2.1 Isolation of PMN and MNC from normal human peripheral blood
Heparinized venous blood obtained from normal individuals was mixed with
one-quarter volume of 2% dextran solution in 37℃ (molecular wight: 425000-575000)
and incubated at room temperature for 30 minutes. The cell suspension was gently
layered over Ficoll-Hypaque density gradient solution (specific gravity 1.077; GE
Healthcare, Waukesha, Wisconsin, USA) and centrifuged at 500 x g for 30 minutes. The
MNC were aspirated from the interphase whereas the PMN were collected from the
bottom. The residual RBC in PMN was lysed in cold 0.85% ammonium chloride
solution. These cells were then rinsed twice with PBS and re-suspended in RPMI 1640
(Gibco/BRL, Grand Island, New York, USA) supplemented with 10% (v/v) fetal bovine
serum (FBS), 2 mM L-glutamine (hereafter referred to as complete medium). The
viability of PMN and MNC were detected by trypan blue dye and the cell concentration
was adjusted to 2×106/ml in complete medium.
2.2 Cell culture
Human promyelocytic leukemia cell line (HL-60) was maintained in complete medium
at 37℃ in a humidified atmosphere containing 5% CO2. HL-60, MNC and PMN (2×106
cells/ml) were treated with LPS (20ng/ml), h-IgG (1μg/ml), anti-CD44 (1μg/ml),
anti-CD45 (1μg/ml), anti-CD3 (1μg/ml)/ anti-CD28 (1μg/ml), and hyaluronic acid (HA
1mg/ml and 2mg/ml) at 37℃, then cultured and harvested at the indicated time points.
Induction of differentiation was obtained by seeding the cells at a concentration of 5 x
105/ml in the presence of DMSO (Microbiological Associates, Rockville, Md.) at a final
concentration of 1.3% (v/v) for 5 days. After exposure, the cells were resuspended in
DMSO-free medium. Next, differentiated-HL60 were treated with LPS (20ng/ml),
anti-CD44 (1μg/ml), and hyaluronic acid (HA 1mg/ml and 2mg/ml) at 37℃, then
cultured and harvested at the indicated time points.
2.3 Detection of CD44 expression treated with inflammatory molecule by flow
cytometry
MNC and PMN were treated with anti-CD3 (1μg/ml)/ anti-CD28 (1μg/ml) or LPS
(20ng/ml) at 37℃, before being cultured and harvested at the indicated time points. The
cells were washed twice with PBS. The cells were then fixed with 4%
paraformaldehyde for 30 minutes at room temperature and incubated with
anti-CD44-FITC overnight to detect the CD44 expression. The percentage (%) and
mean fiuorescence intensity (MFI, denoted by mean channel number) of CD44
expression were determined by FACSort flow cytometry (Becton Dickinson) at wave
length 488nm excitation.
2.4 Cytokine measurements
After treatment with HA and pro-inflammatory molecule at indicated time points, the
cells were centrifuged at 800 x g for 10 minutes for the detectection of cytokine
concentrations in cell culture supernatant. The concentrations of cytokines including
IL-8 were quantified using their respective ELISA kits (R&D Systems, Minneapolis,
Minnesota, USA).
2.5 Detection of PMN phagocytosis-enhancing activity of HA by flow cytometry
Fluoresbrit carboxylate microspheres (0.75 μm in diameter, Polyscience Inc.) were
washed with PBS in advance twice and opsonized by incubation with fresh human
serum at 37℃ for 2 hours. Fresh prepared PMN (2×106 cells/ml) were treated with LPS
(20ng/ml), h-IgG (1μg/ml), anti-CD44 (1μg/ml), anti-CD45 (1μg/ml), and HA (1mg/ml
and 2mg/ml) at 37℃ for 1 hours. The mixture was let reacting with opsonized beads
(1×108 beads/ml) at 37℃ for 1 hour in 5% CO2-95% air. After incubation and wash by
PBS twice, the percentage (%) and mean fluorescence intensity (MFI, denoted by mean
channel number) of PMN phagocytosis were determined by FACSort flow cytometry
(Becton Dickinson) at wave length of 488nm excitation.
2.6 Preparation of whole cell extraction
After treatment with HA or control medium at different time points at 37℃, the cells
were centrifuged at 800 x g for 10 minutes followed by wash with cold PBS. After
centrifugation at 800 x g for 10 minutes, the pelleted cells were lysed with cold RIPA
buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1%NP-40, 1% sodium deoxycholate,
0.1% SDS ) containing protease inhibitor cocktail and phosphatase inhibitor cocktail
(Roche) and kept on ice for 30 minutes. The cell lysates were centrifuged at 10,000 x g
at 4℃ for 20 minutes to remove the debris and the supernatants were applied for
Western blot. The protein concentration of the cell extraction was measured using the
BCA Protein Assay (Pierce).
2.7 Western blotting
Proteins were separated by 10% SDS-PAGE and transfer to polyvinylidene fluoride
(PVDF) membrane (Millipore Inc.) in a Mini Trans-Blot cell (Bio-Rad) for 2 hours at
350 mA. The PVDF membranes were blocked with Tris-buffered saline and Tween 20
(TBST, 50 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.6) containing 1% BSA at
room temperature for 30 minutes, and probed with a specific antibody overnight at 4℃.
After washing the membrane three times with TBST buffer each for 5 minutes, the
complexes were detected by HRP-conjucated-secondary antibody (Jackson ImmunoLab)
and ECL Western Blotting Substrate (Pierce) chemifluorescence detecting system.
2.8 Immunofluorescence microscopic observation of cytoskeleton change in
activated-PMN
After stimulation at 37℃ by HA or LPS for 2 hours, the activated-PMN were wash with
PBS and then fixed for 30 minutes at room temperature with a solution of 4%
paraformaldehyde followed by washing with PBS. Then mixture was centrifuged and
the supernatant was discarded. The cells were resuspended in 200 μL of PBS. 5 to 10
μL of the cell suspension was smeared above a gelatin-coated slide. Placed the slide on
a hot plate (low heat setting), and allowed the liquid to evaporate. The PMN were
permeabilized with 0.1% Triton X-100 for 15 minutes at room temperature, and washed
three times with PBS. The blocking solution (10% FBS in PBS) was added to inhibit the
non-specific binding. PMNs were stained with fluorescent phalloidin (1:200) for
visualization of actin filament at room temperature for 30 to 60 minutes. The slides
were washed three times with PBS and stained with DAPI
(4’,6-diamidino-2-phenylindole, 1:1000) at room temperature for 5 minutes. After three
times washes with PBS, the slides were mounted with glycerol and gently covered the
cover slip avoiding producing the air bubbles. The cells were visualized the
cytoskeleton was observed using a fluorescence microscope (Olympus, Japan).
2.9 Statistical analysis
The statistical analyses were performed using the analysis of variance (ANOVA).
Statistical significance was defined as p＜0.05.