TOXICOLOGICAL SCIENCES 100(1), 203–214 (2007)
Advance Access publication July 28, 2007
Pulmonary and Systemic Immune Response to Inhaled Multiwalled
Leah A. Mitchell,*,† Jun Gao,* Randy Vander Wal,‡ Andrew Gigliotti,† Scott W. Burchiel,* and Jacob D. McDonald†,1
*College of Pharmacy, University of New Mexico, Albuquerque, New Mexico 87131–0001; †Lovelace Respiratory Research Institute, Albuquerque, New Mexico
87108; and ‡The National Center for Microgravity Research, c/o The NASA-Glenn Research Center, Cleveland, Ohio 44135
Received May 27, 2007; accepted July 18, 2007
Inhalation of multiwalled carbon nanotubes (MWCNTs) at
particle concentrations ranging from 0.3 to 5 mg/m3did not result
in significant lung inflammation or tissue damage, but caused
systemic immune function alterations. C57BL/6 adult (10- to 12-
week) male mice were exposed by whole-body inhalation to
control air or 0.3, 1, or 5 mg/m3respirable aggregates of
MWCNTs for 7 or 14 days (6 h/day). Histopathology of lungs
from exposed animals showed alveolar macrophages containing
black particles; however, there was no inflammation or tissue
damage observed. Bronchial alveolar lavage fluid also demon-
strated particle-laden macrophages; however, white blood cell
counts were not increased compared to controls. MWCNT
exposures to 0.3 mg/m3and higher particle concentrations caused
nonmonotonic systemic immunosuppression after 14 days but not
after 7 days. Immunosuppression was characterized by reduced
T-cell–dependent antibody response to sheep erythrocytes as well as
T-cell proliferative ability in presence of mitogen, Concanavalin A.
Assessment of nonspecific natural killer (NK) cell activity showed
that animals exposed to 1 mg/m3had decreased NK cell function.
Gene expression analysis of selected cytokines and an indicator
of oxidative stress were assessed in lung tissue and spleen. No
changes in gene expression were observed in lung; however,
interleukin-10 (IL-10) and NAD(P)H oxidoreductase 1 mRNA
levels were increased in spleen.
Key Words: carbon nanotubes; inhalation; pulmonary pathol-
Carbon nanotubes (CNTs) are among the most promising
and unique engineered nanomaterials. The global production of
CNTs has already reached hundreds of metric tons per year and
is expanding rapidly as new applications and manufacturers
develop (Harper and Vas, 2005). No other material has been
developed that possesses the size (1–20 nm in width, and many
microns in length), strength, and surface chemistry properties
of CNTs. Many of the properties that make CNTs remarkable
for engineering applications have also caused concern for their
biocompatibility, especially in the lung. Their length/width
(aspect) ratios of > 1000, reactive surface chemistry, and poor
solubility raise concerns linked to past experience with hazardous
fibers (e.g., asbestos). Persistent reactive fibers lead to oxidative
reactions that result in lung injury (McClellan, 1994). The
relevance of findings from fiber toxicity studies to the bio-
compatibility of CNTs has been suggested (Huczko et al.,
2001; Magrez et al., 2006) but is not well established.
Early studies demonstrated that CNTs induced pulmonary
injury, including fibrosis, when instilled into rodent lungs. In
2006, Lam et al. published a comprehensive review on the
sources, characteristics, and toxicology of CNTs. Of particular
note is that CNTs exist in many forms, including single-walled
carbon nanotubes (SWCNTs), double-walled CNTs, and
multiwalled carbon nanotubes (MWCNTs). In addition to the
potential for occupational exposure, environmental exposures
to low concentrations of MWCNTs, but not SWCNTs, have
been reported (Murr et al., 2005). The presence of MWCNTs
in the environment was reported to be due to combustion
emissions, as they have been found in the effluent from natural
Biological and toxicological responses to CNTs may vary
by dose, route of dosing, and type of CNTs. Reported research
on the pulmonary in vivo toxicity of CNTs to date has been
conducted by either instillation into the trachea (Lam et al.,
2004; Muller et al., 2005; Warheit et al., 2004) or pharyngeal
aspiration (Shvedova et al., 2005). Each of these publications
reported significant pulmonary effects, including inflammation,
evidence of oxidative stress, fibrosis (Shvedova et al., 2005),
and granuloma formation (Lam et al., 2004; Muller et al.,
2005; Shvedova et al., 2005; Warheit et al., 2004). Warheit
et al. (2004) reported multifocal granulomas in the absence of
a linear dose-response changes in lavage parameters and
uniformity of lesions. These transient, nonmonotonic responses
were potentially attributed to the artificial route of dosing bolus
material to the lung by instillation. Shvedova et al. (2005)
reported improved linearity in dose-response relationships and
the additional observation of fibrotic injury that was attributed
to an improved lung delivery by pharyngeal aspiration.
CNT biocompatibility studies conducted to date form an
initial basis for evaluating the hazard of CNTs. However, the
1To whom correspondence should be addressed at Lovelace Respiratory
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need to conduct inhalation exposures (as opposed to instillation
or aspiration) to CNTs to place these prior results in context has
been stated by several investigators (Kipen and Laskin, 2005;
Lam et al., 2006; Warheit et al., 2004). To this end, we have
developed an inhalation exposure system for the conduct of
whole-body inhalation exposure of rodents to CNTs. The ex-
posure system was developed to produce CNT aerosols that
simulate resuspended CNT powders that may exist in the
workplace. While only limited data exist on the potential for
pulmonary exposure in the workplace, Maynard et al. (2004)
showed the feasibility and characteristics of aerosolized CNTs
produced from mechanical agitation. In that study, approxi-
mately 50 lg/m3of CNTs was produced, and the particle size
changed with the degree of agitation. In general, a bimodal
particle size distribution was observed with some particles
< 100 nm and the majority at approximately 300–600 nm. For
the present study, we attempted to approximate the size distri-
bution observed by Maynard et al. (2004). Although that study
used SWCNTs, the current study used MWCNTs because they
are produced in higher production volumes and we could obtain
large quantities of commercial grade, high-quality MWCNTs.
This study was conducted to assess short-term pulmonary
and systemic immune response to inhaled MWCNTs as a func-
tion of dose and time. Pulmonary toxicity evaluations were
conducted in view of previous reports of significant inflam-
mation and tissue injury observed in short time periods after
dosing. Standard measurements of pulmonary injury, including
histopathology, white blood cell count in bronchial alveolar
lavage fluid (BALF), and some measurements of oxidative
stress and cytokine induction were used. In addition to the
pulmonary response to MWCNTs, we explored the ability of
MWCNTs to cause systemic immune suppression, a response
we have previously reported from inhalation of environmental
pollutants at low concentrations (Burchiel et al., 2004, 2005).
Moreover, immune suppression has also been reported
following asbestos inhalation (Magrez et al., 2006; Rosenthal
et al., 1998). The overall goal of this study was to develop
a suitable aerosol delivery and inhalation system, and apply
that system to characterize the pulmonary and systemic
immune response after short-term (up to 14 days) inhalation
exposures of mice.
MATERIALS AND METHODS
Study design. The study design and a summary of the measurements are
provided in Figure 1. Mice were housed and exposed in whole-body inhalation
exposure chambers for 7 or 14 consecutive days. During exposure, aerosolized
MWCNTs were sampled from within each chamber for characterization.
MWCNT material was imaged by scanning and transmission electron
microscopy (SEM and TEM, respectively). Impurities, functional groups, and
metals were measured by x-ray photoemission and energy-dispersive
spectroscopy. The aerosol particle size distribution was also analyzed. After
exposure, animals were removed from the chamber and sacrificed the following
morning. All pulmonary and systemic end points listed in Figure 1 were carried
out in 7- and 14-day–exposed animals. Lung end points included BALF
cellularity, histopathology, and gene expression analysis. Spleen, a representa-
tive organ for analysis of systemic immunity, was analyzed for T-cell–
dependent antibody response, T- and B-cell proliferative ability, a marker of
innate immune function, and gene expression. Splenic interleukin (IL-10) gene
expression was further validated by IL-10 protein analysis, only 14-day data are
shown. Details on the methodology are described below.
Source and characteristics of MWCNTs. Dispersible MWCNTs were
purchased from Shenzhen Nanotech Port Co. (Shenzhen, China). These
MWCNTs were engineered in high yield from a proprietary chemical vapor
MITCHELL ET AL.
deposition (CVD) process. Additional characterization of the bulk MWCNTs
was conducted, including determination of chemical composition of ash
impurities, residual catalyst, and oxygen functional groups. Functional group
impurities were assessed by x-ray photoemission spectroscopy (XPS). A PHI
500 series XPS with a dual anode x-ray source was used with scans from 0 to
4800 V and a resolution of 0.052 eV. Residual catalyst was determined in the
aerosolized MWCNTs as described below for aerosol characterization. Surface
area was confirmed by gas adsorption analysis (termed BET analysis) using
a MONOSORB Model MS-16 (QuantaChrome Corp., Syosset, NY). Surface
characteristics of the MWCNTs were assessed by both SEM and TEM as
described below for aerosol characterization.
Aerosol generation and exposure. Inhalation exposure atmospheres were
produced by mechanical agitation/resuspension of MWCNTs using a jet mill
(Fluid Energy, Hatfield, PA) coupled to a dry chemical screw feeder (Scheck
AccuRate, Whitewater, WI) (Cheng et al., 1985). Flow through the jet mill was
point calibrated to approximately 30 l/min. Aerosols were size classified through
a 2-lm cut-point cyclone (Fluid Energy) to ensure that the MWCNTs were in
the respirable size range for rodents (approximately < 3 lm). After passing
through the cyclone, the aerosols entered an H-2000 2 m3whole-body inha-
lation exposure chamber (Lab Products, Maywood, NJ). The transit time
between the jet mill, dilution system, and exposure chamber (~3 s) was
minimized to reduce particle agglomeration. Inhalation exposure chambers
were maintained at a flow (~0.5 m3/min) that provided approximately 15 air
exchanges per hour. Approximately 100 g MWCNT was consumed during the
Atmosphere characterization. Aerosol dilutions were monitored and
controlled based on particle mass concentrations measured in both real time
and with integrated filter measurements. All aerosol collections were made
directly from the breathing zone of the rodents. Particle mass was measured
gravimetrically after collection onto Teflon-coated glass fiber filters (Pall-
Gelman T60A20, East Hills, NY). Filters were placed in aluminum filter
holders and flows were point calibrated to 5 l/min. A nephelometer (Dust-
Track, TSI Inc., St Paul, MN) was used for real-time measurement of particle
Aerodynamic particle size distribution was determined using a Mercer
cascade impactor (In-Tox Products, Moriarty, NM) operated at 2 l/min. Particle
number size distribution was determined by a combination of a differential
mobility analyzer (3081 DMA, TSI Inc.) and time of flight (3321 APS, TSI
Inc.). The combination of the DMA and APS allowed integrated analysis of
particle number size distribution from approximately 10 nm to 20 lm.
Particle morphology and structure were analyzed by TEM after collection
(10-min collection) by electrostatic precipitation on copper-coated lacey grids
(Ted-Pella, Inc., Redding, CA). TEM was conducted using high resolution
JEOL 210 at 200 KeV. SEM was conducted using a Hitachi model S-4700 cold
field emission SEM with resolutions of 2.5 nm at 1 KeV and 1.5 nm at 15 KeV.
SEM images were created using Quartz PCI software (Quartz Imaging
Corporation, Vancouver, BC) and TEM images were created using Gatan IV
Digital Imaging software (Gatan Inc., Gatan, UK). Qualitative elemental
composition of samples on the TEM grid was analyzed by energy-dispersive
x-ray spectroscopy (EDS) via an Oxford-LINK Isis instrument. Quantitative
analysis of metal impurities was determined by inductively coupled plasma
mass spectroscopic (ICPMS) analysis of material collected on Teflon-
membrane filters (Pall-Gelman). Approximately 1 mg of material was collected
on the Teflon-membrane filters, which were then subjected to acid digestion
using a MARSXPress microwave digestion system. The acid digestion cocktail
was 14% nitric acid, 6% hydrochloric acid, and 5.0% hydrofluoric acid. Metals
were then detected using a PerkinElmer Elan 6000 ICPMS (PerkinElmer,
Boston, MA) using commercially available analytical standards (Sigma
Aldrich, St Louis, MO).
Estimation of dose. Respiratory minute volume (RMV) was estimated
using an allometric equation described by Bide et al. (2000). Dose was
estimated using the following formula:
Dose ¼ ðC 3RMV3T3DFÞ=BW;
where C is the average concentration of the MWCNTs in the exposure
atmosphere, T is exposure time, and the pulmonary deposition fraction (DF)
was assumed to be 10% (Miller, 2000).
Animals. Male C57BL/6 mice (n ¼ 6 per group) were obtained from
Charles River Laboratories (Portage, MI). Animals were ordered at 8 weeks of
age and quarantined for 2 weeks upon arrival. Animal procedures were
approved by Lovelace Respiratory Research Institute and University of New
Mexico Institutional Animal Care and Use Committees. Animal housing and
husbandry was performed in accordance with the Guide for the Care and Use
of Laboratory Animals (National Research Council, 1996). Mice were exposed
6 h/day for 7 or 14 days and had access to water ad libitum during exposures.
Standard rodent diet (2016 Global Rodent Diet, Harlan Teklad, Madison, WI)
was provided ad libitum during nonexposure hours. The morning following the
last day of exposure, an overdose of pentobarbital and phenytoin was
administered by ip injection. Blood was drawn by cardiac puncture into
heparinized syringes. Lungs were removed with the trachea intact. Spleen was
collected using aseptic technique.
Lung lavage and pathology. The right cranial lung lobe was ligated and
the left lung isolated with a clamp. Remaining right lung lobes were lavaged
three times with 0.5 ml of sterile saline. Lavage fluid from the three washes was
combined. Viable white blood cells were counted using trypan blue exclusion
on a hemocytometer. Cells were spun onto slides using cytofunnels (Shandon,
Thermo Corp., Pittsburgh, PA) and differential populations quantified using
Diff-Quick stain (Baxter Health, McGaw Park, IL). The right lung was then
ligated and removed and the left unclamped and instilled with 10% neutral-
buffered formalin (NBF). Right lungs, with the exception of the right cranial
lobe, were snap frozen in liquid nitrogen and stored at ? 80?C. Right cranial
lobes were stored for future use in Karnovsky’s fixative. Left lungs were
immersed in NBF until sectioning (at least 48 h), and were routinely processed,
paraffin embedded, and sectioned at 5 lm. Slides were stained with
hematoxylin and eosin and examined by a board certified veterinary
Spleen harvest and cell isolation. Spleens were harvested into sterile
Hank’s Balanced Salt Solution (Cambrex, Walkersville, MD) in a sterile 15-ml
centrifuge tube on ice. Using sterile forceps and gauze, spleens were blotted to
remove excess liquid and weighed. Once weights were recorded, spleens were
bathed in approximately 5 ml of supplemented RPMI media in a sterile culture
dish. Supplemented RPMI media contained 10% fetal bovine serum (FBS)
(Hyclone, Logan, UT), 1% penicillin/streptomycin (Cambrex), and 1%
L-glutamine (Cambrex). Microscope slides were flame sterilized and frosted
Ash (catalyst residue)
PULMONARY AND IMMUNOTOXICITY OF MWCNT
ends were used to homogenize the spleen. Isolated splenocytes remained on ice
until all samples had been isolated and then were centrifuged at 280 3 g for
10 min. Splenocyte pellets were resuspended in 2 ml of supplemented RPMI
media. Lymphocytes were counted and viability was determined for each
sample by trypan blue exclusion on a hemocytometer. Counts and viabilities
were recorded and used for normalizing lymphocyte number during cell plating
for immune function assays.
Jerne-Nordin plaque assay. Spleen cells were suspended in 3 ml of
supplemented RPMI media at a concentration of 4 3 106cells/ml. For the
Jerne-Nordin plaque assay, supplemented media contained heat inactivated
FBS (Hyclone), 1% penicillin/streptomycin (Cambrex), 1%
(Cambrex), 0.09% 55mM 2-mercaptoethanol (Gibco, Grand Island, NY), 1%
100mM sodium pyruvate (Cambrex), and 0.5% gentamicin (Gibco).
Sheep red blood cells (SRBC, 1% cells in media) (Colorado Serum, Denver,
CO) were washed and suspended in supplemented RPMI media and added to
the appropriate wells of a 48-well plate. Each sample was immunized with
SRBC in duplicate. SRBC-free media was used for nonimmunized control
wells for each sample. Cells were incubated for 4 days in 5% CO2at 37?C.
Following spleen cell immunization, cells were washed and resuspended in
supplemented media. A 0.8% solution of agarose (SeaPlaque, Cambrex,
Rockland, ME) in 2 3 RPMI medium (Gibco) was warmed to 43?C in glass
tubes. SRBC and spleen cells were added to the tubes and then spread on
agarose-coated microscope slides and incubated face down on custom slide
trays in a humidified plastic box at 37?C for 1 h. Guinea pig complement
(Colorado Serum) was diluted 1:20 in Dulbecco’s phosphate-buffered saline
(PBS) containing Ca2þand Mg2þ(Sigma Aldrich) and warmed to 37?C in
a water bath. Slides were flooded with diluted complement following the 1-h
incubation and then incubated an additional 2 h. Slides were removed from the
incubator and stored in a cold 0.85% sodium chloride solution. SRBC lysis was
quantified by counting plaques in the SRBC/agar lawn using a dissecting
microscope. Control values for the assay are reported and results expressed as
Mitogenesis assay. For each spleen sample, cells were suspended in 5 ml
of supplemented RPMI media at a concentration of 1 3 106cells/ml. Each
spleen sample was tested in replicates of six for three mitogens, for a total of 18
wells per sample. Two hundred microliters (2 3 105cells) were placed in
corresponding wells of sterile flat-bottom 96-well plates. Concanavalin A (Con A)
(Sigma Aldrich) was added to wells at a final concentration of 1 lg/ml.
Lipopolysaccharide (LPS) (Alexis, San Diego, CA) was added to wells at
a final concentration of 10 lg/ml. Supplemented RPMI media were added to
control wells. Plates were briefly mixed on a plate shaker and then placed in
a 5% CO2, 37?C incubator for 48 h. Following incubation, the cells were pulsed
with 1 lCi per well of
incubated for an additional 18 h. Cells were harvested using a Brandel Model
24V cell harvester onto filter paper (Whatman, Maidstone, England) and lysed
with a 0.05% Tween 20 solution (Fisher Scientific, Denver, CO). Once samples
were completely dry, they were placed in liquid scintillation vials containing
3 ml ScintiVerse Scintillation Cocktail (Fisher Scientific), and counted on a
liquid scintillation beta counter. Results for controls are reported and then
expressed as percent control based on counts per minute.
3H-thymidine (MP Biomedicals, Irvine, CA) and
Natural killer cell assay. A mouse lymphoma cell line (Yac-1, ATCC,
Manassas, VA) was used as a target cell in this assay because it is sensitive to
the cytotoxicity of natural killer (NK) cells. Yac-1 cells were incubated with
radioactive chromium-51 (PerkinElmer) (50 lCi/1 3 105Yac-1 cells) for 1 h
prior to plating. Labeled Yac-1 cells were then plated at a concentration of 5 3
103cells per well in round-bottom 96-well plates. Splenocytes were plated at
1 3 106cells/ml for an effector to target ratio (E:T) of 200:1. E:Ts of 200:1,
100:1, 50:1, and 25:1 were plated as an internal control for the assay. In order to
measure maximum chromium release, Triton X-100 was added at a concentra-
tion of 5% to control wells and spontaneous chromium release was measured
by analyzing wells that only contained target and no effector cells. Splenocytes
and Yac-1 cells were coincubated for 4 h at which point plates were centrifuged
at 200 3 g for 10 min. In order to measure chromium release as a result of Yac-1
lysis, supernatants were collected into tubes (PerkinElmer) and analyzed on
a gamma counter.
Real-time RT-PCR. Right lung lobes or spleen samples were transferred
from ? 80?C directly into lysis buffer provided by RNeasy Qiagen Kit
(Valencia, CA), and homogenized manually using RNase-free sterile pellet
pestles (Kimble/Kontes, Vineland, NJ). RNA was isolated using a standard
bench protocol provided with the kit. Total RNA quantity was determined by
spectrophotometer (DNanoDrop Technologies, Wilmington, DE). A reverse
transcription step was performed on total RNA at a concentration of 8 ng/ll
using cDNA archive kit (Applied Biosystems, Foster City, CA). cDNA was
amplified 40 times and detected using Universal PCR master mix (Applied
Biosystems) and TaqMan primer/probe sets (Applied Biosystems) for indicated
Enzyme-linked immunosorbent assay. Isolated splenocytes were centri-
fuged and supernatants collected and frozen for protein detection. Ready Set Go
ELISA kit for IL-10 (eBioscience, San Diego, CA) was purchased and standard
bench protocol provided by the kit was used. Briefly, 96-well plates were
coated overnight at 4?C with mouse IL-10 capture antibody. Plates were
washed five times with a wash buffer containing 0.05% Tween 20 (Fisher
Scientific) in Dulbecco’s PBS (Sigma Aldrich). Plates were then blocked for
1 h with assay diluent provided with the kit and washed five times. One hundred
microliters of sample or IL-10 standard provided by the kit was added per well
and incubated overnight at 4?C followed by five washes. Plates were incubated
for 1 h with 100 ll per well biotin-labeled detection antibody and washed five
times. Plates were incubated with 100 ll per well avidin/HRP for 30 min then
washed 14 times. One hundred microliters per well of substrate solution was
added and incubated for 15 min followed by addition of 50 ll stop solution, 2M
sulfuric acid. Plates were read on a 96-well plate reader at 450 nm with 570-nm
wavelength subtraction. Sample readings were quantified using a standard
curve and expressed as percent control.
Statistical analysis. All assays were run with n ¼ 6 per group with the
exception of the ELISA, where an n ¼ 3 was used due to limited sample.
Differences among treatment groups were tested by one-way ANOVA using
SigmaStat software version 3.5 (Systat Software Inc., Richmond, CA). Error
bars indicate the SE of the samples. Criterion for biostatistical significance was
set at p ¼ 0.05.
MWCNT Bulk Characterization
Table 1 summarizes the manufacturer’s specifications of the
MWCNTs and the additional analytical results we obtained.
(manufacturer’s specifications ranged from 40 to 300 m2/g).
TEM and SEM analysis of the bulk material (Fig. 2) showed
a high purity for the MWCNTs with little indication of
impurities due to metal catalyst, oxide, or ash. The SEM
revealed the clumping of bulk material. TEM showed a range
of diameters (~10–20 nm; ~20 nm shown), with each tube
structure defined by herringbone-shaped carbon layers. The
herringbone structure is one of many carbon layer structures
previously observed in MWCNTs and other carbon nanofibers
(Vander Wal et al., 2002). The length of the individual tubes
were not measured (manufacturer specifications indicate they
are ~5–15 lm in length). It was also noted that nanotubes did
not appear to be rigid, but instead they were flexible and the
majority were coiled into agglomerates that were less than
MITCHELL ET AL.
1 lm. XPS analysis revealed high carbon purity (> 97%),
with little indication of impurities (Table 1). As indicated
below for aerosol characterization, more sensitive methods did
detect small quantities of metal impurities.
Aerosol Concentration and Characteristics
Average MWCNT particle concentrations for the 14-day
inhalation exposure were (mean ± SD) 0 (control), 0.3 ± 0.1,
1.0 ± 0.1, and 5.3 ± 0.6 mg/m3. Electron microscopy (Fig. 3)
showed that the MWCNT aerosols were a mixture of material
in varying states of agglomeration (clumping), including some
tubes that were not agglomerated. The agglomeration increased
with increasing aerosol concentration. The mass median aero-
dynamic particle diameter was approximately 0.7–1 lm (~2.0
geometric SD) for the 0.3- and 1-mg/m3exposure and 1.8 lm
(2.5 geometric SD) for the 5-mg/m3exposure. Particle number
size distribution was smaller, with a median at approximately
350–400 nm at the 1-mg/m3exposure level (Fig. 4).
EDS analysis of MWCNTs on TEM grids (Fig. 5) revealed
the presence of nickel and iron impurities. These metals were
used as catalysts by the manufacturer during the CVD synthesis
of the MWCNTs. ICPMS analysis showed that nickel and iron
each accounted for approximately 0.5 % of the particle mass in
the aerosol. No other metals were observed at concentrations
above the detection limits of the ICPMS.
Bronchial Alveolar Lavage Fluid
Analysis of BALF showed clear evidence of MWCNTs
engulfed by macrophages (Fig. 6). At the 5-mg/m3exposure
level, macrophages appeared to be heavily loaded with particles.
However, there was no increase in inflammatory cells in the
lung as a result of MWCNT exposure, and no change in the
distribution of cell types relative to nonexposed animals
Figure 7 compares control versus the 5-mg/m3exposure
group at increasing magnifications (images taken using 4, 10,
and 40 3 objectives). Control and exposed lung sections
looked very similar at low magnifications (panels A, B, D, and E;
4 and 10 3 objective), illustrating the lack of severe inflam-
mation and tissue injury and an absence of large aggregates of
particles. The inhalation exposure provided good distribution
of MWCNTs throughout the lung. At higher magnification
(40 3 objective, panels C and F) the MWCNTs can be
observed. These MWCNTs were found primarily as aggregates
MWCNT are not completely rigid and bend together into a mesh. Panel B illustrates the diameter and structure of individual MWCNT, showing an approximately
20-nm–wide MWCNT possessing a herringbone-shaped carbon lamella.
Images of MWCNT bulk material by (A) SEM and (B) TEM. MWCNT are provided from the vendor as agglomerated powders. As shown in (A), the
point cyclone, and diluted to 1 mg/m3(A–C). Reference bars are shown for each image. The majority of the MWCNT were agglomerated into clumps. As shown,
the material were not rigid, they were rather flexible. While material agglomerates, the aerosolization procedure did not appear to damage or fracture any of the
MWCNT (compared with Fig. 2).
Electron micrographs of MWCNTs collected after aerosolization from inhalation exposure atmospheres, size selected by passing through a 2-lm cut-
PULMONARY AND IMMUNOTOXICITY OF MWCNT
that were in macrophages. A few free MWCNTs were also
observed. The amount of material apparent on light micro-
scopic exam was qualitatively correlated with exposure con-
centrations. Although, as expected, with increased exposure an
increased number of particles were observed in the lung.
Because no indications of fibrosis, increased cellularity, or
granuloma formation were observed by light microscopic
analysis at the highest exposure group, only sections from that
group are shown. As indicated, most of the visible material was
present within alveolar macrophages, with lesser amounts noted
extracellularly on surfaces, and none noted within other cell
types. There were only minor amounts of the MWCNTs
observed in the conducting airways, and there was a minimal
tendency for macrophages to accumulate in the region of the
alveolar ducts. While macrophages containing MWCNTs were
more visible in histologic sections, a substantiative increase in
macrophage numbers was not observed (similar to the obser-
vation in BALF).
Systemic Immune Function
Mice exposed to MWCNTs for 7 days did not have altered
immune function in any of the treatment groups (data not
shown). Mice exposed for 14 consecutive days, at all MWCNT
concentrations, demonstrated a suppressed T-cell–dependent
antigen response (Fig. 8A). Plaque-forming cell cultures for an
average control-treated animal were in the range of 500–600
plaques per culture, a common range for this assay. Results
are expressed as percent control. Figure 8B shows that exposed
T cells had reduced proliferative ability, as illustrated by
the reduction in response to mitogen (Con A). Results were
collected as counts per minute (average LPS response was
20,000 cpm for control animals) and expressed as percent
control. B-cell proliferation (LPS stimulation) was unaffected
(Fig. 8B). NK cell–mediated lysis of Yac-1 target cells,
a measure of innate immune response, was suppressed by
MWCNT inhalation only at the 1-mg/m3
Gene and Protein Expression
IL-6, IL-10, and NAD(P)H oxidoreductase 1 (NQO1)
mRNA expression was not increased in the lung following
inhalation of MWCNTs for 7 or 14 days (data not shown).
However, spleen mRNA levels for IL-10 and NQO1 were
increased significantly (p < 0.05) with 14-day MWCNT
exposure (Fig. 9A). The mRNA results observed for IL-10
were confirmed with IL-10 protein analysis by ELISA, which
showed an increase in IL-10 protein at 1 and 5 mg/m3
Inhalation of MWCNTs for 14 days up to the current
occupational exposure guideline for human exposure to nuisance
and time of flight for MWCNTs aerosolized, transported through a 2-lm size
selective cyclone, and diluted to 1 mg/m3.
Particle number size distribution analyzed by differential mobility
analysis shows presence of nickel and trace amounts of iron, both catalysts used in the synthesis of MWCNTs. Large carbon peak (left) is from the carbon structure
EDS of TEM sample of MWCNTS from the 1-mg/m3exposure atmosphere. Copper peak is due to the background from the collection grid. Spectral
MITCHELL ET AL.
dusts (5 mg/m3) (NIOSH, 2005) did not result in lung damage
using the measurements described above but did affect
systemic immunity. Immune function measurements on
spleen-derived cells showed suppressed T-cell–dependent
antibody response, decreased proliferation of T cells following
mitogen stimulation, and altered NK cell killing. These results
were accompanied by increased NQO1 and IL-10 gene
expression (indicators of oxidative stress and altered immune
function, respectively) in the spleen, but not in the lung.
The absence of pulmonary effects, even at relatively high
exposure concentrations, contrasts with many of the findings
associated with the pulmonary toxicity of CNTs reported to
date. Others have reported significant inflammation, oxidative
stress, and tissue injury resulting from CNT instillations and
pharyngeal aspirations. These previous studies included rats
and mice receiving instilled or aspirated doses of 1–5 mg/kg of
SWCNTs (Lam et al., 2004; Shvedova et al., 2005; Warheit
et al., 2004) and rats instilled with MWCNTs up to
approximately 10 mg/kg (Muller et al., 2005). All these previous
studies reported significant granuloma formation, and some
studies observed fibrosis in combination with the granulomas
(Shvedova et al., 2005). There was significant variation in the
response to CNTs obtained from different sources, which Lam
et al. (2004) attributed to residual metal content for the
SWCNTs. Muller et al. (2005) reported the only MWCNT
results and showed an increase in lung pathology and inflam-
mation at approximately 10 mg/kg, but not in the 2-mg/kg
range. MWCNT toxicity was increased when physically ground
into smaller agglomerates (Muller et al., 2005). In most cases,
both SWCNTs and MWCNTs at high bolus doses resulted in
significant lung pathology within a short time period (days to
weeks). When the rodents were monitored for several months,
the tissue injury progressed.
The absence of inflammation and significant pathology for the
MWCNT inhalation study described herein is not surprising.
As for silica and some other materials, the response to inhaled
CNTs may manifest over several months (Langley et al., 2004)
and was only analyzed at 14 days. Based on the dramatic (and
early) responses by instillation or aspiration reported in the
literature, it was originally thought that we would observe
some pathology and inflammation even at early time points.
The estimated deposited doses for this study were 0.2, 0.5, and
2.7 mg/kg at the 0.3-, 1-, and 5-mg/m3exposure levels,
respectively. While the highest exposure concentration mimics
the current occupational exposure guideline for nuisance dusts
(applied to CNTs in the absence of a specific standard), it is
approximately 100 times the concentration of CNTs found in
an industrial hygiene report by Maynard et al. (2004). The
justification for higher exposure concentrations may be dif-
ficult, especially considering the potential for false positives
that may result from overload of particles in the lung
(Mossman, 2000; Oberdorster, 2002). The dramatic effects
that have been reported in the literature for MWCNTs have
been typically only reported at doses well above our highest
In addition to differences in response between this study and
previous reports that may be attributed to dose, specific type,
and functionalization of CNTs, an important consideration is
the route of administration. While instillation has commonly
been used to compare particles of different types (Henderson,
1995; Seagrave et al., 2002), the use of instilled or aspirated
CNTs may be complicated by their poor solubility, which
makes creation of homogenous and well-dispersed dosing sus-
pensions difficult. Instillation, and to a lesser extent aspiration,
results in the agglomeration of material (albeit particle size in
solution not reported in aforementioned publications on CNT
instillations and aspirations). This suspension is placed either
directly into the lung or on the back of the tongue and may lead
to granuloma formation because large concentrations of material
collect in specific foci which result in a ‘‘foreign body’’ type
exposed for 14 days to 5-mg/m3(A) MWCNT and (B) controls. BALF from
exposed lungs contained many particle-laden and some enlarged macrophages.
Representative images from BALF collected from animals
BALF Cell Differentials Collected from Three Washes of Right
Lung Lobes Did Not Show a Change in the Composition of
Lavage Cells with MWCNT Exposure
100 ± 0
99.17 ± 1.33
100 ± 0
97.17 ± 2.04
0.67 ± 1.20.17 ± .41
0.99 ± 1.261.84 ± 1.48
Note. Results are expressed as average cell percentage observed in BALF
(n ¼ 6) ± SD.
PULMONARY AND IMMUNOTOXICITY OF MWCNT
A relevant example is that of diesel particles, a carbonaceous
material that is different from CNTs but has similar properties
in solution (forms agglomerates due to poor solubility and Van
der Waal’s forces). Diesel particles will cause granulomas
(similar to what have been reported for CNTs) when
administered by instillation (Seagrave et al., 2002). In contrast,
inhalation of diesel exhaust (DE) at occupational exposure
levels did not lead to persistent lung inflammation, tissue
injury, or granuloma formation (Reed et al., 2004). Further-
more, Reed et al. (2004) did show macrophages laden with
diesel particles, but did not find an increase in macrophage
number in the lung after inhalation exposure durations for
1 week or 6 months. During histopathological analysis, we
observed most of the MWCNTs in the alveolar region and not
in the conducting airways. Deposition in conducting airways is
often observed when particles are administered by instillation.
It is plausible that even with CNTs that may be more toxic than
the MWCNTs we studied here, tissue injury would be dra-
matically reduced if administered by inhalation.
We have previously reported impaired immune function
following inhalation of carbonaceous particles in environmen-
tally relevant materials such as DE and wood smoke. After
6-month exposures to hardwood smoke, AJ mice had sup-
pressed T-cell mitogenic response. This was determined to be
exposed to 5 mg/m3MWCNTs for 14 consecutive days (panels D, E, and F) by inhalation. Note the unremarkable appearance of the lung from the exposed animal
at low magnification (panels D and E), including the alveolar duct regions. At increasing magnification, alveolar macrophages from control lungs are unremarkable
(arrowheads), while dark, particulate-laden macrophages become evident in exposed animals (large arrows). Scattered particulate material is also present on
surfaces (small arrows). However, no substantive lung damage or cellularity increase is apparent with exposure in this study. (V, vessel; TB, terminal bronchial;
AD, alveolar duct. Magnifications as indicated by bars).
Representative hematoxylin and eosin–stained lung sections at equivalent magnifications from a control animal (panels A, B, and C) and an animal
MITCHELL ET AL.
from altered immune cell function and not due to lymphocyte
cell death (Burchiel et al., 2004). Six-month exposures to DE
resulted in suppressed splenic T-cell mitogenic responses as
well (Burchiel et al., 2005). In those studies it was not
determined if the particles or gaseous components of the
exposures caused the immune suppression. If the particles did
play a role, then it may be plausible that the mechanism of
MWCNT-induced immune suppression is similar. However,
this has not been tested.
Others have shown increased oxidative stress and increased
IL-10 levels with carbon-based particle exposures (Harris et al.,
1997a,b; Li et al., 2002, 2003; Pacheco et al., 2001; Ullrich
and Lyons, 2000). Following particle exposures in vivo, Harris
et al. (1997a,b) noted immunosuppression in animals following
inhalation exposures to high doses of aerosolized jet propulsion
fuel (JP-8) that was linked to T-cell dysfunction. This obser-
vation was later expanded by Ullrich and Lyons (2000) and
linked to IL-10 production. IL-10 is an anti-inflammatory
cytokine principally secreted by macrophages and T cells
(T-helper type 2 [TH2]). Its activity serves to downregulate
cytokines such as IL-12, TNF-a, IFN-c, and IL-1b (Yin et al.,
1997a,b). Although the role of IL-10 in modulating the immune
response to MWCNTs has not been confirmed, its upregulation
zation with sheep erythrocytes (SRBC). Plaque number per culture was decreased
with exposure (25–45% of control). Each sample was assayed without SRBC
immunization as a negative control. (B) T- and B-cell proliferation in response
to cell-specific mitogen was analyzed using tritiated thymidine incorporation.
T-cell proliferation in response to Con A was decreased with MWCNT
exposure. (C) NK cell function, as measured by target cell lysis, was suppressed
with exposure. Effector: Target ratios ranging from 200:1 down to 25:1 serve as
a quality control measure for the assay. *Signifies statistically significant dif-
ference (p < 0.05), n ¼ 6.
(A) Jerne-Nordin plaque assay results following 4-day immuni-
gene NQO1 were measured in spleen homogenate. Splenic mRNA expression
for IL-10 and NQO1 was increased with exposure (n ¼ 6). (B) IL-10 protein, as
measured by ELISA with splenocyte supernatant, was increased with exposure
to MWCNTs. *Signifies statistically significant difference (p < 0.05), n ¼ 3.
(A) Interleukin cytokines IL-6 and IL-10 and electrophile response
PULMONARY AND IMMUNOTOXICITY OF MWCNT
in the spleen was intriguing. Furthermore, IL-10 and NQO1
expression in the spleen, not observed in the lung, suggests that
MWCNTs are potentially bypassing pulmonary defense
mechanisms and reaching circulation. This is further evidenced
by a lack of cellular influx and IL-6 expression in the lung.
Within the systemic framework, MWCNTs may induce
oxidative stress and/or activate electrophile response pathways
resulting in NQO1 and simultaneous or consequent IL-10
IL-10 is a very important regulator of the immune system
and serves to maintain homeostatic control of innate and cell-
mediated immune responses. However, if IL-10 is inappropri-
ately expressed, as shown in this study, it may suppress normal
immune response and increase susceptibility to infection and
disease. The complexity of the immune system is such that
suppression on the level of innate immunity results in affects
on a humoral level as well. The Jerne-Nordin plaque assay is an
evidence for immune cell interaction, where T-helper cell
activation in presence of antigen, SRBC, results in communi-
cation with B cells to induce IgM antibody production.
However, IgM induction is a T-helper cell type 1 (TH1)
effect; IL-10 is involved in TH2 polarization and therefore
results in suppression of the IgM response as well as decreased
antiviral, antitumor, and intracellular microbe killing. IL-10
expression has been linked to diabetes promotion (Balasa et al.,
2000) and cancer risk (Mustea et al., 2006) in humans. IL-10
has also been shown to increase atopy by enhancement of IgE
secretion by B lymphocytes (Ohmori et al., 1995; Prasse et al.,
Several questions arise from the observations of immune
suppression related to exposure to CNTs and environmental
pollutants. First, the immune function responses observed in
the spleen have not been evaluated in other parts of the body,
such as in the lymph nodes or in immune cells in the lung.
Many questions exist regarding the mechanism by which this
immune suppression occurs. Certainly, there is evidence that
exposure to inhaled materials may perturb the ability to fight
infection, as evidenced by both animal and epidemiological air
pollution studies (Harrod et al., 2003, 2005; Shay et al., 1999).
While the reports by Shvedova et al. (2005) suggest a decreased
ability to clear Listeria bacteria from the lung after SWCNT
exposure, that study design may have focused more on
evaluating a decreased macrophage function (ability to clear
bacteria), possibly due to particle overload, than on a sup-
pressed immune system.
A key component of our study was development of an
aerosol generation and inhalation exposure system suitable for
CNT rodent exposures. The exposure system was designed to
simulate a potential exposure to MWCNTs in the workplace or
by an end user prior to functionalization and integration into
a product. As such, the approach used powder resuspension of
bulk material using a well-tested aerosol generator (Cheng
et al., 1985). The system was operated with an ~2-lm cut-point
cyclone in line to remove particles that agglomerated and those
larger than the size a rodent can inhale. While the energy and
dilution was designed to minimize excessive agglomeration of
the MWCNTs, there were no attempts to intentionally
aerosolize ‘‘singlet,’’ nonagglomerated MWCNTs. The aerosol
that was produced showed reasonable agreement with the
limited amount of industrial hygiene measurements of CNTs
that have been produced (Maynard et al., 2004). Maynard et al.
(2004) reported a submicron size distribution (based on number)
that changed dependent on the amount of artificial agitation
applied. Our system also showed a submicron aerosol (based on
number), with particles residing primarily at about 300–400 nm.
When sized by mass, they were approximately 1–2 lm in
diameter. The aerosol consisted of a mixture of a small number
of individual tubes and a large amount (by mass) of tubes that
agglomerated. This property was desirable, as particles will
also most likely exist in an agglomerated state when humans
are exposed to them. The agglomeration, as indicated by
particle size, increased at the highest exposure concentration.
Characterization showed approximately 99% pure CNTs,
with only 1% residual metals (nickel and iron) that are used
as catalysts during CVD synthesis. Overall, the system was
judged suitable for application of hazard assessment for a wide
range of CNTs to represent potential occupational exposures.
This study served to improve the overall understanding of
the hazard associated with inhalation of CNTs. While the
results of the MWCNTs used in this study may not represent
the biocompatibility of other CNTs, this work provides a start
toward placing some of the findings presented by instillation in
context of exposure to a well-dispersed aerosol. More work
needs to be done to evaluate the role of CNTs of various
compositions and all CNTs using study designs that evaluate
the potential for progressive injury. Certainly, the findings of
immune suppression, cytokine upregulation, and oxidative
stress in the spleen were intriguing. The immune suppression
was observed even at the lower exposure levels. Further work
is necessary to place these results in context, and to further
elucidate a mechanism for this response.
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