Reagents.

Nickel-nitrilotriacetic acid (Ni-NTA) agarose gel was obtained from Qiagen (Hilden, Germany). N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP) was obtained from Peptide Institute, Inc. (Minoh, Japan). ATRA, nitroblue tetrazolium (NBT), isoluminol, horseradish peroxidase type IV and zymosan A from Saccharomyces cerevisiae, and phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma-Aldrich (St. Louis, MO). Hoechst33342, calcein-AM, and SYTOX orange were obtained from Life Technologies (Carlsbad, CA). Anti-histone H3 Ab (citrulline R17 + R2 + R8) [citrullinated histone H3 (cit-histone H3)] and Alexa Fluor 488–labeled anti-rabbit IgG Ab were obtained from Abcam (Cambridge, UK). Fluorescein isothiocyanate (FITC)-labeled anti-human activated CD11b Ab (clone: CBRM1/5) was obtained from eBioscience (San Diego, CA). Blocking One was obtained from Nacalai Tesque (Kyoto, Japan). Anti-human HRG monoclonal antibody (Rat) and anti-human HRG polyclonal antibody (Rabbit) were synthesized in our laboratory.

Isolation of Human Neutrophils from Human Peripheral Blood.

Peripheral blood polymorphonuclear cells were purified from heatlhy donors using Polymorphprep density gradient media (Alere Technologies AS, Oslo, Norway). In line with the ethical guidelines of Okayama University, written informed consents were obtained from healthy volunteers (n = 5), and we drew blood from the cubital vein. In brief, heparinized peripheral blood was layered over Polymorphprep and centrifuged at 500g for 35 minutes. Polymorphonuclear cells were then harvested and diluted with PBS. After centrifugation at 400g for 10 minutes, the purity of neutrophils was confirmed to be more than 95% under microscope. Subsequently, neutrophils were used at appropriate concentrations for each experiment.

HRG Purification from Human Plasma.

HRG was purified from human plasma (obtained from the Japanese Red Cross Society) by affinity chromatographic methods using Ni-NTA, as previously described (Mori et al., 2003). Briefly, protease inhibitors were added to human plasma. Thereafter, the mixture was centrifuged twice at 10,000 rpm at 4°C for 15 minutes. The supernatant was incubated with Ni-NTA agarose gel, which was pre-equilibrated with 10 mM Tris-buffered saline (TBS) (pH 8.0) and suspended in the same buffer. After gently mixing for 2 hours at 4°C, the gel suspension was transferred into a column, washed with 50 mM imidazole in 10 mM TBS (pH 8.0), 2 M NaCl in 10 mM TBS (pH 8.0), and 100 mM imidazole in 10 mM TBS (pH 8.0) to enable the passage of all unbound proteins through the column. Finally, HRG was eluted with 10 mM TBS (pH 8.0) containing 500 mM imidazole. After exchanging the buffer by dialysis, the eluate was loaded onto a Mono Q column (GE Healthcare, Little Chalfont, UK) and separated by an NaCl gradient. The purified HRG was identified and collected by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using a Western blot with an anti-human HRG polyclonal antibody. The obtained human HRG was stored at −20°C until use.

Differentiation of HL-60 and NB-4 by ATRA.

The human acute myelogenous leukemia (AML) cell lines HL-60 (at the myeloblast stage of development, AML-M2) and NB-4 cells (at the promyelocyte stage of development, AML-M3) were obtained from RIKEN Cell Bank (RCB0041) and CLS Cell Lines Service (GmbH). The cells were cultured in RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% (v/v) FBS (Thermo Fisher Scientific, Waltham, MA), L-glutamine (Sigma-Aldrich), and penicillin streptomycin (Thermo Fisher Scientific) and maintained in a humidified 5% CO₂ environment at 37°C. The media were routinely passaged and diluted every 3 to 4 days to maintain exponential growth. All experiments were performed with cells from passages between 10 and 25.

ATRA (Sigma-Aldrich) was dissolved in 99.5% ethanol as a 10-mM stock solution. The solution was stored at −80°C until use. The final concentration of ethanol in the culture was maintained at less than 0.1% v/v. HL-60 or NB-4 cells were diluted to 2.5 × 10⁵ cells/ml in RPMI-1640 culture medium supplemented with 10% FBS. ATRA was added to the HL-60 and NB-4 cell culture to yield the final concentrations of 10 and 1 mM, respectively. HL-60 and NB-4 were passaged 2 or 3 days after the addition of ATRA. Live cells were isolated with Dead Cell Removal Kit per the manufacturer’s protocol (Miltenyi Biotec Inc., Auburn, CA).

The NBT reduction test is one of the neutrophil function tests that examines the phagocytotic activity and function of neutrophil enzyme systems (Blair et al, 1985). This method was used as a functional measure of the maturation of HL-60 and NB-4 cells. A total of 1.0 × 10⁶ cells was collected by centrifugation and resuspended in 0.25 ml of RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% (v/v) FBS (Thermo Fisher Scientific). Subsequently, 0.25 ml of 0.1% NBT with 5 µl PMA was added to each sample, and the cell suspensions were incubated at 37°C for 30 minutes. Samples were then fixed by the addition of an equal volume of 2% paraformaldehyde in PBS. Cells were observed with a BZ-8000 microscope (Keyence Corporation, Osaka, Japan). If the cells were differentiated into neutrophils, the yellow NBT dye was internalized, thereby resulting in the formation of black formazan due to reduction. The stained and nonstained cells were counted manually under the microscope. Both differentiated neutrophil-like HL-60 cells (dHL-60) and differentiated neutrophil-like NB-4 cells (dNB-4) showed stable differentiation rates (Fig. 1).

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Fig. 1.

Differentiation of the human myeloid cell lines to neutrophil-like cells. HL-60 and NB-4 are human AML cell lines that can be induced by ATRA to form mature neutrophil-like differentiated cells. (A and B) The NBT-stained dHL-60 and dNB-4 cells were induced for 5 days in 10 and 1 μM of ATRA. (C) The ATRA-induced cell differentiation rates were determined by manually counting the NBT positive cells at original magnification, 10×. The results are presented as the average of five different visual fields. Eighty to 90% of HL-60 and NB-4 cells were differentiated to neutrophil-like cells (dHL-60 and dNB-4 cells, respectively) by ATRA. (D) HL-60 or NB-4 cells were diluted to 2.5 × 10⁵ cells/ml (total 2.5 × 10⁶ cells), and ATRA was added to the HL-60 and NB-4 cell culture. After the induction of cell differentiation, the absolute number of cells was counted. The results are the mean ± S.D. of five experiments. *P < 0.05.

Observation of the Morphologic Changes in Differentiated Cells.

To clarify the effects of HRG on the morphology of dHL-60 and dNB-4 cells, cell shape was observed by calcein-AM (for cytosol) and Hoechst33342 (for nuclei) staining as described previously (Wake et al., 2016). Thereafter, 100 μl (5 × 10⁴ cells/well) of the two cell lines and human neutrophils (as a positive control) were aliquoted into each polystyrene well. The cells were incubated with different concentrations of HRG (0.01–1 μM) in the presence of control IgG or anti-HRG Ab, human serum albumin (HSA) (1 μM), bovine serum albumin (BSA) (1 μM), or fMLP (1 μM) at 37°C for 1 hour. The In Cell Analyzer 2000 System (GE Healthcare) was used for morphologic observation. The data were analyzed with the In Cell Investigator Version 1.62 (GE Healthcare). Additionally, the morphologic characteristics of cells were assessed by cell area (square micrometer per cell); the inclusion area per cell in the extended cytoplasm area was automatically determined using the In Cell Analyzer 2000 System.

Microchannel Array Flow Analyzer.

The cell samples were prepared as described above. Briefly, the cells were incubated with either of the following reagents (HRG, HSA, BSA, fMLP: each at a final concentration 1 μM) at 37°C for 1 hour. Thereafter, the passage of cells through the microcapillary slits (7.0-μm width, 4.5-μm depth) was evaluated by Microchannel array flow analyzer (MC-FAN) (MC Laboratory, Japan). Briefly, the cells flowed through the silicon-based artificial microchannels under a constant suction of –20 cm H₂O. Thereafter, the passage time of the 100-μl sample through the microcapillaries was determined. The passage time was dependent on both the cell size and the cell adhesiveness, and the cells adhered to the silicon-coated microchannels based on their adhesiveness.

Expression Analysis of Activated CD11b on the Cell Surface by Flow Cytometry.

Both dHL-60 and dNB-4 samples were stimulated with 100 ng/ml of PMA in the presence of one of the reagents [Hank’s balanced salt solution (HBSS), HSA, or HRG, each at a concentration of 1 μM] for 1 hour at 37°C in a 5% CO₂ atmosphere. Thereafter, the cells were stained with FITC-labeled anti-human activated CD11b antibody for 25 minutes at 4°C and fixed with 0.5% paraformaldehyde in PBS for 30 minutes at 4°C. The extent of activated CD11b expression in cells was analyzed using MACSQuant Analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany).

Determination of Extracellular ROS Production.

Because of the short half-life and reactivity of ROS, direct observation of the amount of ROS production is challenging. Under the presence of ROS, horseradish peroxidase type IV oxidizes isoluminol and induces luminescence. Our method harnessed the reaction and observed the amount of ROS production indirectly. Briefly, isoluminol (final concentration, 50 µM) and horseradish peroxidase type IV (final concentration, 4 U/ml) were added to the dHL-60 and dNB-4 cell suspensions. Thereafter, the suspensions (100 µl; 5 × 10⁵ cells) were aliquoted into a 96-well plate (BD Biosciences). Subsequently, HRG, HSA (each at a final concentration of 1 µM), or HBSS was added to each well. In addition, the zymosan suspension (final concentration of 100 ng/ml) was simultaneously added to each well. Extracellular ROS production was measured 10 times (every 5-minute interval) from the beginning of incubation at 37°C by detecting chemiluminescence intensity using Flexstation 3 (Molecular Devices, Sunnyvale, CA).

Observation of NET Formation.

Citrullination of histone H3 and H4 by peptidylarginine deaminase 4 is required for chromatin decondensation, which is an essential step for NET formation. We observed NET formation using cit-histone H3 with immunocytochemical analysis. The dHL-60 and dNB-4 cells were prepared, as mentioned above. For dead cell imaging, the cells were treated in advance with a cell-impermeable DNA fluorescent dye (5 μM SYTOX orange). PMA 100 ng/ml and one of the reagents (HBSS, HSA, or HRG, each at concentration of 1 μM) were added to the cell suspensions. Thereafter, the cell suspensions (100 μl; 5 × 10⁵ cells) were aliquoted into a 96-well plate (BD Biosciences). The cells were incubated for 2 (neutrophils) or 4 hours (dHL-60 and dNB-4 cells) at 37°C in 5% CO₂ to stimulate NETs formation. Subsequently, the supernatant was gently removed, and the cells were fixed with 4% of paraformaldehyde for 20 minutes at 4°C. To evaluate NET formation by immunocytochemical analysis, the samples were permeabilized with 0.1% Triton X-100 in TBS for 1 minute and blocked with Blocking One for 10 minutes at room temperature. After the addition of the primary antibody (anti–cit-histone H3 Ab at 10 μg/ml) to the samples, the samples were incubated for 2 hours at room temperature. After three rounds of washing with TBS, the secondary antibody (Alexa Fluor 488–labeled anti-rabbit IgG at 2 μg/ml) was added to the samples, and this was followed by incubation for 1 hour at room temperature. Dead cells (SYTOX orange positive site) and NET formation (cit-Histon H3 positive site) were observed using the In Cell Analyzer 2000 System.

Cell Viability.

Purified human neutrophils, dHL-60 cells, and dNB-4 cells were prepared, as mentioned above. To evaluate cell viability, live cells were stained by calcein-AM (for cytosol) for 15 minutes at 37°C in 5% CO₂. PMA 100 ng/ml was added to the cell suspensions in the presence of one of the reagents (HBSS, HSA, or HRG, each at a concentration of 1 μM). Each suspension (100 μl; 5 × 10⁵ cells) was aliquoted into a 96-well plate (BD Biosciences) and incubated for 8 hours at 37°C in 5% CO₂. Cell viability was defined as the percentage of live cells at 8 hours postincubation. The number of live cells was calculated using the In Cell Analyzer 2000 System based on the presence of calcein-AM staining.

Statistical Analysis.

Statistical significance was evaluated by Student’s t test for comparisons between two groups or Tukey’s test for multiple comparison. All data are presented as the mean value ± S.D. The P values less than 0.05 were considered to be statistically significant. The analyses were conducted with SPSS software version 24 (SPSS, Chicago, IL).