Abstract
The aim of this study was to investigate the characteristics of the immune function of monocytes in different stages of the patients with acute on chronic liver failure (ACLF). Human leucocyte antigen (HLA)-DR and Toll-like receptor 4 (TLR-4) expression on monocytes in early and late stages of acute on chronic liver failure were detected by flow cytometry. The secretion function of monocytes was measured by cytometric bead array. Compared with healthy controls, the levels of HLA-DR expression on monocytes in patients with chronic hepatitis B, liver cirrhosis and acute on chronic liver failure were gradually decreased, especially in the late stage of acute on chronic liver failure (P < 0·001). TLR-4 expression on monocytes in patients with liver cirrhosis and acute on chronic liver failure were higher than the healthy controls. The concentrations of interleukin (IL)-1β, tumour necrosis factor (TNF)-α and IL-12p70 in early-stage ACLF were significantly higher compared with healthy controls and lower in late-stage ACLF (P < 0·01, 0·05). However, a significantly lower amount of IL-10 was found on monocytes in early-stage ACLF than that of late-stage ACLF and healthy controls (P < 0·01). Monocyte HLA-DR expression in patients who died was significantly lower compared with patients who survived in the early and late stages of ACLF (P < 0·01). The dynamic detection of HLA-DR expression or cytokines secreted from monocytes could contribute to the estimation of the status of the immune function of patients with acute on chronic liver failure.
Keywords: acute on chronic liver failure, HLA-DR, monocytes, Toll-like receptor
Introduction
Endotoxin syndrome is a systemic inflammatory response mediated by several of the cytokines produced by lymphocytes and macrophages [1]. It is a particularly grave complication, because bacteriologically proven infection occurs in up to 80% of patients with acute liver failure (ALF) [2]. The prevailing theory has been that sepsis represents an uncontrolled inflammatory response [3]. Recent studies have shown that sepsis may be characterized initially by increased inflammatory mediators, but as sepsis persists there is a shift toward an anti-inflammatory immunosuppressive state [4].
The incidence and significance of the systemic inflammatory response syndrome (SIRS) in acute liver failure has been studied by Rolando et al. [5]. In 887 patients with ALF, 504 (56·8%) patients manifested a SIRS during their illness. The SIRS in ALF, whether or not precipitated by infection, appear to be implicated in the progression of encephalopathy, reducing the chances of transplantation and conferring a poor prognosis. Some studies have demonstrated elevated levels of proinflammatory cytokines in patients with liver failure, which were correlated with microbial parameters and clinical complications [6–8].
Monocytes are central to SIRS secreting large quantities of proinflammatory cytokine and are responsible for antigen presentation through the surface expression of the human leucocyte antigen (HLA) class II molecule. Several investigators have shown that low levels of HLA-DR expression in patients with infection and sepsis are linked to recovery and mortality rates [9,10]. There is evidence showing that lipopolysaccharide (LPS)-stimulated whole blood from patients with sepsis releases markedly smaller quantities of the inflammatory cytokines than that of control patients [11].
Acute on chronic liver failure (ACLF) is believed to be an acute decompensation of chronic liver disease due to the effects of a precipitating event such as complications of sepsis, upper gastrointestinal (UGI) bleeding, ischaemia or additional superimposed liver injury due to alcohol, hepatotoxic drugs or hepatitis virus infection [12]. Wasmuth et al. [13] reported that patients with ACLF and severe sepsis show a similar degree of cellular immune depression. The reduced cellular immune function in subjects with ACLF could contribute to the increased infectious morbidity of these patients.
So far, dynamic changes in monocyte HLA-DR and TLR-4 expression and the secretion function of patients with ACLF has not been reported. In the present study, monocyte HLA-DR and TLR-4 expression and the secretion function in different stages of patients with ACLF were detected by flow cytometry and cytometric bead array(CBA). The correlation between recovery and mortality rates and the levels of HLA-DR expression was investigated.
Subjects and methods
Subjects
Blood samples were collected with informed consent from 23 patients with ACLF, 27 patients with chronic hepatitis B, 21 patients with liver cirrhosis and 25 healthy controls (Table 1). Those patients treated with anti-viral or immunomodulatory drugs in the most recent 6 months were excluded. All patients were seronegative for markers of hepatitis A, C, D and E viruses and HIV viruses. ACLF was defined as the following: (a) well-compensated chronic liver disease based on compatible laboratory data and sonographic findings; and (b) recent development of severe jaundice, coagulopathy and/or hepatic encephalopathy [13]. The emergence of more than grade II hepatic encephalopathy and/or other complications (such as severe haemorrhage, ascites, etc.) were defined as late-stage ACLF. The study protocol was approved by the local ethics committee.
Table 1.
Characteristics of HBV patients and controls.
| HC n = 25 | CHB n = 27 | LC n = 21 | ACLF n = 23 | |
|---|---|---|---|---|
| Sex | 17 male | 21 male | 18 male | 19 male |
| Age (years) | 33 ± 6 | 35 ± 7 | 42 ± 8 | 44 ± 11 |
| ALT (units/l) | n.a. | 289 ± 103 | 149 ± 72 | 345 ± 197 |
| HBV DNA (log copies/ml) | n.a. | 6·1 ± 2·7 | 3·8 ± 1·4 | 5·2 ± 2·3 |
| PT (%) | n.a. | 81·2 ± 9·3 | 53·4 ± 11·6 | 23·9 ± 7·1 |
| ALB (g/l) | n.a. | 39·2 ± 6·4 | 28·7 ± 4·7 | 31·2 ± 5·5 |
| TBil (μmol/l) | n.a. | 116·7 ± 57·8 | >39·4 ± 21·6 | 415 ± 167·2 |
HC, healthy controls; CHB, chronic hepatitis B; LC, liver cirrhosis; ACLF, acute on chronic liver failure; n.a., not applicable. ALT: alanine aminotransferase; ALB: albumin; PT: prothrombin time; TBil: total bilirubin.
Isolation and stimulation of monocytes
Peripheral blood mononuclear cells (PBMC) were isolated from fresh heparinized blood via Ficoll-Paque (StemCell Technologies Inc., Vancouver, BC, Canada) separation. Cells were washed twice with phosphate-buffered saline (PBS) and suspended in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS) for further use. PBMCs (1 × 106/well) were cultured in 24-well plates (Corning, Acton, MA, USA). After 3 h incubation, adherent cells were collected by washing with PBS. The purity of the cell fractions was determined by flow cytometry analysis with antibodies against CD14 (more than 85%). Monocytes were resuspended at 5 × 105/ml and cultured in 24-well plates (Corning). LPS (Sigma, St Louis, MO, USA) at 10 μg/ml was added. After 12 h incubation, microtiter plates were centrifuged and the supernatants were harvested, divided into aliquots, and kept at −70°C.
Cytokine bead assay
The quantification of interleukin (IL)-1, IL-6, IL-10, IL-12p70 and tumour necrosis factor (TNF)-α in the supernatants of cultured monocytes was determined by the human cytometric bead array kit (CBA; BD Bioscience, San Jose, CA, USA). The operations were performed according to the manufacturer's instructions. The intensity of the fluorescence signal was acquired on a fluoresence activated cell sorter (FACS) flow cytometer (BD Becton Dickinson), and analysed using CBA software. The concentration range for detection using this assay is 20–5000 pg/ml for each of the five cytokines.
Detection of HLA-DR and TLR-4 expression on monocytes
Quantitative HLA-DR and TLR-4 expression of monocytes was determined by flow cytometic assay (BD Becton Dickinson). Briefly, 100 μl of heparin-treated whole blood was added to 20 μl antibody, mixed and incubated for 30 min at room temperature. Erythrocytes were lysed by adding 2 ml of FACS lysing solution. The samples were measured on the FACS using cellquest software.
Statistical analysis
All values are expressed as mean ± standard deviation (s.d.). Data analysis was performed using spss version 11·0. One-way analysis of variance (anova) following the Student-Newman-Keuls (SNK)-q-test was used among groups. Student's t-test was used for impaired data. Spearman's correlation was used between variables. P-values < 0·05 were considered statistically significant.
Results
Expression of HLA-DR and TLR-4 on monocytes
The surface expression of HLA-DR and TLR-4 on monocytes was quantified in different categories of patients with chronic HBV infection and healthy controls by flow cytometry. As shown in Fig. 1a, a gradually lower percentage of HLA-DR was shown in chronic hepatitis B, liver cirrhosis and different stages of ACLF compared with healthy controls (84·1 ± 6·5%, 65·7 ± 11·8%, 53·2 ± 8·7% and 38·9 ± 9·3% versus 93·5 ± 2·4%; P < 0·001). As shown in Fig. 1b, patients with liver cirrhosis and different stages of ACLF showed a significantly higher percentage of TLR-4 compared with healthy controls (50·1 ± 16·9%, 47·2 ± 11·3% and 42·8 ± 10·4% versus 35·9 ± 9·8%; P < 0·01).
Fig. 1.
Expression of human leucocyte antigen (HLA)-DR and Toll-like receptor 4 (TLR-4) on monocytes in study groups. (a) HLA-DR; (b) TLR-4. The HLA-DR and TLR-4 percentage is the percentage of monocytes expressing HLA-DR and TLR-4 molecules within the gated CD14-positive population. HC, healthy controls; CHB, chronic hepatitis B; LC, liver cirrhosis; ACLF-E, early-stage acute on chronic liver failure; ACLF-L, late-stage acute on chronic liver failure.
Cytokine production after LPS stimulation
The production of cytokines after stimulation with LPS was measured using the CBA kit, as shown in Table 2. Concentrations of IL-1β, TNF-α and IL-12p70 in early-stage ACLF were significantly higher compared with healthy controls and lower in late-stage ACLF (P < 0·01, 0·05). However, significantly lower amounts were found of IL-10 on monocytes in early-stage ACLF than in late-stage ACLF and healthy controls (P < 0·01). Concentrations of IL-6 in the early stage of ACLF were slightly higher compared with healthy controls (P > 0·05) and lower in late-stage ACLF (P < 0·01)
Table 2.
Comparison of cytokines secreted on monocyte in study groups (pg/ml).
| Groups | n | IL-β | IL-6 | TNF-α | IL-12p70 | IL-10 |
|---|---|---|---|---|---|---|
| HC | 25 | 498·2 ± 167·9 | 3256·3 ± 1021·8 | 1890·3 ± 442·6 | 239·7 ± 54·8 | 67·2 ± 16·5 |
| CHB | 27 | 671·3 ± 218·6 | 3471·5 ± 890·4 | 1608·2 ± 599·3 | 172·6 ± 63·2 | 84·9 ± 23·6 |
| LC | 21 | 713·5 ± 239·2 | 2832·1 ± 768·3 | 1354·8 ± 323·4 | 153·2 ± 49·5 | 46·1 ± 11·2 |
| ACLF-E | 23 | 985·2 ± 196·8* | 3789·2 ± 1272·6 | 2305·4 ± 732·7† | 298·4 ± 76·9* | 53·9 ± 19·8* |
| ACLF-L | 23 | 378·3 ± 88·2‡ | 1821·4 ± 447·9‡ | 1173·1 ± 513·9‡ | 97·8 ± 38·3‡ | 78·3 ± 27·4‡ |
LC: liver cirrhosis; CHB: chronic hepatitis B; PT: prothrombin time; IL: interleukin; TNF: tumour necrosis factor. ACLF: acute on chronic liver failure; ACLF-E: early stage of ACLF; ACLF-F: late stage of ACLF.
P < 0·01 versus healthy controls (HC)
P < 0·05 versus HC
P < 0·01 versus ACLF-E.
Expression of HLA-DR on monocyte correlation with clinical parameters
Prothrombin time (PT) is one of the important indexes measuring the severity of hepatic injury. A significantly positive correlation was found between the expression of HLA-DR and PT (rs = 0·73, P < 0·01). Seventeen patients with infection and 27 patients with no infection were included among 44 patients with LC and ACLF. There was a significant difference in the expression of HLA-DR on monocytes between patients in the infective groups and patients in the non-infective groups (36·3 ± 6·6% versus 61·4 ± 12·9%, P < 0·01). Twenty-three patients with ACLF included 11 patients who died and 12 patients who survived. The monocyte HLA-DR expression in patients who died was significantly lower compared with patients who survived in the early and late stages of ACLF (39·4 ± 7·2% versus 65·9 ± 10·1%, 26·8 ± 6·4% versus 49·9 ± 11·7%; P < 0·01).
Discussion
The capacity of mononuclear cells to secrete cytokines ex vivo has been established as a marker of immune activation at the defined disease stage [14,15]. In the present study, we showed that the ex vivo production of IL-1β, TNF-α and IL-12p70 in early-stage ACLF were significantly higher compared with healthy controls and lower in late-stage ACLF. These results suggest that the function of monocyte-secreted inflammatory cytokines is enhanced in early-stage ACLF, but decreased in late-stage ACLF. IL-10 is one of the immunosuppressive mediators postulated to play an important role in down-regulation of the immune response. Elevated levels of IL-10 have been associated with increased mortality from septic shock [16,17]. A significantly lower amount of IL-10 secretion on monocytes was shown in early-stage ACLF compared with healthy controls; however, IL-10 secretion was restored in late-stage ACLF. The continued release of IL-10 may contribute to the impairment of monocyte proinflammatory cytokine release and the development of immune dysfunction in patients with ACLF.
The expression of MHC class II on monocytes is a prerequisite for effective antigen presentation to CD4+ T cells, an important component of the immune response to infection [18]. HLA-DR expression on peripheral blood monocytes has been found to correlate highly with infection in many clinical scenarios [19]. Recent reports show a markedly reduced expression of HLA-DR molecules on monocytes from patients with ACLF compared to healthy controls and patients with stable liver cirrhosis. A gradually lower percentage of HLA-DR has been shown in chronic hepatitis B, liver cirrhosis and different stages of ACLF compared with healthy controls (P < 0·001). Furthermore, a significant correlation was found between the expression of HLA-DR and severity index such as PTA (P < 0·01). The monocyte HLA-DR expression in patients who died was significantly lower compared with patients who survived in the early and late stages of ACLF (P < 0·01). These results suggest that the expression of HLA-DR molecules on monocytes might serve as dynamic prognosis markers not only in the early stage, but also in the late stage in patients with ACLF.
LPS recognition is mediated predominantly by TLR-4, which involves the binding of LPS with the LPS-binding protein and subsequently with CD14 that associates physically with a complex including TLR-4. Formation of the TLR-4-centred LPS receptor complex induces the production of proinflammatory cytokines through the MyD88 pathway [20,21]. Riordan et al. have reported that TLR-4 expression on peripheral blood monocytes (PBM) did not differ significantly between control and cirrhotic groups. In contrast, TLR-2 expression on PBM was increased significantly in patients with cirrhosis [22]. However, Mao et al. have reported that similar levels of TLR-2 and TLR-4 on PBM were shown in primary biliary cirrhosis and controls [23]. In the present study, patients with liver cirrhosis and different stages of ACLF showed a significantly higher percentage of TLR-4 compared with healthy controls. These results suggest that PBM from patients with ACLF might be more sensitive to injury induced by LPS. This disparity may be associated with the different aetiology of the liver cirrhosis and ACLF and/or different severe degrees of disease.
In conclusion, analysis of our data indicates that there were significant differences in cytokines secreted from monocytes during the different stages of patients with ACLF. Decreased monocytic HLA-DR expression seems to be a prognostic marker for survival of patients with ACLF. Dynamic monitoring of the immune status of ACLF may contribute to the estimation of risk of death in relation to other clinical or laboratory markers.
Acknowledgments
This study was supported by the National High Technology R&D Program of China (2003AA205150) and China Postdoctoral Science Foundation (2005037827).
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