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Changes in cytokine levels during admission and mortality in acute alcoholic hepatitis
Alcohol 46 (2012) 433e440
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Changes in cytokine levels during admission and mortality in acute alcoholic hepatitis E. González-Reimers a, *, M.J. Sánchez-Pérez a, F. Santolaria-Fernández a, P. Abreu-González b, M.J. De la Vega-Prieto c, J. Viña-Rodríguez a, M.R. Alemán-Valls a, M. Rodríguez-Gaspar a a b c
Servicio de Medicina Interna, Hospital Universitario de Canarias, Tenerife, Canary Islands, Spain Dpto. De Fisiología, Facultad de Medicina, Universidad de La Laguna, Tenerife, Canary Islands, Spain Servicio de Laboratorio, Hospital Universitario de Canarias, Tenerife, Canary Islands, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 January 2011 Received in revised form 21 September 2011 Accepted 3 October 2011
Cytokine levels are raised in acute alcoholic hepatitis. However, there are disparate results regarding the duration of altered plasma levels, and there are also discrepancies about the relation of changes during the first 15 days after admission with short-term (in-hospital) or long-term mortality. In 56 patients with acute alcoholic hepatitis we found that IL-8, IL-4, Interferon-g (IFN-g), malondialdehyde and C-reactive protein remained higher in patients than in 18 age- and sex-matched controls at admission, at the 7th day and at the 15th day after admission. Moreover, IL-4 levels (and to a lesser extent, IL-10 and IFN-g ones) increased along the three determinations. However, comparing patients who died during the admission with those who did not, there were no statistically significant differences, but there was a nearly significant trend for MDA (Z ¼ 1.89; p ¼ 0.059), with higher levels among those who died. When changes between the first and the second determinations were compared with long-term survival, only IL-8 and IFN-g showed a relation with mortality. IFN-g values increased among those who survived and decreased among those who died (p ¼ 0.048). IFN-g values at the first determination also showed a relation with long-term mortality, especially when patients with IFN-g values in the first quartile were compared with those of the 4th one (log rank ¼ 5.64; p ¼ 0.018; Breslow ¼ 4.64; p ¼ 0.031). Besides Interferon-g, only C-reactive protein showed differences between the first and the 4th quartile regarding mortality (Log rank ¼ 4.50; p ¼ 0.034; Breslow 4.33; p ¼ 0.038). In contrast with other studies, no relation was found between TNF-a or IL-6 and mortality. Ó 2012 Elsevier Inc. All rights reserved.
Keywords: Alcoholic hepatitis Cytokines Lipid peroxidation Acute phase reaction Short term survival Cytokine sequential
Introduction Since about 20 years ago it is known that cytokines play outstanding roles in the development and progression of chronic alcoholic liver disease (Bird, 1994; Bird, Sheron, Goka, Alexander, & Williams, 1990; Fujimoto et al., 2000; Hill et al., 1992, Hill, Marsano, & McClain, 1993; Huang et al., 1996; Khoruts, Stahnke, McClain, Logan, & Allen, 1991; Sheron et al., 1993). The main mechanisms seem to be related to the activation of Kupffer cells by lipopolisaccaride (LPS), derived from intestinal gram negative bacteria. Theoretically, this feature could be dependent on intestinal bacterial overgrowth, increased intestinal permeability, or to failure of Kupffer cells to neutralize LPS; the bulk of studies have shown that increased intestinal permeability plays an outstanding, primary role (Rao, Seth, & Sheth, 2004). After binding to toll-like receptors * Corresponding author. Tel.: þ34 922 67 80 00; fax: þ34 922 31 93 79. E-mail address: [email protected] (E. González-Reimers). 0741-8329/$ e see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.alcohol.2011.10.001
(especially TLR-4), cytokines are released by Kupffer cells, and initiate the cascade of events which ultimately leads to the development of hepatocyte necrosis, recruitment of neutrophils and immune cells, and liver fibrosis. During this process, increased freeradical (ROS) production aggravates the scenario (Kono et al., 2000; Loguercio & Federico, 2003; Poli, 2000), since they yield both harmful and immunogenic effects. Later in the evolution of alcoholic hepatitis, an increase in TH-2 derived cytokines, such as IL-4 and IL13, has been reported, and is presumably related to liver fibrogenesis (Crews et al., 2006). The duration of the effect of released cytokines is uncertain, but some authors have reported prolonged increase of TNF-a levels in some tissues, such as brain (Qin et al., 2008). Within 6 days after alcohol abstinence levels of IL-6 and IL-10 show a marked decrease (González-Quintela et al., 2000). In the recovery phase of severe alcoholic hepatitis, IL-6 and IL-8 tend to decrease (Fukui, 2005) in survivors, but not in those who subsequently died. Indeed, ethanol is known to alter cytokine levels in a variety of tissues including
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liver, lung, and brain, which may contribute to long-term organ dysfunction (Crews et al., 2006), and, probably, to altered immune response and susceptibility to infection, which may persist for as long as three years after abstinence (Eggers et al., 2006). In a previous report we showed that levels of some cytokines, such as IL-6, still remained high in already stable alcoholics (GonzálezReimers et al., 2007). Therefore, it seems that several cell types are involved in cytokine production. Some cytokines directly derive from activated macrophages, others from activated T-cells; some of them are proinflammatory, but others may exert protective functions. In addition, as commented, there is controversy regarding the evolution of cytokine levels in alcoholics. Based on these facts, in the present study we analyse the sequential behaviour of proinflammatory, anti-inflammatory, Th-1 and Th-2 cytokines among patients affected by alcoholic hepatitis, and their relation with inhospital and long-term mortality. Methods Patients and controls Fifty-six heavy alcoholics (6 women), consecutively admitted to the hospitalization unit of the Internal Medicine Service, entered the study. Ethanol intake was assessed by recall and calculated as follows: grams of ethanol ¼ volume of beverage (cm3) strength (v/v, as %) 0.8. All the patients were drinkers of more than 100 g ethanol/day (193 93 g/day; median ¼ 150 g; (interquartile range) ¼ 120e250 g) during more than 10 years (27.8 10.5 years; 30 (20e30.25). Mean age was 47.09 9.86 years; 45 (40.3e52.75) years. Cytokines and biochemical parameters outlined below were determined at admission, one week later, and at the day 15th among those who were not already discharged (or had died before). Written consent was obtained by each of the patients, and the study protocol was approved by the local ethical committee of our Hospital and conforms to the ethical guidelines of the 1975 Declaration of Helsinki. All the patients were admitted with acute alcoholic hepatitis, but the following conditions were also present at admission: pancreatitis, 5 cases (one also with urinary tract infection); pneumonia, 11 cases (one also with urinary tract infection), one with cutaneous infection; another, with acute cholecystitis; 2 more with urinary tract infection, and 3, with spontaneous bacterial peritonitis. Owing to the well described alteration of cytokines in inflammatory conditions, we grouped our patients in two groups: those with only alcoholic hepatitis (32 cases), and those with alcoholic hepatitis and another inflammatory process (24 cases). Thirty one patients developed a florid ethanol withdrawal syndrome. Twelve patients were placed on corticosteroids at admission (prednisone, 40 mg/ day) due to a high Maddrey’s index (Maddrey et al., 1978). Treatment also included fluidotherapy and thiamine, and broad spectrum antibiotics in patients with infections (usually third generation cephalosporins, with or without levofloxacin) and benzodiacepine in the cases of withdrawal syndrome. However, treatment always began when blood was extracted for the basal biochemical assessment, so treatment did not influence on the first determination. The control group was composed of 18 healthy sanitary workers (2 women), drinkers of less than 10 g ethanol day, aged 45.33 6.80 years. Fifty-five patients were followed up for a median period of 85 months (interquartile range, 32e101 months). During this period, 24 died, 9 of them during the admission episode or shortly after (within 2 months after admission). The relationship between interleukins and malondialdehyde (MDA), all of them classified in quartiles and tertiles and survival was analysed by means of
KaplaneMeyer curves and log-rank test. A Cox regression analysis was also performed including age, interleukins, MDA, classic parameters of liver function, including Maddrey’s index, and nutritional parameters such as body mass index and a previously validated subjective nutritional assessment, which consisted in physical examination of the muscle masses of the upper and lower limbs and of the temporal muscle, defining two degrees of atrophy (severe, moderate) and absence of atrophy, and fat loss on the cheek and abdomen (Bichat’s fat and subcutaneous fat atrophy) classified in a similar way. Each patient was assigned a score (SNS), based on the sum of the assigned points, for which the poorest value was 10 and 0 the best one (Hernández-Plasencia et al., 1991). Cytokines and biochemical parameters Blood samples were taken at 8.00 am in fasting conditions, immediately frozen at 80. Blood sample were extracted A) Shortly after admission (maximum interval, 48 h); B) In survivors, one week later; C) by the 15th day after admission if the patient was still hospitalized. In addition to routine laboratory tests and C-reactive protein determination, the following parameters were measured (Table 1). In a few cases, some of these parameters were not recorded (loss of sample, not enough blood extracted, etc.). Tumour necrosis factor (TNF-a) by immunometric chemiluminiscent assay (intra-assay variation coefficient ranging 4e6.5%, inter-assay variation coefficient ranging 2.6e3.6%, recovery 92e112%, Diagnostic Products Corporation (DPC), Los Angeles, CA, USA); IL-6, by chemiluminiscent assay (inter-assay variation coefficient ranging 5.3e7.5%, recovery ¼ 85e104%, DPC, Los Angeles, CA, USA); IL-4, by enzyme-linked immunosorbent assay (ELISA); (interand intra-assay coefficient of variation <10%; sensitivity <2 pg/ml; recovery ¼ 101%, Bender MedSystems Diagnostics GmbH, Vienna, Austria); IL-8, by chemiluminiscent assay (inter-assay variation coefficient ranging 5.3e7.5%, DPC, Los Angeles, CA, USA); IL-10, by enzyme immunometric assay (sensitivity ¼ 3 pg/ml, inter- and intra-assay coefficient of variation ranging from 3.9 to 7.3%; recovery ranging from 86 to 94%; DPC, Los Angeles, CA, USA); interferon gamma (IFN-g), by ELISA (inter- and intra-assay coefficient of variation 0.3e10.7%; sensitivity <1.5 pg/ml; recovery ¼ 90e112%, Bender MedSystems Diagnostics GmbH, Vienna, Austria). In addition, patients underwent routine laboratory evaluation. Lipid peroxidation (LPX) products Serum MDA levels, referred to as thiobarbituric acid-reactive substance (TBARS), were measured according to the method described by Kikugawa, Kojima, Yamaki, and Kosugi (1992). A volume sample of 0.2 ml of plasma was added to 0.2 ml of H3PO4 (0.2 M) and the colour reaction was initiated by the addition of 25 ml of 0.11 M thiobarbituric acid (TBA) solution. Samples were placed in a 90 C heating block for 45 min. After the samples were cooled, the TBARS (pink complex colour) were extracted with 0.4 ml of n-butanol. Butanol phase was separated by centrifugation at 6000 g for 10 min. Aliquots of the n-butanol phase were placed in a 96 well plate and read at 535 nm in a microlate spectrophotometer reader (Benchmark Plus, Bio-Rad, Hercules, CA, USA). The calibration curve was prepared with authentic MDA standards ranging from 0 to 20 mM. The intra-and inter-assay variation coefficients were 1.82 and 4.01, respectively. Statistics The KolmogoroveSmirnov test was used to test normality, a condition not fulfilled by most of the cytokines analysed. Therefore, non-parametric tests, such as ManneWhitney’s U test and
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Table 1 Cytokines, MDA and C-reactive protein levels values in patients and controls at admission, the 7th day (2nd) and the 15th day (3rd). Results are given as mean standard deviation and in the second subfile, median (interquartile range). Number of patients included are given in brackets.
C-reactive protein IL-8 (pg/ml) IL-10 (pmol/l) IL-4 (ng/ml) IFN-g (pg/ml) TNF-alfa (pg/ml) IL-6 (pg/ml) Malon-dialdehyde IFN-g/IL-4
Patients (1st) (56)
Patients (2nd) (48)
Patients (3rd) (24)
Controls (18)
1st/ctrl
2nd/ctrl
3rd/ctrl
3.68 6.09 0.87 (0.47e4.81) 57.80 97.05 20.65 (9.7e59.8) 5.13 0.59 5 (5e5) 6.88 10.20 3.91 (2.4e6.2) 4.74 12.60 0.87 (0.46e1.82) 7.22 7.21 6.8 (4.0e9.3) 39.82 81.85 9.4 (0.0e38.0) 8.34 6.76 6.55 (4.1e9.8) 1.02 2.60 0.22 (0.16e0.49)
1.82 0.73 0.73 (0.26e3.02) 31.59 47.04 11.8 (7e29.6) 5.0.7 0.42 5.0 (5.0e5.0) 4.14 2.74 4.14 (1.8e6.4) 4.13 11.40 1.00 (0.49e1.52) 6.40 7.00 7.0 (0e8.50) 17.50 23.92 8.3 (0e22.1) 6.81 5.77 5.34 (4.3e7.62) 1.57 5.35 0.21 (0.12e0.62)
1.34 1.87 0.67 (0.36e1.75) 26.60 35.15 13.8 (5.58e33.05) 5.90 4.10 5.0 (5.0e5.0) 27.49 42.90 7.81 (3.58e36.5) 2.94 3.68 1.93 (1.09e2.84) 6.97 3.84 7.3 (4.9e9.5) 11.87 21.38 4.0 (0.0e12.3) 7.16 5.38 4.99 (3.83e9.15) 0.49 0.95 0.16 (0.05e0.34)
0.36 1.11 0.02 (0.01e0.1) 6.75 1.79 6.95 (5.0e7.75) 8.22 10.69 6.0 (5.0e6.0) 2.28 1.72 2.32 (.9e3.2) 0.66 0.50 0.5 (0.3e0.87) 5.83 1.77 5.1 (4.4e8.0) 5.77 1.40 5 (5.0e6.35) 1.31 0.69 1.1 (0.8e1.96) 0.60 0.79 0.28 (0.08e0.79)
Z ¼ 4.93 p < 0.001 Z ¼ 4.66 p < 0.001 Z ¼ 5.01 p < 0.001 Z ¼ 2.54 p ¼ 0.011 Z ¼ 2.58 p ¼ 0.010 Z ¼ 1.52 NS Z ¼ 1.63 NS Z ¼ 6.50 p < 0.001 Z ¼ 0.16 NS
Z ¼ 4.48 p < 0.001 Z ¼ 3.69 p < 0.001 Z ¼ 5.41 p < 0.001 Z ¼ 2.19 p ¼ 0.027 Z ¼ 2.00 p ¼ 0.045 Z ¼ 0.84 NS Z ¼ 1.96 p ¼ 0.05 Z ¼ 6. 23 p < 0.001 Z ¼ 0.04 NS
Z ¼ 4.05 p < 0.001 Z ¼ 2.82 p ¼ 0.004 Z ¼ 4.09 p < 0.001 Z ¼ 3.85 p < 0.001 Z ¼ 3.84 p < 0.001 Z ¼ 1.75 NS Z ¼ 1.18 NS Z ¼ 5.57 p < 0.001 Z ¼ 0.75 NS
KruskalleWallis were used to analyse between group differences in these parameters. Also, non-parametric tests such as Friedman’s test and Wilcoxon’s test were used to compare variations of cytokines values along the three determinations. Spearman’s correlation analysis was used to compare quantitative parameters. Relationships between cytokines and MDA (classified in tertiles or quartiles) with survival were analysed using log-rank test and KaplaneMeier curves. Multivariate Cox regression analysis was used to discern which parameters yield prognostic value.
Results Cytokine values in patients and controls are shown in Table 1. Differences are clearly evident for all the cytokines except for IL-6 and TNF-a, which were non-significantly higher among patients. When patients were grouped in those affected by an infectious or inflammatory condition besides acute alcoholic hepatitis, these two cytokines were significantly higher (Z ¼ 2.28, p ¼ 0.023 and Z ¼ 2.21, p ¼ 0.027, respectively) among those with an inflammatory process at admission than those without it. No differences were observed among those placed or not on corticosteroids, except for IL-6, slightly higher among the former (Z ¼ 2.08; p ¼ 0.037). As shown, cytokines (besides IL-10) still remained high at the second determination (Table 1). No differences were observed among patients treated or not with steroids, except for slightly higher values of IL-8 among those who received prednisone (Z ¼ 1.97; p ¼ 0.048). Regarding the third determination, no differences were observed among those treated or not with steroids, but, indeed, between patients and controls. In general, cytokine values were lower in the second and third determinations than in the first one, with the notable exceptions of IL-4 and, to a lesser extent, IL-10 and INF-g. Analysed by means of Friedman test, differences along the study were significant for IL-8 (c2 ¼ 13.76; p < 0.001), INF-g (c2 ¼ 7.55, p ¼ 0.023) and CRP (c2 ¼ 6.08, p ¼ 0.048). However, using Wilcoxon test to compare changes between point 1 and point 3, differences were also significant for IL-4 (Z ¼ 2.44, p ¼ 0.015) (which levels rose), and nearly significant, for MDA (Z ¼ 1.89, p ¼ 0.059), which levels decreased. We calculated the ratio INF-g/IL-4 (Th-1 and Th-2 main cytokines, respectively). As shown in Table 1, no differences were observed in this ratio between patients and controls. There was a non-significant trend to lower values in the third determination compared with the first and second ones.
As shown in Table 2, significant correlations were observed between TNF-a and IL-6, IL-8, IFN-g, IL-10, and C-reactive protein. C-reactive protein was also directly related with IL-6 and IL-8, and also, a direct correlation was observed between IL-4 and IFN-g. When data obtained at the 7th day were compared to each other, IL-6 still showed a significant correlation with CRP (r ¼ 0.58, p < 0.001, Fig. 1), TNF-a (r ¼ 0.30, p ¼ 0.047) and IL-8 (r ¼ 0.35, p ¼ 0.027)) A significant correlation was also observed between IL-8 and CRP (r ¼ 0.43; p ¼ 0.006). At the third determination, the only significant correlation still observed was between MDA and CRP (r ¼ 0.43; p ¼ 0.036). Changes in CRP values between the first and the second determination showed a significant correlation with changes in IL-6 (r ¼ 0.52, p ¼ 0.001), whereas changes in IL-8 showed an inverse correlation with changes in IFN-g (r ¼ 0.46; p ¼ 0.003). Changes in TNF-a values between the first and the third determinations showed a significant relationship with changes in IL-6 (r ¼ 0.69, p ¼ 0.007) and CRP (r ¼ 0.48, p ¼ 0.021) and an inverse one with changes in IL-10 (r ¼ 0.57, p ¼ 0.035); in addition, a close relationship was observed between changes in IL-6 and in CRP (r ¼ 0.81, p < 0.001). As shown in Figs. 2 and 3, evolution of cytokine values among patients with or without infectious and/or inflammatory conditions other than alcoholic hepatitis was similar, with the notable exception of IL-6, which showed a trend (p ¼ 0.10) to increase among non-infected patients, and to decrease among infected ones. Also, IL-4 levels showed a non-significant trend to increase among non-infected patients. However, these different behaviour was not statistically significant (F ¼ 3.99; p ¼ 0.086). Relationship with liver function Il-6 and IL-8 kept a close relationship with liver function, assessed all by Maddrey index (r ¼ 0.29; p ¼ 0.047 and r ¼ 0.40, p ¼ 0.004), Pugh’s score (among cirrhotics; only IL-6, r ¼ 0.59, p < 0.001), albumin, bilirubin, and prothrombin activity (data not shown). MDA was also significantly correlated with Maddrey index (r ¼ 0.32, p ¼ 0.021). Pugh’s score (r ¼ 0.43, p ¼ 0.044), and bilirubin (r ¼ 0.47, p < 0.001). Changes in Maddrey index between the first and the second determination were significantly related to changes in MDA (r ¼ 0.30, p ¼ 0.05), as well as changes between MDA at the 1st and 15th day and changes in Maddrey index during the same period (r ¼ 0.58, p ¼ 0.009).
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Table 2 Correlations among cytokines and MDA at admission. IL-6 IL-6
IL-8
TNF-a
IFN-g
IL-10
IL-4
MDA
CRP
b
IL-8
1.000 47 .222 .153 43 .431a .003 45 .031 .844 43 .002 .989 34 .244 .114 43 .077 .617 44 .504a .000 46
.222 .153 43 1.000 48 .368b .013 45 .143 .354 44 .164 .361 33 .047 .761 44 .190 .211 45 .306b .041 45
TNF-a
IFN-g
IL-10
IL-4
MDA
CRP
.431a .003 45 .368b .013 45 1.000
.031 .844 43 .143 .354 44 .322b .033 44 1.000
.002 .989 34 .164 .361 33 .338b .041 37 .109 .534 35 1.000
.244 .114 43 .047 .761 44 .186 .226 44 .426a .003 46 .232 .180 35 1.000
.077 .617 44 .190 .211 45 .061 .677 49 .076 .629 43 .003 .986 37 .033 .835 43 1.000
.504a .000 46 .306b .041 45 .329b .020 50 .045 .772 44 .036 .840 35 .080 .605 44 .109 .462 48 1.000
52 .322b .033 44 .338b .041 37 .186 .226 44 .061 .677 49 .329b .020 50
46 .109 .534 35 .426a .003 46 .076 .629 43 .045 .772 44
38 .232 .180 35 .003 .986 37 .036 .840 35
46 .033 .835 43 .080 .605 44
52 .109 .462 48
51
Correlation is significant at the 0.01 level (2-tailed). Correlation is significant at the 0.05 level (2-tailed).
Relation with mortality Of the parameters recorded at admission, only IFN-g showed a relation with mortality. When classified in tertiles, there was a nearly significant trend to a lower mortality among those with higher values (tertiles: log rank ¼ 5.86; p ¼ 0.053; Breslow 5.0, p ¼ 0.082). Differences were more marked when patients belonging to the first quartile were compared with those of the 4th quartile (Fig. 4; log rank ¼ 5.64; p ¼ 0.018; Breslow ¼ 4.64; p ¼ 0.031). Besides IFN-g, only C-reactive protein showed differences between the first and the 4th quartile regarding mortality (Log rank ¼ 4.50; p ¼ 0.034; Breslow 4.33; p ¼ 0.038); Fig. 5. Comparing patients who died during the admission with those who did not, there were no statistically significant differences, although there was a nearly significant trend for MDA (Z ¼ 1.89; p ¼ 0.059), with higher levels among those who died. When changes between the first and the second determinations were compared with long-term survival, only IL-8 and IFN-g showed a relation with mortality. IFN-g values increased among those who survived and decreased among those who died (p ¼ 0.048, Fig. 6). However, in a survival analysis using Cox regression model, introducing the variables age, prothrombin activity, bilirubin, albumin and Maddrey index in addition to cytokines, only Maddrey index (p ¼ 0.02) and prothrombin activity (p ¼ 0.013) were related to mortality during admission or during the whole follow-up period, respectively. SNS score also displaced cytokines when nutritional parameters were also included in the Cox regression analysis. Discussion
2nd and 3rd determinations. IL-10 is an anti-inflammatory cytokine able to limit the inflammatory response and hepatotoxicity in chronic liver disease (Louis et al., 1998). It is upregulated in patients with alcoholic liver disease, but, in severe cases, it is insufficient to modulate TNF-a mediated (through TNFrp55) liver toxicity. Our
Re lationship be tween IL-6 and C-reactive protein at the 7th day
100,00
75,00
IL-6 (pg/ml)
a
Correlation Coefficient Sig. (2-tailed) N Correlation Coefficient Sig. (2-tailed) N Correlation Coefficient Sig. (2-tailed) N Correlation Coefficient Sig. (2-tailed) N Correlation Coefficient Sig. (2-tailed) N Correlation Coefficient Sig. (2-tailed) N Correlation Coefficient Sig. (2-tailed) N Correlation Coefficient Sig. (2-tailed) N
50,00
25,00
0,00
0,00
2,50
5,00
7,50
10,00
C-reactive protein(ng/ml) In this study we have shown that MDA and all the cytokines besides TNF-a and IL-6 were different among patients and controls. In general, cytokine values were higher among patients, with the notable exception of IL-10, which was initially lower when the patients were admitted to the hospital, and remained lower at the
rho =0.58; p<0.001 Fig. 1. Relationship between IL-6 and C-reactive protein at the 7th day after admission.
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437
Fig. 2. Evolution of TNF-a, IFN-g, IL-4 and MDA among patients with or without infectious/inflammatory conditions.
results do not agree with those of Naveau et al. (2005), who found higher levels of IL-10 among patients with alcoholic hepatitis, but not sufficient enough to counteract the effect of proinflammatory cytokines. The behaviour of IL-6 may be viewed in a similar fashion. IL-6 has a dual effect, acting as proinflammatory, as well as antiinflammatory cytokine. It is assumed that IL-6 plays a role in liver regeneration (Drucker, Gewiese, Malchow, Scheller, & Rose-John, 2010). Other researchers (El-Assal, Hong, Kim, Radaeva, & Gao, 2004) have shown that IL-6 protects mice from ethanol-induced liver injury. In addition, recent research has shown that IL-6 plays an important role in mitochondrial DNA repair (Zhang et al., 2010). Mitochondrial DNA becomes damaged by ethanol-induced production of reactive oxygen species (Bansal et al., 2010). In any case, it is well known that IL-6 also exerts proinflammatory effects, inducing liver synthesis of several acute phase reactants. In our study it is clear that IL-6 and CRP are closely related at the different points at which blood was extracted, and also that changes in CRP parallel those of IL-6. It is therefore not surprising the relationship observed between IL-6 and Maddrey index ewhich assess the severity of alcoholic hepatitis, although, given the hepatoprotective
effect of IL-6 mentioned before, this relation may be also interpreted as a result of a compensatory mechanism. As commented, macrophage activation leads to increased lipid peroxidation. ROS are continuously produced in the respiratory chain of mitochondria by one-electron reduction of molecular oxygen. Major enzymatic sources of ROS are NADPH oxidases, xanthine oxidases, lipo- and cyclooxygenase, and myeloperoxidase (Steinbrenner & Sies, 2009). Excessive ROS production can damage proteins, lipids, and DNA, and may lead eventually to cell death. Peroxydized fatty acids stimulate IL-8 production by peripheral blood monocytes (Jayatilleke & Shaw, 1998). In addition, oxidized products may act as neoantigens and therefore, they may trigger a specific immune response (Stewart, Vidali, Day, Albano, & Jones, 2004; Tuma, 2002). MDA is a lipid peroxidation product which was increased in patients included in this study. As expected, it was related to CRP and with liver function derangement, a result in agreement with the current knowledge about the role of cytokines and ROS in acute alcoholic hepatitis. The importance of lipid peroxidation products is stressed in this study by the nearly significant trend observed between MDA levels and mortality during admission.
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Fig. 3. Variations of serum IL-6, IL-8 and IL-10 levels during the first 15 days after admission.
Of course, ethanol-mediated increased intestinal permeability is not the only factor involved in raised cytokine levels, but also other inflammatory conditions which, in some cases, led to admission of our patients. As expected, patients with superimposed inflammatory diseases showed a further increase of cytokines such as TNF-a and IL-6, but no differences were observed with respect to MDA. However, the nearly significant relationship between MDA and mortality disappeared when only those patients without infectious/ inflammatory diseases at admission (besides alcoholic hepatitis) were included. With the exceptions of IL-10, which was lower, and TNF-a and IL-6, (in this last cases, partly because of the high variability of the values), which differences did not reach statistical significance, cytokines in our patients remained higher than in controls even at he 15th day after admission. This result is in disagreement with that of others who studied this item, such as González-Quintela et al. (2000), who found that in patients with alcohol withdrawal, serum IL-6, IL-10, and IL-8 decreased 6 days after withdrawal in patients with abstinence, but agrees with the current theory of activation of the immune system in alcoholic hepatitis. In this sense it is interesting the rise in the levels of IL-4. In alcoholic hepatitis, initially, innate immune activation of monocytes and macrophages by LPS acting on their toll receptors leads to enhance IL-12 secretion and activation of TH-1 cells, IFN-g being the main
cytokine secreted. An interesting issue is related to the so called LPS tolerance. This is based on the observation that LPS stimulatory effect on monocytes to produce cytokines is smaller after repeated injections of LPS, particularly regarding IL-12 production. It is well known that IL-12 is a key cytokine involved in Th-1 stimulation (Frank, Witte, Schrödl, & Schütt, 2004), and that it regulates Th-2 activation. Its decrease with time leads to an enhanced secretion of Th-2 cytokines, such as IL-4. The finding of a progressive increase in IL-4 levels is fully in accordance with this theory. Also in agreement with this theory is the finding of a lower IFN-g/IL-4 ratio at the third determination, although differences in this ratio along the study were not statistically significant. It is worth of note that the decrease in IFN-g levels showed a relation with mortality. It is said that IFN-g inhibits fibrosis (Jeong, Park, & Gao, 2008), and that ethanol inhibits this inhibitory effect; therefore, there is a theoretical support for the finding of a relation between decreasing IFN-g levels and long-term mortality in the patients included in this study. It is also noteworthy that the only other factor related with prognosis was MDA, stressing the importance of lipid peroxidation in the natural history of alcoholic hepatitis. In conclusion, our study shows that in alcoholic hepatitis, there is a persistent cytokine secretion; although most cytokines tend to decrease slowly, IL-4 and IL-10 levels rise along the study period. In
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Survival Functions
0,8
Cum Survival
Interferon-γ variations in dead and survivors (in pg/ml) Quartiles INF-g 1,00 4,00 1,00-censored 4,00-censored
1,0
439
14,00
Survivors Dead
12,00
10,00 0,6
8,00 0,4
6,00 0,2
4,00 0,0
2,00 0,00
20,00
40,00
60,00
80,00 100,00 120,00
0,00
Months Fig. 4. Different survival rates among patients with interferon-g values in the first quartile and those belonging to the 4th quartile.
1
2
1=first determination; 2=2nd determination Fig. 6. Differences in the behaviour of interferon-g values in patients who died compared with those who survived.
the first case, this may be interpreted as expression of the described shift in T-cell response from Th-1 to Th-2; in the case of the increase in IL-10 levels, it may represent a compensatory mechanism of an anti-inflammatory cytokine, opposed to the harmful effect of the others, which still remain in abnormal values. Probably as a consequence of ongoing inflammation, MDA levels remain high at all the points of the study, stressing the importance of lipid peroxidation in the pathogenesis of alcoholic liver disease. High MDA levels at admission showed a trend to be related with short-term mortality, whereas IFN-g was related to long-term mortality, although analysing survival using Cox regression model, classic variables such as prothrombin activity and Maddrey index, and subjective nutritional evaluation, displaced cytokines and MDA as prognostic indicators.
Survival Functions PCR quartile 1,00 4,00 1,00-censored 4,00-censored
1,0
Cum Survival
0,8
0,6
0,4
0,2
0,0 0,00
20,00
40,00
60,00
80,00 100,00 120,00
Months Fig. 5. Different survival rates among patients with C-reactive protein values in the first quartile and those belonging to the 4th quartile.
References Bansal, S., Liu, C. P., Sepuri, N. B., Anandatheerthavarada, H. K., Selvaraj, V., Hoek, J., et al. (2010). Mitochondria-targeted cytochrome P450 2E1 induces oxidative damage and augments alcohol-mediated oxidative stress. Journal of Biological Chemistry, 285, 24609e24619. Bird, G. (1994). Interleukin-8 in chronic alcoholic liver disease. Acta Gastroenterologica Belgica, 57, 255e259. Bird, G. L., Sheron, N., Goka, A. K., Alexander, G. J., & Williams, R. S. (1990). Increased plasma tumor necrosis factor in severe alcoholic hepatitis. Annals of Internal Medicine, 112, 917e920. Crews, F. T., Bechara, R., Brown, L. A., Guidot, D. M., Mandrecker, P., Oak, S., et al. (2006). Cytokines and alcohol. Alcoholism: Clinical & Experimental Research, 30, 720e730. Drucker, C., Gewiese, J., Malchow, S., Scheller, J., & Rose-John, S. (2010). Impact of interleukin-6 classic and trans-signaling on liver damage and regeneration. Journal of Autoimmunity, 34, 29e37. Eggers, V., Pascher, A., Althoff, H., Thiele, S., Mütze, J., Selignow, J., et al. (2006). Immune reactivity is more suppressed in patients with alcoholic liver disease than in patients with virus-induced cirrhosis after CRH stimulation. Alcoholism: Clinical & Experimental Research, 30, 140e149. El-Assal, O., Hong, F., Kim, W. H., Radaeva, S., & Gao, B. (2004). IL-6 deficient mice are susceptible to ethanol-induced hepatic steatosis: IL-6 protects against ethanolinduced oxidative stress and mitochondrial permeability transition in the liver. Cellular and Molecular Immunology, 1, 205e211. Frank, J., Witte, K., Schrödl, W., & Schütt, C. (2004). Chronic alcoholism causes deleterious conditioning of innate immunity. Alcohol and Alcoholism, 39, 386e392. Fujimoto, M., Uemura, M., Nakatani, Y., Tsujita, S., Hoppo, K., Tamagawa, T., et al. (2000). Plasma endotoxin and serum cytokine levels in patients with alcoholic hepatitis: relation to severity of liver disturbance. Alcoholism: Clinical & Experimental Research, 24, 48Se54S. Fukui, H. (2005). Relation of endotoxin, endotoxin binding proteins and macrophages to severe alcoholic liver injury and multiple organ failure. Alcoholism: Clinical & Experimental Research, 29(11 Suppl.), 172Se179S. González-Quintela, A., Domínguez-Santalla, M. J., Pérez, L. F., Vidal, C., Lojo, S., & Barrio, E. (2000). Influence of acute alcohol intake and alcohol withdrawal on circulating levels of IL-6, IL-8, IL-10 and IL-12. Cytokine, 12, 1437e1440. González-Reimers, E., García-Valdecasas, E., Santolaria-Fernández, F., de la VegaPrieto, M. J., Ros-Vilamajó, R., Martínez-Riera, A., et al. (2007). Proinflammatory cytokines in stable chronic alcoholics: relationships with lean and fat mass. Food & Chemical Toxicology, 45, 904e909. Hernández-Plasencia, D., Santolaria-Fernández, F., Hernández-García, M. T., González-Reimers, E., Batista-López, N., Jorge-Hernández, J. A., et al. (1991). Subjective nutritional assessment and short term prognosis. Journal of Nutritional and Environmental Medicine, 2, 151e162. Hill, D. B., Marsano, L. S., Cohen, D., Allen, J., Shedlofsky, S., & McClain, C. J. (1992). Increased plasma interleukin-6 concentrations in alcoholic hepatitis. Journal of Laboratory and Clinical Medicine, 119, 547e552.
440
E. González-Reimers et al. / Alcohol 46 (2012) 433e440
Hill, D. B., Marsano, L. S., & McClain, C. J. (1993). Increased plasma interleukin-8 concentrations in alcoholic hepatitis. Hepatology, 18, 576e580. Huang, Y. S., Chan, C. Y., Wu, J. C., Pai, C. H., Chao, Y., & Lee, S. D. (1996). Serum levels of interleukin-8 in alcoholic liver disease: relationship with disease stage, biochemical parameters and survival. Journal of Hepatology, 24, 377e384. Jayatilleke, A., & Shaw, S. (1998). Stimulation of monocyte interleukin-8 by lipid peroxidation products: a mechanism for alcohol-induced liver injury. Alcohol, 16, 119e123. Jeong, W. I., Park, O., & Gao, B. (2008). Abrogation of the antifibrotic effects of natural killer cells/interferon-gamma contributes to alcohol acceleration of liver fibrosis. Gastroenterology, 134, 248e258. Khoruts, A., Stahnke, L., McClain, C. J., Logan, G., & Allen, J. I. (1991). Circulating tumor necrosis factor, interleukin-1 and interleukin-6 concentrations in chronic alcoholic patients. Hepatology, 13, 267e276. Kikugawa, K., Kojima, T., Yamaki, S., & Kosugi, H. (1992). Interpretation of the thiobarbituric acid reactivity of rat liver and brain homogenates in the presence of ferric ion and ethylediaminotetraacetic acid. Analytical Biochemistry, 202, 249e255. Kono, H., Rusyn, I., Yin, M., Gäbele, E., Yamashina, S., Dikalova, A., et al. (2000). NADPH oxidase-derived free radicals are key oxidants in alcohol-induced liver disease. Journal of Clinical Investigation, 106, 867e872. Loguercio, C., & Federico, A. (2003). Oxidative stress in viral and alcoholic hepatitis. Free Radical Biology and Medicine, 34, 1e10. Louis, H., Van Laethem, J. L., Wu, W., Quertinmont, E., Degraef, C., Van den Bergh, K., et al. (1998). Interleukin 10 controls neutrophilic infiltration, hepatocyte proliferation, and liver fibrosis induced by carbon tetrachloride in mice. Hepatology, 28, 1607e1615.
Maddrey, W. C., Boitnott, J. K., Bedine, M. S., Weber, F. L., Mezey, E., & White, R. I. (1978). Corticosteroid therapy of alcoholic hepatitis. Gastroenterology, 75, 193e199. Naveau, S., Balian, A., Capron, F., Raynard, B., Fallik, D., Agostini, H., et al. (2005). Balance between pro and anti-inflammatory cytokines in patients with acute alcoholic hepatitis. Gastroenterologie clinique et biologique, 29, 269e274. Poli, G. (2000). Pathogenesis of liver fibrosis: role of oxidative stress. Molecular Aspects of Medicine, 21, 49e98. Qin, L., He, J., Hanes, R. N., Pluzarev, O., Hong, J. S., & Crews, F. T. (2008). Increased systemic and brain cytokine production and neuroinflammation by endotoxin following ethanol treatment. Journal of Neuroinflammation, doi:10.1186/17422094-5-10. Rao, R. K., Seth, A., & Sheth, P. (2004). Recent advances in alcoholic liver disease I. Role of intestinal permeability and endotoxemia in alcoholic liver disease. American Journal of Physiology e Gastrointestinal and Liver Physiology, 286, G881eG884. Sheron, N., Bird, G., Koskinas, J., Portmann, B., Ceska, M., Lindley, I., et al. (1993). Circulating and tissue levels of the neutrophil chemotaxin interleukin-8 are elevated in severe acute alcoholic hepatitis, and tissue levels correlate with neutrophil infiltration. Hepatology, 18, 41e46. Steinbrenner, H., & Sies, H. (2009). Protection against reactive oxygen species by selenoproteins. Biochimica et Biophysica Acta, 1790, 1478e1485. Stewart, S. F., Vidali, M., Day, C. P., Albano, E., & Jones, D. E. (2004). Oxidative stress as a trigger for cellular immune responses in patients with alcoholic liver disease. Hepatology, 39, 197e203. Tuma, D. J. (2002). Role of malondialdehydeeacetaldehyde adducts in liver injury. Free Radical Biology and Medicine, 32, 303e308. Zhang, X., Tachibana, S., Wang, H., Hisada, M., Williams, G. M., Gao, B., et al. (2010). Interleukin-6 is an important mediator for mitochondrial DNA repair after alcoholic liver injury in mice. Hepatology, 52, 2137e2147.
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Changes in cytokine levels during admission and mortality in acute alcoholic hepatitis E. González-Reimers a, *, M.J. Sánchez-Pérez a, F. Santolaria-Fernández a, P. Abreu-González b, M.J. De la Vega-Prieto c, J. Viña-Rodríguez a, M.R. Alemán-Valls a, M. Rodríguez-Gaspar a a b c
Servicio de Medicina Interna, Hospital Universitario de Canarias, Tenerife, Canary Islands, Spain Dpto. De Fisiología, Facultad de Medicina, Universidad de La Laguna, Tenerife, Canary Islands, Spain Servicio de Laboratorio, Hospital Universitario de Canarias, Tenerife, Canary Islands, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 January 2011 Received in revised form 21 September 2011 Accepted 3 October 2011
Cytokine levels are raised in acute alcoholic hepatitis. However, there are disparate results regarding the duration of altered plasma levels, and there are also discrepancies about the relation of changes during the first 15 days after admission with short-term (in-hospital) or long-term mortality. In 56 patients with acute alcoholic hepatitis we found that IL-8, IL-4, Interferon-g (IFN-g), malondialdehyde and C-reactive protein remained higher in patients than in 18 age- and sex-matched controls at admission, at the 7th day and at the 15th day after admission. Moreover, IL-4 levels (and to a lesser extent, IL-10 and IFN-g ones) increased along the three determinations. However, comparing patients who died during the admission with those who did not, there were no statistically significant differences, but there was a nearly significant trend for MDA (Z ¼ 1.89; p ¼ 0.059), with higher levels among those who died. When changes between the first and the second determinations were compared with long-term survival, only IL-8 and IFN-g showed a relation with mortality. IFN-g values increased among those who survived and decreased among those who died (p ¼ 0.048). IFN-g values at the first determination also showed a relation with long-term mortality, especially when patients with IFN-g values in the first quartile were compared with those of the 4th one (log rank ¼ 5.64; p ¼ 0.018; Breslow ¼ 4.64; p ¼ 0.031). Besides Interferon-g, only C-reactive protein showed differences between the first and the 4th quartile regarding mortality (Log rank ¼ 4.50; p ¼ 0.034; Breslow 4.33; p ¼ 0.038). In contrast with other studies, no relation was found between TNF-a or IL-6 and mortality. Ó 2012 Elsevier Inc. All rights reserved.
Keywords: Alcoholic hepatitis Cytokines Lipid peroxidation Acute phase reaction Short term survival Cytokine sequential
Introduction Since about 20 years ago it is known that cytokines play outstanding roles in the development and progression of chronic alcoholic liver disease (Bird, 1994; Bird, Sheron, Goka, Alexander, & Williams, 1990; Fujimoto et al., 2000; Hill et al., 1992, Hill, Marsano, & McClain, 1993; Huang et al., 1996; Khoruts, Stahnke, McClain, Logan, & Allen, 1991; Sheron et al., 1993). The main mechanisms seem to be related to the activation of Kupffer cells by lipopolisaccaride (LPS), derived from intestinal gram negative bacteria. Theoretically, this feature could be dependent on intestinal bacterial overgrowth, increased intestinal permeability, or to failure of Kupffer cells to neutralize LPS; the bulk of studies have shown that increased intestinal permeability plays an outstanding, primary role (Rao, Seth, & Sheth, 2004). After binding to toll-like receptors * Corresponding author. Tel.: þ34 922 67 80 00; fax: þ34 922 31 93 79. E-mail address: [email protected] (E. González-Reimers). 0741-8329/$ e see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.alcohol.2011.10.001
(especially TLR-4), cytokines are released by Kupffer cells, and initiate the cascade of events which ultimately leads to the development of hepatocyte necrosis, recruitment of neutrophils and immune cells, and liver fibrosis. During this process, increased freeradical (ROS) production aggravates the scenario (Kono et al., 2000; Loguercio & Federico, 2003; Poli, 2000), since they yield both harmful and immunogenic effects. Later in the evolution of alcoholic hepatitis, an increase in TH-2 derived cytokines, such as IL-4 and IL13, has been reported, and is presumably related to liver fibrogenesis (Crews et al., 2006). The duration of the effect of released cytokines is uncertain, but some authors have reported prolonged increase of TNF-a levels in some tissues, such as brain (Qin et al., 2008). Within 6 days after alcohol abstinence levels of IL-6 and IL-10 show a marked decrease (González-Quintela et al., 2000). In the recovery phase of severe alcoholic hepatitis, IL-6 and IL-8 tend to decrease (Fukui, 2005) in survivors, but not in those who subsequently died. Indeed, ethanol is known to alter cytokine levels in a variety of tissues including
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liver, lung, and brain, which may contribute to long-term organ dysfunction (Crews et al., 2006), and, probably, to altered immune response and susceptibility to infection, which may persist for as long as three years after abstinence (Eggers et al., 2006). In a previous report we showed that levels of some cytokines, such as IL-6, still remained high in already stable alcoholics (GonzálezReimers et al., 2007). Therefore, it seems that several cell types are involved in cytokine production. Some cytokines directly derive from activated macrophages, others from activated T-cells; some of them are proinflammatory, but others may exert protective functions. In addition, as commented, there is controversy regarding the evolution of cytokine levels in alcoholics. Based on these facts, in the present study we analyse the sequential behaviour of proinflammatory, anti-inflammatory, Th-1 and Th-2 cytokines among patients affected by alcoholic hepatitis, and their relation with inhospital and long-term mortality. Methods Patients and controls Fifty-six heavy alcoholics (6 women), consecutively admitted to the hospitalization unit of the Internal Medicine Service, entered the study. Ethanol intake was assessed by recall and calculated as follows: grams of ethanol ¼ volume of beverage (cm3) strength (v/v, as %) 0.8. All the patients were drinkers of more than 100 g ethanol/day (193 93 g/day; median ¼ 150 g; (interquartile range) ¼ 120e250 g) during more than 10 years (27.8 10.5 years; 30 (20e30.25). Mean age was 47.09 9.86 years; 45 (40.3e52.75) years. Cytokines and biochemical parameters outlined below were determined at admission, one week later, and at the day 15th among those who were not already discharged (or had died before). Written consent was obtained by each of the patients, and the study protocol was approved by the local ethical committee of our Hospital and conforms to the ethical guidelines of the 1975 Declaration of Helsinki. All the patients were admitted with acute alcoholic hepatitis, but the following conditions were also present at admission: pancreatitis, 5 cases (one also with urinary tract infection); pneumonia, 11 cases (one also with urinary tract infection), one with cutaneous infection; another, with acute cholecystitis; 2 more with urinary tract infection, and 3, with spontaneous bacterial peritonitis. Owing to the well described alteration of cytokines in inflammatory conditions, we grouped our patients in two groups: those with only alcoholic hepatitis (32 cases), and those with alcoholic hepatitis and another inflammatory process (24 cases). Thirty one patients developed a florid ethanol withdrawal syndrome. Twelve patients were placed on corticosteroids at admission (prednisone, 40 mg/ day) due to a high Maddrey’s index (Maddrey et al., 1978). Treatment also included fluidotherapy and thiamine, and broad spectrum antibiotics in patients with infections (usually third generation cephalosporins, with or without levofloxacin) and benzodiacepine in the cases of withdrawal syndrome. However, treatment always began when blood was extracted for the basal biochemical assessment, so treatment did not influence on the first determination. The control group was composed of 18 healthy sanitary workers (2 women), drinkers of less than 10 g ethanol day, aged 45.33 6.80 years. Fifty-five patients were followed up for a median period of 85 months (interquartile range, 32e101 months). During this period, 24 died, 9 of them during the admission episode or shortly after (within 2 months after admission). The relationship between interleukins and malondialdehyde (MDA), all of them classified in quartiles and tertiles and survival was analysed by means of
KaplaneMeyer curves and log-rank test. A Cox regression analysis was also performed including age, interleukins, MDA, classic parameters of liver function, including Maddrey’s index, and nutritional parameters such as body mass index and a previously validated subjective nutritional assessment, which consisted in physical examination of the muscle masses of the upper and lower limbs and of the temporal muscle, defining two degrees of atrophy (severe, moderate) and absence of atrophy, and fat loss on the cheek and abdomen (Bichat’s fat and subcutaneous fat atrophy) classified in a similar way. Each patient was assigned a score (SNS), based on the sum of the assigned points, for which the poorest value was 10 and 0 the best one (Hernández-Plasencia et al., 1991). Cytokines and biochemical parameters Blood samples were taken at 8.00 am in fasting conditions, immediately frozen at 80. Blood sample were extracted A) Shortly after admission (maximum interval, 48 h); B) In survivors, one week later; C) by the 15th day after admission if the patient was still hospitalized. In addition to routine laboratory tests and C-reactive protein determination, the following parameters were measured (Table 1). In a few cases, some of these parameters were not recorded (loss of sample, not enough blood extracted, etc.). Tumour necrosis factor (TNF-a) by immunometric chemiluminiscent assay (intra-assay variation coefficient ranging 4e6.5%, inter-assay variation coefficient ranging 2.6e3.6%, recovery 92e112%, Diagnostic Products Corporation (DPC), Los Angeles, CA, USA); IL-6, by chemiluminiscent assay (inter-assay variation coefficient ranging 5.3e7.5%, recovery ¼ 85e104%, DPC, Los Angeles, CA, USA); IL-4, by enzyme-linked immunosorbent assay (ELISA); (interand intra-assay coefficient of variation <10%; sensitivity <2 pg/ml; recovery ¼ 101%, Bender MedSystems Diagnostics GmbH, Vienna, Austria); IL-8, by chemiluminiscent assay (inter-assay variation coefficient ranging 5.3e7.5%, DPC, Los Angeles, CA, USA); IL-10, by enzyme immunometric assay (sensitivity ¼ 3 pg/ml, inter- and intra-assay coefficient of variation ranging from 3.9 to 7.3%; recovery ranging from 86 to 94%; DPC, Los Angeles, CA, USA); interferon gamma (IFN-g), by ELISA (inter- and intra-assay coefficient of variation 0.3e10.7%; sensitivity <1.5 pg/ml; recovery ¼ 90e112%, Bender MedSystems Diagnostics GmbH, Vienna, Austria). In addition, patients underwent routine laboratory evaluation. Lipid peroxidation (LPX) products Serum MDA levels, referred to as thiobarbituric acid-reactive substance (TBARS), were measured according to the method described by Kikugawa, Kojima, Yamaki, and Kosugi (1992). A volume sample of 0.2 ml of plasma was added to 0.2 ml of H3PO4 (0.2 M) and the colour reaction was initiated by the addition of 25 ml of 0.11 M thiobarbituric acid (TBA) solution. Samples were placed in a 90 C heating block for 45 min. After the samples were cooled, the TBARS (pink complex colour) were extracted with 0.4 ml of n-butanol. Butanol phase was separated by centrifugation at 6000 g for 10 min. Aliquots of the n-butanol phase were placed in a 96 well plate and read at 535 nm in a microlate spectrophotometer reader (Benchmark Plus, Bio-Rad, Hercules, CA, USA). The calibration curve was prepared with authentic MDA standards ranging from 0 to 20 mM. The intra-and inter-assay variation coefficients were 1.82 and 4.01, respectively. Statistics The KolmogoroveSmirnov test was used to test normality, a condition not fulfilled by most of the cytokines analysed. Therefore, non-parametric tests, such as ManneWhitney’s U test and
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Table 1 Cytokines, MDA and C-reactive protein levels values in patients and controls at admission, the 7th day (2nd) and the 15th day (3rd). Results are given as mean standard deviation and in the second subfile, median (interquartile range). Number of patients included are given in brackets.
C-reactive protein IL-8 (pg/ml) IL-10 (pmol/l) IL-4 (ng/ml) IFN-g (pg/ml) TNF-alfa (pg/ml) IL-6 (pg/ml) Malon-dialdehyde IFN-g/IL-4
Patients (1st) (56)
Patients (2nd) (48)
Patients (3rd) (24)
Controls (18)
1st/ctrl
2nd/ctrl
3rd/ctrl
3.68 6.09 0.87 (0.47e4.81) 57.80 97.05 20.65 (9.7e59.8) 5.13 0.59 5 (5e5) 6.88 10.20 3.91 (2.4e6.2) 4.74 12.60 0.87 (0.46e1.82) 7.22 7.21 6.8 (4.0e9.3) 39.82 81.85 9.4 (0.0e38.0) 8.34 6.76 6.55 (4.1e9.8) 1.02 2.60 0.22 (0.16e0.49)
1.82 0.73 0.73 (0.26e3.02) 31.59 47.04 11.8 (7e29.6) 5.0.7 0.42 5.0 (5.0e5.0) 4.14 2.74 4.14 (1.8e6.4) 4.13 11.40 1.00 (0.49e1.52) 6.40 7.00 7.0 (0e8.50) 17.50 23.92 8.3 (0e22.1) 6.81 5.77 5.34 (4.3e7.62) 1.57 5.35 0.21 (0.12e0.62)
1.34 1.87 0.67 (0.36e1.75) 26.60 35.15 13.8 (5.58e33.05) 5.90 4.10 5.0 (5.0e5.0) 27.49 42.90 7.81 (3.58e36.5) 2.94 3.68 1.93 (1.09e2.84) 6.97 3.84 7.3 (4.9e9.5) 11.87 21.38 4.0 (0.0e12.3) 7.16 5.38 4.99 (3.83e9.15) 0.49 0.95 0.16 (0.05e0.34)
0.36 1.11 0.02 (0.01e0.1) 6.75 1.79 6.95 (5.0e7.75) 8.22 10.69 6.0 (5.0e6.0) 2.28 1.72 2.32 (.9e3.2) 0.66 0.50 0.5 (0.3e0.87) 5.83 1.77 5.1 (4.4e8.0) 5.77 1.40 5 (5.0e6.35) 1.31 0.69 1.1 (0.8e1.96) 0.60 0.79 0.28 (0.08e0.79)
Z ¼ 4.93 p < 0.001 Z ¼ 4.66 p < 0.001 Z ¼ 5.01 p < 0.001 Z ¼ 2.54 p ¼ 0.011 Z ¼ 2.58 p ¼ 0.010 Z ¼ 1.52 NS Z ¼ 1.63 NS Z ¼ 6.50 p < 0.001 Z ¼ 0.16 NS
Z ¼ 4.48 p < 0.001 Z ¼ 3.69 p < 0.001 Z ¼ 5.41 p < 0.001 Z ¼ 2.19 p ¼ 0.027 Z ¼ 2.00 p ¼ 0.045 Z ¼ 0.84 NS Z ¼ 1.96 p ¼ 0.05 Z ¼ 6. 23 p < 0.001 Z ¼ 0.04 NS
Z ¼ 4.05 p < 0.001 Z ¼ 2.82 p ¼ 0.004 Z ¼ 4.09 p < 0.001 Z ¼ 3.85 p < 0.001 Z ¼ 3.84 p < 0.001 Z ¼ 1.75 NS Z ¼ 1.18 NS Z ¼ 5.57 p < 0.001 Z ¼ 0.75 NS
KruskalleWallis were used to analyse between group differences in these parameters. Also, non-parametric tests such as Friedman’s test and Wilcoxon’s test were used to compare variations of cytokines values along the three determinations. Spearman’s correlation analysis was used to compare quantitative parameters. Relationships between cytokines and MDA (classified in tertiles or quartiles) with survival were analysed using log-rank test and KaplaneMeier curves. Multivariate Cox regression analysis was used to discern which parameters yield prognostic value.
Results Cytokine values in patients and controls are shown in Table 1. Differences are clearly evident for all the cytokines except for IL-6 and TNF-a, which were non-significantly higher among patients. When patients were grouped in those affected by an infectious or inflammatory condition besides acute alcoholic hepatitis, these two cytokines were significantly higher (Z ¼ 2.28, p ¼ 0.023 and Z ¼ 2.21, p ¼ 0.027, respectively) among those with an inflammatory process at admission than those without it. No differences were observed among those placed or not on corticosteroids, except for IL-6, slightly higher among the former (Z ¼ 2.08; p ¼ 0.037). As shown, cytokines (besides IL-10) still remained high at the second determination (Table 1). No differences were observed among patients treated or not with steroids, except for slightly higher values of IL-8 among those who received prednisone (Z ¼ 1.97; p ¼ 0.048). Regarding the third determination, no differences were observed among those treated or not with steroids, but, indeed, between patients and controls. In general, cytokine values were lower in the second and third determinations than in the first one, with the notable exceptions of IL-4 and, to a lesser extent, IL-10 and INF-g. Analysed by means of Friedman test, differences along the study were significant for IL-8 (c2 ¼ 13.76; p < 0.001), INF-g (c2 ¼ 7.55, p ¼ 0.023) and CRP (c2 ¼ 6.08, p ¼ 0.048). However, using Wilcoxon test to compare changes between point 1 and point 3, differences were also significant for IL-4 (Z ¼ 2.44, p ¼ 0.015) (which levels rose), and nearly significant, for MDA (Z ¼ 1.89, p ¼ 0.059), which levels decreased. We calculated the ratio INF-g/IL-4 (Th-1 and Th-2 main cytokines, respectively). As shown in Table 1, no differences were observed in this ratio between patients and controls. There was a non-significant trend to lower values in the third determination compared with the first and second ones.
As shown in Table 2, significant correlations were observed between TNF-a and IL-6, IL-8, IFN-g, IL-10, and C-reactive protein. C-reactive protein was also directly related with IL-6 and IL-8, and also, a direct correlation was observed between IL-4 and IFN-g. When data obtained at the 7th day were compared to each other, IL-6 still showed a significant correlation with CRP (r ¼ 0.58, p < 0.001, Fig. 1), TNF-a (r ¼ 0.30, p ¼ 0.047) and IL-8 (r ¼ 0.35, p ¼ 0.027)) A significant correlation was also observed between IL-8 and CRP (r ¼ 0.43; p ¼ 0.006). At the third determination, the only significant correlation still observed was between MDA and CRP (r ¼ 0.43; p ¼ 0.036). Changes in CRP values between the first and the second determination showed a significant correlation with changes in IL-6 (r ¼ 0.52, p ¼ 0.001), whereas changes in IL-8 showed an inverse correlation with changes in IFN-g (r ¼ 0.46; p ¼ 0.003). Changes in TNF-a values between the first and the third determinations showed a significant relationship with changes in IL-6 (r ¼ 0.69, p ¼ 0.007) and CRP (r ¼ 0.48, p ¼ 0.021) and an inverse one with changes in IL-10 (r ¼ 0.57, p ¼ 0.035); in addition, a close relationship was observed between changes in IL-6 and in CRP (r ¼ 0.81, p < 0.001). As shown in Figs. 2 and 3, evolution of cytokine values among patients with or without infectious and/or inflammatory conditions other than alcoholic hepatitis was similar, with the notable exception of IL-6, which showed a trend (p ¼ 0.10) to increase among non-infected patients, and to decrease among infected ones. Also, IL-4 levels showed a non-significant trend to increase among non-infected patients. However, these different behaviour was not statistically significant (F ¼ 3.99; p ¼ 0.086). Relationship with liver function Il-6 and IL-8 kept a close relationship with liver function, assessed all by Maddrey index (r ¼ 0.29; p ¼ 0.047 and r ¼ 0.40, p ¼ 0.004), Pugh’s score (among cirrhotics; only IL-6, r ¼ 0.59, p < 0.001), albumin, bilirubin, and prothrombin activity (data not shown). MDA was also significantly correlated with Maddrey index (r ¼ 0.32, p ¼ 0.021). Pugh’s score (r ¼ 0.43, p ¼ 0.044), and bilirubin (r ¼ 0.47, p < 0.001). Changes in Maddrey index between the first and the second determination were significantly related to changes in MDA (r ¼ 0.30, p ¼ 0.05), as well as changes between MDA at the 1st and 15th day and changes in Maddrey index during the same period (r ¼ 0.58, p ¼ 0.009).
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Table 2 Correlations among cytokines and MDA at admission. IL-6 IL-6
IL-8
TNF-a
IFN-g
IL-10
IL-4
MDA
CRP
b
IL-8
1.000 47 .222 .153 43 .431a .003 45 .031 .844 43 .002 .989 34 .244 .114 43 .077 .617 44 .504a .000 46
.222 .153 43 1.000 48 .368b .013 45 .143 .354 44 .164 .361 33 .047 .761 44 .190 .211 45 .306b .041 45
TNF-a
IFN-g
IL-10
IL-4
MDA
CRP
.431a .003 45 .368b .013 45 1.000
.031 .844 43 .143 .354 44 .322b .033 44 1.000
.002 .989 34 .164 .361 33 .338b .041 37 .109 .534 35 1.000
.244 .114 43 .047 .761 44 .186 .226 44 .426a .003 46 .232 .180 35 1.000
.077 .617 44 .190 .211 45 .061 .677 49 .076 .629 43 .003 .986 37 .033 .835 43 1.000
.504a .000 46 .306b .041 45 .329b .020 50 .045 .772 44 .036 .840 35 .080 .605 44 .109 .462 48 1.000
52 .322b .033 44 .338b .041 37 .186 .226 44 .061 .677 49 .329b .020 50
46 .109 .534 35 .426a .003 46 .076 .629 43 .045 .772 44
38 .232 .180 35 .003 .986 37 .036 .840 35
46 .033 .835 43 .080 .605 44
52 .109 .462 48
51
Correlation is significant at the 0.01 level (2-tailed). Correlation is significant at the 0.05 level (2-tailed).
Relation with mortality Of the parameters recorded at admission, only IFN-g showed a relation with mortality. When classified in tertiles, there was a nearly significant trend to a lower mortality among those with higher values (tertiles: log rank ¼ 5.86; p ¼ 0.053; Breslow 5.0, p ¼ 0.082). Differences were more marked when patients belonging to the first quartile were compared with those of the 4th quartile (Fig. 4; log rank ¼ 5.64; p ¼ 0.018; Breslow ¼ 4.64; p ¼ 0.031). Besides IFN-g, only C-reactive protein showed differences between the first and the 4th quartile regarding mortality (Log rank ¼ 4.50; p ¼ 0.034; Breslow 4.33; p ¼ 0.038); Fig. 5. Comparing patients who died during the admission with those who did not, there were no statistically significant differences, although there was a nearly significant trend for MDA (Z ¼ 1.89; p ¼ 0.059), with higher levels among those who died. When changes between the first and the second determinations were compared with long-term survival, only IL-8 and IFN-g showed a relation with mortality. IFN-g values increased among those who survived and decreased among those who died (p ¼ 0.048, Fig. 6). However, in a survival analysis using Cox regression model, introducing the variables age, prothrombin activity, bilirubin, albumin and Maddrey index in addition to cytokines, only Maddrey index (p ¼ 0.02) and prothrombin activity (p ¼ 0.013) were related to mortality during admission or during the whole follow-up period, respectively. SNS score also displaced cytokines when nutritional parameters were also included in the Cox regression analysis. Discussion
2nd and 3rd determinations. IL-10 is an anti-inflammatory cytokine able to limit the inflammatory response and hepatotoxicity in chronic liver disease (Louis et al., 1998). It is upregulated in patients with alcoholic liver disease, but, in severe cases, it is insufficient to modulate TNF-a mediated (through TNFrp55) liver toxicity. Our
Re lationship be tween IL-6 and C-reactive protein at the 7th day
100,00
75,00
IL-6 (pg/ml)
a
Correlation Coefficient Sig. (2-tailed) N Correlation Coefficient Sig. (2-tailed) N Correlation Coefficient Sig. (2-tailed) N Correlation Coefficient Sig. (2-tailed) N Correlation Coefficient Sig. (2-tailed) N Correlation Coefficient Sig. (2-tailed) N Correlation Coefficient Sig. (2-tailed) N Correlation Coefficient Sig. (2-tailed) N
50,00
25,00
0,00
0,00
2,50
5,00
7,50
10,00
C-reactive protein(ng/ml) In this study we have shown that MDA and all the cytokines besides TNF-a and IL-6 were different among patients and controls. In general, cytokine values were higher among patients, with the notable exception of IL-10, which was initially lower when the patients were admitted to the hospital, and remained lower at the
rho =0.58; p<0.001 Fig. 1. Relationship between IL-6 and C-reactive protein at the 7th day after admission.
E. González-Reimers et al. / Alcohol 46 (2012) 433e440
437
Fig. 2. Evolution of TNF-a, IFN-g, IL-4 and MDA among patients with or without infectious/inflammatory conditions.
results do not agree with those of Naveau et al. (2005), who found higher levels of IL-10 among patients with alcoholic hepatitis, but not sufficient enough to counteract the effect of proinflammatory cytokines. The behaviour of IL-6 may be viewed in a similar fashion. IL-6 has a dual effect, acting as proinflammatory, as well as antiinflammatory cytokine. It is assumed that IL-6 plays a role in liver regeneration (Drucker, Gewiese, Malchow, Scheller, & Rose-John, 2010). Other researchers (El-Assal, Hong, Kim, Radaeva, & Gao, 2004) have shown that IL-6 protects mice from ethanol-induced liver injury. In addition, recent research has shown that IL-6 plays an important role in mitochondrial DNA repair (Zhang et al., 2010). Mitochondrial DNA becomes damaged by ethanol-induced production of reactive oxygen species (Bansal et al., 2010). In any case, it is well known that IL-6 also exerts proinflammatory effects, inducing liver synthesis of several acute phase reactants. In our study it is clear that IL-6 and CRP are closely related at the different points at which blood was extracted, and also that changes in CRP parallel those of IL-6. It is therefore not surprising the relationship observed between IL-6 and Maddrey index ewhich assess the severity of alcoholic hepatitis, although, given the hepatoprotective
effect of IL-6 mentioned before, this relation may be also interpreted as a result of a compensatory mechanism. As commented, macrophage activation leads to increased lipid peroxidation. ROS are continuously produced in the respiratory chain of mitochondria by one-electron reduction of molecular oxygen. Major enzymatic sources of ROS are NADPH oxidases, xanthine oxidases, lipo- and cyclooxygenase, and myeloperoxidase (Steinbrenner & Sies, 2009). Excessive ROS production can damage proteins, lipids, and DNA, and may lead eventually to cell death. Peroxydized fatty acids stimulate IL-8 production by peripheral blood monocytes (Jayatilleke & Shaw, 1998). In addition, oxidized products may act as neoantigens and therefore, they may trigger a specific immune response (Stewart, Vidali, Day, Albano, & Jones, 2004; Tuma, 2002). MDA is a lipid peroxidation product which was increased in patients included in this study. As expected, it was related to CRP and with liver function derangement, a result in agreement with the current knowledge about the role of cytokines and ROS in acute alcoholic hepatitis. The importance of lipid peroxidation products is stressed in this study by the nearly significant trend observed between MDA levels and mortality during admission.
438
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Fig. 3. Variations of serum IL-6, IL-8 and IL-10 levels during the first 15 days after admission.
Of course, ethanol-mediated increased intestinal permeability is not the only factor involved in raised cytokine levels, but also other inflammatory conditions which, in some cases, led to admission of our patients. As expected, patients with superimposed inflammatory diseases showed a further increase of cytokines such as TNF-a and IL-6, but no differences were observed with respect to MDA. However, the nearly significant relationship between MDA and mortality disappeared when only those patients without infectious/ inflammatory diseases at admission (besides alcoholic hepatitis) were included. With the exceptions of IL-10, which was lower, and TNF-a and IL-6, (in this last cases, partly because of the high variability of the values), which differences did not reach statistical significance, cytokines in our patients remained higher than in controls even at he 15th day after admission. This result is in disagreement with that of others who studied this item, such as González-Quintela et al. (2000), who found that in patients with alcohol withdrawal, serum IL-6, IL-10, and IL-8 decreased 6 days after withdrawal in patients with abstinence, but agrees with the current theory of activation of the immune system in alcoholic hepatitis. In this sense it is interesting the rise in the levels of IL-4. In alcoholic hepatitis, initially, innate immune activation of monocytes and macrophages by LPS acting on their toll receptors leads to enhance IL-12 secretion and activation of TH-1 cells, IFN-g being the main
cytokine secreted. An interesting issue is related to the so called LPS tolerance. This is based on the observation that LPS stimulatory effect on monocytes to produce cytokines is smaller after repeated injections of LPS, particularly regarding IL-12 production. It is well known that IL-12 is a key cytokine involved in Th-1 stimulation (Frank, Witte, Schrödl, & Schütt, 2004), and that it regulates Th-2 activation. Its decrease with time leads to an enhanced secretion of Th-2 cytokines, such as IL-4. The finding of a progressive increase in IL-4 levels is fully in accordance with this theory. Also in agreement with this theory is the finding of a lower IFN-g/IL-4 ratio at the third determination, although differences in this ratio along the study were not statistically significant. It is worth of note that the decrease in IFN-g levels showed a relation with mortality. It is said that IFN-g inhibits fibrosis (Jeong, Park, & Gao, 2008), and that ethanol inhibits this inhibitory effect; therefore, there is a theoretical support for the finding of a relation between decreasing IFN-g levels and long-term mortality in the patients included in this study. It is also noteworthy that the only other factor related with prognosis was MDA, stressing the importance of lipid peroxidation in the natural history of alcoholic hepatitis. In conclusion, our study shows that in alcoholic hepatitis, there is a persistent cytokine secretion; although most cytokines tend to decrease slowly, IL-4 and IL-10 levels rise along the study period. In
E. González-Reimers et al. / Alcohol 46 (2012) 433e440
Survival Functions
0,8
Cum Survival
Interferon-γ variations in dead and survivors (in pg/ml) Quartiles INF-g 1,00 4,00 1,00-censored 4,00-censored
1,0
439
14,00
Survivors Dead
12,00
10,00 0,6
8,00 0,4
6,00 0,2
4,00 0,0
2,00 0,00
20,00
40,00
60,00
80,00 100,00 120,00
0,00
Months Fig. 4. Different survival rates among patients with interferon-g values in the first quartile and those belonging to the 4th quartile.
1
2
1=first determination; 2=2nd determination Fig. 6. Differences in the behaviour of interferon-g values in patients who died compared with those who survived.
the first case, this may be interpreted as expression of the described shift in T-cell response from Th-1 to Th-2; in the case of the increase in IL-10 levels, it may represent a compensatory mechanism of an anti-inflammatory cytokine, opposed to the harmful effect of the others, which still remain in abnormal values. Probably as a consequence of ongoing inflammation, MDA levels remain high at all the points of the study, stressing the importance of lipid peroxidation in the pathogenesis of alcoholic liver disease. High MDA levels at admission showed a trend to be related with short-term mortality, whereas IFN-g was related to long-term mortality, although analysing survival using Cox regression model, classic variables such as prothrombin activity and Maddrey index, and subjective nutritional evaluation, displaced cytokines and MDA as prognostic indicators.
Survival Functions PCR quartile 1,00 4,00 1,00-censored 4,00-censored
1,0
Cum Survival
0,8
0,6
0,4
0,2
0,0 0,00
20,00
40,00
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80,00 100,00 120,00
Months Fig. 5. Different survival rates among patients with C-reactive protein values in the first quartile and those belonging to the 4th quartile.
References Bansal, S., Liu, C. P., Sepuri, N. B., Anandatheerthavarada, H. K., Selvaraj, V., Hoek, J., et al. (2010). Mitochondria-targeted cytochrome P450 2E1 induces oxidative damage and augments alcohol-mediated oxidative stress. Journal of Biological Chemistry, 285, 24609e24619. Bird, G. (1994). Interleukin-8 in chronic alcoholic liver disease. Acta Gastroenterologica Belgica, 57, 255e259. Bird, G. L., Sheron, N., Goka, A. K., Alexander, G. J., & Williams, R. S. (1990). Increased plasma tumor necrosis factor in severe alcoholic hepatitis. Annals of Internal Medicine, 112, 917e920. Crews, F. T., Bechara, R., Brown, L. A., Guidot, D. M., Mandrecker, P., Oak, S., et al. (2006). Cytokines and alcohol. Alcoholism: Clinical & Experimental Research, 30, 720e730. Drucker, C., Gewiese, J., Malchow, S., Scheller, J., & Rose-John, S. (2010). Impact of interleukin-6 classic and trans-signaling on liver damage and regeneration. Journal of Autoimmunity, 34, 29e37. Eggers, V., Pascher, A., Althoff, H., Thiele, S., Mütze, J., Selignow, J., et al. (2006). Immune reactivity is more suppressed in patients with alcoholic liver disease than in patients with virus-induced cirrhosis after CRH stimulation. Alcoholism: Clinical & Experimental Research, 30, 140e149. El-Assal, O., Hong, F., Kim, W. H., Radaeva, S., & Gao, B. (2004). IL-6 deficient mice are susceptible to ethanol-induced hepatic steatosis: IL-6 protects against ethanolinduced oxidative stress and mitochondrial permeability transition in the liver. Cellular and Molecular Immunology, 1, 205e211. Frank, J., Witte, K., Schrödl, W., & Schütt, C. (2004). Chronic alcoholism causes deleterious conditioning of innate immunity. Alcohol and Alcoholism, 39, 386e392. Fujimoto, M., Uemura, M., Nakatani, Y., Tsujita, S., Hoppo, K., Tamagawa, T., et al. (2000). Plasma endotoxin and serum cytokine levels in patients with alcoholic hepatitis: relation to severity of liver disturbance. Alcoholism: Clinical & Experimental Research, 24, 48Se54S. Fukui, H. (2005). Relation of endotoxin, endotoxin binding proteins and macrophages to severe alcoholic liver injury and multiple organ failure. Alcoholism: Clinical & Experimental Research, 29(11 Suppl.), 172Se179S. González-Quintela, A., Domínguez-Santalla, M. J., Pérez, L. F., Vidal, C., Lojo, S., & Barrio, E. (2000). Influence of acute alcohol intake and alcohol withdrawal on circulating levels of IL-6, IL-8, IL-10 and IL-12. Cytokine, 12, 1437e1440. González-Reimers, E., García-Valdecasas, E., Santolaria-Fernández, F., de la VegaPrieto, M. J., Ros-Vilamajó, R., Martínez-Riera, A., et al. (2007). Proinflammatory cytokines in stable chronic alcoholics: relationships with lean and fat mass. Food & Chemical Toxicology, 45, 904e909. Hernández-Plasencia, D., Santolaria-Fernández, F., Hernández-García, M. T., González-Reimers, E., Batista-López, N., Jorge-Hernández, J. A., et al. (1991). Subjective nutritional assessment and short term prognosis. Journal of Nutritional and Environmental Medicine, 2, 151e162. Hill, D. B., Marsano, L. S., Cohen, D., Allen, J., Shedlofsky, S., & McClain, C. J. (1992). Increased plasma interleukin-6 concentrations in alcoholic hepatitis. Journal of Laboratory and Clinical Medicine, 119, 547e552.
440
E. González-Reimers et al. / Alcohol 46 (2012) 433e440
Hill, D. B., Marsano, L. S., & McClain, C. J. (1993). Increased plasma interleukin-8 concentrations in alcoholic hepatitis. Hepatology, 18, 576e580. Huang, Y. S., Chan, C. Y., Wu, J. C., Pai, C. H., Chao, Y., & Lee, S. D. (1996). Serum levels of interleukin-8 in alcoholic liver disease: relationship with disease stage, biochemical parameters and survival. Journal of Hepatology, 24, 377e384. Jayatilleke, A., & Shaw, S. (1998). Stimulation of monocyte interleukin-8 by lipid peroxidation products: a mechanism for alcohol-induced liver injury. Alcohol, 16, 119e123. Jeong, W. I., Park, O., & Gao, B. (2008). Abrogation of the antifibrotic effects of natural killer cells/interferon-gamma contributes to alcohol acceleration of liver fibrosis. Gastroenterology, 134, 248e258. Khoruts, A., Stahnke, L., McClain, C. J., Logan, G., & Allen, J. I. (1991). Circulating tumor necrosis factor, interleukin-1 and interleukin-6 concentrations in chronic alcoholic patients. Hepatology, 13, 267e276. Kikugawa, K., Kojima, T., Yamaki, S., & Kosugi, H. (1992). Interpretation of the thiobarbituric acid reactivity of rat liver and brain homogenates in the presence of ferric ion and ethylediaminotetraacetic acid. Analytical Biochemistry, 202, 249e255. Kono, H., Rusyn, I., Yin, M., Gäbele, E., Yamashina, S., Dikalova, A., et al. (2000). NADPH oxidase-derived free radicals are key oxidants in alcohol-induced liver disease. Journal of Clinical Investigation, 106, 867e872. Loguercio, C., & Federico, A. (2003). Oxidative stress in viral and alcoholic hepatitis. Free Radical Biology and Medicine, 34, 1e10. Louis, H., Van Laethem, J. L., Wu, W., Quertinmont, E., Degraef, C., Van den Bergh, K., et al. (1998). Interleukin 10 controls neutrophilic infiltration, hepatocyte proliferation, and liver fibrosis induced by carbon tetrachloride in mice. Hepatology, 28, 1607e1615.
Maddrey, W. C., Boitnott, J. K., Bedine, M. S., Weber, F. L., Mezey, E., & White, R. I. (1978). Corticosteroid therapy of alcoholic hepatitis. Gastroenterology, 75, 193e199. Naveau, S., Balian, A., Capron, F., Raynard, B., Fallik, D., Agostini, H., et al. (2005). Balance between pro and anti-inflammatory cytokines in patients with acute alcoholic hepatitis. Gastroenterologie clinique et biologique, 29, 269e274. Poli, G. (2000). Pathogenesis of liver fibrosis: role of oxidative stress. Molecular Aspects of Medicine, 21, 49e98. Qin, L., He, J., Hanes, R. N., Pluzarev, O., Hong, J. S., & Crews, F. T. (2008). Increased systemic and brain cytokine production and neuroinflammation by endotoxin following ethanol treatment. Journal of Neuroinflammation, doi:10.1186/17422094-5-10. Rao, R. K., Seth, A., & Sheth, P. (2004). Recent advances in alcoholic liver disease I. Role of intestinal permeability and endotoxemia in alcoholic liver disease. American Journal of Physiology e Gastrointestinal and Liver Physiology, 286, G881eG884. Sheron, N., Bird, G., Koskinas, J., Portmann, B., Ceska, M., Lindley, I., et al. (1993). Circulating and tissue levels of the neutrophil chemotaxin interleukin-8 are elevated in severe acute alcoholic hepatitis, and tissue levels correlate with neutrophil infiltration. Hepatology, 18, 41e46. Steinbrenner, H., & Sies, H. (2009). Protection against reactive oxygen species by selenoproteins. Biochimica et Biophysica Acta, 1790, 1478e1485. Stewart, S. F., Vidali, M., Day, C. P., Albano, E., & Jones, D. E. (2004). Oxidative stress as a trigger for cellular immune responses in patients with alcoholic liver disease. Hepatology, 39, 197e203. Tuma, D. J. (2002). Role of malondialdehydeeacetaldehyde adducts in liver injury. Free Radical Biology and Medicine, 32, 303e308. Zhang, X., Tachibana, S., Wang, H., Hisada, M., Williams, G. M., Gao, B., et al. (2010). Interleukin-6 is an important mediator for mitochondrial DNA repair after alcoholic liver injury in mice. Hepatology, 52, 2137e2147.