NK-4 was synthesized at Functional Dyes Unit, Hayashibara Co., Ltd. (Okayama Japan). A stock solution of 10 mM NK-4 was prepared with DMSO and stored frozen at -80°C. Endotoxin content in 100 mM NK-4 was below detection limits (0.00625 endotoxin units/ml), as determined according to a protocol listed in the Japanese Pharmacopoeia using an Endospecy ES-50M set (Seikagaku Co., Tokyo, Japan). Final concentrations of DMSO at 0.05% or less did not affect the results of the experiments. PMA and LPS (E. coli O55:B5) were purchased from Sigma-Aldrich Japan (Tokyo, Japan). Pam3CSK4 hydrochloride was purchased from InvivoGen (San Diego, CA). Poly(I:C) was purchased from Calbiochem (La Jolla, CA). Recombinant human IL-4 and IL-13 were purchased from R&D Systems (Minneapolis, MN). Recombinant annexin V was purchased from BD Biosciences (Franklin Lakes, NJ). Wortmannin was purchased from Wako Pure Chemical (Osaka, Japan). Human TNF-a, and monoclonal antibodies (mAb) for the human TNF-a ELISA were prepared and purified in our laboratories.
Culture and differentiation of THP-1 cells
The human monocytic cell line, THP-1 (ATCC, Manassas, VA), was maintained in complete medium comprised of RPMI 1640 (Sigma-Aldrich) supplemented with 10% FCS (GE Healthcare Life Sciences, South Logan, UT) and 1% Penicillin-Streptomycin (Wako Pure Chemical) in a 5% CO2 humidified atmosphere at 37°C.
For differentiation to a macrophage phenotype, THP-1 cells (1.1 x 105 /well) were incubated with 100 nM PMA in 48-well tissue culture plates (Corning, Kennebunk, ME) at 37°C for 2 days. Following differentiation, PMA-containing media was replaced with complete medium and cells were rested for 24 h.
Stimulation of THP-1 macrophages
After the resting period, THP-1 macrophages were washed once with RPMI1640 medium supplemented with 1% FCS and 1% Penicillin-Streptomycin (conditioned medium) and incubated with 4 mM NK-4, unless otherwise stated, and/or with 20 ng/ml human IL-4 and IL-13 at 37°C for 2-3 days. After treatment with NK-4 for 3 days, cells were washed twice with conditioned medium and stimulated with 1 mg/ml LPS for 2 days. In some experiments, THP-1 macrophages were stimulated with various concentrations of LPS, Pam3CSK4, or poly(I:C) in the presence or absence of 4 mM NK-4 at 37°C for 1-3 days.
Morphological features of THP-1 macrophages were examined by an inverted microscope (ECLIPSE TS100, Nikon, Tokyo, Japan).
Cell growth was assessed by cell counting kit-8 (Wako Pure Chemical, Osaka, Japan). Briefly, 15 ml WST-8 reagent, a redox indicator, was added to each well for the last 2 to 3 h of the incubation period. The optical density of the culture supernatants was measured at 450 nm.
THP-1 cells were seeded at 7.5 x 104 cells per well in the 8-well chamber slide (LAB-TEK, Rochester, NY) and were differentiated as described above. THP-1 macrophages were cultured with 4 or 5 mM NK-4 or with 20 ng/mL human IL-4/IL-13 in conditioned medium for 3 days at 37°C. Cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, washed and permeabilized with 0.1% (v/v) Triton X-100 for 30 min. After overnight incubation in PBS containing 3% BSA at 4°C, cells were treated with human FcR blocking reagent (Miltenyi Biotech, Auburn, CA) and were stained with the following antibodies: FITC-labelled mouse anti-human CD38 mAb (303504, BioLegend CNS, Inc., San Diego, CA), FITC-labelled mouse anti-human CD86 mAb (555657, BD PharMingen), Alexa Fluor 488-labelled mouse anti-human CD206 mAb (FAB25342G, R&D Systems). Nuclei were detected with Hoechst 33258. Stained cells were detected with an inverted fluorescence microscope (BX53F-B, OLYMPUS, Tokyo, Japan).
Phagocytosis of rabbit IgG-FITC conjugated latex beads
3 days after incubation with varying concentrations (0, 1, 2, or 4 mM) of NK-4, cells were recovered by pipetting with ice-cold phosphate-buffered saline (PBS) containing 5 mM EDTA and were washed once with conditioned medium. The cells were then assessed for their phagocytic activity using a Phagocytosis Assay kit (Cayman Chemical, Ann Arbor, MI). Briefly, latex beads with rabbit IgG-FITC conjugates (1:100) were incubated with vehicle- or NK-4-treated cells for 2 h at 37°C, followed by 2 minutes of incubation with trypan blue to quench non-phagocytosed bead fluorescence. Cells were washed twice with PBS containing 0.1% bovine serum albumin (BSA) and analyzed by flow cytometry (Gallios, Beckman Coulter Japan, Tokyo). The percent phagocytosis was expressed as the proportion of THP-1 macrophages that phagocytosed fluorescent beads.
Analysis of the phenotypic switch from M1-like to M2-like THP-1 macrophages after co-culture with apoptotic cells
THP-1 macrophages were treated with various concentrations (0, 1, 3, or 5 mM) of NK-4 for 3 days in 48-well tissue culture plates as described above. After the treatment, cells were washed two times with the conditioned medium, and fresh medium was added to each well.
Jurkat E6.1 (ATCC) cells were suspended at 1 x 106 cells/ml in complete medium and treated with 50 mM H2O2 for 6 h at 37°C to specifically induce apoptosis as described previously . Cells were washed three times with the conditioned medium, suspended in the same medium, and counted with trypan blue. Apoptotic Jurkat E6.1 (Apo-J) cells were then added to the culture of NK-4-pretreated THP-1 macrophages at various ratios, and the mixed cells were incubated for 1 h at 37°C. Since cell growth of THP-1 macrophages was comparable between control and NK-4 cultures after 3 days of treatment (Fig. 1c), the number of THP-1 macrophages (1.1 x 105 /well) added to each well of 48-well plates before PMA treatment was used when calculating the ratios. After the incubation period, the mixed cells were stimulated with 1 mg/ml LPS for 2 days at 37°C. Levels of TNF-a at day 1 and levels of IL-10 and TGF-b1 on day 2 were measured by ELISA in the culture supernatants.
In some experiments, Apo-J cells were pretreated with 10 mg/ml annexin V for 30 min at 37°C before co-culture with NK-4 (5 mM)-treated THP-1 macrophages. In other experiments, NK-4 (5 mM)-treated THP-1 macrophages were pretreated with 10 mM cytochalasin D or wortmannin (1 – 5 mM) for 30 min before co-culture with Apo-J cells.
Cytokines (human TNF-a, IL-10 and TGF-b1) in culture supernatants were measured by two-site sandwich ELISA. Levels of human TNF-a were determined by a sandwich ELISA system that was developed in our laboratory. Briefly, this ELISA system utilizes mouse anti-TNF-a mAb (MAb-TNFa-5) and HRPO-conjugated mouse anti-TNF-a mAb (MAb-TNFa-1) for capture and detection of human TNF-a, respectively. The detection limit is 3 pg/ml. Levels of human IL-10 were determined with a human IL-10 ELISA set (Diaclone SAS, Cedex, France). Human TGF-b1 levels were determined with human TGF-b1 DuoSet ELISA Development Systems (DY240-05, R&D Systems, Minneapolis, MN).
Phagocytosis of Apo-J cells
Jurkat E6.1 cells (1 x 106 cells/ml) were stained with 7 mM Cell Tracker Red (excitation 577/emission 602) (Thermo Fisher Scientific, Waltham, MA) in serum-free RPMI1640 medium for 30 min at 37°C. Cells were washed twice with complete medium, and apoptosis was induced in the Cell Tracker Red-treated Jurkat E6.1 cells by treatment with 50 mM H2O2 as described above. The Cell Tracker Red-treated Apo-J cells were incubated with 5 mM NK-4-treated THP-1 macrophages at a 1:1 ratio in conditioned medium for 1 h at 37°C in the 4-well chamber slide (LAB-TEK). After the incubation period, the mixed cells were washed sequentially by complete medium and PBS to remove non-phagocytosed Apo-J cells. Cells were then fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, washed and permeabilized with 0.1% (v/v) Triton X-100 for 30 min. After overnight incubation in PBS containing 3% BSA at 4°C, THP-1 macrophages were treated with human FcR blocking reagent and were stained with FITC-labelled mouse anti-human CD86 mAb. The phagocytosis percentage was calculated in 5 random fields per each well. The number of macrophages that engulfed Apo-J cells in each field was counted and then divided by the total number of THP-1 macrophages in the same field. Results were expressed as mean ± SD of triplicate wells (15 fields in total).
Western immunoblotting analysis of Akt
NK-4 (5 mM)-treated THP-1 macrophages were incubated with or without Apo-J cells at a ratio of 1:1 for 45 min at 37°C. Whole-cell extracts were prepared with RIPA buffer (Wako Pure Chemical) containing phosphatase inhibitor (Nacalai Tesque Inc., Kyoto, Japan) and protease inhibitor (Roche Diagnostics, Mannheim, Germany) and subjected to western immunoblotting. Membranes were probed with a 1:1000 dilution of anti-phospho-Akt (Ser473) rabbit pAb (9171; Cell Signaling Technology, Danvers, MA). Specific bands were detected using an ECL™ Plus Western Blotting System (Immobilon Western Chemiluminescent HRP substrate; GE Healthcare, UK). After treatment with a reprobing solution (Restore Western Blot Stripping Buffer; Pierce Biotechnology, Rockford, IL) for 15 min at room temperature, the membrane was used for secondary detection with a 1:1000 dilution of anti-Akt rabbit mAb (C67E7; Cell Signaling Technology). Band density was measured using ImageQuant TL software (GE Healthcare).
Statistical analyses were performed using Statcel 4 software (OMS Publishing Inc., Saitama, Japan). Phagocytic data were evaluated using the non-parametric Mann-Whitney U test. Other data analyses were conducted by one-way ANOVA followed by Dunnett’s test for multiple comparisons or by the Student t-test for comparison between two variables. P values < 0.05 were considered statistically significant.