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Abstract

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Systemic inflammation and susceptibility to developing sepsis is common in acute liver failure (ALF) resulting in tissue damage and organ failure. This study characterized the function of circulating neutrophils in 25 patients with ALF and subacute liver failure (SALF). ALF (n = 15) / SALF (n = 10) patients were prospectively studied and compared with 11 healthy (HC) and 6 septic controls (SC). Neutrophils were isolated on admission to intensive care and every 3-4 days until death / liver transplantation / recovery. Neutrophil phenotype was determined using fluorochrome-labeled antibodies to CD16 and CD11b and assessed by flow cytometry. Neutrophil phagocytic activity (NPA) was determined using fluorescein isothiocyanate-labeled opsonized Escherichia coli and oxidative burst (OB) was determined by the percentage of neutrophils producing reactive oxygen species (ROS) at rest and after stimulation with opsonized E. coli. Physiological variables, biochemistry, arterial ammonia, microbiology, and outcomes were collected. Plasma pro- and antiinflammatory cytokine profiles were performed by enzyme-linked immunosorbent assay. Neutrophil expression of CD16 which recognizes the FcγRIII region of immunoglobulin G was significantly reduced in the ALF cohort (P < 0.001) on day 1 compared to HC. NPA was significantly impaired in the SALF cohort compared to HC (P < 0.01). Impaired NPA in the ALF and SALF cohorts on admission predicted nonsurvival without liver transplantation (P = 0.01). Spontaneous neutrophil production of ROS was not significantly increased in any of the cohorts. E. coli-stimulated OB was preserved in ALF/SALF cohorts but was significantly impaired in the SC group (P < 0.05). Conclusion: Circulating neutrophils in ALF/SALF have impaired bacteriocidal function similar to that seen in severe sepsis. Neutrophil function indices are important biomarkers in ALF and may be implicated in the development of organ dysfunction and the increased susceptibility to developing sepsis. (HEPATOLOGY 2013)

Acute liver failure (ALF) is a rare but frequently catastrophic consequence of an acute primary hepatic injury arising from a wide variety of insults. It is characterized by coagulopathy and encephalopathy, with a variable dynamic of progression to multiple organ dysfunction syndrome (MODS) and death.1 Liver transplantation (LT) remains the only curative option for advanced ALF with poor prognostic criteria and contributes to ∼10% of LT in the Western world.2

Neutrophils are a major innate immune cell subset involved in the first line of defense against infection. Neutrophils are produced in vast numbers in the bone marrow (1-2 × 1011 per day) and have a short half-life of 12-18 hours. They are rapidly recruited to sites of infection and inflammation. Neutrophils phagocytose invading microbes3 and proceed to kill them by generating superoxide anions and hydrogen peroxide along with other reactive oxygen species (ROS) through activation of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, a process termed respiratory or oxidative burst (OB).4 The OB products are effective in eradicating invading microorganisms, but unfortunately may damage “innocent bystanders,” leading to tissue destruction, inflammation, and organ failure. Neutrophils possess receptors for the Fc region of immunoglobulin G (FcγRIII/CD16 and FcγRII/CD32) and for complement molecules such as iC3b (MAC-1/CD11b-CD18), which bind to the surface of the microbe (opsonization). Complement-opsonized particles are gently internalized within the neutrophil with Fcγ receptor ligation augmenting the process through the extension of pseudopods which surround and engulf the microbe.3

Neutrophils are rapidly recruited to the liver in response to hepatic injury in ALF,5 and once there they become activated by cytokines (e.g., interleukin [IL]-8 and tumor necrosis factor alpha [TNF-α]), and may contribute to further tissue damage by release of proteolytic enzymes and ROS.6 An exaggerated systemic inflammatory response (SIRS) is frequently present in ALF and increasingly it is being recognized to play a key role in the pathogenesis and outcome.7 Systemic neutrophil activation with associated immune paresis is well recognized in severe sepsis, a condition that shares many phenotypic features with ALF including microvascular dysfunction, hemodynamic instability, coagulopathy, encephalopathy, and high levels of both proinflammatory and antiinflammatory cytokines.8 In severe sepsis excessive activation of neutrophils has been implicated in the pathogenesis of acute lung and kidney injury.9 Neutrophils might therefore serve as critical effector cells of the progressive parenchymal liver damage and MODS in ALF.

There is a high incidence of bacterial and fungal infection early in the course of ALF (10) which may preclude listing for LT. ALF is also associated with an acute and often precipitous increase in plasma ammonia levels.2 A recent study has shown that neutrophils exposed to ammonia have reduced phagocytic activity of opsonized E. coli and high spontaneous production of ROS, suggesting a direct toxic effect of ammonia on neutrophils.11 Neutrophil dysfunction has also been previously reported in ALF with reduced complement expression,12 impaired neutrophil adhesion,13 decreased production of ROS,14 and decreased neutrophil phagocytosis and intracellular killing.

We postulate that circulating neutrophil dysfunction is present in ALF and may add value as a prognostic marker of severity and outcome. The aim of this prospective case-control longitudinal study was therefore to characterize circulating neutrophil phenotype, phagocytic activity (NPA) and production of ROS in neutrophils isolated from the peripheral blood of patients with ALF and comparing it to healthy (HC) and septic controls (SC). Indices of neutrophil phenotype and function were examined with respect to severity and nature of liver injury, severity of organ failure, liver prognostic criteria of survival, and eventual outcome. The relationship between plasma-derived factors and neutrophil function was also examined in order to aid identification of other associated biomarkers in ALF.

Patients and Methods

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Study Design.

A cross-sectional case-control cohort study was performed. Patients with ALF (n = 15) and subacute liver failure (SALF) (n = 10) were prospectively studied. Neutrophil phenotype, NPA, and OB (spontaneous and stimulated with opsonized E. coli) were determined and compared to n = 11 HC and n = 6 SC. The dynamics of neutrophil function during the course of the illness were compared between patient groups and in relation to those who survived compared to those who did not survive. Patients who were transplanted were considered nonsurvivors. Baseline sampling was performed within 24 hours of admission to an intensive care (ICU) and every 3-4 days until spontaneous recovery, death, or LT. In those who underwent LT further sampling was performed 72 hours post-LT. Subjects were followed up for 90 days.

Patients.

Twenty-five patients with ALF or SALF were recruited nonconsecutively on admission to the liver ICU at King's College Hospital between October 2008 and August 2010. ALF was defined by the onset of hepatocellular dysfunction in the absence of preexisting liver disease characterized by coagulopathy and encephalopathy and an illness of less than 26 weeks duration. ALF was further subclassified according to the criteria defined by O'Grady et al.15 depending on the time between the onset of jaundice and encephalopathy. (1) Hyperacute (jaundice to encephalopathy time <7 days) consisting predominantly of patients with acetaminophen-induced liver failure (AALF). (2) Acute liver failure (jaundice to encephalopathy time 8-28 days) typified by patients presenting with fulminant viral hepatitis. (3) SALF (jaundice to encephalopathy time 5-12 weeks) typified by those presenting with nonacetaminophen drug-induced liver injury and seronegative/acute autoimmune hepatitis.

Inclusion Criteria.

Patients with ALF/SALF were included if they were age >18 years and <80 years. Healthy age- and sex-matched nonsmoking volunteers with no history of liver disease were used as HC. The HC alcohol intake was <20 g/day and volunteers had not drunk alcohol or exercised excessively in the 24 hours prior to blood being drawn. SC patients were recruited from the general ICU and had severe sepsis with MODS. Severe sepsis was defined by the presence of an SIRS score ≥2,16 with radiological and/or laboratory evidence of infection and one or more extrahepatic organ failure(s). Patients presenting with ALF/SALF were given empirical intravenous antibiotic and antifungal cover as standard of care. This consisted of tazocin 4.5 g every 8 hours (substituted for meropenem 1 g every 8 hours if penicillin allergic) and fluconazole 400 mg once daily.

Exclusion Criteria.

Patients were excluded from the ALF/SALF cohorts if on presentation they had evidence of bacterial, fungal, or viral infection on clinical examination, radiological or laboratory investigation, malignancy, and any coexisting history of immunodeficiency including human immunodeficiency virus (HIV) and glycogen storage disease. Patients with preexisting liver disease, a history of alcohol intake >20g/day, or who were on immunosuppressive therapies such as steroids or azathioprine were also excluded.

Consent and Data Collection.

The study was performed in accordance with the Declaration of Helsinki and ethical permission was granted from the North East London Research Ethics Committee (Ref. No. 08/H0702/52). Following obtaining fully informed consent/assent, clinical, biochemical, and physiological data were collected. Data included tobacco and alcohol use, arterial ammonia (μmol/L), serum sodium levels (mmol/L), arterial blood gas analysis including lactate (mmol/L), differential leukocyte count (×109), complement, and immunoglobulin and lipoprotein levels. SIRS score16 was also calculated on admission and on subsequent neutrophil sampling days. A number of organ failure scores were also quantified including the model of endstage liver disease score (MELD), sequential organ failure assessment score (SOFA),17 and the acute physiology and chronic health evaluation (APACHE) II score.18 Length of ICU stay, survival, and number of days requiring vasopressors, ventilation, or hemofiltration were also recorded.

Antibiotic use and details of potentially immunomodulatory therapies such as corticosteroids, hypothermia, hemofiltration, and plasmapheresis were recorded. The occurrence of bacterial and fungal infection was recorded along with other relevant patient outcomes including the development of organ failure and 90-day survival.

Blood Sampling.

Venous blood was collected aseptically from patients/volunteers into heparinized pyrogen-free tubes and was immediately precooled to 0-4°C for 10 minutes. Neutrophil phenotype and function test analyses were performed within 1 hour of blood being drawn. Plasma was obtained by centrifugation at 4,500 rpm for 10 minutes at 4°C and stored at −80°C for subsequent cytokine determination by enzyme-linked immunosorbent assay (ELISA).

Characterization of Neutrophil Phenotype.

Polymorphonuclear granulocytes (PMN) were isolated using a blood lysis method. One hundred microliter aliquots of whole blood were placed in flow cytometry tubes and 1 mL of lysis solution (containing <15 mL formaldehyde and <50 mL diethylene glycol; Becton Dickinson, UK) was added to each tube at room temperature for 15 minutes. The solution was centrifuged at 1,600 rpm for 5 minutes at 18°C and the supernatant discarded leaving the PMN pellet at the bottom of the tube. Twenty microliters of each of two antibody stains (antihuman CD16-phycoerythrin(PE)IgG1κ and anti-human CD11b-APCCy7IgG1κ; Becton Dickinson, UK) was then added and incubated at room temperature in darkness for 25 minutes. The cells were then washed twice with sterile phosphate-buffered saline (PBS) prior to analysis by fluorescence activated cell sorting (FACS) using a FACS Canto II analyzer and FACS Diva 6.0 software (Becton Dickinson, San Jose, CA). Neutrophils were gated from the PMNs on forward and side-scatter characteristics and the percentage of CD16/CD11b-positive cells were calculated along with the mean fluorescence intensity (MFI).

Ex Vivo Neutrophil Function Studies.

The neutrophil function studies were performed as described.11 All ex vivo studies were performed in pyrogen-free conditions. Neutrophil function was examined in fresh neutrophils isolated from whole blood to more closely resemble physiological conditions and to prevent neutrophil activation during separation, with all samples being performed in triplicate.

(1) Phagocytic Activity.

Phagocytosis was quantified using the Phagotest (Orpegen Pharma), which uses fluorescein isothiocyanate (FITC)-labeled opsonized E. coli bacteria and analyzed using flow cytometery. In brief, 100 μL of heparinized whole blood was mixed with 20 μL of FITC-labeled opsonized E. coli (2 × 107) (opsonized with immunoglobulin and complement of pooled sera) and incubated in a water bath at 37°C for 20 minutes. Fluorescence of bacteria at the cell surface was quenched using ice-cold Trypan blue solution. Red blood cells were lysed and PMNs were washed twice with sterile PBS prior to analysis. Neutrophils were gated on forward and side-scatter characteristics and stained with anti-CD16-Phycoerythrin(PE)IgG1 κ and analyzed by FACS. NPA was expressed as the percentage of neutrophils undergoing phagocytosis along with the MFI. The interassay and intraassay coefficient of variance for triplicate samples were 1.6% and 10.1%, respectively.

(2) Oxidative Burst.

Neutrophil OB was quantified using the Burstest (Orpegen Pharma) which measures the percentage of phagocytic cells that produce ROS. In brief, 100 μL of heparinized whole blood was incubated for 20 minutes with 20 μL of opsonized E. coli (2 × 107), or without stimulus at 37°C. Neutrophil high burst capacity was assessed by adding 5 μL of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, to 100 μL of heparinized whole blood. Neutrophil low burst was assessed by adding 5 μL of the chemotactic synthetic peptide formyl-Met-Leu-Phe (fMLP) for 20 minutes at 37°C. fMLP is a synthetic peptide that mimics the activity of bacterially derived peptides with formylated N-terminal methionine groups. The formation of ROS was detected using the oxidation of dihydrorhodamine-123 to rhodamine-123 which emits green fluorescence. Red blood cells were lysed and PMNs were washed with sterile PBS prior to analysis. Neutrophils were gated on forward and side-scatter characteristics and stained with anti-CD16-Phycoerythrin(PE)IgG1 κ and analyzed by FACS. OB was determined by the percentage of CD16-positive cells producing ROS, which was calculated along with the MFI. The interassay coefficient of variance was 4.7% and 2.4% for spontaneous and stimulated OB, respectively. The intraassay coefficient of variance was 5.4% and 4.2% for spontaneous and stimulated OB, respectively.

Cytokine Measurement.

Plasma levels of the pro- and antiinflammatory cytokines (TNF-α, IL-1β, IL-6, CXC8/IL-8, IL-10, and IL-17) were determined from samples previously stored at −80°C using sandwich ELISA (R&D Systems DuoSets, UK).

Statistics.

Where appropriate, values are expressed as median and interquartile range (IQR). Group comparisons were performed using the chi-squared test for categorical and Mann-Whitney U test for continuous variables. When comparing three or more groups simultaneously, the Kruskal-Wallis test was utilized with Dunn's multiple comparison test. Comparisons of paired observations were performed using Wilcoxon matched pairs test. P < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism 4.0 (GraphPad Software, San Diego, CA).

Results

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Patient Baseline Demographics and Clinical Parameters.

Fifteen nonconsecutive patients with ALF and 10 patients with SALF were recruited. Baseline (on admission to ICU) patient demographics, biochemical, and physiological parameters are detailed in Tables 1 and 2, respectively. The ALF group was heterogeneous in terms of etiology and severity of liver injury (acetaminophen n = 6; acute viral hepatitis n = 3; other n = 6). The predominant etiology in SALF was seronegative/acute autoimmune hepatitis n = 7.

Table 1. Baseline Demographic Data for Patients With ALF/SALF
 Severe SepsisAcute Liver FailureSubacute Liver FailureLiver Failure Spontaneous SurvivorsLiver Failure Death / Liver Transplantation
  • *

    Fulfilled King's College Hospital poor prognostic criteria (19).

  • This patient had comorbidities that included hypertension, diabetes mellitus, hypercholesterolemia, and a previous cerebral vascular accident.

  • This patient had preexisting chronic obstructive pulmonary disease.

  • ALF, acute liver failure; SALF, subacute liver failure; HAV, hepatitis A virus; HBV, hepatitis B virus; HEV, hepatitis E virus; LT, liver transplantation.

Number615101015
Median age (range)40.5 (24-70)33 (26-48)52.5 (44-60)34 (28-44)51 (32-59)
Female (%)2 (33)10 (67)6 (60)7 (70)9 (60)
Etiology (%)     
Acetaminophen 6 (40)3 (30)3 (20)
Viral hepatitis (HAV, HBV, HEV). 3 (20)1 (10)2 (20)2 (13)
Seronegative hepatitis 1 (7)7 (70)3 (30)5 (33)
Drug-induced (nonacetaminophen) 2 (13)2 (20)4 (27)
Ischemia/Budd-Chiari/eosinophilic. 3 (20)2 (20)1 (7)
Etiology of sepsis (%)     
Infected pancreatic pseudocyst3 (50)    
Aspiration pneumonia1 (17)    
Infective endocarditis1 (17)    
Fecal peritonitis1 (17)    
Transplant-free 90 day survival (%) 7/15 (40)3/10 (30)10/10 (100)0 (0)
Met King's criteria for LT (%)* 9/15 (60)8/10 (80)2/10 (20)15 (100)
Declined LT due to comorbidity (%) 4/9 (44)1/10 (10)2/2 (100)3/15 (20)
Underwent LT (%) 4/9 (44)6/10 (60) 10/15 (67)
Listed for LT but died before graft became available (%) 1/9 (12) 1/15 (7)
Listed but survived without LT 1/10 (10) 1/10 (10)
Died (%)4 (67)4/15 (27)2/10 (20) 6/15 (40)
Table 2. Clinical, Biochemical, Neutrophil, and Organ Failure Parameters of Patients With ALF/SALF
Clinical/Biochemical/Organ Failure VariablesSevere Sepsis n=6Acute Liver Failure n=15Subacute Liver Failure n=10P value
  • Data presented as number of cases (percentage) or median (IQR) for the variables as appropriate.

  • ALF, acute liver failure; SALF, subacute liver failure; MELD, model of endstage liver disease; SOFA, sequential organ failure score; APACHE II, acute physiology and chronic health evaluation II; SIRS, systemic inflammatory response syndrome score

  • *

    Patients presenting with ALF/SALF were given empirical intravenous antibiotic and antifungal cover as standard of care. This was tazocin 4.5 g every 8 hours (substituted for meropenem 1 g every 8 hours in those who were penicillin allergic) and fluconazole 400 mg once daily.

  • Values in both ALF and SALF cohorts were significantly increased compared with septic controls.

  • ‡, †

    C-reactive protein was significantly greater in septic controls compared with both ALF and SALF cohorts.

  • §

    HDL values were significantly lower in SALF cohort compared to the ALF cohort. The Kruskal-Wallis test was utilized with Dunn's multiple comparison test and P < 0.05 was considered statistically significant.

Body mass index24.5 (20.8-29.7)21.8 (21.4-24.7)25.3 (23.2-27.4)0.74
Empirical antibiotics and antifungals*6 (100%)13 (87%)9 (90%)0.65
Grade of encephalopathy    
Grade 0-2 4 (27%)5 (50%) 
Grade 3-4 11 (73%)5 (50%)0.62
Mean arterial pressure (mmHg)83 (74-88)71.5 (67-81.5)80 (73-94)0.05
SIRS score3 (2-3)2 (1-2.5)1 (1-2)0.22
MELD score13 (9-14)34.2 (31.7-41.4)42.4 (28.1-48.8)0.001
SOFA score6 (4.5-6.5)16 (14.5-17.0)16 (15.5-17.5)0.04
APACHE II score14 (11-17)21 (17.5-24.5)22 (18.5-23.5)0.17
Number requiring invasive ventilation3 (50%)9 (60%)6 (60%)0.91
Number requiring vasopressors4 (66%)6 (40%)3 (30%)0.36
Number requiring hemofiltration2 (33%)10 (66%)6 (60%)0.37
Days on hemofiltration3 (3-5)4.0 (3.25-7.25)4.0 (1.5-5.0)0.63
Hemoglobin (g/dL)9.0 (8.2-9.7)9.5 (8.2-11)9.3 (8.7-12.8)0.44
White cell count (x 109/L)13.6 (9.7-18.7)12.7 (5.3-13.7)9.6 (6.4-12.9)0.38
Neutrophil count (x 109L)11.7 (8.2-15.5)7.5 (5.5-11.0)7.9 (5.1-10.1)0.31
Lymphocyte count (x 109/L)1.1 (0.8-1.2)1.0 (0.5-2.0)0.7 (0.5-1.7)0.77
Platelets (x 109/L)319 (161-508)92 (60-197)89.5 (75.5-180.5)0.06
INR1.32 (1.19-1.38)3.21 (2.25.4.70)3.18 (2.27-7.15)0.002
Aspartate aminotransferase (IU/L)35 (32-42)386 (237-2577)203 (131-1062)0.001
Bilirubin (μmol/L)9 (7-10)106 (80.0-210.5)189 (136.8-266.0)0.001
Albumin (g/L)27 (20-29)21 (19.5-25.0)17 (13.8-27.0)0.39
Sodium (mmol/L)142 (137-146)142 (140.5-145.5)143 (136.5-144.8)0.90
Creatinine (μmol/L)105 (69-138)161 (89.5-214.0)132 (89.3-120.5)0.51
Glucose (mmol/L)7.8 (6.8-8.6)7 (6.1-9.1)5.7 (4.7-10.1)0.65
Ferritin (μg/L)198 (198-198)1665 (170-20,853)2177 (1453-10,401)0.30
Lactate (mmol/L)0.9 (0.7-1.2)2 (1.4-3.0)2 (1.65-2.5)0.025
C-reactive protein (mg/L)226 (116-349)17.3 (5.3-31.8)23 (8.1-29.5)0.001
Arterial ammonia (μmol/L)22 (19-25)75 (62-107)61.5 (44-84)0.001
High density lipoprotein (mmol/L)0.5 (0.35-0.65)0.35 (0.18-0.50)0.1 (0.10-1.13)0.02§
Arterial pH7.4 (7.38-7.43)7.4 (7.37-7.46)7.49 (4.43-7.52)0.15
% Neutrophil phagocytic activity70.2 (55.6-78.3)66.0 (48.8-81.5)39.6 (32.5-63.9)0.16
% Neutrophil resting burst6.8 (6.3-8.1)13.8 (6.4-22.8)6.3 (2.7-10.0)0.35
% Neutrophil stimulated burst52.2 (39.3-75.3)85.4 (76.3-92.0)85.8 (71.8-90.5)0.11

Within the ALF cohort 9/15 (60%) fulfilled King's College Hospital criteria for poor prognosis,19 of whom 4/9 (44%) underwent successful LT, 4/9 (44%) were declined LT due to comorbidity, and 1/9 (12%) was listed but died of cerebral edema before a graft became available. One patient met poor prognostic criteria but was declined due to psychiatric comorbidity and survived following plasmapheresis. In the SALF cohort 8/10 (80%) fulfilled poor prognostic criteria, of whom 6/10 (60%) underwent LT, 1/10 (10%) was declined due to comorbidity, and 1/10 (10%) recovered and was delisted. Two SALF patients died (one post-LT from MODS).

All patients with ALF/SALF were significantly unwell with MODS and, indeed, MELD and SOFA scores were significantly higher in the ALF and SALF cohorts compared to the SC (P = 0.001 and P = 0.0035, respectively) (Table 2). Patients with SALF had a tendency to be older, with higher bilirubin and lower arterial ammonia, but due to the small numbers in the groups these comparisons did not reach significance.

Neutrophil Phenotype.

Neutrophil surface receptor expression of CD16 (FcγRIII) and CD11b (Mac-1) was performed on days 1, 4, and 7 in 8/15 of the ALF cohort and compared to HC (n = 8) and SC (n = 5). Neutrophil expression of CD16 was significantly reduced in the ALF cohort compared to HC (P < 0.001) on day 1 (Fig. 1). CD16 expression was also reduced in the SC group compared to HC but this did not reach statistical significance. The CD16 downregulation persisted in the ALF group on days 4 and 7 regardless of outcome but normalized within 72 hours post-LT. No differences were observed in neutrophil surface receptor expression of CD11b in patients with ALF/SALF or in SC (data not shown).

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Figure 1. Neutrophil CD16 expression (Immunoglobulin G: FcγRIII) in healthy controls (n = 8) compared to septic controls (n = 5) and patients with ALF (n = 8) on day 1 of ICU admission. Differences between healthy controls and patients were calculated using the Kruskal-Wallis test (P = 0.0006) with Dunn's multiple comparison test. ALF patients had significantly reduced neutrophil CD16 expression compared to controls (***P < 0.001).

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Neutrophil Phagocytic Activity (NPA).

Neutrophils isolated from the ALF [Fig. 2(b)i], SALF and SC cohorts on day 1 all demonstrated reduced NPA compared to HC (median [IQR] NPA in the cohorts were as follows: HC 77.7% [72.8-83.7], SC 70.2% [55.6-78.3], ALF 66% [48.8-81.5], and SALF 39.6% [32.5-63.9]). The SALF group showed the greatest reduction in NPA (SALF versus HC P < 0.01) (Fig. 3). NPA in the SC cohort showed a nonsignificant reduction in NPA compared to HC's. Overall, NPA remained depressed on follow-up ICU admission days (P = 0.047) in the ALF/SALF cohorts compared to HC. Figure 4C charts the typical NPA trend observed on admission and on days 5 and day 9 in an ALF and SALF survivor compared to that observed in an ALF who was transplanted and an SALF who died. NPA was significantly improved 72 hours post-LT compared to pre-LT levels; P = 0.03 (Fig. 4B).

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Figure 2. Representative FACS plots of neutrophil function tests in (a) an (HC), (b) a patient with AALF, and (c) the AALF patient 72 hours post-LT. (i) The plots obtained for phagocytic activity with opsonized E. coli labeled with FITC; (ii) spontaneous (unstimulated) oxidative burst; (iii) for oxidative burst with fMLP; (iv) oxidative burst with PMA; and (v) oxidative burst with opsonized E. coli. The x axis of the plots represents FITC/rhodamine fluorescence intensity and the y axis represents CD16 fluorescence intensity. Gates were set using the patients unlabeled (double negative) neutrophils. Neutrophils that have undergone phagocytosis/burst appear in the upper right quadrant of each plot and the overall percentage of neutrophils is labeled. These plots show that in this representative ALF patient there is reduced phagocytic activity compared to an HC (54.7 versus 92.7%) and increased spontaneous oxidative burst (29.6 versus 6.5%) which is normalized post-LT.

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Figure 3. The median neutrophil phagocytic activity (%) of 11 HCs, 6 SCs, 15 ALF, and 10 SALF patients. Differences between HC and patients were calculated using the Kruskal-Wallis test (P = 0.0015) with Dunn's multiple comparison test. Neutrophil phagocytic activity was significantly reduced in the SALF group compared to HC (**P < 0.01).

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Figure 4. (A) The median NPA (%) of those who spontaneously survived without LT (n = 9) is higher compared to those who died or underwent LT (n = 10); **P = 0.01 using the Mann-Whitney test. Those who died are represented by circles and those who were transplanted are represented by triangles. (B) The impact of LT on the median NPA (%) on six ALF/SALF patients who underwent LT; P = 0.03 using Wilcoxon matched pairs test. (C) The typical NPA trend observed in an ALF and SALF spontaneous survivor compared to that observed in an ALF (transplanted) survivor and SALF nonsurvivor on admission, day 5, and day 9.

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Neutrophil OB.

Neutrophil spontaneous production of ROS was increased in the sickest patients with ALF compared to HC who went on to require LT which was reversed within 72 hours post-LT (Fig. 2ii). However, spontaneous OB was statistically unchanged overall when the ALF/SALF cohorts were compared with the HC and SC groups (P = 0.11) (Fig. 5A). No difference in neutrophil spontaneous OB was seen when comparing AALF to non-AALF etiologies (P = 0.99) and remained unchanged during the course of the illness (P = 0.24).

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Figure 5. (A) The median neutrophil spontaneous (unstimulated) oxidative burst of 11 HCs, 6 SCs, 15 ALF, and 10 SALF patients. Differences between groups were calculated using the Kruskal-Wallis test (P = 0.112) with Dunn's multiple comparison test (all comparisons P > 0.05). (B) The median neutrophil stimulated oxidative burst with opsonized E. coli of 11 HCs, 6 SCs, 15 ALF, and 10 SALF patients. Differences between groups were calculated using the Kruskal-Wallis test (P = 0.014) with Dunn's multiple comparison test. The SC group had significantly lower stimulated OB with opsonized E. coli compared to both ALF and SALF groups (both *P < 0.05).

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Neutrophil stimulated OB with opsonized E. coli was significantly reduced in the SC cohort (P < 0.05), while ALF/SALF neutrophils killed E. coli as effectively as HC [Figs. 2(v), 5b].

Neutrophil Function, Organ Failure Scores, and Plasma-Derived Factors in ALF and SALF.

In the ALF cohort, there was no association seen between neutrophil function and SIRS score, MELD and SOFA score, and absolute neutrophil count. Patients with AALF (hyperacute) had higher plasma levels of the proinflammatory cytokines TNF-α, IL-6, and IL-8 (all P < 0.05) compared to non-AALF. IL-17 was significantly elevated in the AALF patients who died or underwent LT compared to spontaneous survivors (P = 0.008). In the ALF cohort spontaneous OB did not correlate with serum biochemistry, arterial ammonia, or organ failure scores.

In the SALF cohort decreasing NPA correlated with increasing peak arterial ammonia concentration (P = 0.001; r2 = 0.677) (Supporting Fig. 6a), increasing concentration of plasma IL-10 (P = 0.019; r2 = 0.407) (Supporting Fig. 6b), and plasma IL-17 (P = 0.0003; r2 = 0.708) (Supporting Fig. 6c). In the SALF cohort, increased neutrophil ROS production correlated with higher APACHE II score (P = 0.003; r2 = 0.635) and SOFA score (P = 0.01; r2 = 0.561) and increased serum high density lipoprotein levels (P = 0.001; r2 = 0.763).

When stimulated OB with E. coli was impaired in the ALF cohort it correlated with lower plasma concentrations of IL-6 (P = 0.038; r2 = 0.190), IL-10 (P = 0.047; r2 = 0.175) and IL-17 (P = 0.007; r2 = 0.301) and in the SALF cohort, correlated with higher plasma concentrations of IL-8 (P = 0.007; r2 = 0.465) and lower plasma concentration of IL-17 (P = 0.025; r2 = 0.354).

Neutrophil Function and Patient Outcomes.

Patients with ALF/SALF who died or were transplanted (considered nonsurvivors in the analysis) had lower NPA on day 1 than spontaneous survivors (P = 0.01) (Table 3; Fig. 4A). Impaired NPA predicted nonsurvival in ALF/SALF even when the transplanted patients were removed from the analysis (P = 0.029). No difference was observed when comparing admission neutrophil spontaneous OB in spontaneous survivors to patients who died or underwent LT (P = 0.5).

Table 3. Clinical, Biochemical, Neutrophil, and Organ Failure Parameters of Survivors Versus Nonsurvivors with ALF/SALF
Clinical/Biochemical/Organ Failure VariablesLiver Failure Spontaneous Survivors n=10Liver Failure Death / Liver Transplantation n=15P value
  • Data presented as number of cases (percentage) or median (IQR) for the variables as appropriate.

  • MELD, model of endstage liver disease; SOFA, sequential organ failure score; APACHE II, acute physiology and chronic health evaluation II; SIRS, systemic inflammatory response syndrome score

  • *Patients presenting with ALF/SALF were given empirical intravenous antibiotic and antifungal cover as standard of care. This was tazocin 4.5 g every 8 hours (substituted for meropenem 1 g every 8 hours in those who were penicillin allergic) and fluconazole 400 mg once daily.

  • Significant differences between spontaneous survivors versus those who died or were transplanted are shown using the Mann-Whitney U test; P < 0.05 was considered statistically significant.

Grade of encephalopathy  0.19
Grade 0-26 (60%)5 (33%) 
Grade 3-44 (40%)10 (67%) 
Mean arterial pressure (mmHg)77 (70-95)74 (68.5-79)0.52
SIRS score1 (1-2)2 (1-2.5)0.60
MELD score32 (26-42)42 (33-49)0.71
SOFA score16 (14-16)16 (15-17)0.50
APACHE II score22 (10-22)21 (19-26)0.39
Number requiring invasive ventilation4 (40%)11 (73%)0.10
Number requiring vasopressors2 (20%)8 (53%)0.10
Number requiring hemofiltration5 (50%)11 (73%)0.23
Days on hemofiltration7 (4-8)4 (3-5)0.15
Hemoglobin (g/dL)10.7 (8.1-11.5)9.3 (8.9-10.9)0.98
White cell count (x 109/L)6.7 (5.4-10.4)12.7 (9.6-13.3)0.06
Neutrophil count (x 109/L)5.5 (4.1-6.1)10.1 (6.8-12.1)0.01
Lymphocyte count (x 109/L)1.4 (0.8-1.9)0.7 (0.4-1.1)0.13
Platelets (x 109/L)100 (84-225)84 (63-101)0.18
INR4.6 (2.9-6.6)3.3 (.8-5.6)0.24
Aspartate aminotransferase (IU/L)317 (164-1047)386 (192-1362)0.57
Bilirubin (μmol/L)106 (82-125)204 (134-285)0.048
Albumin (g/L)21 (18.5-28.5)20 (16-26)0.50
Sodium (mmol/L)141 (138-142)144 (141-146)0.12
Creatinine (μmol/L)121.5 (73-191)140 (116-166)0.72
Glucose (mmol/L)6.3 (4.8-8.2)7.0 (4.9-9.1)0.78
Ferritin (μg/L)1812 (216-4118)2274 (1453-5207)0.76
Lactate (mmol/L)2.1 (1.2-2.8)1.95 (1.7-2.5)0.64
C-reactive protein (mg/L)26 (20-33)11 (5-28)0.11
Arterial ammonia (μmol/L)69 (60-94)144 (91-199)0.81
High density lipoprotein (mmol/L)0.3 (0.1-0.4)0.1 (0.1-0.3)0.35
Arterial pH7.47 (7.4-7.5)7.43 (7.38-7.47)0.22
% Neutrophil phagocytic activity78.2 (56.3-83.3)53.6 (36.6-69.0)0.01
% Neutrophil resting burst10.1 (3-18)10.4 (4-25)0.77
% Neutrophil stimulated burst85.3 (66-91)85.8 (76-94)0.37

Four deaths occurred in the ALF cohort; three patients died of MODS (two on day 10 of ICU admission and one on day 45) and one died of uncontrolled intracranial hypertension. In the SALF cohort, two patients died from MODS on days 15 and 17 of the ICU admission, respectively. The incidence of culture-positive sepsis in the ALF/SALF cohorts overall was low, with one patient with seronegative SALF developing an episode of Staphylococcus epidermidis bacteremia on day 3 (day 1 NPA 29.7% improving to 40.3% post-LT on day 6) and in another seronegative SALF a Klebsiella spp. urinary tract infection developed on day 18 (patient had an NPA of 61%).

Discussion

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

This prospective study is the most comprehensive performed to date characterizing circulating neutrophil dysfunction in patients admitted to ICU with ALF and SALF until spontaneous recovery, death, or LT. It demonstrates significant reduction in neutrophil surface expression of CD16 (FcγRIII), which may contribute to the reduced ability of the neutrophil to bind to an opsonized microbe, akin to patients with sepsis and MODS. Pronounced impaired phagocytic activity of opsonized E. coli was also observed in neutrophils isolated from patients with ALF and particularly SALF. Lower NPA at presentation predicted poor outcomes, with patients who died or required LT showing a lower NPA compared to non-LT survivors.

Neutrophil dysfunction has been implicated both in the immune paresis observed in ALF13, 14 and in direct injury to the liver6 and extrahepatic organs.9 Neutrophil-driven hepatocellular injury has also been shown to contribute to hepatocellular damage in models of ischemia-reperfusion injury,20 alcoholic hepatitis,21 and endotoxemia.22 Neutrophil recruitment is seen within the liver in AALF23 and there is evidence to suggest that hepatocyte injury is amplified by neutrophil infiltration in a mouse model of AALF.24 Neutrophils are also likely to contribute to liver injury in ALF, resulting from their overwhelming capacity to produce large quantities of ROS and proteases following recruitment and activation within the hepatic sinusoids. For this reason, the relationship between circulating neutrophil function and the progression and outcomes of acute liver injury was therefore explored in this study.

The observation of a reduction in NPA in ALF/SALF cohorts akin to that frequently observed in sepsis8 may explain why patients with ALF exhibit phenotypic features of septic shock with microvascular dysfunction, hemodynamic instability, coagulopathy, encephalopathy, MODS, and high levels of circulating proinflammatory cytokines. Why the severity of NPA is less so in those presenting with ALF compared to SALF is less clear but the development of impaired NPA may occur in a time-dependent manner, as evidenced by the most severe reduction in phagocytic ability seen in cases of SALF, where the liver injury takes on a more insidious course over several weeks. Indeed, many patients with established SALF may present with moderate portal hypertension with features of splenomegaly and ascites. Nevertheless, NPA on admission appears to be a predictor of spontaneous survival compared to conventional organ failure scores such as SOFA and MELD, which did not predict poor outcome in this study. Trying to understand the relationship between neutrophil phagocytic dysfunction and poor prognosis therefore seems critical. LT resulted in rapid improvement of neutrophil phagocytic function within 72 hours but not complete reversal, which could be the result of ischemia-reperfusion phenomena, ongoing production of proinflammatory cytokines/SIRS, or sepsis.

The incidence of “culture-positive” sepsis was low in this ALF/SALF cohort overall, and indeed the deaths could not be directly attributed to infection, suggesting phagocytic dysfunction is either a reflection of general immune activation or a specific factor related to liver failure. Peak plasma ammonia levels demonstrated a robust correlation with poor phagocytic function in SALF and high circulating levels of IL-10 and IL-17. Ammonia was previously shown to impair NPA and induce spontaneous OB in healthy neutrophils exposed to supraphysiological concentrations of ammonia ex vivo and in rats fed an ammonia-rich diet.11 The peak arterial ammonia concentration did not, however, correlate with impaired NPA in the ALF cohort but might be attributed to the impact of rapid ammonia reduction by continuous veno-venous hemofiltration prior to neutrophil sampling.

Pro- and antiinflammatory cytokine profiles might be expected to show a closer association with neutrophil OB than phagocytic activity, although this was not generally the case. Higher plasma IL-10 and IL-17 concentrations correlated with impaired NPA and may suggest the development of a compensatory antiinflammatory response syndrome (CARS) in this condition25 with involvement of T-regulatory cells.26 However, the condition with the highest levels of proinflammatory cytokines, AALF demonstrated only modest neutrophil dysfunction. CD4+CD25+CD127-FOXP3+ T-regulatory cells directly inhibit neutrophil function, promoting apoptosis and death when exposed to lipopolysaccharide through TLR4 expressed on their surface which inhibits proinflammatory activities.27 This is an important role in the direct control of innate immune responses. Upon activation, these T-regulatory cells can either induce themselves or CD4+CD25-FOXP3-T effector cells to differentiate into IL-17A-producing cells, Th17, in the presence of TGF-beta, and/or IL-6.28 In contrast to the role of T-regs on neutrophils, one of the functions of Th17 is to recruit neutrophils into inflamed tissue, further increasing the antimicrobial response in vitro and in vivo.29, 30

The evidence for a role of increased circulating neutrophil production of ROS as a contributor to the development of MODS and poor outcomes in ALF in this study is less clear than that of NPA. Interestingly, in the SALF cohort increased spontaneous OB correlated with increased serum high density lipoprotein levels and higher SOFA and APACHE II scores. High-density lipoprotein plays an important role in the transport of cholesterol to the adrenal gland for steroidogenesis, which may modulate the response to sepsis and critical illness. Low concentrations of high-density lipoprotein have recently been shown to be a predictor of poor outcome in ALF but were not associated with an increased risk of sepsis.31 The problem with measuring spontaneous neutrophil ROS production in isolated circulating cells is that this may not reflect the production within the hepatic parenchyma or other organs, so it is difficult to draw firm conclusions. In addition, ALF and SALF patients are a heterogeneous patient group who are prone to deteriorating rapidly, necessitating a number of invasive interventions such as high flow hemofiltration and mild hypothermia potentially influencing neutrophil function and which are difficult to control for, constituting the main weakness of this study. Furthermore, the empirical use of potent broad-spectrum antibiotics and antifungals as standard of care in this study is also likely to have abrogated any increased susceptibility to developing sepsis in this cohort.

Neutrophil stimulated OB with E. coli was significantly reduced in the SC group, while ALF/SALF neutrophils killed E. coli as effectively as HC. This may represent the fact that neutrophils in patients with sepsis have been exhausted fighting the infection and have very little capacity left for responding to the E. coli. Alternatively, it could result from the development of CARS.

In summary, circulating neutrophils in patients with ALF/SALF have impaired bacteriocidal function similar to that seen in patients with severe sepsis and MODS. Neutrophil function indices are important biomarkers of poor prognosis in ALF/SALF and can be implicated as important mediators in the development of cellular and organ dysfunction and the increased susceptibility to developing sepsis. Clearly these neutrophil function tests in their present format are cumbersome to perform and cannot be performed at the bedside, but development of a rapid test of neutrophil dysfunction may offer the possibility for refinement of current prognostic criteria and might tailor therapy to those at highest risk. These data also support the circulating neutrophil as a novel therapeutic target in ALF.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We are indebted to Dr. Lee Markwick for invaluable input into article preparation.

References

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_26102_sm_SuppFig6.tif8507KSupporting Information Figure 6a demonstrates the correlation between impaired neutrophil phagocytic activity (%) and elevated arterial ammonia (μmol/L) in the SALF cohort (r2=0.677; p=0.001). Figure 6b demonstrates the correlation between impaired neutrophil phagocytic activity (%) and plasma IL-10 (pg/ml) in the SALF cohort (r2=0.407; p=0.019). Figure 6c demonstrates the correlation between impaired neutrophil phagocytic activity (%) and plasma IL-17 (pg/ml) in the SALF cohort (r2=0.403; p=0.02).

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