Abstract
Serum levels and liver expression of CCL2 are increased in patients with alcoholic hepatitis (AH). In an experimental model of alcoholic liver disease (ALD), CCL2 was implicated in proinflammatory cytokines activation and hepatic lipid metabolism, but its role in human disease is currently unknown. In a large cohort of ALD patients, we analysed plasma levels and liver expression of CCL2 and their association with liver disease severity and histological lesions. We also studied the relationship between −2518 A > G CCL2 and CCR2 190 A/G polymorphisms and severity of ALD. We show that CCL2 plasma levels are increased in ALD patients compared with healthy subjects. AH patients had significantly higher plasma levels and hepatic expression of CCL2 than patients without AH. Plasma levels and hepatic expression of CCL2 were associated with disease severity. CCL2 liver expression was correlated with neutrophil infiltrate and interleukin (IL)-8 expression, but not with steatosis. Moreover, there were more G-allele carriers of −2518 A > G CCL2 polymorphism in severe AH patients than in other ALD patients. Our results demonstrate that CCL2 is increased in ALD, particularly in severe forms, and suggest a role for CCL2 in the pathogenesis of ALD via neutrophil recruitment.
Keywords: alcoholic hepatitis, alcoholic liver disease, CCL2/MCP-1, neutrophil
Introduction
Alcoholic liver diseases (ALD) are the most common cause of cirrhosis in the western world [1]. A subset of ALD patients will develop alcoholic hepatitis (AH) characterized by hepatocellular damage and liver neutrophil infiltrates [2]. Severe forms of AH are associated with poor short-term prognosis [3]. Moreover, AH is an independent predictive factor in liver fibrosis progression [4]. Treatments for ALD are currently limited, and better understanding of the pathogenesis of this disease may provide new therapeutic targets. Immune dysregulation occurs in ALD and serum levels of proinflammatory cytokines such as tumour necrosis factor (TNF)-α and interleukins (IL)-1 and -6 are associated with disease severity [5–7]. Chemokines are small proteins that direct the movement of circulating leucocytes to sites of inflammation and injury. CXC chemokines, including IL-8, attract neutrophils and are correlated with prognosis of patients with AH [8]. CCL2, also referred to as monocyte chemotactic peptide-1 (MCP-1), is a member of the beta (C-C) chemokine family. Its expression can be induced in many cell types, including inflammatory cells, hepatocytes and stellate cells [9,10]. CCR2 is the only known receptor for CCL2 and is expressed on monocytes, T lymphocytes and basophils [11,12]. CCL2 protein and mRNA liver expression have been reported previously in alcoholic liver disease [8,9,13]. In patients with AH, CCL2 plasma levels are increased, and spontaneous and/or lipopolysaccharide (LPS)-stimulated mononuclear cell secretion of CCL2 is higher in severe AH subjects than in healthy controls [14,15]. Moreover, a recent study has shown that CCL2-deficient mice are protected against alcoholic liver injury, independently of CCR2, by inhibition of proinflammatory cytokines and induction of genes related to fatty acid oxidation [16]. Therefore, in a large cohort of patients with biopsy-proven ALD, we analysed plasma levels and liver expression of CCL2 and studied their relationship with severity of liver disease and histological damage. Moreover, to emphasize the involvement of CCL2 in ALD in humans, we also studied the association between −2518 A > G CCL2 and CCR2 190 A/G polymorphisms and severity of ALD.
Materials and methods
Patients
CCL2 genotyping was performed on 235 consecutive ALD patients undergoing liver biopsy at our institution between 2003 and 2008. Patients suffering from ALD had a history of excessive alcohol ingestion of >30 g/day for males and >20 g/day for females in the absence of other causes of liver disease. The diagnosis of cirrhosis was based on liver biopsy or unequivocal clinical and biochemical data and compatible findings on imaging techniques. The presence of AH was based on histological definition [17,18]. Severe AH was defined as a modified Maddrey discriminant function (Mdf) higher than 32.
Frequencies of CCL2 genotypes were compared with those of 224 healthy controls without excessive alcohol intake, recruited from the Occupational Medicine Department. Patients and controls were European Caucasians.
Among these 235 ALD patients, we studied the 122 available plasma samples. Clinical characteristics of these patients are shown in Table 1. Snap-frozen liver fragments were available for 74 of these 122 ALD patients and included seven steatofibrosis, four steatofibrosis with AH, 27 cirrhosis and 36 cirrhosis with AH.
Table 1.
Characteristics of alcoholic liver disease (ALD) patients
| ALD (n = 122) | |||
|---|---|---|---|
| No AH foci (n = 49) | AH foci (n = 73) | P-value | |
| Age | 53·5 (29–78) | 54 (35–72) | 0·910 |
| Sex (F/M) | 12/37 | 24/49 | 0·288 |
| Cirrhosis (N/Y) | 4/45 | 9/64 | 0·443 |
| Child score (A/B/C)% (cirrhosis) | 27·5/42·5/30 | 13·3/33·3/53·4 | 0·07 |
| MELD score (cirrhosis) | 14 (6–32) | 16 (6–44) | 0·082 |
| Mdf (Mdf<32/Mdf ≥ 32) | 28·9 (0–119·7) (39/34) | ||
| Total bilirubin level (mg/dl) | 2·2 (0·4–12) | 3·9 (0·4–52) | 0·009 |
| INR* | 1·4 (0·9–3·3) | 1·5 (0·8–5·5) | 0·306 |
| Albumin level (g/dl) | 3·4 (0·7–4·9) | 3 (1·5–5·9) | 0·035 |
| ALT level (IU/l) | 29 (10–234) | 40 (14–137) | 0·051 |
| AST level (IU/l) | 48 (24–172) | 91·5 (25–333) | <0·001 |
| CRP (mg/dl) | 0·7 (0–17) | 3·1 (0·1–12) | <0·001 |
| WBC count (103 cells/mm3) | 6·5 (2·8–11) | 9·8 (3·1–24·9) | <0·001 |
| Neutrophil count (103 cells/mm3) | 3·7 (1·2–16·6) | 6·3 (1·1–20·9) | <0·001 |
| Hepatic venous pressure gradient (mm Hg) | 16 (2–26) | 16 (1–30) | 0·514 |
INR: International Normalized Ratio used to standardize blood coagulation test. AH: alcoholic hepatitis; ALT: alanine aminotransferase; AST: aspartate aminotransferase; CRP: C-reactive protein; F/M: female/male; N/Y: no/yes; Mdf: Maddrey discriminant function; MELD: model for end-stage liver disease; WBC: white blood cell.
To determine whether steroid therapy reduces CCL2 plasma levels, we quantified CCL2 plasma levels before and after 7 days of steroid therapy in 16 patients with severe AH.
The study was performed after approval by the Erasme Hospital Ethics Committee. Written informed consent was obtained from each participant (Clinical Trials number NCT01128010).
CCL2 plasma level measurements
Plasma was stored at −20°C until assay. CCL2 plasma level measurements were assayed by the enzyme-linked immunosorbent assay (ELISA) using the commercially available Quantikine assay system (R&D Systems, Abingdon, UK). This assay had a sensitivity of 5 pg/ml.
RNA extraction and RT-PCR
A snap-frozen fragment of each liver biopsy was stored at −80°C. Snap-frozen liver biopsies were crushed with a MagNalyser (Roche Diagnostics, Vilvoorde, Belgium). PolyA-mRNA was extracted using Magnapure (Roche Diagnostics) according to the manufacturer's instructions, including DNase treatment. RNA was quantified using a Lightcycler 480 system (Roche Diagnostics) with a one-step quantitative reverse transcription–polymerase chain reaction (qRT–PCR). Hypoxanthine–guanine phosphoribosyltransferase (HPRT) was used as a housekeeping gene. Primers and probes were designed with primer 3 software (Whitehead Institute for Biomedical Research, Cambridge, MA, USA): CCL2 sense 5′-ACTTCACCAATAGGAAG ATCTCAGT-3′; anti-sense 5′-TGAAGATCACAGCTTCTTTGG-3′; probe 5′-(6Fam)-TCGGGAGCTATAGAAGAATCACCAGCA-(Tamra)-3′; IL-8 sense 5′-CTCTCT TGGCAGCCTTCCT-3′; anti-sense 5′-TCTAAGTTCTTTAGCACTCCTTGG-3′; probe 5′-(6Fam)-TCTGCAGCTCTGTGTGAAGGTGCA-(Tamra)-3′. Copy numbers were calculated as described previously [19].
Liver histology
Paraffin-embedded liver biopsy sections were stained with haematoxylin and eosin and Sirius red. AH was defined by the presence of hepatocytes with ballooning degeneration with or without Mallory's hyaline surrounded by polymorphonuclear leucocytes [17,18].
Immunohistochemistry
Paraffin-embedded formalin-fixed liver biopsies were deparaffinized in xylene and rehydrated in graded alcohol and water. Tissue slides were incubated with monoclonal anti-myeloperoxidase (MPO) (clone 59A5, 1/200; Leica-Ménarini, Florence, Italy), anti-CD3 (clone PS1, 1/300; Leica-Ménarini) and anti-CD68 (clone KP1,1/1000; Dako, Glostrup, Denmark) antibodies for detection of neutrophils, T lymphocytes and macrophages, respectively. Diaminobenzidine (DAB) (Dako) was used as chromogen. Immunohistochemistry for IL-17 was performed as described previously [20]. The numbers of positive cells for different staining were counted by two independent investigators (D.D, L.V.) in a blinded manner on 20 fields per sample (original magnification ×400).
Flow cytometry analysis
Granulocyte pellets obtained after a Ficoll gradient of blood from ALD patients were depleted of erythrocytes by hypotonic saline lysis (NH4CL 15 mM, NaHCO310 mM, ethylenediamine tetra-acetic acid 0·1 mM, pH 7·4). Neutrophils were identified by their light-scattering properties and expression of CD15 and CD16. Surface staining was performed with phycoerythrin-labelled anti-CD15, fluorescein-isothiocyanate-labelled anti-CD16 and Alexa fluor-647-labelled anti-CCR2 (clones HI98, 3G8 and 48607, respectively; BD Biosciences, Erembodegem, Belgium) mouse anti-human antibodies. Cell analysis by flow cytometry was performed using FACScalibur (BD Biosciences). At least 100 000 events were acquired on gated neutrophils. Positive staining cut-off was determined in comparison to the control isotype (clones 27–35; BD Biosciences) following the manufacturer's instructions (BD Biosciences).
DNA isolation and CCL2 genotyping
For each patient, genomic DNA was isolated by the phenol–chloroform method [21] from a whole blood sample collected on the day of the liver biopsy. Twenty nanograms of DNA were used to assay CCL2 rs1024611 A > G with the TaqMan assay ID C_2590362_10 and CCR2 190 A/G rs1799864 assays (Applied Biosystems, Foster City, CA, USA) on a LightCycler® 480-real-time PCR System (Roche Diagnostics GmbH, Mannheim, Germany). We included DNA samples of known genotypes as internal positive and negative (water) controls to secure the genotyping procedure. Plates were run as follows: initial denaturation and enzyme activation at 95°C for 5 min, followed by 45 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 30 s. CCL2 rs1024611 polymorphism was determined by an allelic discrimination assay run on the LightCycler® 480-System (Roche Diagnostics). Allele frequencies were in Hardy–Weinberg equilibrium.
Statistical analysis
Data are expressed as medians (minimum–maximum). Multiple comparisons were performed using the Kruskal–Wallis test. The Mann–Whitney U-test was then used for post-hoc analysis. Non-parametric correlations were performed using the Spearman test. Results are shown as box-plots. Genotype frequencies are reported with their group percentages. A two-sided χ2 test was used for comparison of qualitative variables. Kaplan–Meir survival curves were compared using the log-rank test.
A P-value <0·05 was considered statistically significant. Calculations were performed with spss version 17·0 software (Chicago, IL, USA).
Results
Plasma levels and hepatic expression of CCL2 are increased in ALD and are associated with disease severity
CCL2 plasma levels were increased in patients with ALD [229·7 (20·4–1563) pg/ml; n = 122] compared to healthy subjects (HS) [139 (61·4–294·1) pg/ml; n = 10] (P = 0·003). Among ALD patients, those with AH had higher CCL2 plasma levels [284·5 (74·9–1563) pg/ml; n = 73] than those without AH [188·4 (20·4–523·2) pg/ml; n = 49] (P < 0·001), Fig. 1a. Patients with severe AH (Mdf ≥ 32) had higher CCL2 plasma levels than those with non-severe AH [368·2 (77·8–1563) pg/ml; n = 34]versus[245·8 (74·9–1371·4) pg/ml; n = 39] (P = 0·016), Fig. 1b. No difference in CCL2 plasma levels was observed between patients with cirrhosis [226·6 (20·4–1563) pg/ml; n = 109] and those without [280·9 (109·1–523·2) pg/ml; n = 13] (P = 0·526). CCL2 plasma concentrations showed an association with parameters of liver disease severity (Table 2a).
Fig. 1.
CCL2 plasma levels and liver expression in alcoholic liver disease (ALD) patients. (a) Plasma CCL2 levels were compared in 10 healthy subjects (HS) and 122 ALD patients measured by enzyme-linked immunosorbent assay (ELISA): 49 patients without alcoholic hepatitis (AH) and 73 patients with AH. Results are presented as box-plots; line represents median value; outer limits of the box represent 25th and 75th percentile values (*P < 0·05). (b) CCL2 plasma levels were compared in 34 patients with severe AH defined by a Maddrey discriminant function (Mdf) ≥32 and 39 patients with non-severe AH (*P < 0·05). (c) Liver expression of CCL2 was measured by quantitative reverse transcription–polymerase chain reaction (qRT–PCR) and was compared in 34 patients with AH and 40 patients without AH: results are presented as box-plots; line represents median value, and outer limits of the box represent 25th and the 75th percentile values (*P < 0·05). (d) Liver expression of CCL2 measured by qRT–PCR was compared in 18 patients with severe AH defined by an Mdf ≥ 32 and 17 patients with non-severe AH (*P < 0·05).
Table 2a.
Correlation of indexes of disease severity with CCL2 plasma concentrations in patients with alcoholic liver disease (ALD)
| r | P | |
|---|---|---|
| AST | 0·337 | <0·001 |
| ALT | 0·292 | 0·002 |
| Bilirubin plasma level | 0·262 | 0·004 |
| INR | 0·061 | 0·513 |
| MELD | 0·235 | 0·015 |
| Mdf | 0·305 | 0·009 |
| HVPG | −0·130 | 0·166 |
ALT: alanine aminotransferase; AST: aspartate aminotransferase; INR: International Normalized Ratio; Mdf: Maddrey discriminant function; MELD: model for end-stage liver disease; HVPG: hepatic venous pressure gradient.
We also performed a qRT–PCR for CCL2 on mRNA extracts obtained from transjugular liver biopsies. CCL2 plasma levels were correlated with liver CCL2 mRNA (r = 0·288 P = 0·033). Liver CCL2 mRNA levels were higher in patients with AH [6·4 102 (44–1·1 104) mRNA copies/105 copies HPRT] than in those without AH [2·2 102 (3·5-2·4 103) mRNA copies/105 copies HPRT] (P < 0·005), Fig. 1c. Among patients with AH, those with an Mdf ≥ 32 had higher liver CCL2 mRNA expression than those without severe AH [1·4 103 (43·9-1·1 104)]versus[2·9 102 (96·3–3·1 103) mRNA copies/106 copies HPRT] (P = 0·005), Fig. 1d. There was no statistically significant difference in CCL2 liver expression between cirrhotic patients [4·4 102 (26·5-1·1 104) mRNA copies/106 copies HPRT; n = 62] and those without cirrhosis [2·4 102 (3·5-3·1 103) mRNA copies/106 copies HPRT; n = 12] (P = 0·071). Liver CCL2 mRNA expression also showed an association with parameters of disease severity (Table 2b).
Table 2b.
Correlation of indexes of disease severity with liver CCL2 expression
| r | P | |
|---|---|---|
| AST | 0·377 | 0·002 |
| ALT | 0·112 | 0·367 |
| Bilirubin plasma level | 0·468 | <0·001 |
| INR | 0·404 | 0·001 |
| MELD | 0·403 | 0·002 |
| Mdf | 0·464 | 0·005 |
| HVPG | 0·248 | 0·036 |
ALT: alanine aminotransferase; AST: aspartate aminotransferase; INR: International Normalized Ratio; MELD: model for end-stage liver disease; Mdf: Maddrey discriminant function; HVPG: hepatic venous pressure gradient.
We studied plasma levels and hepatic CCL2 expression according to short-term prognosis defined by 90-day survival. We did not find higher plasma levels in patients who died within 90 days [2·1 102 (90·5–1·6 103) pg/ml; n = 12] compared to those who survived [2·3 102 (20·4-1·4 103) pg/ml; n = 79] (P = 0·769). Nor was CCL2 liver expression higher in patients who died within 90 days [3·5 102 (38·6-1·1 104) mRNA copies/106 copies HPRT; n = 11] than in those who survived [3·1 102 (3·5–4·3 103) mRNA copies/106 copies HPRT; n = 51] (P = 0·950).
We sought to determine whether steroid therapy reduces CCL2 plasma levels, and we showed a trend towards decreased CCL2 plasma levels after 7 days of treatment (P = 0·056) (Supplementary Fig. S1).
In the liver, CCL2 expression is correlated with IL-8 expression and neutrophil infiltrates
To further unravel the role of CCL2 in the pathogenesis of ALD, we quantified inflammatory infiltrates of liver biopsy for which we had performed qRT–PCR for CCL2 (n = 74) (Fig. 2). Liver CCL2 mRNA levels in ALD patients were correlated specifically with neutrophil infiltrates (r = 0·411; P < 0·005), Fig. 3a, but neither with T lymphocyte nor with mononuclear cell infiltrates [(r = 0·226; P = 0·058) and (r = −0·229; P = 0·055), respectively]. Moreover, we showed that liver CCL2 mRNA expression was correlated highly with liver IL-8 mRNA levels (r = 0·895; P < 0·001), Fig. 3b. As expected, IL-8 mRNA levels were correlated with neutrophil infiltration (r = 0·446; P = 0·002), Fig. 3c.
Fig. 2.
Immunohistochemical analysis of (a) monoclonal anti-myeloperoxidase (MPO), (b) CD3, (c) CD68 and (d) interleukin (IL)-17 liver expression (×200). This case is representative of alcoholic hepatitis (AH) patients.
Fig. 3.
Correlation between CCL2 liver expression, neutrophil infiltrates and IL-8 liver expression. Correlation between (a) liver CCL2 expression and neutrophil infiltrates, (b) CCL2 and interleukin (IL)-8 liver expression and (c) IL-8 liver expression and neutrophil infiltrates. CCL2 and IL-8 liver expression were measured by quantitative reverse transcription–polymerase chain reaction (qRT–PCR), while neutrophil infiltrates were quantified by immunohistochemistry using anti-myeloperoxidase antibodies.
To determine whether CCL2 plays a role in neutrophil recruitment, we analysed circulating neutrophils of ALD patients (alcoholic cirrhosis with or without AH) by flow cytometry and we found that these cells did not express CCR2, Fig. 4.
Fig. 4.
CCR2 expression in circulating neutrophils. CCR2 expression in circulating neutrophils was analysed by flow cytometry and compared to the control isotype. Data are from one alcoholic donor with cirrhosis and were representative of seven experiments on five cirrhotic patients without alcoholic hepatitis (AH) and two with AH. These data are comparable to those obtained in healthy subjects (data not shown).
Because T helper type 17 (Th-17) cells play a role in neutrophil recruitment and express CCR2 [22], we evaluated, by immunohistochemistry, liver expression of IL-17 in patients for whom we had performed quantification of liver CCL2 mRNA. We found that CCL2 liver expression was associated with the number of IL-17+ cells (r = 0·339; P = 0·013). Moreover, Il-17+ cell infiltrates were correlated strongly with neutrophil infiltrates (r = 0·715; P < 0·001) and with IL-8 liver expression (r = 0·346; P = 0·038).
CCL2 mRNA liver expression was not correlated with the degree of steatosis (r = 0·057 P = 0·637).
G-allele carriers of functional – 2518 A > G CCL2 promoter polymorphism were more frequent among patients with severe AH
We performed −2518 A > G CCL2 genotyping in 235 patients with ALD (109 cirrhosis without AH, 84 cirrhosis with AH, 13 steatofibrosis with AH and 29 steatofibrosis) and in 224 healthy controls. The frequency of G-carriers did not differ between patients with ALD and healthy controls (P = 0·442). Frequencies of individual genotypes were similar to those reported previously in other Caucasian control populations [23–25]. We observed more G-allele carriers in the severe AH patient group than in other ALD patients. Moreover, among AH patients, the G-allele was more frequent in the severe form of the disease (Table 3a). However, the CCL2 polymorphism −2518G-allele was not associated with patient survival. Indeed, there was no difference in 90-day survival between G-carriers and non-G-carriers patients in the entire population of ALD (88·1% ± 3·5% versus 88·4% ± 3·2%, P = 0·909), nor in a subgroup of patients with alcoholic hepatitis (83·8% ± 5·6% versus 81·6% ± 5·6%, P = 0·792) and severe alcoholic hepatitis (75·9% ± 9·4% versus 64·3% ± 12·8%, P = 0·528). We performed CCR2 190 A/G polymorphism genotyping in this cohort of ALD patients and we found no difference between genotypes (Table 3b).
Table 3a.
Frequency of −2518 CCL2 polymorphism G-carriers
| % of G-non-carriers | % of G-carriers | P | |
|---|---|---|---|
| ALD patients (n = 235)/controls (n = 224) | 57·4/52·7 | 42·6/47·3 | 0·44 |
| Cirrhosis (n = 193)/no cirrhosis (n = 42) | 57·5/57·1 | 42·5/42·9 | 0·965 |
| AH (n = 97)/no AH (n = 138) | 55·7/58·7 | 44·3/41·3 | 0·644 |
| Severe AH (n = 38)/non-severe AH (n = 43) | 42·1/65·1 | 57·9/34·9 | 0·038 |
| Severe AH (n = 38)/other ALD (n = 197) | 42·1/60·4 | 57·9/39·6 | 0·037 |
AH: alcoholic hepatitis; ALD: alcoholic liver disease.
Table 3b.
Frequency of V64I CCR2 polymorphism-A-carriers
| % of A-non-carriers | % of A-carriers | P | |
|---|---|---|---|
| ALD patients (n = 235)/controls (n = 224) | 81·9/78·6 | 18·1/21·4 | 0·415 |
| Cirrhosis (n = 193)/no cirrhosis (n = 42) | 83·8/80 | 16·2/20 | 0·498 |
| AH (n = 97)/no AH (n = 138) | 84·5/80·6 | 15·5/19·4 | 0·51 |
| Severe AH (n = 38)/non-severe AH (n = 43) | 92·3/81·6 | 7·7/18·4 | 0·214 |
| Severe AH (n = 38)/other ALD (n = 197) | 92·3/81·1 | 7·7/18·9 | 0·105 |
AH: alcoholic hepatitis; ALD: alcoholic liver disease.
Discussion
In the present study, we show that plasma levels and hepatic expression of CCL2 are increased in a large cohort of biopsy-proven ALD patients, particularly those with severe AH. Interestingly, this CCL2 over-expression is associated with parameters of disease severity such as hepatic venous pressure gradient and model for end-stage liver disease (MELD) score. We found no relationship between plasma levels or hepatic expression of CCL2 and 90-day survival. Nevertheless, these results should be viewed with caution, as many patients were lost to follow-up. We also measured CCL2 plasma levels in patients with severe AH before and after 7 days of steroid therapy, and we showed a trend towards decreased CCL2 plasma levels after treatment. However, the reason why the CCL2 plasma level decreased after steroid treatment is not clear, and further studies on a large cohort of AH patients are required.
Moreover, we demonstrated that CCL2 liver expression is correlated with neutrophil infiltrates and IL-8 liver expression. CCL2 is a CC chemokine which is chemotactic for monocytes and lymphocytes. Arguments in the literature suggest that, under inflammatory conditions, neutrophils undergo phenotypic changes enabling them to respond to chemokines that are functionally inactive under resting conditions [26,27]. However, we showed that circulating neutrophils of ALD patients did not express CCR2, suggesting that CCL2 does not directly recruit neutrophils via this receptor. Nevertheless, CCL2 could play a role in neutrophil recruitment via a receptor other than CCR2; indeed, a recent study showed, in an experimental model of ALD, that CCL2-deficient mice were protected against alcoholic liver injury independently of CCR2. Interestingly, KC/IL-8 mRNA liver expression was decreased significantly in alcohol-fed CCL2-deficient mice [16]. In agreement with those results, but in humans, we show a very strong correlation between CCL2 and IL8 mRNA liver expression.
Another hypothesis is that CCL2 plays a role in the pathogenesis of ALD by recruiting CCR2+-expressing cells. These cells could, in turn, recruit neutrophils. Because livers of ALD patients, particularly those with AH, are infiltrated by IL-17+ cells [20], and because Th-17 cells play a role in neutrophil recruitment and express CCR2 [22], we correlated CCL2 liver expression with IL-17+ cell infiltrates. We found that CCL2 liver expression was correlated with numbers of IL-17+ cells. Furthermore, IL-17+ cell infiltrates were correlated strongly with neutrophil infiltrates and with IL-8 liver expression. These results suggest that CCL2 plays a role in the pathogenesis of ALD by recruitment of Th17 cells which, in turn, would recruit neutrophils via an IL-8 effect. However, IL-17+ cell infiltrates may, in part, reflect neutrophil infiltrates. Indeed, we have shown previously, using confocal microscopy, that among liver-infiltrating IL-17+, T lymphocytes and neutrophils were represented most frequently [20].
As each AH episode is thought to be profibrogenic [4], we speculate that CCL2 secreted during the AH inflammatory burden could enhance the fibrogenesis process. However, we found no difference in liver CCL2 expression between ALD patients with and without cirrhosis; nevertheless, this result should be viewed with caution, as non-cirrhotic patients in our cohort were scarce.
We found no correlation between CCL2 liver expression and hepatic steatosis in our patient cohort, whereas CCL2 was involved in hepatic lipid metabolism in an experimental model of alcoholic liver disease [16]. This relationship between CCL2 liver expression and steatosis may be present in the beginning of ALD, but not in severe disease such as cirrhosis.
Patients with the G-allele for −2518 A > G CCL2 polymorphism were present more frequently in the severely ill AH group than in other ALD patients. Moreover, among AH patients, the G-allele was more frequent in the severe form of the disease. It was shown previously that the presence of the −2518 G-allele resulted in significantly greater CCL2 secretion than that found in patients with the A/A homozygous genotype in response to a given inflammatory stimulus [23], and this polymorphism has been implicated in numerous inflammatory diseases, including hepatitis C, acute pancreatitis, Crohn's disease and, more recently, spontaneous bacterial peritonitis [24,25,28,29]. However, we did not find higher CCL2 plasma levels or liver expression in G-allele carriers in our cohort of patients (data not shown). It is possible that G-allele carriers are more likely to develop a severe form of AH, but that the levels of CCL2 at the time of alcoholic hepatitis are the same as in G-non-carriers.
Our finding suggests that G-allele carriers are more likely to develop a severe form of AH than patients without the G-allele when exposed to alcohol.
This result reinforces the potential role of CCL2 in the pathogenesis of ALD, but this observation needs to be validated in a larger independent cohort of alcoholic patients. We did not find any association between CCR2 190 A/G polymorphism and ALD severity. In line with these results, it was demonstrated recently in an animal model that CCL2 plays a role in alcoholic liver injury independently of CCR2 [16].
In conclusion, plasma levels and hepatic expression of CCL2 are increased in patients with ALD, particularly in severe forms of AH. Our results further support the potential role of CCL2 in the pathogenesis of ALD, probably through neutrophil recruitment. CCL2 may in the future constitute an attractive therapeutic target in patients with severe AH.
Acknowledgments
This study was supported in part by grants from the Erasme Foundation and from the Belgian National Fund for Scientific Research (FNRS). A. Lemmers is a post-doctoral researcher and R. Ouziel is a research fellow; D. Degré is an MD postdoctoral fellow (FRSM). The authors thank I. Roland for help in treating pathological tissues.
Disclosure
None of the authors has any potential financial conflict of interest related to this manuscript.
Supporting Information
Additional Supporting Information may be found in the online version of this article:
Fig. S1. Evolution of CCL2 plasma levels after 7 days of steroids therapy in 16 patients with severe alcoholic hepatitis (AH).
Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
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