Figure 1.
The counts of peripheral monocyte were increased in association with plasma L-Cystine in patients with advanced cirrhosis.
A, B, The concentrations of plasma L-Cys in patients with cirrhosis was increased and that of plasma L-Glu was decreased acccording to Child-Pugh grade. The levels of plasma L-Cys and L-Glu in patients with cirrrhosis (n = 130) were measured using HPLC and classfied by the Child-Pugh classfication. C, Nonliner regression model was used to model variation in plasma L-Cys and eGFR. D, E,F, Linear regression model was used to model variation in plasma L-Cys and monocyte and lymphocyte counts. Individual correlations between plasma L-Cys levels and monocyte counts in patients with early cirrhosis (D), that in patients with advanced cirrhosis (E), and lymphocyte counts in patients with advanced cirrhosis (F). A,B, **, p<0.01, _*, p<0.05 vs Child-Pugh grade A. Statistical significance was determined by one-way ANOVA and Dunnett's post-hoc procedure. C, D, E, F, R2 represents coefficient of determination.
Figure 2.
L-Cystine dose-dependently increased TNF-alpha from CD14+ monocytes with LPS under the amino acid environment of patients with advanced cirrhosis.
A, Isolated CD14+ monocytes (purity >90%) were cultured at a density of 2.5×105 cells/well in 96-well plates containing in ACM and ACM plus L-Cys (L-Cys : 150 nmol/mL) with 1,000 U/mL M-CSF. One half the amount of culture fluid was exchanged every one day. Cells were maintained for 20 days and the proliferation rate of the cells was measured using CFSE staining. B, Influence of L-Cys on microscopic appearance of monocyte proliferation under serum-free conditions. Day 20, cells in firmly adherent clusters in both ACM and ACM+Cys. C, Monocytes were cultured under CCM, HCM, ACM and ACM plus L-Cys (100–300 nmol/mL). Cells were pre-incubated at a density of 2.5×105 cells/well in 96-well flat-bottom plates for 2 hours in each of the media, and 100 ng/mL LPS was added. The supernatants were collected after 24 hours and immediately TNF-alpha was determined by specific cytokine ELISA kits. D,E, Similarly as in Fig. 2C, IL-10, IFN gamma from monocytes and GM-CSF from PBMCs under CCM, HCM, ACM, ACM+Cys (L-Cys 150 nmol/mL) were measured with ELISA. F, Cells were harvested after 24 hours, stained with different mAbs, and analyzed using flow cytometry. Cells were stained with FITC-labeled anti-CD14, -CD80, -CD86, and -HLA-DR. A, B and F results are representative of four experiments from three different donors. C, D and E, Mean ± SEM values from five different donors are shown. C,D,E *, p<0.05 vs ACM (paired Student's t test, two-tailed).
Figure 3.
High levels of extracellular L-Cystine promoted the L-Cystine-L-Glutamate antiport via xCT and decreased the intracellular GSH/GSSG ratio in monocyte under the amino acid environment of patients with advanced cirrhosis.
A, THP-1, Jurkat and Molt-4 cultured under CCM were harvested and labeled with antibodies (CD98, xCT or the relevant isotype controls). Using flow cytometry, surface marker expressions were analyzed. The figure expresses the mean fluorescence intensity. Data shown are representative of three independent experiments with cells. B, xCT relative mRNA levels of these cell lines were determined by real time PCR: delta-delta CT method. All mRNA expression levels were normalized to GAPDH. C, D, The THP-1 cells were pre-incubated for 2 hours in ACM, then resusupended with LPS (100 ng/mL) in 1 mL of ACM, L-Cys-free ACM and ACM plus L-Cys. The concentration of extracellular (C) and intracellular (D) amino acid was determined as described in material and methods. E, The CD14+ monocytes, cultured under ACM and ACM plus L-Cys for 24 hours, were harvested and labeled with antibodies (CD98, xCT or the relevant isotype controls). Using flow cytometry, surface marker expressions were analyzed. The figure expresses the mean fluorescence Intensity. Data shown are representative of three independent experiments with cells. F, Similarly as in Fig. 4C, the monocytes were cultured for 24 hours in ACM, L-Cys-free ACM and ACM plus L-Cys. The supernatants were measured by HPLC as extracellular amino acids quantification. G, Monocytes were pre incubated at a density of 2.0×105 cells/well in 96-well flat-bottom plates for 2 hours in HCM, and then cultured in HCM, ACM and ACM plus L-Cys (200 nmol/mL) for an additional 2 hours. These intracellular glutathione levels were measured by GSH-Glo™ at the time point indicated. C and D, Mean ± SD values from five independent experiments are shown. B *, p<0.05 vs Molt-4 (the Mann-Whitney U-test.). C,F **, p<0.01 *,P<0.05 (mean change vs ACM) D *, p<0.05 (paired Student's t test, two-tailed).
Figure 4.
The plasma L-Cys/L-Glu ratio significantly correlated with the plasma TNF-alpha level in patients with advanced cirrhosis.
A, CD14+monocytes were isolated from healthy volunteers (n = 5) and patients with adcanced cirrhosis (Table S2 : Patient 1–19). the TNF-alpha relative mRNA levels of CD14+monocytes were determined by real time PCR : delta-delta CT method. All mRNA expression levels were normalized to GAPDH. B, Similarly as in Fig. 4A, the xCT relative mRNA levels of monocytes were determined by real time PCR. C, These patients were separated into a high L-Cys/L-Glu ratio group (≥1) and low ratio group (<1). In healthy control (HC), the average plasma L-Cys/L-Glu ratio was 0.61±0.21. D, Linear regression model was used to model variation in plasma L-Cys/L-Glu ratio and plasma TNF-alpha. R2 represents coefficient of determination. E, The schematic diagram of the present study concerning monocytes abnormality in patients with decompensated cirrhosis. A,B,C *, p<0.05 vs HC (the Mann-Whitney U-test.).