Toxicology 262 (2009) 207–214

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Toxicology
journal homepage: www.elsevier.com/locate/toxicol

High-dose, short-term exposure of mice to perfluorooctanesulfonate (PFOS) or

perfluorooctanoate (PFOA) affects the number of circulating neutrophils
differently, but enhances the inflammatory responses of macrophages to
lipopolysaccharide (LPS) in a similar fashion
Mousumi R. Qazi a , Jasna Bogdanska a , John L. Butenhoff b , B. Dean Nelson a , Joseph W. DePierre a ,
Manuchehr Abedi-Valugerdi a,∗
a
Department of Biochemistry and Biophysics, Arrhenius Laboratories for the Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden
b
3M Medical Department, 3M Company, St. Paul, MN 55144, USA

a r t i c l e i n f o a b s t r a c t

Article history: Having found previously that high-dose, short-term dietary exposure of mice to perfluorooctanesulfonate
Received 30 April 2009 (PFOS) or perfluorooctanoate (PFOA) suppresses adaptive immunity, in the present study we characterize
Received in revised form 10 June 2009 the effects of these fluorochemicals on the innate immune system. Male C57BL/6 mice receiving 0.02%
Accepted 11 June 2009
(w/w) PFOS or PFOA in their diet for 10 days exhibited a significant reduction in the numbers of total white
Available online 21 June 2009
blood cells (WBC), involving lymphopenia in both cases, but neutropenia only in response to treatment
with PFOA. Moreover, both compounds also markedly reduced the number of macrophages (CD11b+ cells)
Keywords:
in the bone marrow, but not in the spleen or peritoneal cavity. The ex vivo production of tumor necrosis
Innate immunity
Lipopolysaccharide
factor-␣ (TNF-␣) and interleukin 6 (IL-6) by peritoneal macrophages isolated from animals treated with
Perfluorooctanesulfonate PFOA or PFOS was increased modestly. Moreover, both fluorochemicals markedly enhanced the ex vivo
Perfluorooctanoate production of these same cytokines by peritoneal and bone marrow macrophages stimulated either in
Tumor necrosis factor alpha vitro or in vivo with lipopolysaccharide (LPS); whereas there was no such effect on splenic macrophages.
Interleukin 6 The serum levels of these inflammatory cytokines observed in response to in vivo stimulation with LPS
were elevated substantially by prior exposure to PFOA, but not by PFOS. None of these parameters of
innate immunity were altered in animals receiving a dietary dose of these compounds that was 20-fold
lower (0.001%, w/w). These findings reveal that in addition to suppressing adaptive immunity, high-dose,
short-term exposure of mice to either PFOS or PFOA augments inflammatory responses to LPS, a potent
activator of innate immunity.
© 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction In large part because of their long elimination half-lives

In perfluorooctanesulfonate (PFOS, C8 F17 SO3 − ), perfluorooc-
tanoate (PFOA, C7 F15 COO− ) and their salts, all hydrogen atoms
bonded to carbon have been replaced by fluorine. The unique
properties of these perfluorochemicals, including potent surfac-
tant activity, chemical inertness and exceptional stability to both
metabolic and environmental degradation have been exploited in
numerous industrial and consumer applications (Kissa, 2001; Lau
et al., 2007; Lehmler, 2005), while, at the same time, posing envi-
ronmental concerns.
Abbreviations: IL-6, Interleukin 6; LPS, Lipopolysaccharide; PFOS, Perfluorooc-
tanesulfonate; PFOA, Perfluorooctanoate; PPAR-␣, Peroxisome proliferator-activated
receptor-alpha; TNF-␣, Tumor necrosis factor alpha.
∗ Corresponding author. Tel.: +46 8 164240; fax: +46 8 153679.
E-mail address: abedi@dbb.su.se (M. Abedi-Valugerdi).
in humans (approximately 4 years; see Olsen et al., 2007;
Spliethoff et al., 2008), more and more focus is being placed
on research concerning the environmental toxicology of PFOS,
PFOA and their congeners (OECD, 2002, 2007; United States
Environmental Protection Agency, 2002, 2006; Lau et al., 2007;
Canadian Government Department of the Environment, 2008). The
numerous effects that high-dose, short-term exposure to these
compounds have on rodents, characterized in our own laboratory
(Sohlenius et al., 1992, 1993, 1994) and others (Kissa, 2001; Lau
et al., 2007; Martin et al., 2007; Andersen et al., 2008), include
pronounced hepatomegaly and peroxisome proliferation; reduced
appetite and loss of body weight and fat; changes in hormonal sta-
tus; reproductive and neurotoxicity; as well as, following long-term
exposure, hepatocarcinogenesis.
Moreover, our studies, as well as those of other investigators,
have shown that these compounds can suppress the adaptive
immune system of rodents (Yang et al., 2000, 2001, 2002a,b;

0300-483X/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.tox.2009.06.010
208 M.R. Qazi et al. / Toxicology 262 (2009) 207–214
Peden-Adams et al., 2008; Zheng et al., 2008; DeWitt et al.,
2009; Dong et al., 2009; Qazi et al., 2009). For instance, 7–10
days of dietary or gavage exposure (approximately 40 mg/kg/day)
of mice to either PFOS or PFOA causes severe atrophy of the
thymus (a 63–67% reduction in size) and spleen (a 34–50% reduc-
tion), two central organs of adaptive immunity (Yang et al., 2000,
2001; Zheng et al., 2008; Qazi et al., 2009). At the same time,
the populations of splenocytes and thymocytes of all phenotypes
are reduced dramatically in size (by approximately 23–78% and
48–99%, respectively). The most pronounced reduction in the case
of the thymus involves immature cells, indicating that prolifer-
ation and differentiation are disturbed. With both compounds,
the reduction in splenic cellularity is associated with a signif-
icant reduction in specific humoral immune responses against
foreign antigens, e.g., horse or sheep red blood cells (Yang et al.,
2002a; Zheng et al., 2008; Peden-Adams et al., 2008; Dong et al.,
2009).
We have also observed that the immunotoxic, but not hep-
atotoxic effects of PFOA are largely reversed within 7–10 days
after termination of exposure, revealing that no permanent dam-
age to the thymus or spleen has occurred (Yang et al., 2001).
Furthermore, it has been shown that the developing adap-
tive immune system is also sensitive to the effects of PFOS,
which results in functional defects in specific humoral antibody
responses detectable in adulthood (Keil et al., 2008). Moreover,
both PFOS and PFOA appear to exert their effects on the murine
adaptive immune system at least partially via the peroxisome
proliferator-activated receptor-alpha (Yang et al., 2002b; Qazi et al.,
2009).
In addition to their adaptive immunity, higher vertebrates
possess an innate branch of the immune system that responds
rapidly and non-specifically to invading microorganisms, lacks
memory, and involves a small number of germ-line encoded
receptors (collectively referred to as Toll-like receptors, TLR) that
recognize conserved molecular patterns associated with microbial
pathogens (PAMPs) (e.g., bacterial lipopolysaccharide (LPS), viral
single- and double-stranded RNA, and CpG motifs in viral DNA
(Medzhitov and Janeway, 1998; Kawai and Akira, 2005; Borghesi
and Milcarek, 2007). Previous studies on the effects of perfluo-
rinated compounds on this branch of the immune system have
focused primarily on the effects of PFOS on the function of Nat-
ural Killer (NK) cells, which spontaneously recognize and destroy
both virus-infected and tumor cells (Cerwenka and Lanier, 2001).
High doses of this compound decrease, while low doses enhance
the activity of these cells (Dong et al., 2009; Peden-Adams et
al., 2008; Zheng et al., 2008). It has also been shown that ges-
tational exposure to PFOS can suppress the function of NK cells
in adult offspring, suggesting that the development of NK activ-
ity is susceptible to the effects of this compound (Keil et al.,
2008).
The present study was designed to determine whether expo-
sure to PFOS and/or PFOA also influences other cells of the innate
immune system. To this end, we examined potential alterations in
the numbers of the different types of circulating innate immune
cells, as well as the functional capacity of the macrophages (key
cellular components of innate immune responses) present in
the peritoneal cavity, bone marrow and spleen to produce the
pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-␣)
and interleukin-6 (IL-6), the key mediators of inflammation (Van
Amersfoort et al., 2003). In addition, these same parameters were
monitored in association with activation of these innate immune
cells by bacterial LPS, both in vitro and in vivo. Our present find-
ings demonstrate that in contrast to their suppressive effects on the
adaptive branch of the murine immune system, high-dose, short-
term exposure to either PFOS or PFOA activates certain components
of innate immunity in mice.
2. Materials and methods
2.1. Animals
Male C57BL/6 (H-2b ) mice (6–8 weeks old at the beginning of each experi-
ment) were obtained from the Microbiology and Tumorbiology Center at Karolinska
Institute (Stockholm, Sweden) or from B&K (Solna, Sweden). This mouse strain was
chosen because of its wide use and paradigmatic status in immunological research.
Animals were housed in the animal facilities at the Wenner-Gren Institute, Stock-
holm University, at 22 ◦ C with a 12-h light/12-h dark cycle, 50% humidity and access
to the diets indicated below and tap water ad libitum. Upon arrival, each mouse was
placed in a separate cage (for individual monitoring of food intake) and allowed
to acclimatize to this environment for at least 5 days before starting the dietary
exposure to PFOS or PFOA. In order to mitigate the stress associated with single
housing, each cage was provided with white paper towels and toilet paper rolls. All
of the experiments performed on these animals were pre-approved by the North-
ern Stockholm Ethical Committee for Animal Experimentation (approval numbers
N380/03, N299/05, and N300/05).
2.2. Preparation of the diet
PFOS (the tetraammonium salt, 98% purity) and PFOA (the free acid, 96% purity)
were purchased from Sigma–Aldrich Sweden AB, Stockholm, Sweden. These sub-
stances were dissolved in 20 ml acetone and mixed with 100 g powdered RMI (E) FG
SQC diet (containing 2.71% fat, 14.38% protein and 61.73% carbohydrate; SDS, Special
Diets Services, Essex, UK) to obtain a concentration of 0.02% (w/w) (the dose that
elicits maximal responses, as demonstrated in our previous experiments (Yang et
al., 2000, 2001, 2002a; Qazi et al., 2009)) or 0.001% (w/w). This chow was subse-
quently dried for 12–24 h in a ventilated hood, after which no smell of acetone was
detectable. Control food was prepared in the same manner, except that no xenobiotic
was added. Thereafter, the chow was shaped into cakes to facilitate quantitation of
food consumption (since mice scatter powdered food all over their cages). All diets
were stored at 4 ◦ C prior to use.
2.3. Dietary exposure to either PFOS or PFOA with and without induction of
endotoxemia
In the first series of experiments, our objective was to characterize the direct
effects of PFOS and PFOA on the unactivated innate immune system. In this case,
groups of four mice each received chow containing 0.02% or 0.001% PFOS or PFOA or
a control diet (without added xenobiotic) for 10 consecutive days.
Subsequently, we examined whether exposure to PFOS or PFOA influences innate
immune responses to LPS. For this purpose, the mice were again treated as described
above and, thereafter, on day 10, each of the three groups was divided randomly into
two subgroups, one of which was injected through the tail vein with 0.1 ml sterile
physiological saline containing 300 ␮g LPS (Escherichia coli 055:B5; Sigma–Aldrich
Sweden, Stockholm), the optimal dose for induction of acute inflammation (Redl et
al., 1993), while the other group received the same volume of the vehicle alone via
the same route.
2.4. Collection of blood, organs and peritoneal cells
Either directly following the period of exposure (in the first series of studies) or
2 h after administration of LPS (in the second series), the mice were bled by retroor-
bital puncture under light isoflurane anesthesia and thereafter sacrificed by cervical
dislocation. Blood samples for preparation of plasma or serum were collected in cap-
illary blood collection tubes (Microtainer BD Bioscience, NJ, USA) containing either
dipotassium EDTA or no additive. The blood samples collected in the latter tubes
were allowed to clot at 4 ◦ C and then centrifuged in order to obtain serum, which
was stored at −20 ◦ C until being assayed for cytokines. The liver, epididymal fat,
spleen and thymus were collected and weighed.
Resident peritoneal exudate cells (primarily macrophages; see Section 3) were
collected by flushing the peritoneal cavities of the mice immediately following sac-
rifice with 10 ml Dulbecco’s Modified Eagle Medium (D-MEM), pH 7.0, containing
4500 mg glucose and 110 mg sodium pyruvate/l and 4 mM l-glutamine (Invitrogen
AB, Stockholm, Sweden) and supplemented with 0.02% (w/v) sodium bicarbonate
and 100 IU penicillin and 100 ␮g streptomycin/ml. These cells were washed once
with culture medium prior to subsequent manipulations.
2.5. Preparation of cell suspensions from the spleen and bone marrow
Spleens were dissected out aseptically and thereafter gently teased apart
with forceps in RPMI 1640 medium, pH 7.0, containing Glutamax (Invitrogen
AB, Stockholm, Sweden) supplemented with 15 mM HEPES, 100 IU penicillin and
100 ␮g streptomycin/ml, and 0.02% (w/v) sodium bicarbonate. The spleen cells thus
obtained were washed twice with culture medium prior to subsequent manipula-
tions.
Bone marrow cells were flushed out of the tibia with sterile PBS, using a syringe
with a 27-gauge needle. These cells were also washed twice with RPMI 1640 medium
prior to use.
 M.R. Qazi et al. / Toxicology 262 (2009) 207–214
2.6. Cell cultures and collection of culture media
Peritoneal cells, splenocytes and bone marrow cells were re-suspended in
the same D-MEM or RPMI 1640 medium utilized for obtaining these cells (see
above) with additional supplementation with 10% heat-inactivated fetal calf serum
(FCS) (Invitrogen AB, Stockholm, Sweden) and 5 × 10−5 M 2-mercaptoethanol
(Sigma–Aldrich Sweden AB, Stockholm, Sweden). After adjusting these suspensions
to contain 1 × 106 viable (as determined by Trypan blue exclusion) peritoneal or
2 × 106 viable bone marrow or spleen cells per ml, duplicate 2-ml aliquots were
plated into the wells of 12-well flat-bottom culture plates (Costar, Corning Incor-
porated, Corning NY, USA). Following incubation for 18 h (4–18 h being the period
routinely employed for monitoring cytokine production (Redl et al., 1993) at 37 ◦ C
under a humidified atmosphere containing 5% CO2 , the culture media were har-
vested and stored at −20 ◦ C for subsequent analysis. In experiments designed to
examine whether in vivo treatment with PFOS or PFOA altered the direct response of
macrophages to LPS, these cell cultures were first incubated in this standard manner;
the media then harvested; the cultures washed twice with fresh medium; and then,
fresh culture medium containing 100 ng LPS was added to each well and incubation
continued for an additional 18 h, following which the media were again harvested
and stored at −20 ◦ C for subsequent assay of cytokines.
2.7. Determination of TNF-˛ and IL-6
The levels of TNF-␣ and IL-6 in serum samples and culture media were quan-
titated employing commercially available ELISA kits (eBiosciense, San Diego, CA,
USA) in accordance with the manufacturer’s instructions. In all cases, quantitative
values were obtained by comparison with a standard curve constructed employing
cytokines provided by the manufacturer.
2.8. Total and differential white blood cell counts (WBC)
To obtain the total WBC count, 10 ␮l blood was diluted and homogenized in
190 ␮l Turk’s solution (3 ml glacial acetic acid and 1 ml 1% genetian violet dissolved
in 100 ml distilled deionized water) and placed in a Neubauer chamber (Improved
Neubauer, Levy Hemacytometer, Hausser Scientific, Gaithersburg, MD, USA) for cell
counting under a light microscope (Zeiss, Jena, Germany, 120×). For differential
counting of lymphocytes, neutrophils, monocytes, eosinophils and basophils, an
aliquot of each blood sample was smeared onto a glass slide, air-dried, and sub-
sequently stained with Wright stain. Oil immersion was used to count a total of 100
cells.
2.9. Immunofluorescent staining and flow cytometric analysis of peritoneal, bone
marrow and spleen macrophages
One million peritoneal, bone marrow or spleen cells suspended in 50 ␮l FACS
buffer (PBS containing 1% FCS and 0.1% NaN3 ) were added to each individual well of
96-well round-bottom tissue culture plates (Corning, NY) and thereafter incubated
with rat anti-mouse CD11b (an antigen expressed at high levels on the surface of
macrophages) labeled with (−) fluorescein isothiocyanate (FITC) (BD PharMingen;
San Diego, CA) in the dark and on ice for 30 min, in accordance with the recom-
mendations of the manufacturer (BD PharMingen; San Diego, CA). Subsequently,
the cells were washed by centrifugation at 200 × gav for 10 min, re-suspended in
400 ␮l FACS buffer and analyzed utilizing a single-laser FACSCalibur flow cytometer
(Becton Dickinson, San Jose, CA) equipped with a 15-mW, air-cooled 488 nm argon-
ion laser. The signals emitted by FITC were detected at 530 nm. For each sample, the
data from 10,000 events (individual cells) were collected and analyzed using the
CellQuest Software.
2.10. Expression of the data and statistical analysis
The data are expressed as means ± SE (standard errors) and evaluated by anal-
ysis of variance (ANOVA) or the Mann–Whitney (U-test) at the significance level
2˛ = 0.05, as indicated in the legends to the figures and tables. All statistical analy-
ses were performed utilizing the WinSTAT software (R. Fitch Software, Medina AB,
Vänerborg, Sweden).
3. Results
3.1. Effects of high-dose, short-term exposure to PFOS or PFOA on
the numbers of circulating white blood cells
As expected (Yang et al., 2000, 2002a; Qazi et al., 2009), dietary
administration of 0.02% PFOS or PFOA to mice for 10 days signif-
icantly enhanced the weight of their livers, whereas the weights
of the whole body, thymus, spleen and epididymal fat depots were
reduced (not shown). Moreover, food consumption by the animals
exposed to PFOS or PFOA was reduced by approximately 25% (to
approximately 3 g/day) and 35% (2.6 g/day), respectively, in com-
209
Table 1
Effects of a 10-day dietary exposure of male C57BL/6 mice to 0.02% (w/w) of PFOS
or PFOA on the numbers of circulating white blood cells (WBC), lymphocytes and
neutrophils.a .
Treatment Number × 109 of white blood cells/l
Total WBC Lymphocytes Neutrophils
None (control) 8.080 ± 0.059 7.120 ± 0.521 0.777 ± 0.114
PFOS 3.700 ± 1.030* , b 3.460 ± 0.912* 0.617 ± 0.107
PFOA 2.300 ± 0.412* 1.900 ± 0.383* 0.371 ± 0.046*
a
Blood samples were analyzed for the total numbers of white blood cells (WBC),
as well as lymphocytes and neutrophils using Turk’s solution and Wright staining,
respectively.
b
All values are means ± SE (standard error) for eight animals in each group. Dif-
ferences were analyzed for statistical significance using the U-test.
*
Statistically significant as compared with control group.
parison to untreated mice (4.1 g/day). Thus, total intake of PFOS
and PFOA in 10 days would be about 6 mg and 5.2 mg, respectively.
The concentrations of PFOS and PFOA in the serum following this
regimen of treatment have been determined previously employ-
ing mass spectrophotometric analysis to be 340 ± 16 ␮g/ml = ppm
(680 ± 32 ␮M) and 152 ± 8.6 ␮g/ml = ppm (367 ± 20.8 ␮M), respec-
tively (Qazi et al., 2009).
As a first indicator of possible effects of PFOS and/or PFOA on
the innate immune system, we analyzed the total number of cir-
culating WBC and subpopulations thereof following treatment. As
depicted in Table 1, both compounds reduced the total numbers
of circulating WBC (leukopenia) and lymphocytes (lymphopenia),
whereas the number of circulating neutrophils was decreased only
by PFOA. Neutrophils are key cellular mediators of innate immune
responses and these findings thus provide an initial indication that
at least PFOA may exert an adverse effect on these responses. The
numbers of circulating monocytes, eosinophils and basophils were
too small to be determined reliably.
3.2. Effects of dietary exposure of mice to 0.02% (w/w) PFOS or
PFOA on the total numbers of macrophages among cells isolated
from the peritoneal cavity, bone marrow and spleen
Macrophages are broadly distributed in various body organs
and tissues (Gordon, 1998; Naito et al., 1996), where they play a
key role in defences against invading microorganisms, clearance of
altered host cells, activation of inflammatory responses and reac-
tions against xenobiotics (Gordon, 1998, 2002; Xia and Triffitt,
2006). Exposure to PFOS or PFOA elevated the proportion (percent-
age), but not the total number of macrophages (CD11b+) among
the cells isolated from the peritoneal cavity; reduced both the pro-
portion and total number of these cells in the bone marrow; and
had no significant effect on the size of the splenic population of
macrophages, despite the fact that the overall cellularity of the
spleen was strikingly reduced (Table 2). The significant elevation
in the percentage of macrophages in the spleen of PFOA-treated
animals appears to be simply a mathematical consequence of the
pronounced decreases in the populations of the other types of cells
present in this organ (Yang et al., 2000, 2001; Qazi et al., 2009). Thus,
the numbers and relative proportions of macrophages in these three
immune compartments are influenced differently by exposure to
PFOS or PFOA.
3.3. The inflammatory responses of macrophages isolated from
the peritoneal cavity, bone marrow and spleen of mice upon
exposure to lipopolysaccharide (LPS) in vitro are altered by prior
in vivo treatment with PFOS or PFOA
To examine whether PFOS and/or PFOA might also influence
the functional properties of peritoneal, bone marrow and splenic
210 M.R. Qazi et al. / Toxicology 262 (2009) 207–214

Table 2
Effects of a 10-day dietary exposure of male C57BL/6 mice to 0.02% (w/w) of PFOA or PFOS on the numbers and relative proportions of macrophages (CD11b+ cells) among
the cells isolated from the peritoneal cavity, bone marrow and spleen.a .

Compartment Treatment

None (control) 0.02% PFOS 0.02% PFOA

Total cells (106 ) Macrophages % of total Total cells (106 ) Macrophages % of total Total cells (106 ) Macrophages % of total
(106 ) (106 ) (106 )

Peritoneal cavity 3.7 ± 0.5 2.9 ± 0.4 78.1 ± 1.9 3.3 ± 0.6 2.8 ± 0.5 84.2 ± 1.2* , b 2.3 ± 0.5 2.0 ± 0.4 85.6 ± 1.0*
Bone marrow 8.9 ± 0.7 1.8 ± 0.2 20.1 ± 1.5 7.0 ± 0.4 0.8 + 0.1* 12.1 + 1.4* 4.9 ± 0.6* 0.6 ± 0.1* 12.2 ± 0.9*
Spleen 81.4 ± 6.9 10.7 ± 2.4 12.7 ± 1.7 50.5 ± 1.5* 7.3 + 0.7 14.4 + 1.2 36.2 ± 4.0* 7.9 ± 0.1 21.5 ± 1.5* , c
a
Peritoneal, bone marrow and spleen cells were isolated and incubated with a monoclonal antibody directed towards CD11b, a selective cell-surface marker for macrophages.
Subsequently, the numbers and percentages of macrophages present were determined by flow cytometry.
b
All values are means ± SE (standard error) for eight animals in each group. Differences were analyzed for statistical significance employing the U-test.
c
Significantly different (p < 0.05) from the corresponding values for both control and PFOS-treated animals (ANOVA test).
*
Statistically significant as compared with the corresponding control group.

macrophages, we determined the ability of these cells to produce
TNF-␣ and IL-6 ex vivo with and without stimulation with LPS
(100 ng/ml). Since the isolation of macrophages would introduce
uncertainties (i.e., concerning the extent of recovery and the func-
tional state of the isolated cells) into the analysis and since in
response to LPS macrophages are the primary producers of TNF-
␣ and IL-6 (Freudenberg et al., 1986; Salkowski et al., 1995), we
performed these experiments on crude cell suspensions. As shown
in Fig. 1, in the absence of this potent activator of macrophages,
peritoneal cells isolated from mice treated with either compound
produced significantly enhanced levels of both of these cytokines
(Fig. 1a and d); the bone marrow cells from animals administered
PFOA secreted elevated levels of TNF-␣ (Fig. 1b); whereas the pro-
duction of this latter cytokine by spleen cells was reduced by both
PFOS and PFOA (Fig. 1c and f).
As expected, in vitro stimulation of cells isolated from the
peritoneal cavity, bone marrow and spleen of control mice with
LPS potently enhanced the production of both TNF-␣ and IL-6
(Fig. 1a–f). Moreover, in vivo exposure to PFOS potentiated these ex
vivo responses of peritoneal and bone marrow cells to LPS (Fig. 1a,
b, d, and e), while, at the same time, attenuating the correspond-
ing production of TNF-␣ by spleen cells (Fig. 1c). A similar pattern
was observed following treatment with PFOA, except that in this
case, the ex vivo production of TNF-␣ and IL-6 by spleen cells in
response to in vitro stimulation with LPS was also significantly
enhanced (Fig. 1a–f). This latter enhancement can be accounted for
partially by the increase in the proportion of macrophages among
the splenocytes. These findings indicate that in vivo treatment
with either of these fluorochemicals alters the ex vivo responses
of macrophages located in all three of these compartments to LPS.

Fig. 1. High-dose, short-term exposure of mice to PFOS or PFOA alters the ex vivo production of TNF-␣ and IL-6 by cells isolated from the peritoneal cavity, bone marrow and
spleen of mice, both with and without subsequent stimulation in vitro by lipopolysaccharide (LPS). Male C57BL/6 mice received a normal (control) diet or diet containing
0.02% PFOS or PFOA for 10 days. Peritoneal, bone marrow and spleen cells were then collected and cultured in D-MEM or RPMI 1640 medium for 18 h as described in Section
2. Thereafter, culture media were collected, fresh medium containing 100 ng LPS/ml added and the cells then incubated for an additional 18 h, following which the culture
media were harvested. The levels of TNF-␣ (a–c) and IL-6 (d–f) in all of the culture media collected (both prior to and after activation by LPS) were determined by ELISA
procedures. The values presented are means ± SE (standard errors) for a total of eight mice in each group, examined in two independent experiments. For clarity, the actual
concentrations of TNF-␣ or IL-6 for unstimulated cells are shown in parentheses above the corresponding bars. *Statistically significant as compared with the corresponding
control group.
 M.R. Qazi et al. / Toxicology 262 (2009) 207–214 211

Fig. 2. Exposure of mice to PFOS or PFOA prior to induction of endotoxemia by intravenous injection of lipopolysaccharide (LPS) potentiates the ex vivo production of TNF-␣
and IL-6 by cells isolated from the peritoneal cavity and bone marrow, but not from the spleen. Male C57BL/6 mice received a normal (control) diet or diet containing 0.02%
PFOS or PFOA for 10 days. On day 10, half of the mice receiving each treatment were injected (inj.) through the tail vein with 300 ␮g lipopolysaccharide (LPS) and 2 h later
peritoneal, bone marrow and spleen cells were collected from all of the animals and cultured as described in Section 2. The levels of TNF-␣ (a–c) and IL-6 (d–f) in the culture
media collected were then determined by ELISA. The values presented are means ± SE (error bars) for a total of eight mice in each group, examined in two independent
experiments. *Statistically significant as compared with the corresponding control group.

3.4. In vivo cytokine responses associated with
lipopolysaccharide-induced inflammation are also altered by
high-dose, short-term exposure to PFOS or PFOA
The observations documented above raised the question as to
whether exposure to PFOS and/or PFOA renders mice more sus-
ceptible to inflammatory responses associated with infection. As
shown in Fig. 2a–f, when, as a positive control, mice not exposed to
a perfluorochemical were challenged with an intravenous injection
of LPS (300 ␮g/mouse) 2 h prior to sacrifice, the ex vivo produc-
tion of both TNF-␣ and IL-6 by peritoneal and bone marrow cells
els of both cytokines, as expected; and pretreatment with either
PFOS or PFOA potentiated these responses to LPS, although not to
a statistically significant extent in the case of PFOS (Table 3). Thus,
the enhancement by these fluorochemicals of the ex vivo respon-
siveness of peritoneal and bone marrow macrophages to LPS is,
Table 3
Prior 10-day dietary exposure of male C57BL/6 mice to 0.02% (w/w) of PFOS or PFOA
potentiates the elevations in circulating levels of the pro-inflammatory cytokines,
tumor necrosis factor-alpha (TNF-␣) and interleukin-6 (IL-6) that occur in response
to challange with bacterial lipopolysaccharide (LPS).a .

and splenocytes was, as expected, enhanced markedly (compare Treatment Serum levels (pg/ml)
all bars labelled Control + LPS inj. in Fig. 2a–f to all Control bars in
TNF-␣ IL-6
Fig. 1a–f). At the same time, in essential agreement with our in
None (control) 1.3 ± 0.6 2.0 ± 0.6
vitro findings (see above), pretreatment with PFOS or PFOA prior
PFOS 2.0 ± 0.5 24 ± 10* , b
to the challenge with LPS further potentiated production of these PFOA 2.1 ± 0.6 5.0 ± 2.0*
cytokines by both peritoneal and bone marrow cells (Fig. 2a, b, d None (control) + LPS injection 4900 ± 1640 212,000 ± 23,000
and e), while attenuating the ex vivo responses of splenocytes to PFOS + LPS injection 6810 ± 1430 251,000 ± 29,000
LPS-induced inflammation (Fig. 2c and f). PFOA + LPS injection 28,100 ± 6830* 407,000 ± 51,000*

Subsequently, the possible influence of these changes on the cir- a

culating levels of these inflammatory cytokines was evaluated. As
documented in Table 3, mice administered either xenobiotic (with-
out being subsequently challenged with LPS) exhibited a relatively
limited, but statistically significant increase in their serum levels of
IL-6; a challenge with LPS alone (without previous treatment with
PFOS or PFOA) elicited pronounced elevations in the circulating lev-
Mice were administered either a normal diet (control) or a diet containing 0.02%
PFOS or PFOA for 10 days. After this period, half of the animals in each group were
injected intravenously with LPS (as described in detail in Section 2). Two hours
after this injection, the animals were bled and the serum levels of TNF-␣ and IL-6
determined.
b
All values are means ± SE (standard error) for eight animals in each group. Dif-
ferences were analyzed for statistical significance employing the U-test.
*
Statistically significant as compared with the corresponding control group.
212 M.R. Qazi et al. / Toxicology 262 (2009) 207–214
indeed, reflected in changes in the circulating levels of these two
inflammatory cytokines in vivo.
3.5. Dietary exposure to a 20-fold lower dose (0.001%, w/w) of
PFOS or PFOA does not alter any of these parameters of innate
immunity in mice
When the dietary dose of PFOS or PFOA to which mice were
exposed was reduced 20-fold, no alterations in organ weights, cir-
culating numbers of blood cells or plasma levels of cytokines or
the ex vivo production of cytokines by cells isolated from the peri-
toneal cavity, bone marrow or spleen, with or without stimulation
by LPS, were observed (data not shown). Thus, the effects of short-
term treatment with PFOS or PFOA on murine innate immunity
described here appear to be a high-dose phenomenon.
4. Discussion
We demonstrate here that dietary treatment of mice with 0.02%
PFOS or PFOA for 10 days results in significant alterations in a num-
ber of parameters associated with the innate immune system. Our
initial observation that both of these treatments lead to severe
leukopenia combined with lymphopenia was expected, since it is
well established that this regimen of exposure induces pronounced
atrophy of the thymus and spleen (Yang et al., 2000, 2001; Zheng et
al., 2008; Dong et al., 2009; Qazi et al., 2009). It is noteworthy, how-
ever, that only PFOA causes neutropenia under these experimental
conditions, which indicates that there are differences in the mech-
anisms by which these compounds exert their biological effects.
Moreover, it implies that at least certain fluorochemicals can reduce
the number of circulating neutrophils, the cellular mediators of
innate immunity that first encounter infectious agents and play a
pivotal role in limiting the spread of microbial infections (Nathan,
2006). Circulating neutrophils have a half-life of only 11.4 h in mice
and must constantly be produced in large numbers in the bone mar-
row (von Vietinghoff and Ley, 2008). PFOA may elicit neutropenia
through direct effects on the bone marrow itself. This suggestion
gains some support from our observation that exposure to PFOA,
but not PFOS reduces the total number of cells in the bone mar-
row.
At the same time, it is possible that PFOA induces neutropenia
indirectly by the induction of severe food restriction. Indeed, our
studies have demonstrated that dietary exposure of mice to 0.02%
(w/w) PFOS or PFOA reduces their food intake to approximately
65% and 75%, respectively, of the amount consumed by untreated
animals (Qazi et al., 2009; and present study). Severe caloric restric-
tion is known to substantially reduce the numbers, chemotaxis and
phagocytic activity of neutrophils in the bone marrow pool, as well
as in the peripheral blood of rats (Suda et al., 1976; Ogawa et al.,
1985; Garcia and Barbieri, 1986; Levin et al., 1993; Slapnickova and
Berger, 2002). In addition, moderate caloric restriction is known
to reduce thymic and splenic weight and cellularity, as well as
the numbers of CD4+ CD8+ thymocytes and splenic B-lymphocytes
(Hishinuma et al., 1990; Poetschke et al., 2000; Savino, 2002; Martin
et al., 2008) and to alter the nucleolar structure and function in
lymphocytes (Berger et al., 2005). Together, these observations
indicate that while moderate food restriction affects primarily lym-
phocytes, severe food restriction can exert adverse effects on both
lymphocytes and neutrophils. We are now examining the poten-
tial contribution of caloric restriction to the reduced number of
circulating neutrophils observed in mice exposed to PFOA.
Our second major finding is that high-dose, short-term expo-
sure to PFOS or PFOA alters the absolute and relative sizes of the
macrophage populations in the peritoneal cavity, bone marrow and
spleen in an organ-dependent manner. For example, upon exposure
to either of these compounds, both the relative proportion and total
number of macrophages in the bone marrow are reduced, whereas
no such effects are observed in the peritoneal cavity and spleen.
This organ specificity may reflect the heterogeneity of macrophage
populations, which contain subpopulations that may be derived
from different precursor cells (Naito et al., 1996; Gordon, 2002;
Gordon and Taylor, 2005). For instance, macrophages derived from
promonocytes in the bone marrow are short-lived and do not pos-
sess the capacity to proliferate; whereas the resident macrophages
in peripheral tissues, derived from granulocyte/macrophage colony
forming cells are long-lived and can replenish themselves through
proliferation (Naito et al., 1996; Gordon, 2002; Gordon and Taylor,
2005). Thus, it is certainly possible that bone marrow macrophages
are susceptible to, e.g., induction of necrosis and/or apoptosis by
PFOS and PFOA, whereas the resident macrophages in the peri-
toneal cavity and spleen are resistant.
Our third significant finding is that cells isolated from the peri-
toneal cavity and bone marrow, but not from the spleen of mice
subjected to high-dose, short-term exposure to either PFOS or
PFOA produce enhanced levels of the pro-inflammatory cytokines
TNF-␣ and IL-6 in response to stimulation by LPS in vitro. Since
macrophages are the major producers of these cytokines in these
three compartments, we conclude that exposure to either PFOS or
PFOA alters the functionality of these cells.
Together, these findings suggest that PFOS and PFOA may sen-
sitise macrophages to external insults, in particular by infectious
agents. In fact, this proposal receives strong support from two
additional observations described here. First, exposure of mice
to PFOS or PFOA prior to the induction of endotoxemia through
intravenous injection of LPS significantly enhances the ex vivo pro-
duction of TNF-␣ and IL-6 by the cells subsequently isolated from
the peritoneal cavity and bone marrow, but does not alter the cor-
responding production by splenocytes.
This lack of potentiation of inflammatory responses in the spleen
is consistent with the general concept that macrophages adapt
functionally to the requirements of the tissues in which they inhabit
(Naito et al., 1996; Takahashi et al., 1996; Gordon and Taylor, 2005).
The spleen functions as a secondary immune organ that responds to
blood-born antigens and, at the same time, serves as a graveyard for
aging erythrocytes. These functions are performed by several spe-
cific subpopulations of macrophages, e.g., red pulp (F4/80 positive),
marginal metallophilic (MOMA-1-positive), marginal zone (ER-TR-
positive) and white pulp (MOMA-2-positive) macrophages (Naito
et al., 1996; Takahashi et al., 1996).
Of considerable interest in this context is the observation that
CD14, a 55-kDa glycoprotein which, together with TLR-4 and MD-
2, acts as a co-receptor for the detection of LPS (Kitchens, 2000),
is expressed constitutively on the surface of bone marrow and
peritoneal, but not splenic macrophages (Matsuura et al., 1994).
Moreover, LPS can induce the expression of this molecule by splenic
macrophages (Matsuura et al., 1994). We are presently testing
the hypothesis that PFOS and PFOA prevent or, at least, attenu-
ate the expression of the CD14 receptor by splenic macrophages,
thereby inhibiting their ability to produce inflammatory cytokines
in response to activation by LPS.
The second and more significant indication that PFOS and PFOA
can sensitize macrophages to external insults is our finding that the
increases in circulating levels of TNF-␣ and IL-6 caused by an in vivo
challenge with LPS can be enhanced even further by prior exposure
to either of these perfluorochemicals. In fact, this observation sug-
gests that not only macrophages in many different organs (with
the exception of the spleen), but also possibly the entire mononu-
clear phagocyte system (MPS) is sensitized by such exposure. This
proposal could be tested by determining the LD50 for LPS in mice
with and without exposure to PFOS or PFOA, but this approach is
ethically unacceptable.
 M.R. Qazi et al. / Toxicology 262 (2009) 207–214
The mechanism(s) via which high-dose, short-term expo-
sure of mice to PFOS or PFOA enhances cytokine production by
macrophages in response to LPS remains to be elucidated. How-
ever, one possibility is that this phenomenon may reflect elevated
levels of apoptosis and/or necrosis induced by high doses of PFOS or
PFOA (Martin et al., 2007; Cui et al., 2009). Under normal physiolog-
ical conditions, macrophages employ various cell surface molecules
and receptors to internalise apoptotic cells and thereby remove
them from the system without causing inflammation (Krysko et
al., 2006). In contrast, in connection with their internalization
and clearance of necrotic cells, which involves macropinocytosis,
macrophages are provoked to increase their production of pro-
inflammatory cytokines (Krysko et al., 2006). In addition, release
of heat-shock proteins and/or large amounts of uric acid by the
necrotic cells themselves can elicit pro-inflammatory responses in
macrophages via both TLR2 (the receptor that recognizes bacterial
lipoproteins) and TLR4 (which recognizes LPS) (Krysko et al., 2006).
This possibility that clearance of excessive numbers of necrotic or,
perhaps, even apoptotic cells by macrophages during high-dose
exposure to PFOS or PFOA renders the macrophages highly respon-
sive to LPS obviously requires further investigation. Although the
potential consequences of such sensitization for the animal itself
remain to be elucidated, potentiation of the production of TNF-␣
and IL-6 in response to LPS by pretreatment with PFOS or PFOA
suggests that these compounds might render the animal more sus-
ceptible to disease conditions (e.g., systemic autoimmune diseases)
in which TNF-␣ and IL-6 play a pivotal role (Ishihara and Hirano,
2002; Aringer and Smolen, 2003).
One general observation here is that following short-term
dietary administration to mice at a dose of 0.02% (w/w), in almost
all respects the effects of PFOS on the innate branch of the immune
system are less pronounced than those of PFOA. Furthermore, we
have recently demonstrated that with this same short-term, high-
dose regimen of exposure, PFOA causes more severe deleterious
effects on the adaptive immune system than does PFOA (Qazi et al.,
2009). Together, these findings indicate that with this regimen of
treatment, PFOS is less potent than PFOA in causing immunotoxi-
cological effects in mice.
Finally, we found that dietary exposure of mice to a 20-fold lower
dose (i.e., 0.001%, w/w) of PFOS or PFOA for 10 days does not alter
any of the parameters examined here. Clearly, as is also the case
with respect to adaptive immunity (Qazi et al., 2009), the short-
term effects of these compounds on the innate immune system of
mice is a high-dose phenomenon. With respect to environmental
relevance, it should be noted that the levels of PFOS or PFOA in our
mice receiving a dietary dose of 0.001% are about 1000 times higher
than those observed in general human populations (Lau et al.,
2007) and, furthermore, several recent biomonitoring studies have
demonstrated that the serum concentrations of these compounds
in the general population have declined significantly in recent years
(Calafat et al., 2007; Olsen et al., 2008; Spliethoff et al., 2008). Thus,
although experiments analogous to the ones described here, but
involving low-dose, long-term exposure are obviously warranted,
it appears unlikely that the effects on the innate immune system
of mice documented here represent an immediate environmental
concern.
The present findings demonstrate for the first time that at least
certain perfluorinated compounds can exert adverse effects on the
innate branch of the immune system, an important defence system
found in all plants and animals. The mechanisms underlying the
immunotoxic effects of both PFOS and PFOA on both the adaptive
and innate branches of the immune system require further elu-
cidation. Toxicokinetic investigations will help elucidate whether
these compounds exert their immunotoxic effects directly (e.g., by
being accumulated in primary and/or secondary immune organs)
or indirectly (e.g., by acting on the endocrine organs whose hor-
213
mone products influence immune responses). Investigations of this
nature are currently being performed in our laboratory.
Conflicts of interest
None of the authors have any conflicting interests.
Acknowledgments
This study was financed by an unrestricted research grant from
the 3M Company (St. Paul, Minnesota, USA). We would also like to
thank Jarl Olsson for his skilful technical assistance in diet prepara-
tion and animal care.
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