Cell culture, cell stimulation and co-culture
Human acute monocytic leukemic cell line (THP-1) cells (ATCC, Manassas, VA, USA) and lymphocytic leukemic cell line (Jurkat) cells (ATCC, Manassas, VA, USA) each were maintained in RPMI-1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 mM HEPES, 100 U/mL penicillin/streptomycin at 37oC in 5% carbon dioxide incubator. Cells were cultured to a density of 5 × 105 cells/mL. Cell viability, as determined by tyropan blue dye exclusion, was > 99%. For macrophage differentiation, the THP-1 cells (5 × 105 cells/mL) were prepared in a 75T-flask (corning Co, USA) and 1 µL/mL of PMA (sigma-Aldrich Co., St. Louis, MO, USA) was added for 3days. The supernatant was discarded and washed with 10 mL of phosphate buffer saline (PBS), followed by the addition of 5 mL of trypLETM express (Gibco Co, Denmark) to take the attached cells off the floor. After the addition of 10 mL of fresh medium and the reaction mixture was centrifuged for 5 min at 500 × g get the differentiated THP-1 cells. Therefore, THP-1-derived macrophages and Jurkat cells were used for the experiments. Prostaglandin E2 (PGE2) (1μg/mL) (Sigma-Aldrich Co., St. Louis, MO, USA) stimulation was used to demonstrate the effect of immunosuppression. Lipopolysaccharide (LPS) (1μg/mL) (Sigma-Aldrich Co., St. Louis, MO, USA) induction was used to simulate the effect of endotoxin..
First, the differentiated THP-1 cells were plated at a density of 1 × 104 cells/mL in 24 well plates and were placed under normoxia (20% O2) followed by hypoxia (1% O2) for 30min. After that, macrophages were stimulated with LPS (1μg/mL) followed by variable treatments such as PTX, glycerol, HTS, protease inhibitor, and DEXA, followed by normoxia (20% O2) or hyperoxia (80% O2) for 2 hrs. After 20hrs or overnight under normoxic conditions, the levels of macrophage migration inhibiting factor (MIF) and inducible nitric oxide synthase (iNOS) expression were measured by western blots and concentration of MIF in supernatant was measured by ELISA. Second, Jurkat cells (2 × 106 cells/mL) were subjected to normoxia followed by hypoxia for 30 min. After that, Jurkat cells induced with PGE2 (1μg/mL) with various treatments such as PTX, glycerol, HTS, protease inhibitor, and DEXA, followed by either normoxia or hyperoxia for 2 hrs. After 20hrs or overnight, their metabolic state, as indicated by cell survival, was measured using an MTT assay (with dimethyl thiazolyl diphenyl tetrazolium salt). The levels of MIF, interleukin 2 (IL-2), and interleukin 8 (IL-8) expression were measured by western blots. Third, in the event of hypoxia and trauma or infection, macrophages that are important to the initial responses were cocultured with T cells to understand the effects of T cells that are important to the immunity in the hypoxic condition. In addition, we want to find out the changes in oxygen supply and injection of variable treatments such as PTX, glycerol, HTS, protease inhibitor, and DEXA in normoxia or hyperoxia. For coculture, differentiated THP-1 cells (1 × 104 cells/mL × 0.5ml) / well were prepared on a bottom floor pf 24-well plate and Jurkat cells (2×106 cells/ml X 0.5mL) / well were prepared in a top floor of transwell plate. In order to determine the proper incubation time for the coculture of macrophages and T cells in hypoxia, it was cocultured for 1 minute, 5 minutes, 30 minutes, 1 hour, and 2 hours based on the values of the appropriate MTT value and IL-2 of the T cells that are important for immunity. And cells were incubated under hypoxic conditions for 30 min. After that, cells in transwell plate were stimulated with LPS (1μg/mL) followed by variable treatments such as PTX, glycerol, HTS, protease inhibitor, and DEXA, followed by normoxia or hyperoxia for 2 hrs and cells were incubated overnight under normoxic conditions. The concentrations of MIF in the supernatant was measured, and MTT, IL-2, IL-8 were measured using the western blots method using the Jurkat cells at the top of the transwell plate (Fig. 1). Hypoxic insult is referred to as cells cultured under hypoxic conditions. Hyperoxic treatment is administered to cells with hypoxic insult followed by hyperoxia. This study was carried out with the approval of the Korea University Guro Hospital Institutional Review Board (approval number: 2019GR0188).
Enzyme‑linked immunosorbent assay (ELISA) for MIF.
The MIF concentration in the culture supernatants was measured by sandwich enzyme‑linked immunosorbent assay (ELISA). Briefly, 2μg/ml of monoclonal capture antibody (R&D Systems, Minneapolis, MN, USA) was added to a 96‑well plate and incubated for one day at room temperature. After incubation, the plates were incubated in a blocking solution of phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) and 0.05% Tween 20 for 2hrs at room temperature. Test samples and standard recombinant MIF (R&D Systems) were added to the plates and incubated for 2hrs at 4˚C. Plates were washed three times with PBS containing Tween 20, and after the addition of 200ng/ml of biotinylated detection monoclonal goat‑antihuman antibodies (R&D Systems), the plates were incubated for 2hrs at room temperature. The plate were washed, streptavidin‑alkaline‑phosphatase (1:2000; Sigma‑Aldrich Co.) was added and the reaction was allowed to proceed for 20min at room temperature. The plates were washed three times and 1mg/ml of p‑nitrophenylphosphate dissolved in diethanolamine (Sigma‑Aldrich Co.) was added to induce a color reaction, which was stopped with 50μl of 1M NaOH. The optical density at 450nm was measured on an automated microplate reader (Bio‑Rad Laboratories Inc., Hercules, CA, USA). A standard curve was generated by plotting the optical density vs. the log of the MIF concentration.
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) viability assay
The tetrazolium dye, MTT, is widely used to assess the viability or the metabolic state of the cells. The MTT-colorimetric monocyte mediated cytotoxicity assay, is based on the ability of living cells to reduce MTT into formazan by mitochondrial succinate dehydrogenase in viable cells. After treatment at the different culture conditions, Jurkat cells were plated in 96-well flat-bottom tissue culture plates to attain a final concentration of 2 × 106 cells/mL. After incubation for 12 hours at 37oC, the resultant Jurkat cell viability was determined by the MTT viability assay (ATCC, Manassas, VA, USA)
Western blot analysis for iNOs, MIF, IL-2 and IL-8 expression
The cells were washed 2 times in cold PBS and then centrifuged for 10 minutes. Cells pellet were suspended in 10 μL per 2 × 106 cell/mL pro prep protein extraction buffer. Incubated on ice for 10 minutes, and then centrifuged at 3,000 × g for 15 minutes at 4℃. The supernatant was then transferred to a new tube and used for assay. The total protein concentration was determined by the Bradford method using a Bradford solution (Sigma Co.). The prepared protein were used for western blot analysis. Expression of iNOs, MIF, IL-2 and IL-8 protein was quantified by western blot analysis. Proteins (20μg/sample) were fractionated on a 15% sodium dodecyl sulfate-polyacrylamide gel (Bio-Rad Laboratories Inc.) and transferred onto a nitrocellulose membrane. Membranes were blocked for 1 hour in 5% skim milk (Bio-rad Co.), and then incubated with a primary antibody, anti-human IL-8, iNOS, MIF, IL-2 (1:500; R&D systems). After washing, membranes were incubated with 1:1000 horseradish peroxidase-labeled anti-rabbit antibody (R&D systems) as the secondary antibody. The proteins were detected using ECL (Cyanagen) chemiluminescence kit.
Data and Statistical analysis
All results were expressed as the mean ± SD. Statistical significance was performed using a t-test, one-way ANOVA, and the Mann-Whitney U test using SPSS 18.0 (SPSS Inc, Chicago, IL). Each experiment was repeated tweleve times at least. p < 0.05 was considered statistically significant.