Polymorphonuclear neutrophils are a source of hepatocyte growth factor in patients with severe alcoholic hepatitis

Journal of Hepatology 36 (2002) 342–348 www.elsevier.com/locate/jhep

Polymorphonuclear neutrophils are a source of hepatocyte growth factor in patients with severe alcoholic hepatitis Julien Taı¨eb 1,2,3, Charlotte Delarche 3, Vale´rie Paradis 1,2, Philippe Mathurin 1,2, Alain Grenier 3,4, Bruno Crestani 4, Monique Dehoux 4, Dominique Thabut 1,2, Marie-Anne Gougerot-Pocidalo 3, Thierry Poynard 1,2, Sylvie Chollet-Martin 3,* 1

Hepatology Department, Pitie´-Salpeˆtrie`re Hospital, 47, boulevard de l’hoˆpital, 75651 Paris, cedex 13, France 2 URA CNRS 1484, 4 avenue de l’observatoire, 75006 Paris, France 3 Service d’He´matologie et d’Immunologie Biologiques, INSERM U479, Hoˆpital Bichat, 16, rue Henri-Huchard, 75877 Paris cedex 18, France 4 INSERM U408, Bichat-Claude Bernard Hospital, 46 rue Henri Huchard, 75877 Paris cedex 18, France

Background/Aims: Hepatocyte growth factor (HGF) is a pleiotropic cytokine involved in liver regeneration. Plasma HGF levels correlate with survival and hepatocyte proliferation in alcoholic hepatitis (AH). As AH is accompanied by inflammation, neutrophilia and polymorphonuclear neutrophil (PMN) infiltration of the liver, we postulated that PMN could be a source of HGF in such patients. Methods: We studied 25 patients with severe AH in comparison with 20 alcoholic cirrhotic patients without AH and 20 healthy controls; the impact of a 28-day course of corticosteroids was evaluated in patients with AH. Results: On day 0, HGF plasma and homogenized liver tissue levels were markedly increased in AH patients as compared to controls. The role of PMN in HGF production during AH was confirmed by a significantly higher ex-vivo HGF production capacity of lipopolysaccharide-stimulated blood PMN from AH patients relative to both control groups. Formyl-Methionyl-Leucyl-Phenylalanine-induced PMN release of HGF (degranulation conditions) was also higher in AH patients. In this setting, we found that HGF release by PMN ex vivo correlated strongly with HGF plasma levels, and that the degree of hepatic PMN correlated strongly with hepatic HGF levels. HGF plasma levels and ex-vivo HGF release by PMN were unaffected by steroid therapy. Conclusions: These findings suggest that, by releasing HGF, PMN could participate in liver regeneration during severe alcoholic hepatitis. q 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. Keywords: Cytokine; Alcoholic liver disease; Liver regeneration; Corticosteroids

1. Introduction Hepatocyte growth factor (HGF) is a pleiotropic substance with mitogenic, morphogenic, motogenic and tumor-suppressor activity. HGF and its receptor c-met are key factors in liver regeneration [1]. HGF administration reduces hepatotoxicity and increases survival in various animal models of liver damage [2–4]. HGF also has antiReceived 10 August 2001; received in revised form 18 October 2001; accepted 4 November 2001 * Corresponding author. Tel.: 133-1-40-25-85-21; fax: 133-1-40-25-8853. E-mail address: [email protected] (S. Chollet-Martin).

fibrotic properties: in a rat model of dimethylnitrosamineinduced fibrosis, HGF infusion improved liver histology and collagen content [5]. Little is known of the role of HGF in alcoholic liver disease. In animal models of neutrophilic hepatitis with high levels of circulating TNFa, which resemble human alcoholic hepatitis (AH), HGF prevents lethal hepatic failure [4]. HGF infusion also promotes rat recovery from alcohol-induced fatty liver [6]. In humans, plasma HGF levels are elevated during alcoholic liver disease, and correlate with disease severity and outcome [7], together with hepatocyte proliferation [8]. Fang et al also found that the number of liver cells expressing HGF correlated with the

0168-8278/02/$20.00 q 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. PII: S01 68- 8278(01)0027 6-8

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expression of proliferation markers and survival in patients with AH [9]. In various liver diseases, hepatic HGF is located in non-parenchymal cells such as hepatic stellate cells, and also in endothelial and Kuppfer cells [10,11]. There is also some evidence that polymorphonuclear neutrophil (PMN) could be a source of HGF. Wolf et al. reported immunocytochemical data showing the presence of HGF in human bone-marrow PMN [12]. Likewise, Sakagushi found HGF-immunostained PMN in the sinusoids of hepatic lobules and in the portal area of diseased liver [13]. There is increasing evidence that PMN play a role in the pathophysiology of AH. Indeed, neutrophilia is often frequent, and the liver is infiltrated by PMN. We recently showed that blood PMN are hyperactivated during AH, as they produce high amounts of reactive oxygen species (ROS), proinflammatory cytokines and chemokines maintaining liver inflammation [14]. PMN infiltration is also a factor of good prognosis, as corticosteroid therapy – the reference treatment of severe acute AH [15–17] – is particularly effective in patients with marked neutrophilia or liver PMN infiltration [18]. We postulated that activated PMN were a source of HGF in AH, and could therefore participate in tissue repair. Blood PMN from cirrhotic patients with and without AH were isolated and compared for their capacity to secrete HGF ex vivo in various conditions of stimulation. HGF was also quantified in plasma and liver homogenates of patients with AH. Finally, we evaluated the impact of a 28-day course of steroids on the HGF production by blood PMN from patients with AH. Our data suggest that, in addition to their known proinflammatory effects on liver cells, PMN, through HGF synthesis, could also modulate hepatic parenchyma repair and limit fibrosis, and thus play a previously undescribed role in homeostasis recovery after AH.

2. Patients and methods 2.1. Patients and healthy controls 65 subjects forming three groups were prospectively enrolled: 25 heavy drinkers with severe biopsy-proven AH; 20 heavy drinkers with alcoholic cirrhosis but free of AH; and 20 healthy abstinent staff members of our department with no history of alcoholism. Written informed consent was obtained from all the patients or their closest relative, and from the healthy controls. All procedures were conducted in strict accordance with the ethical standards of our institution. The patients with alcoholic liver disease (AH and/or cirrhosis) had consumed more than 50 g of alcohol per day for at least 5 years. Some patients with alcoholic cirrhosis without AH stopped drinking a few months before blood sampling. The diagnosis of AH was based on transjugular vein liver biopsy in all 25 patients; a combination of acidophil necrosis, ballooned hepatocytes and PMN infiltrate was required. The severity of AH was defined by a Maddrey score (discriminant function) above 32 [19]. The discriminant function was as follows: 4.6 [Prothrombin Time – Control Time (s)] 1 Serum Bilirubin (mmol/l)/17. All patients with severe AH were treated with 40 mg/day methylprednisolone for 28 days. AH was ruled out in the cirrhotic controls by the absence of relevant clinical and biological findings;

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liver biopsy was performed in equivocal cases. Patients were excluded if they had hepatitis B antigen, anti-HCV or anti-HIV antibodies, proven sepsis or gastrointestinal bleeding at entry, or hepatocellular carcinoma.

2.2. Study design The 65 subjects were enrolled in a two-phase study. The first 15 patients with severe AH were studied for HGF plasma and liver-tissue levels and 20h blood PMN cultures; they were compared to ten AH-free patients with alcoholic cirrhosis and to ten healthy volunteers. Then, to better understand the mechanism of PMN HGF production, a further ten AH patients were studied for plasma HGF levels and for PMN HGF preformed stores and their mobilization. These ten AH patients were compared to a further ten AH-free cirrhotic patients and a further ten healthy controls. All the patients with AH received corticosteroid therapy, starting the day after transjugular vein liver biopsy (referred to as day 0). Day 0 blood samples were collected within 4 days following admission. HGF plasma levels were measured on day 0 in all the subjects and on days 3, 7, 14, 21 and 28 of corticosteroid therapy. Ex-vivo HGF secretion by isolated PMN was assessed on days 0 and 28 in the first 15 patients with AH.

2.3. Plasma and liver sample preparation Blood was collected in sterile EDTA-treated vacuum tubes, transported on ice to the laboratory and immediately centrifuged at 48C to avoid cytokine synthesis or degradation in vitro. Plasma samples were stored at 2708C until HGF assay. Transjugular vein liver biopsy specimens were obtained from patients with AH. Control liver tissue were obtained during hepatic lobectomy performed for hepatic metastasis of colorectal cancer; the samples were taken at least 3 cm from all pathological lesions and were considered as normal tissue. Liver fragments were transported on ice to the laboratory and stored at 2708C. They were subsequently weighed, homogenized and solubilized as previously described [20]. After 20 min of ultracentrifugation at 100 000 £ g, supernatants were assayed for HGF.

2.4. Quantification of hepatic PMN infiltration HGF levels in liver homogenates could be studied in 14 patients. Paraffin-embedded liver biopsy specimens from these patients were retrospectively analyzed by a pathologist who was unaware of all clinical and biological data. A semi-quantitative assessment of PMN infiltration was made on hematoxylin- and eosin-stained sections. The PMN infiltrate was graded as follows: 0 ¼ none; 1 ¼ mild (,2 foci/biopsy); 2 ¼ moderate (.)–6 , foci/biopsy), 3 ¼ marked (.6 foci/biopsy).

2.5. Purification of blood PMN Blood was collected in sterile EDTA-treated vacuum tubes. Leukocytes were rapidly isolated in endotoxin-free conditions by sedimentation on a separating medium containing 9% Dextran T500 (Pharmacia, Uppsala, Sweden) and 38% Radioselectan (Schering Plough, Lannoy, France). The leukocyte-rich suspension was then centrifuged on a Ficoll-Paque density gradient (Pharmacia). PMN from the pellet were further purified as follows: after removal of contaminating erythrocytes by hypotonic lysis, the PMN preparation was incubated with pan anti-human HLA class II-coated magnetic beads (Dynal, Oslo, Norway) for 30 min at 48C with gentle rotation, to deplete monocytes, B cells and activated T cells [20].

2.6. Twenty hours-blood PMN culture Highly purified blood PMN were resuspended in RPMI 1640 medium containing 2 mmol/l glutamine, antibiotics and 5% heat-inactivated fetal calf serum (Biowhittacker) at a final density of 10 7/ml. The cells were then cultured at 378C with 5% CO2, with or without 100 ng/ml lipopolysaccharide (LPS, E. coli 055:B5, Sigma) and 250 IU/ml human IFNg (Genzyme Diagnostics, MA, USA), a very potent stimulus for the production of

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Table 1 Clinical and biological characteristics of the patients (values are means and ranges, unless otherwise indicated)

Male/female (no.) Age (y) Ascites (no. of patients) Encephalopathy (no of patients) Child–Pugh Score Maddrey’s discriminant function Prothrombin time (% of normal) Albumin (g/l) ASAT (IU/l) Serum bilirubin (mmol/l) g-glutamyl-transpeptidase (IU/l) Serum creatinine (mmol/l) White blood cells (£10 3/mm 3) Polymorphonuclear neutrophils (£10 3/mm 3)

Alcoholic hepatitis before steroid therapy (day 0, n ¼ 25)

Alcoholic cirrhosis without AH ðn ¼ 20Þ

18/7 54 (40–72) 16 3 11 (9–14) 62 (32–101) 36 (22–50) 25 (17–37) 155 (51–739) 305 (60–770) 310 (36–1433) 104 (42–237) 10 (3–26) 8 (1.9–21)

15/5 57 (40–79) 3 0 8 (5–13) / 60 (29–100) 33 (19–48) 81 (20–223) 53 (11–190) 308 (16–1618) 83 (41–140) 5.8 (3.8–8.3) 3.6 (1.8–5.9)

various cytokines. After 20 h of culture, cell-free supernatants were harvested and stored at 2708C until HGF assay.

2.7. Thirty minutes-blood PMN stimulation Highly purified blood PMN were resuspended in Hanks buffered salt solution at a density of 10 6/ml (HBSS-Gibco, Life Technologies, Grand Island, NY, USA). Part of the cell suspension was kept on ice, and the remainder was incubated at 378C for 10 min alone, then for 5 min with 5 mg/ml cyclochalasin B (Sigma Chemical Co, St Louis, MO, USA), and finally with or without 1 mM fMLP (fMet-Leu-Phe) for 10 min. All the tubes were then immediately centrifuged (400 £ g, 10 min, 48C) and the cell-free supernatants were collected and kept on ice. The cell pellets were washed with phosphate-buffered saline and centrifuged (400 £ g, 10 min, 48C); the supernatants were discarded and the PMN pellets were resuspended in RPMI 1640 medium (Biowhittaker). The supernatants and PMN pellets were stored at 2708C until HGF assay.

2.8. HGF assay HGF was measured by using a ELISA kit with a detection limit of 40 pg/ ml (Quantikine Human HGF kit, R&D Systems, Abingdon, UK). HGF was assayed in plasma, liver homogenates, and PMN culture supernatants and pellets. Cell pellets were sonicated three times (10 s, 48C) just before HGF assay.

patients with AH were not significantly different, and neither were those of the two cirrhotic control groups. The demographic, clinical and biochemical characteristics of these groups were thus combined and are shown in Table 1. Liver biopsy confirmed underlying cirrhosis in all the AH patients. During the study period, three patients with AH died, one on day 2 of massive gastrointestinal bleeding, one on day 7 of hepatocellular insufficiency and sepsis, and one on day 17 of diffuse alveolar hemorrhage. 3.2. Plasma and liver tissue HGF levels on day 0 As shown in Fig. 1, day-0 plasma HGF levels were significantly higher ðP , 0:00001Þ in the AH patients (6070 ^ 738 pg/ml) than in the two control groups (3243 ^ 438 pg/ml in AH-free cirrhotic patients, and 407 ^ 27 pg=ml in healthy volunteers). The difference between the AH-free cirrhotic patients and the healthy controls was also significant ðP , 0:0001Þ. HGF concentrations in liver tissue were significantly

2.9. Statistical analysis All results are expressed as means ^ standard error of the mean (SEM), or as ranges when appropriate. Because the variables studied were not normally distributed, non-parametric statistical methods were used; the Wilcoxon two-sample rank sum test was used to compare the values of continuous variables between two groups. When three or more groups were compared, the Kruskall–Wallis test was used. Associations between two continuous variables were evaluated with the Spearman rank correlation method. A paired t-test was used to compare day 0 and day 28 results. P values of ,0.05 were considered significant.

3. Results 3.1. Patients The characteristics of the two chronological groups of

Fig. 1. Mean and individual plasma levels of HGF in patients with AH, AH-free patients with cirrhosis, and healthy controls. *Significantly higher than healthy controls, P , 0.05; #significantly higher than cirrhotic controls, P , 0.05.

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Fig. 2. Mean and individual tissue levels of HGF in patients with AH and controls. *Significantly higher than controls (i.e. lobectomy specimens performed for hepatic metastasis of colorectal cancer; samples taken at least 3 cm from pathological lesions and considered as normal liver).

higher in patients with AH than in controls (1017 ^ 168 pg/ mg vs. 165 ^ 28 pg/mg of liver, respectively; P , 0.0001) (Fig. 2). 3.3. Hepatic PMN infiltration Hepatic PMN infiltration was always present in the 14 AH patients in whom hepatic HGF levels were also available. Five AH patients had mild PMN infiltration (grade 1), seven had moderate infiltration (grade 2) and two had marked infiltration (grade 3). 3.4. HGF release by 20 h-cultured blood PMN As shown in Fig. 3, basal HGF production by PMN cultured for 20 h was similar in the three groups. After exvivo stimulation by LPS and IFNg, HGF levels increased in the three groups but were significantly higher in the AH patients than in the healthy controls ðP , 0:05Þ. 3.5. HGF release by 30 min-stimulated blood PMN As an increasing number of cytokines are being described in the intracellular compartment of human PMN, we postu-

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Fig. 4. Effect of 30-min stimulation by cytochalasin B plus fMLP on HGF secretion by blood PMN from patients with AH, AH-free patients with cirrhosis, and healthy controls. HGF was assayed in cell-free supernatants. Values are means ^ SEM. *Significantly higher than in cirrhotics and healthy controls, P , 0.05.

lated that HGF could be stored as a preformed pool that could be mobilized upon stimulation. As shown in Fig. 4, after 30 min of stimulation with a combination of cytochalasin B and fMLP at 378C, HGF release by PMN ex vivo was significantly increased in AH patients compared with the other two groups ðP , 0:05Þ. HGF release was total, as no HGF was found in the cell pellets (data not shown). Incubation at 378C in the absence of stimuli induced little HGF release, as no HGF was detected in the supernatants when cells were maintained at 48C (Fig. 4). 3.6. Impact of 28-day corticosteroid therapy on HGF levels in patients with AH HGF levels were studied during corticosteroid therapy in the first 15 AH patients enrolled in the study. As shown in Fig. 5A, plasma HGF levels were unaffected by the treatment. Likewise, neither basal nor LPS 1 IFN g-induced HGF production by blood PMN cultured for 20 h differed between day 0 and day 28 (Fig. 5B). 3.7. Correlations

Fig. 3. Basal and LPS 1 IFNg-induced HGF release by blood PMN cultured for 20 h from patients with AH, AH-free patients with cirrhosis, and healthy controls. HGF was assayed in cell-free supernatants. Values are means ^ SEM. *Significantly higher than healthy controls, P , 0.05.

HGF plasma levels positively correlated with the serum bilirubin level, the prothrombin time and the Child–Pugh score (P ¼ 0:0003, 0.008 and 0.006; r ¼ 0:52, 0.24 and 0.42, respectively) in both alcoholic patient groups with or without AH. In the subgroup of patients with AH, some of these correlations were stronger (r ¼ 0:57 or 0.49, for bilirubin levels and prothrombin time, respectively). In AH patients, HGF plasma levels did not correlate with clinical outcome (6-month survival). In contrast, HGF plasma levels in these patients positively correlated with the amount of HGF release by 30 min-stimulated blood PMN (P ¼ 0:02 and 0.04; r ¼ 0:57 and 0.55, respectively). Finally, as

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Fig. 5. (A) Serial plasma HGF levels in patients with severe AH during 28-day corticosteroid therapy (n ¼ 15). Values are means ^ SEM. No significant change was observed during therapy. (B) Basal and LPS 1 IFNg-induced HGF release by blood PMN cultured for 20 h from patients with severe AH, before (day 0) and after a 28-day course of corticosteroids (day 28). Values are means ^ SEM. No significant difference was observed between day 0 and day 28.

shown in Fig. 6, hepatic HGF levels correlated with the degree of hepatic PMN infiltration (P ¼ 0:0015, r ¼ 0:76).

4. Discussion The pathophysiology of AH involves increased gutderived endotoxin translocation with subsequent Kuppfer cell activation, PMN recruitment and activation, and a cytokine cascade leading to liver injury [21]. Although the initial tissue injury may not be caused by PMN, AH-associated liver damage is thought to be strongly enhanced by liverinfiltrating PMN. Recent data suggest that PMN could also be involved in modulating inflammation and tissue repair in other inflammatory diseases [20,22,23]. Recovery from alcohol-related liver injury reflects the repair-enhancing capacity of several liver-derived mediators. In particular, HGF is involved in liver regeneration [1]. Our findings suggest for the first time that blood and liver PMN are a source of HGF in patients with alcoholic liver disease and may play a role in liver regeneration during AH. Plasma HGF levels were high in all the alcoholic patients we studied relative to the healthy controls. Although the

high interindividual variations well known in cytokine production, plasma HGF levels were significantly higher in patients with AH than in AH-free cirrhotic patients. These results are in keeping with previous studies [7–9]. Moreover, we found that HGF plasma levels in both groups correlated with several parameters currently used to evaluate the severity of liver injury, namely the serum bilirubin level, the prothrombin time, and the Child–Pugh score; stronger correlations were found in the AH group. Marked hepatic production of HGF was also observed, with HGF concentrations in liver homogenates six fold higher in AH patients than in controls. Previous in situ hybridization and immunohistochemical studies have suggested that the HGFproducing cells are Kuppfer cells and hepatic stellate cells [24–28]. In our study, liver homogenate HGF levels correlated with the degree of hepatic PMN infiltration, suggesting that PMN could also be a source of HGF particularly important in case of large PMN infiltrate. Human PMN can produce and release numerous cytokines and growth factors [29]. We used two complementary approaches to assess the potential role of PMN in HGF synthesis during AH, namely the capacity of blood PMN to release HGF in culture, and their capacity to release a preformed intracellular stock of HGF. Only blood PMN were studied for their HGF release capacity, as functional studies cannot be performed on liver PMN in humans. However, in endotoxic rats with and without acute ethanol intoxication, circulating PMN were found to be at least as activated as liver PMN [30,31]; moreover, a good correlation between blood and tissue PMN functions has been observed in several other injured tissues such as lung and gums, from which PMN can be readily obtained [32,33]. After 20 h of culture, we found that blood PMN from patients with AH, AH-free cirrhotic patients, and healthy controls were able to release HGF, but PMN from patients with AH were hyperresponsive to LPS 1 IFNg stimulation ex vivo. These results confirm the key role of primed PMN in cytokine production during AH, as already suggested by us [14]. Our second experimental approach, using fMLP 1 cytochalasin B (which stimulate degranulation of both specific and azurophil granules [34]), showed

Fig. 6. Correlation between HGF tissue levels and the degree of hepatic PMN infiltration in patients with AH. Hepatic PMN infiltration, assessed semi-quantitatively as: (1) mild; (2) moderate; or (3) marked, correlated positively with hepatic HGF concentrations (r ¼ 0.76, P , 0.05).

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that blood PMN from patients with AH were able to rapidly release a preformed pool of HGF ex vivo. Interestingly, HGF release after 30 min of stimulation was higher than that observed after 20 h of culture, possibly owing to HGF degradation by proteases in the culture medium. Moreover, the amount of HGF released after PMN degranulation ex vivo correlated with the corresponding plasma HGF levels, further supporting a role of PMN in HGF release into the circulation during AH. The respective importance of degranulation and de novo protein synthesis by normal human PMN is currently under investigation. Hepatic HGF levels correlated with the degree of hepatic PMN infiltration, suggesting that these cells are involved in the production of this cytokine. The consequences in patients with AH are in cascade. In addition to direct HGF production, PMN can release potent proteases leading to rapid secretion of HGF from the hepatic biomatrix [35]. Moreover, hepatocytes in normal liver are not ready to respond to mitogenic signals without a priming event such as collagenase [36,37]. It is also noteworthy that, through an autocrine mechanism, HGF can augment the fMLP-induced PMN oxidative burst by up to 200% [38] and that HGF promotes PMN adhesion and transmigration through cytoskeletal rearrangements and adhesion molecule modulation, particularly by increasing integrin avidity [39–41]. HGF could thus also increase the recruitment of activated PMN to the liver, thereby maintaining local inflammation. However, the major role of HGF in damaged liver is to promote development, regeneration and reconstruction of the normal liver structures. Among the mechanisms recently forwarded for the repair-promoting effects of HGF, induction of hepatocyte-derived metalloproteinases [42] and inhibition of hepatocyte apoptosis [4] seem to be of particular importance. Intravenous HGF injection limits tissue damage and improves animal survival numerous models of liver injury [2–4]. Thus, even if HGF may have a role in PMN recruitment and activation, its overall effect could be beneficial in AH. Recent studies have suggested that PMN may be involved in tissue recovery in other acute inflammatory diseases, via oncostatin M or VEGF secretion [20,23,43]. We studied HGF plasma levels and PMN HGF release during 28 days of corticosteroid therapy (the reference treatment for severe AH) as, in a previous study, we observed a normalization of PMN activation parameters and cytokine levels in AH patients [14]. However, in the present study we observed no significant change in HGF plasma levels or in PMN HGF release capacity ex vivo. Likewise, dexamethasone failed to modulate in vitro HGF release by PMN from healthy controls (data not shown). We can thus assume that the beneficial role of HGF in liver regeneration in patients with AH could be maintained during the 28 days of corticosteroid therapy. PMN migrating towards hepatic lesions are usually considered to be deleterious for adjacent tissues. However, our findings in patients with AH show that PMN participate

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in the systemic and, probably, the local secretion of HGF, a growth factor with antifibrotic properties involved in hepatic regeneration. We also show that corticosteroids, which normalize PMN proinflammatory functions, do not affect PMN HGF secretion or HGF plasma levels during severe AH.

Acknowledgements The authors thank David Young for linguistic advice. This work was supported by a grant from Schering Plough Research Institute, Kenilworth, NJ, USA.

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