Frequency and phenotype of human circulating and intrahepatic natural killer cell subsets is differentially regulated according to stage of chronic liver disease.

BACKGROUND/AIMS: Experimental liver injury models have indicated that natural killer (NK) cells are critical regulators of inflammation and fibrosis. However, data on NK cells and subsets in patients with liver diseases are limited. We thus comprehensively characterized peripheral and hepatic NK cell subsets in patients with chronic liver diseases (CLDs) of different etiologies and fibrosis stages. METHODS: NK cells and other lymphocyte populations were characterized by FACS in 189 CLD patients (71 non-cirrhosis, 118 cirrhosis) and 153 healthy controls in blood and liver biopsies (n = 40). RESULTS: In contrast to other lymphocyte subsets, circulating NK cells were generally reduced in CLD patients. Patients with fibrosis displayed a distinct increase of CD16- NK cells in blood and of the CD16+ NK cell subset in liver. Patients with cirrhosis had overall lymphopenia, including reduced peripheral NK cells. Most pronounced shifts in NK cell subsets in blood and liver were found in cholestatic and autoimmune CLDs. Blood NK cells and subsets correlated with liver function, and inversely with fibrosis markers and inflammatory cytokines. CONCLUSIONS: The close association of human NK cells with disease severity and the intrahepatic accumulation of CD16+ NK cells in early fibrosis favor the concept of beneficial NK cell functions in hepatofibrogenesis.

Elevated circulating soluble interleukin-2 receptor in patients with chronic liver diseases is associated with non-classical monocytes.

BACKGROUND: The soluble interleukin-2 receptor (sIL-2R, sIL2R, sTAC, sCD25) is a reliable biomarker for disease activity in inflammatory disorders such as sarcoidosis. Based on the essential pathogenic role of inflammation for progression of liver diseases, we hypothesized that sIL-2R might be an indicator of inflammatory cell activation and disease severity in patients with chronic liver diseases (CLD). METHODS: We measured sIL-2R serum levels in 71 patients with different stages and etiologies of CLD in comparison to 41 healthy controls. Serum sIL-2R concentrations were correlated with laboratory markers of liver diseases, cytokine / chemokine levels and circulating immune cell subpopulations as simultaneously assessed by FACS analysis from peripheral leukocytes. RESULTS: CLD patients showed significantly elevated serum sIL-2R levels compared with controls. sIL-2R was significantly higher in patients with compared to patients without established liver cirrhosis and increased with the Child-Pugh stage of cirrhosis, independent of the underlying etiology. sIL-2R levels correlated inversely with parameters indicating the hepatic biosynthetic capacity, such as albumin or international normalized ratio, and positively with non-invasive markers of liver fibrosis such as hyaluronic acid or procollagen-III-peptide. Circulating immune cells might represent a major source of sIL-2R. In fact, sIL2-R levels correlated closely with circulating monocytes, especially non-classical CD14+ CD16+ monocytes, which were found to express high levels of CD25 by FACS. Pro-inflammatory cytokines, including IL-2, IFNγ or IL-6, and chemokines were also associated with sIL2-R. In addition, renal failure was an important confounder of sIL-2R levels independent of liver dysfunction and inflammation. CONCLUSIONS: sIL-2R is elevated in patients with liver diseases and cirrhosis, is associated with circulating inflammatory cells and is increased in concomitant renal failure. These data indicate that sIL-2R might be a potential marker for immune cell activation in CLD, especially for proinflammatory and profibrogenic non-classical CD14 + CD16+ monocytes.

Circulating soluble urokinase plasminogen activator is elevated in patients with chronic liver disease, discriminates stage and aetiology of cirrhosis and predicts prognosis.

BACKGROUND: Inflammation is a major factor for the progression of chronic liver diseases. Interactions between urokinase plasminogen activator (uPA) and its receptor (uPAR) have been functionally linked to hepatic inflammation and fibrosis in mice. High serum concentrations of soluble uPAR (suPAR) are suggested to reflect activated immune cells. AIMS: We evaluated suPAR serum levels as a diagnostic and prognostic biomarker in patients with chronic liver diseases. METHODS: Prospective, cross-sectional cohort study of 159 patients with chronic liver diseases (61 without, 98 with established cirrhosis) and 43 healthy controls. Transplant-free survival was monitored for up to 3 years. RESULTS: Soluble urokinase plasminogen activator serum concentrations were significantly elevated in patients with chronic liver diseases compared with controls. Cirrhotic patients displayed higher levels than non-cirrhotics, closely depending on stage of fibrosis or cirrhosis. suPAR levels had high diagnostic power to identify established cirrhosis in chronic liver diseases. Circulating suPAR closely correlated with liver function, fibrosis markers, but also with systemic inflammation and renal function. A distinct suPAR elevation was noticed in patients with alcoholic aetiology of liver disease. suPAR identified alcoholic origin more precisely compared with classical indicators of alcoholism (mean corpuscular volume, gamma glutamyl transpeptidase). Strikingly, elevated suPAR levels were identified as a strong predictor of mortality or need for transplantation. suPAR levels >9 ng/ml indicated adverse prognosis (sensitivity: 70.7%, specificity: 77.8%, relative risk: 8.5; 95% confidence interval: 3.5-20.3). CONCLUSIONS: Serum suPAR is a potential novel biomarker for the diagnosis of cirrhosis, identification of alcoholic origin and for determining prognosis in patients with chronic liver disease.

Interleukin-8 is activated in patients with chronic liver diseases and associated with hepatic macrophage accumulation in human liver fibrosis.

BACKGROUND: Interleukin-8 (IL-8, CXCL8) is a potent chemoattractant for neutrophils and contributes to acute liver inflammation. Much less is known about IL-8 in chronic liver diseases (CLD), but elevated levels were reported from alcoholic and hepatitis C-related CLD. We investigated the regulation of IL-8, its receptors CXCR1 and CXCR2 and possible IL-8 responding cells in CLD patients. METHODOLOGY: Serum IL-8 levels were measured in CLD patients (n = 200) and healthy controls (n = 141). Intrahepatic IL-8, CXCR1 and CXCR2 gene expression was quantified from liver samples (n = 41), alongside immunohistochemical neutrophil (MPO) and macrophage (CD68) stainings. CXCR1 and CXCR2 expression was analyzed on purified monocytes from patients (n = 111) and controls (n = 31). In vitro analyses explored IL-8 secretion by different leukocyte subsets. PRINCIPAL FINDINGS: IL-8 serum levels were significantly increased in CLD patients, especially in end-stage cirrhosis. Interestingly, patients with cholestatic diseases exhibited highest IL-8 serum concentrations. IL-8 correlated with liver function, inflammatory cytokines and non-invasive fibrosis markers. Intrahepatically, IL-8 and CXCR1 expression were strongly up-regulated. However, intrahepatic IL-8 could only be associated to neutrophil infiltration in patients with primary biliary cirrhosis (PBC). In non-cholestatic cirrhosis, increased IL-8 and CXCR1 levels were associated with hepatic macrophage accumulation. In line, CXCR1, but not CXCR2 or CXCR3, expression was increased on circulating monocytes from cirrhotic patients. Moreover, monocyte-derived macrophages from CLD patients, especially the non-classical CD16⁺ subtype, displayed enhanced IL-8 secretion in vitro. CONCLUSIONS: IL-8 is strongly activated in CLD, thus likely contributing to hepatic inflammation. Our study suggests a novel role of IL-8 for recruitment and activation of hepatic macrophages via CXCR1 in human liver cirrhosis.

Age-dependent alterations of monocyte subsets and monocyte-related chemokine pathways in healthy adults.

BACKGROUND: Recent experimental approaches have unraveled essential migratory and functional differences of monocyte subpopulations in mice. In order to possibly translate these findings into human physiology and pathophysiology, human monocyte subsets need to be carefully revisited in health and disease. In analogy to murine studies, we hypothesized that human monocyte subsets dynamically change during ageing, potentially influencing their functionality and contributing to immunosenescence. RESULTS: Circulating monocyte subsets, surface marker and chemokine receptor expression were analyzed in 181 healthy volunteers (median age 42, range 18-88). Unlike the unaffected total leukocyte or total monocyte counts, non-classical CD14+CD16+ monocytes significantly increased with age, but displayed reduced HLA-DR and CX(3)CR1 surface expression in the elderly. Classical CD14++CD16- monocyte counts did not vary dependent on age. Serum MCP-1 (CCL2), but not MIP1alpha (CCL3), MIP1beta (CCL4) or fractalkine (CX(3)CL1) concentrations increased with age. Monocyte-derived macrophages from old or young individuals did not differ with respect to cytokine release in vitro at steady state or upon LPS stimulation. CONCLUSIONS: Our study demonstrates dynamic changes of circulating monocytes during ageing in humans. The expansion of the non-classical CD14+CD16+ subtype, alterations of surface protein and chemokine receptor expression as well as circulating monocyte-related chemokines possibly contribute to the preserved functionality of the monocyte pool throughout adulthood.

Functional contribution of elevated circulating and hepatic non-classical CD14CD16 monocytes to inflammation and human liver fibrosis.

BACKGROUND: Monocyte-derived macrophages critically perpetuate inflammatory responses after liver injury as a prerequisite for organ fibrosis. Experimental murine models identified an essential role for the CCR2-dependent infiltration of classical Gr1/Ly6C(+) monocytes in hepatic fibrosis. Moreover, the monocyte-related chemokine receptors CCR1 and CCR5 were recently recognized as important fibrosis modulators in mice. In humans, monocytes consist of classical CD14(+)CD16(-) and non-classical CD14(+)CD16(+) cells. We aimed at investigating the relevance of monocyte subpopulations for human liver fibrosis, and hypothesized that 'non-classical' monocytes critically exert inflammatory as well as profibrogenic functions in patients during liver disease progression. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed circulating monocyte subsets from freshly drawn blood samples of 226 patients with chronic liver disease (CLD) and 184 healthy controls by FACS analysis. Circulating monocytes were significantly expanded in CLD-patients compared to controls with a marked increase of the non-classical CD14(+)CD16(+) subset that showed an activated phenotype in patients and correlated with proinflammatory cytokines and clinical progression. Correspondingly, CD14(+)CD16(+) macrophages massively accumulated in fibrotic/cirrhotic livers, as evidenced by immunofluorescence and FACS. Ligands of monocyte-related chemokine receptors CCR2, CCR1 and CCR5 were expressed at higher levels in fibrotic and cirrhotic livers, while CCL3 and CCL4 were also systemically elevated in CLD-patients. Isolated monocyte/macrophage subpopulations were functionally characterized regarding cytokine/chemokine expression and interactions with primary human hepatic stellate cells (HSC) in vitro. CD14(+)CD16(+) monocytes released abundant proinflammatory cytokines. Furthermore, CD14(+)CD16(+), but not CD14(+)CD16(-) monocytes could directly activate collagen-producing HSC. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate the expansion of CD14(+)CD16(+) monocytes in the circulation and liver of CLD-patients upon disease progression and suggest their functional contribution to the perpetuation of intrahepatic inflammation and profibrogenic HSC activation in liver cirrhosis. The modulation of monocyte-subset recruitment into the liver via chemokines/chemokine receptors and their subsequent differentiation may represent promising approaches for therapeutic interventions in human liver fibrosis.