Plasma L-Cystine/L-Glutamate Imbalance Increases Tumor Necrosis Factor-Alpha from CD14+ Circulating Monocytes in Patients with Advanced Cirrhosis
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).