytes from these 15 volunteers showed a basal nitrite production with a mean value of 7.5 pmol&g protein and a range of 0.0-11 .Opmo&g (Table 1). The addition of endotoxin led to a marked stimulation of nitrite production by the cells (Table l), with a mean’of 55.5 pmol&g protein and a range of 25.7-96.8 pmollpg. T-test analysis showed the difference between these means to be highly significant p 0.5). (b) The monocyte samples isolated from 22 patients with alcoholic hepatitis showed a mean basal nitrite production of 35.3 pmol/pg protein with a range of 18.1-121.3 pmol&g (Table 1). The difference between the mean in this case and that of the normal basal production was compared using t-test analysis and found to be highly significant @
146 HEPAT 00995
Nitric oxide production by monocytes in alcoholic liver disease
N.C.A. Hunt and R.D. Goldin Department of Histopathology, St. Mary’s Hospital Medical School, London, United Kingdom
(Received 7 January 1990)
Nitric oxide, initially described as an endothelial-derived relaxing factor, has recently been recognised as a mediator of macrophage function. We have studied the production of nitric oxide by peripheral blood monocytes from both normal volunteers and alcoholics. This was measured indirectly by assessing nitrite formation. Normal monocytes were found to produce a basal level of nitrite, which could be stimulated more than 6-fold using endotoxin. This effect was abrogated by the addition of nitric oxide synthesis inhibitor, L-n-monomethyl-arginine. A striking difference was observed in the monocytes obtained from alcoholics with and without evidence of alcoholic hepatitis. Whereas the latter behaved in a similar manner to the controls, the former had markedly increased basal levels. In the hepatitis group there was also substantial inhibition of production by t_-n-monomethyl-arginine. We believe that these results indicate that nitric oxide derived from monocytes may play a role in the pathogenesis of alcoholic liver disease, especially alcoholic hepatitis.
Much work has been carried out on the role of various cells, including hepatocytes, neutrophils and lymphocytes, in the pathogenesis of alcoholic liver disease (ALD) (1). There is increasing evidence to suggest that cells of the mononuclear phagocyte family are involved, as effector cells, in this process (2). Of particular interest are the hepatic sinusoidal macrophages (Kupffer cells) and, more recently, peripheral blood monocytes; both, when stimulated, are known to produce a variety of mediators including interleukin-1, and tumour necrosis factor (cachectin) (3). Early reports suggested a decrease in the number of hepatic sinusoidal macrophages, as assessed by measuring the number of lysozyme-positive cells, in patients with increasingly severe ALD (4). Thus, the decrease was greatest in patients with cirrhosis and least in those with steatosis alone. Later work supported this decrease in lysozyme-positive cells. However, using a number of other antibodies it was also shown that certain classes of hepatic sinusoidal macrophages were in fact more abundant and overall there is an increase in num-
bers of hepatic sinusoidal macrophages, even in the earliest stages of ALD (5). It has been suggested that functional impairment of hepatic sinusoidal macrophages may be of greater importance in ALD than the decrease in cell number. In support of this it has been shown that alcohol-exposed macrophages produce decreased amounts of lysozymes (6). Decreased macrophage function in conjunction with portosystemic shunting, in certain groups of ALD patients, is believed to contribute to the endotoxaemia seen in these people (6,7). Endotoxin is itself a known activator of monocytes and macrophages (8). The fact that endotoxin stimulates the production of nitric oxide by macrophages, including hepatic sinusoidal macrophages has been well documented (9). It has been shown that endotoxin can stimulate monocytes to produce increased amounts of tumour necrosis factor and, moreover, the monocytes of alcoholics show an increased basal production of this mediator (10). The object of the present study was to investigate the production of nitric oxide
Correspondence: R.D. Goldin, Department of Histopathology, St. Mary’s Hospital Medical School, London W2, U.K.
MONOCYTJZS, NITRIC OXIDE
AND ALCOHOLICS
147
by monocytes in patients with ALD and to see to what extent they, and normal monocytes, responded to endotoxin.
NMMA: Sigma). All samples were incubated for 24 h at 37 “C, 5% CO,/95% air, before the supematants were removed for subsequent nitrite assay
Materiais and Methods
Nitrite assay Nitric oxide production was measured indirectly using a quantitative, calorimetric assay based on the Griess reaction, a sensitive assay for nitrite ions (11). Nitrite and nitrate ions are the stable, anionic, products formed in a molar ratio of 3:2 from nitric oxide as a result of its decompos8ttion (12). In the present study triplicate 100~,ulaliquots of supernatants were added to an equal volume of freshly prepared Griess reagent. In this system nitrite ions react with 1% sulphanilamide in 5% ortho-phosphoric acid/O. 1% N-l-naphthylethylenediamine dihydrochloride to yield an azo-chromophore, the absorbance of which was measured at 550 nm using a Titretek 96well plate reader. All samples were assayed against a blank comprising RPMI-1640 medium incubated for 24 h in the same plates as the samples but in the absence of cells.
Subjects .4 total of 54 subjects were recruited for the study. The control group consisted of 15 normal volunteers (mean age = 37.7 years, age range = 19-61, eight males and seven females). The alcoholic hepatitic group consisted of 22 patients, all with histories of excessive alcohol intake (mean age = 42.8 years, age range = 18-65,14 males and eight females). All were out-patients with histological diagnosis of alcoholic hepatitis with varying degrees of fibrosis. In addition, a third group of 17 alcohol abusers with non-hepatic liver disease were studied (mean age = 39.0 years, age range = 23-57, ten males and seven females). These patients ail had liver biopsies showing fatty change with varying degrees tif fibrosis but no evidence of alcoholic hepatitis. In each case serological markers of viral hepatitis, including hepatitis C virus, were negative. All biopsies were reviewed prior to inclusion in the study in order to confirm the histological diagnosis (RDG.) Monocyte extraction and culture Monocytes were extracted from peripheral blood on a density gradient materiai (IEstopaque, Sigma). This and all subsequent procedures required specific precautions in order to minimise contamination by LPS. Such measures included baking of all laboratory glassware, use of pyrogen-free disposables and endotoxin-free culture media. Briefly, peripheral blood mononuclear cells were removed from the density gradient, washed twice in endotoxin-free Hanks’ Balanced Salts Solution, and finally resuspended in RPMI-1640 (RPM]-1640, endotoxin-free. Phenol red-free; Sigma) supplemented with 10% decomplemented fetal calf serum (low endotoxin; Flow, U.K.) and 2 mmol t_-glutamine (Sigma). Cells were then plated out at a concentration of 2-4-l@ per ml in individual 16mm wells of tissue cuiture plates (24 well; Nunclon). A Trypan blue exclusion analysis was performed at this stage and showed a mean viability of 95% with a range of 92-97%. Monocyte enrichment was achieved by allowing the cells to adhere to the plates for 2 h at 37 “C, after which time the medium was removed by aspiration after vigorous washing to remove any non-adherent cells. For the final incubation cells were cultured in either RPMI-1640 (supplemented as above) alone, medium with the addition of 1 &ml endotoxin (Escherichia co/i lipopolysaccharide, L4516; Sigma), or medium with 1 ,@ml endotoxin plus 300 ,uM t-n-monomethyl-arginine (L-
Protein assay Briefly, PBMCs were removed from the density gradient, washed twice in endotoxin-free Hanks’ Balanced Salts Solution, and finally resuspended in RMPI-1640 medium (RMPI-1640, low endotoxin, Phenol red-free; Sigma Chemicals) supplemented with 10% decomplemented fetal bovine serttm (low endotoxin; Flow, U.K.). Cells iyere then plated out at a concentration of plates (Nunclan, 24 well). A Trypan blue exclusion analysis was performed at this stage and showed a mean viability of 95% with a range of 92-97%. Monocyte enrichment was achieved by allowing the cells to adhere to the plates for 2 h at 37 “C, after which time the medium was removed by aspiration after vigorous washing to displace any non-adherent cells. For the final incubation cells were cultured in either RPM&1640 alone, RPMI-1640 with the addition of 1 ,ug/ml endotoxin (addition of 1 pg/ml endotoxin, E. coli, L4516; Sigma) or RPMI-1640 with the addition of endotoxin (as before), plus 3OOpM L-NMMA (Sigma). All samples were incubated for 24 h at 37 “C, 5% C02/95% air before the supematants were removed for subsequent nitrite (NO,-) assay. The concentration of endotoxin used, 1 @ml, stimulates maximal nitrite production. Concentrations of endotoxin in the range O.l-lOO&ml were also studied, but produced no increase in nitrite production (results not shown). Statktical analysis To assess the significance of the differences means a modified Student’s t-test was used.
between
N.C.A. HUNT AND R.D. GOLDIN
148 Results Normal monocytes The monocytes from these 15 volunteers showed a basal nitrite production with a mean value of 7.5 pmol&g protein and a range of 0.0-11 .Opmo&g (Table 1). The addition of endotoxin led to a marked stimulation of nitrite production by the cells (Table l), with a mean’of 55.5 pmol&g protein and a range of 25.7-96.8 pmollpg. T-test analysis showed the difference between these means to be highly significant p 0.5). (b) The monocyte samples isolated from 22 patients with alcoholic hepatitis showed a mean basal nitrite production of 35.3 pmol/pg protein with a range of 18.1-121.3 pmol&g (Table 1). The difference between the mean in this case and that of the normal basal production was compared using t-test analysis and found to be highly significant @
