Nanoparticles and characterization
NiO NPs were purchased from Nanostructured Amorphous Materials, Inc. (Houston, TX,
USA). Physicochemical characterization including surface area, primary size, hydrodynamic
size, polydispersity, zeta potential, solubility, and levels of endotoxin contamination
were determined. Briefly, the surface area was measuring using the BET (Brunauer,
Emmett, and Teller) method in ParticleCIC Ltd. (Leeds, UK) with a Micromeritics TriStar
3000 analyzer (Micromeritics Ltd., Bedfordshire, UK). The shape and primary size of
the NiO NPs was measured using TEM (JEM-1200EX II, JEOL, Tokyo, Japan) and FE-SEM
(Hitachi, Tokyo, Japan) and the average size was calculated by measuring at least
200 separate particles using a built-in program (JEOL). The hydrodynamic size and
zeta potential of NiO NPs in DW or in PBS with/without dispersion medium (3 % heat-activated
rat serum) was measured by Zetasizer Nano-ZS (Malvern, Malvern Hill, UK) according
to the manufacturer’s instruction. To measure the solubility of the NiO NPs, NPs were
suspended in various conditions including PBS (pH 7.4), PBS with 3 % rat serum, and
artificial lysosomal fluid (pH 5.5) 33] at 1 mg/mL and incubated for 24 h at room temperature with continuous stirring. The
NP-free supernatants were collected by three rounds of centrifugation at 15000 g for
30 min and the absence of NPs were confirmed by dynamic light scattering using Zetasizer
Nano-ZS (Malvern). The concentration of dissolved metal ions was measured by the Center
for Collaborative Instruments at Dong-A University using inductively coupled mass
spectrometry (ICP-MS; Agilent Technologies, Seoul, Korea). Solubility was calculated
and expressed as a percentage of the detected mass of nickel over the initial mass
of nickel in NiO NPs. The concentration of endotoxin of NiO NPs at 400 cm
2
/mL in sterile PBS, the equivalent dose for animal experimentation, was evaluated
by an endpoint chromogenic Limulus Amoebocyte Lysate (LAL) assay kit (Cambrex, Walkersville, MD, USA) with a detection
limit of 0.1 EU/mL.
Dispersion of NPs for animal experiment
Previous studies showed that NPs are tend to agglomerate when dispersed in medium
and agglomerations are accelerated in high salt conditions such as PBS and saline
34]. Therefore, we used serum protein as a dispersion medium to provide protein corona
formation, which can improve dispersion of NPs in physiological condition as previously
described 34]. Briefly, the stock solution of NiO NPs was prepared by dispersion at 4000 cm
2
/mL (4360 ?g/mL) in DW. The solution was sonicated for 10 min using a bath sonicator
(Saehan-Sonic, Seoul, Korea) and heat-inactivated rat serum collected from healthy
6-week old female Wistar rats (Samtako, Gyeonggi-do, Korea) was added at 3 % of end
concentration and sonicated for 5 min. Finally, Ca
2+
– and Mg
2+
-free PBS (Life Technologies, Gaithersburg, MD, USA) was added to the NP suspension
to achieve pre-determined final concentrations (100, 200, and 400 cm
2
/mL) for animal experimentation, followed by sonication for 5 min in a bath sonicator
(Saehan-Sonic).
Intratracheal instillation of NiO NPs
Animal protocols were reviewed and approved by the Institutional Animal Care and Use
Committee in Dong-A University. Six-week old female Wistar rats (Samtako) were acclimatized
for 7 days before experimentation. Rats were housed in an individually ventilated
cage system with controlled temperature and humidity (22?±?1 °C and 50?±?10 %) with
a 12 h light/dark cycle. NiO NP suspensions were intratracheally instilled into the
lungs of rats at surface area doses of 50, 100, and 200 cm
2
/rat (54.5, 109, and 218 ?g/rat) according to the previously described method 8]. Briefly, rats were anesthetized by isoflurane inhalation (Piramal Critical Care,
Bethlehem, PA, USA) using a rodent anesthesia system (VetEquip, Pleasanton, CA, USA),
and intubated with a 16-gauge blunt polycarbonate catheter. Then 500 ?L of NP suspension
or vehicle control (PBS with 3 % rat serum) was instilled once using a sterile syringe.
OVA-induced allergic airway inflammation model
As a positive control for IgE-mediated allergic inflammation, the OVA-induced allergic
airway inflammation model was established according to the previously described method,
with slight modification 35]. Briefly, 6-week old female Wistar rats were sensitized on days 0, 7, and 14 with
an intraperitoneal injection of 1 mg OVA (Grade V, Sigma-Aldrich, St Louis, MO, USA)
and 100 mg aluminum hydroxide (Sigma-Aldrich) in 1 mL sterile saline. On day 21, rats
were challenged with 1 mg OVA by intratracheal instillation and then sacrificed on
day 22 to collect the BALF and serum.
Intratracheal instillation of nickel ion (NiCl
2
)
Because we hypothesized that NiO NPs can produce eosinophilia via the accumulation
of nickel ions in the phagolysosomes of phagocytes, NiCl
2
were instilled into lungs of rats to demonstrate the direct effect of nickel ions.
Our previous studies also showed that instillation CoCl
2
and ZnCl
2
produced similar magnitude of eosinophilia as observed in CoO and ZnO at an equal
metal concentrations 8], 11]. Although NiO NPs in the lung might not completely dissolved in the lung within 3 days
after instillation, we selected the dose of NiCl
2
at 378.0 ?g/rat (171.1 ?g Ni/rat), which is equivalent nickel concentration of 200 cm
2
/rat of NiO NPs to evaluate whether the dissolved nickel from NiO NPs directly induce
pulmonary eosinophilia or not. NiCl
2
(Sigma-Aldrich) dissolved in sterile Ca
2+
– and Mg
2+
-free PBS (Life Technologies) was intratracheally instilled into the lungs of rats
and then sacrificed at 24 h post-instillation to collect the BALF and serum.
Preparation of BALF
At each time point, rats were anesthetized by isoflurane inhalation (Piramal Critical
Care) and euthanized by removing blood from the inferior vena cava. The trachea was
then cannulated by 14-gauge blunt stainless-steel needle and lavaged in-situ with
8 mL sterile cold Ca
2+
– and Mg
2+
-free PBS (Life Technologies) 4 times. The recovery volumes of the first lavage were
about 7 mL and the subsequent lavages were about 8 mL. The first lavage was collected
separately, and the cell-free supernatant was kept at ?70 °C for further assays. Cell
pellets from 4 lavages were pooled for total cell counts and cytological evaluation.
The total number of cells was counted using a NucleoCounter (Chemometec, Allerod,
Denmark); 4?×?10
4
cells were attached to glass slides by spinning at 27?g for 5 min using a cytospin (Hanil, Seoul, Korea). Slides were then dried, fixed with
100 % methanol, and stained with Diff-Quik (Thermo Fisher Scientific, Waltham, MA,
USA). Differential cell counting was performed under a light microscope based on the
morphology of cells. A minimum of 300 cells per slide was counted.
Measurement of nickel levels in BALF
The levels of nickel in BALF were measured to understand the uptake and dissolution
of NiO NPs in the lung. The cell-free first lavage was centrifuged at 15000?g for 30 min to collect NP-free supernatant. Then, the concentration of dissolved nickel
ions in the supernatant was measured using inductively coupled mass spectrometry (ICP-MS;
Agilent Technologies).
Measurement of LDH, total protein, and pro-inflammatory cytokines in BALF
The levels of LDH, a maker for cytotoxicity, were measured in BALF using an LDH assay
kit (Roche Diagnostics, Mannheim, Germany). Total protein, a maker for vascular permeability,
was measured in BALF using a bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific).
To evaluate the underlying mechanism of inflammation by NiO NPs, pro-inflammatory
cytokines related to acute neutrophilic inflammation (IL-1? and CINC-3), eosinophilic
inflammation (eotaxin and IL-4), and Th1 response (IFN-?) were measured in BALF using
a Duoset enzyme-linked immunosorbent assay (ELISA) kit (all from RD systems, Minneapolis,
MN, USA).
Measurement of total IgE, C3a, and C5a in BALF and serum
To evaluate the mechanism of allergic lung inflammation, the level of total IgE was
measured in BALF and serum using an ELISA kit (Komabiotech, Seoul, Korea) according
to the manufacturer’s instruction. The levels of C3a and C5a, marker for complement
activation, were measured in BALF and serum using a C3a ELISA kit (MBS2503333, MyBioSource,
San Diego, CA, USA) and C5a ELISA kit (MBS2506864, MyBioSource).
Measurement of intracellular eotaxin in alveolar macrophages and normal lung tissue
To evaluate whether the direct lysis of cells can release intracellular eotaxin, the
levels of eotaxin were measured using alveolar macrophages and normal lung tissue
before and after addition of cell lysis buffer. To collect the alveolar macrophages,
6-week old female Wistar rats (Samtako) were sacrificed and lungs were lavaged 4 times
with 8 mL of PBS and cell pellets were collected by simple centrifugation and suspended
with 1 mL of PBS. The number of total cells was counted using a NucleoCounter (Chemometec)
and cytospin slides were prepared to confirm that more than 99 % of cells in BALF
are alveolar macrophages. Then, cell lysis buffer (Atto, Tokyo, Japan) was added at
a volume of 1 mL for 1?×?10
7
cells. To evaluate the dose–response relationship, the cells were lysed at different
concentrations of cell lysis buffer by serial dilution with PBS. On the other hand,
normal lung tissue was collected from 6-week old female Wistar rats (Samtako) and
homogenized using tissue homogenizer. Then, cell lysis buffer (Atto) was added at
a volume of 1 mL for 50 mg of lung tissue. Cell lysis buffer was serially diluted
with PBS to evaluate the dose–response relationship. Alveolar macrophages and tissue
homogenates were incubated with cell lysis buffer for 30 min and cell-free supernatants
were collected by centrifugation at 500?g for 5 min followed by centrifugation at 15000?g for 5 min. The levels of LDH and eotaxin levels were then measured using a LDH assay
kit (Roche Diagnostics) and a Duoset ELISA kit (RD systems), respectively.
Statistical analysis
Number of rats for NiO NPs was 10 per group, while the number of rats for NiCl
2
and OVA-induced airway inflammation group was 4 per group. One-way analysis of variance
(ANOVA) was applied to the data having more than 3 groups, while the unpaired student’s
t-test was applied to the OVA-induced airway inflammation group and NiCl
2
treated group. When ANOVA test showed statistical significance (p??0.05), each group was compared using post-hoc Tukey’s pairwise comparisons. Pearson
correlation test was applied to evaluate the correlation between the levels of LDH
and eotaxin in cell lysis experiment. All statistical analysis and graphs were prepared
using GraphPad Prism (version 6.0 for Windows; GraphPad Software, San Diego, CA, USA).
A value of p??0.05 was considered statistically significant.