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. 2010 Jun 10;5(6):e11049.
doi: 10.1371/journal.pone.0011049.

Functional Contribution of Elevated Circulating and Hepatic Non-Classical CD14CD16 Monocytes to Inflammation and Human Liver Fibrosis

Free PMC article

Functional Contribution of Elevated Circulating and Hepatic Non-Classical CD14CD16 Monocytes to Inflammation and Human Liver Fibrosis

Henning W Zimmermann et al. PLoS One. .
Free PMC article


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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. Blood monocytes increase in patients with chronic liver disease and are associated with disease progression.
(A) Monocytes are defined by CD14 staining of PBMC (representative FACS plots shown). (B+C) Relative proportion of monocytes (CD14+) (B) and absolute monocyte numbers (C). (D) Association of circulating monocytes with laboratory parameters in CLD patients. *p<0.05, **p<0.001.
Figure 2
Figure 2. Relative increase of CD14+CD16+ blood monocytes and more activated phenotype in patients with liver cirrhosis.
(A) Representative FACS plots displaying an increase of CD14+CD16+ monocytes (black gate: CD14+CD16, grey gate: CD14+CD16+) among PBMC in patients with cirrhosis compared to healthy controls and non-cirrhotic patients (left). The histograms show the relative distribution of CD16 expression on CD14+ cells (right; grey: isotype control). (B) Statistical analysis of monocyte subsets. HC, healthy controls (n = 181); CLD, patients (n = 226); NC, non-cirrhotic (n = 85); CIR, cirrhotic (n = 141). (C) Patients with liver cirrhosis and hepatocellular carcinoma (HCC) have significantly (p = 0.008) higher CD16+ monocytes than cirrhotics without HCC. (D) Representative FACS staining for HLA-DR on monocyte subsets. (E) Ratio of HLA-DR expression on CD16+ vs. CD16 monocytes. *p<0.05, **p<0.001.
Figure 3
Figure 3. Intrahepatic CD16+ macrophages predominantly increase during liver fibrosis progression.
(A) Representative examples of biopsies from normal liver (upper panel) and cirrhotic liver (lower panel) show mononuclear inflammatory infiltrates in fibrotic periportal regions (left: H&E staining, right: Ladewig staining, in which collagen stains blue). (B) Immunofluoresecent co-staining for CD14 (red) and CD16 (green) identifies CD14+CD16 and CD14+CD16+ macrophages in human liver tissue (blue: nuclei counterstained with DAPI). Bold arrow, CD14+CD16 macrophage; thin arrow, CD14+CD16+ macrophage. (C+D) Semiquantative analysis of CD14+CD16, CD14+CD16+ and total CD14+ intrahepatic cells. *p<0.05, **p<0.001.
Figure 4
Figure 4. Intrahepatic macrophages consist of different subpopulations mirroring blood monocyte subsets.
(A) FACS analysis of intrahepatic monocytes/macrophages, based on >30 fresh liver biopsies. Representative plots are displayed. Among the CD45+ intrahepatic leukocytes, three different populations of intrahepatic CD14+ macrophages can be distinguished based on CD14 and CD16 expression that also differ characteristically in HLA-DR and DC-SIGN expression. (B) Expression levels of monocyte/macrophage activation and differentiation markers were compared in the same patients between blood CD14+CD16 monocytes (dotted line) and liver CD14hiCD16 macrophages (dark grey) as well as CD14+CD16+ monocytes (dashed line) and liver CD14+CD16+ macrophages (light grey); representative analyses from patients with a F0 fibrosis (no fibrosis, upper panel) and a F3 fibrosis (advanced fibrosis, lower panel) are shown. Isotype control, black line. Blood was drawn at the time of liver biopsy, and blood/liver samples were run with the same FACS settings at the same time.
Figure 5
Figure 5. Activation of monocyte-related chemokine pathways and of monocytic chemokine receptors in chronic liver disease.
(A) Intrahepatic gene expression levels of chemokine receptors. (B) Expression of CCR2, CCR1 and CCR5 was assessed by FACS on monocytes/macrophages (CD14+, green), T- (CD3+CD56, light orange), NK- (CD3CD56+, dark orange) and NKT-cells (CD3+CD56+, red) from freshly isolated liver tissue. Representative histograms are shown, isotype control in grey. (C) Intrahepatic gene expression levels of chemokines. (D) Serum concentrations of monocyte-related chemokines in patients with chronic liver diseases and healthy controls. Abbreviations are: HC, healthy control; CLD, chronic liver disease; NC, no cirrhosis; CIR, cirrhosis. *p<0.05, **p<0.001.
Figure 6
Figure 6. Regulation of chemokine receptors on circulating monocytes in chronic liver disease.
(A) Monocytic chemokine receptor gene expression by real-time PCR after purification of circulating monocytes by CD14 microbeads (MACS). (B) CCR2 expression (MFI, mean fluorescence intensity) on blood monocyte subsets by FACS. Abbreviations are: HC, healthy control; CLD, chronic liver disease; NC, no cirrhosis; CIR, cirrhosis. Representative histograms are shown, comparing either CCR2 expression levels between both monocyte subsets as well as between healthy controls and CLD patients on the two monocyte subpopulations in peripheral blood. *p<0.05, **p<0.001.
Figure 7
Figure 7. Monocytes are functionally active in liver cirrhosis with a differential release of distinct cytokines/chemokines by monocyte subsets.
(A+B) Cytokine/chemokine release of monocyte-derived macrophages (2 days in culture) without stimulation (A) and after stimulation with 1 mg/ml LPS (B). (C) Cytokine/chemokine release of purified monocyte subsets after 5 days of culture without stimulation. *p<0.05, **p<0.001.
Figure 8
Figure 8. CD14+CD16+, but not CD14+CD16 monocytes directly activate hepatic stellate cells.
(A) Primary human HSC were isolated and co-cultured for 5 days with CD14+CD16, CD14+CD16+ monocytes or lymphocytes. No morphological differences were noted on HSC in these conditions. (B) HSC activation was determined by collagen-1A (col1A) mRNA expression, normalized to the ‘HSC-house keeping gene’ Acta2. (C) Expression of surface molecules by FACS (MFI, mean fluorescence intensity) on CD14+CD16 (CD14) and CD14+CD16+ (CD16) monocytes/macrophages at day 0, and after 5 days in culture or co-culture with HSC. All results derived from three independent experiments. *p<0.05, **p<0.001.

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