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Am J Physiol Lung Cell Mol Physiol 286: L354–L362, 2004.
First published October 3, 2003; 10.1152/ajplung.00380.2002.
Environmental oxygen tension affects phenotype in cultured
bone marrow-derived macrophages
Jean C. Pfau, Jordan C. Schneider, Amy J. Archer,
Jami Sentissi, Francisco J. Leyva, and Jennifer Cramton
Center for Environmental Health Sciences, Department of Biomedical and
Pharmaceutical Sciences, The University of Montana, Missoula, Montana 59812
Submitted 7 November 2002; accepted in final form 1 October 2003
exposed to the environment, the
lung contends with unique challenges on a constant basis and
is protected from damage by respired microbes and other
particles through unique immunomodulatory capabilities.
However, the prevalence and severity of chronic lung inflammatory disorders, such as asthma and fibrosis, demonstrate the
susceptibility of this organ to dysfunction from which it often
cannot recover. The lung maintains a fairly immunosuppressed
environment, while retaining the ability to mount innate and
specific immune responses. This allows most contaminants to
be cleared by mechanical means, avoiding constant inflammatory reactions that would result in chronic damage. The alveolar macrophage (AM) is in close contact with the environment, making it one of the major cells involved in both
mechanical clearance and maintaining immune homeostasis.
Compared with other tissue macrophages such as peritoneal
macrophages (PM), AM have been shown to be relatively poor
in antigen presenting activity (5, 14) and to actively suppress
some dendritic cell (DC) and T cell activities (3, 10, 23).
Studies have suggested that the factors mediating this suppres-
sion may include macrophage-derived products (2, 15, 19, 24)
and low expression of the B7 costimulatory molecules for
antigen presenting cell (APC) activity (3). However, there is
very little information regarding how the AM is maintained in
this immune regulatory state. Therefore, a study of AM phenotypic regulation is critical to an understanding of lung
immunotoxicology.
Although many factors contribute to tissue-specific differentiation, one of the critical factors unique to AM might be the
high oxygen tension present in the lung. Compared with other
tissue macrophages, AM are exposed to much higher oxygen
partial pressures (PO2), which might require compensatory
mechanisms not needed in other tissues, such as increased
production of antioxidants. In addition, the AM are phagocytic,
leading to oxidative activities within the cells themselves.
Because oxidative stress can be described as the inability of a
cell’s antioxidant systems to deal adequately with levels of
oxygen and its radicals, prevention of oxidative stress in this
environment requires a different antioxidant status from that in
other tissues. In macrophages, the same transcription factors
that respond to oxidative stress (for example NF-␬B and
activator protein-1) are also involved in the regulation of many
macrophage functions, including production of inflammatory
mediators (20, 21). Nevertheless, normal AM often seem to
have an anti-inflammatory role. Therefore, a mechanism
clearly exists to protect the cells from oxidative stress in the
absence of true stress signaling, and this suggests that there are
unique settings for the transcription factors involved in these
activities.
As stated above, a major transcription factor in immune cells
is NF-␬B, which has been shown to be redox sensitive due to
reactive thiol groups on signal pathway components (11).
Genes that contain NF-␬B response elements appear generally
to be involved in proinflammatory activities or responses to
stress. Several characteristics of bone marrow-derived DC that
lead to highly immune active APC have recently been shown
to be coordinately regulated through NF-␬B (27). These include upregulation of major histocompatibility complex
(MHC) II, B7 costimulatory molecules (CD80, CD86), and
cytokines. This is consistent with the prevailing notion that
NF-␬B activity plays a critical role in macrophage function and
suggests that this transcription factor might be part of the
mechanism whereby redox changes could alter phenotype.
The hypothesis for this study was based on the proposal that
AM must compensate for their toxic environment by upregu-
Address for reprint requests and other correspondence: J. C. Pfau, SB154,
Center for Environmental Health Sciences, Univ. of Montana, Missoula, MT
59812 (E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
redox; alveolar macrophage; glutathione; nuclear factor-␬B
DUE TO ITS LARGE SURFACE AREA
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Pfau, Jean C., Jordan C. Schneider, Amy J. Archer, Jami
Sentissi, Francisco J. Leyva, and Jennifer Cramton. Environmental oxygen tension affects phenotype in cultured bone marrow-derived
macrophages. Am J Physiol Lung Cell Mol Physiol 286: L354–L362,
2004. First published October 3, 2003; 10.1152/ajplung.00380.2002.—
This study tested the hypothesis that the unique phenotype of alveolar
macrophages (AM) is maintained through adaptation to the relatively
high oxygen partial pressure (PO2) of the lung, through modification
of redox-sensitive transcription factors. BALB/c mouse bone marrowderived macrophages (BMC) were differentiated under different PO2
and compared functionally to AM and peritoneal macrophages (PM).
BMC differentiated in normoxia (PO2 140 Torr, BMChigh) were
similar to AM in having low phagocytic and antigen presenting cell
(APC) activities. However, BMC grown in low oxygen tension as
found in other tissues (⬍40 Torr, BMClow) were better phagocytes
and APCs, similar to PM. BMChigh were more oxidative intracellularly than BMClow, based on oxidation of dichlorofluorescein and
higher glutathione disulfide/glutathione (GSH) ratios, despite having
more GSH. Finally, lipopolysaccharide-induced nuclear factor-␬B
translocation, measured by laser scanning cytometry, was reduced in
BMChigh and AM, compared with BMClow and PM, respectively.
These data suggest that regulation of the AM phenotype may occur, at
least in part, via inhibition of NF-␬B by the unique redox environment.
OXYGEN TENSION AFFECTS MACROPHAGE PHENOTYPE
lation of antioxidants to prevent constant oxidant stress, but
this must occur without the normally coincident expression of
proinflammatory mediators. By suppressing the latter, redox
control of gene expression in the lung may be a critical factor
in maintaining the AM phenotype. For this study, differentiation of murine bone marrow-derived macrophages (BMC) in
different oxygen tension environments was used to demonstrate redox regulation of macrophage phenotypes, and that
adaptation to extracellular oxygen tension is linked to transcriptional mechanisms that would affect phenotype. Ultimately, this new understanding of redox-dependent macrophage responsiveness will have implications for intervention in
the pathological progression of lung inflammatory conditions.
METHODS
AJP-Lung Cell Mol Physiol • VOL
than 95% of the resulting cells stained positive for the macrophage
marker F4-80 (Caltag, Burlingame, CA) (data not shown).
Oxygen-controlled cell culture. To establish culturing conditions to
mimic the oxygen tensions of alveolar spaces and other tissues, we set
up two Thermo-Forma (Marietta, OH) tissue culture incubators side
by side. One was left at the normal settings of 5% CO2 in ambient
oxygen (21%), and the other was set to provide 5% CO2 and 5% O2
with N2 to flush out the excess oxygen. Using an Orion (Beverly, MA)
model 830A dissolved oxygen meter, we have shown that in the 5%
O2 incubator at 37°, RPMI culture medium containing 10% FBS
developed a PO2 of ⬃25 Torr, which corresponds with tissue values of
⬍40 Torr (Fig. 1). This condition is referred to as “low oxygen”
(BMClow) and approximates the PO2 that would be expected in many
tissues of the body. In the regular 5% CO2 incubator, RPMI with FBS
had a PO2 of 140–150 Torr, which falls between reported PO2 for
atmospheric vs. alveolar conditions but is much higher than tissue PO2
(⬍40). Throughout this study, to distinguish this from the low-oxygen
condition, we refer to this as the “high oxygen” condition (BMChigh),
even though it is actually normoxic for the lung.
Dichlorofluorescein diacetate assay. Dichloro-dihydro-fluorescein
diacetate (10 ␮M H2DCFDA; Molecular Probes, Eugene, OR) was
added to the BMC in six-well culture plates for uptake, and after 1 h
cells were washed with sterile PBS. The dye was oxidized gradually,
with the rate dependent on the intracellular redox state. Cells without
dye were used to subtract background. Readings for fluorescein
fluorescence intensity were measured by flow cytometry (AM and
PM) counting 104 cells in each sample, on a Becton-Dickinson
FACSCalibur (San Jose, CA) or, for BMC, taken hourly for 3 h using
a SpectraMax fluorescence plate reader set at 485-nm excitation/
530-nm emission (Molecular Devices, Sunnyvale, CA).
Glutathione disulfide/glutathione analysis. Glutathione (GSH) levels and glutathione disulfide (GSSG)/GSH ratios were determined in
a microtiter assay as described by Vandeputte et al. (25). Briefly, the
cells were lysed in dilute HCl (10 mM), a small aliquot was removed
for protein quantitation by Bradford protein assay from Pierce (Rockford, IL), and proteins were removed from the remaining lysate in
1.3% sulfosalicylic acid (SSA) precipitation. The supernatants were
plated in 96-well plates and neutralized with a sodium phosphate/
EDTA buffer, and then 5,5⬘-dithiobis-2-nitrobenzoic acid (DTNB)
and NADPH were added at room temperature. The enzymatic reaction
was started by addition of GSSG reductase. The plate was read
immediately on a colorimetric plate reader for a kinetic analysis for 2
min at 405 nm with mixing. Final concentrations of the reagents were
0.73 mM DTNB, 0.24 mM NADPH, 0.09% SSA, and 1.2 IU/ml
GSSG reductase. GSSG was measured after derivatization of GSH by
Fig. 1. Oxygen controlled cell culture. Two Thermo-Forma tissue culture
incubators were set as follows: 1) 5% CO2 in ambient oxygen (21%, “high
oxygen”) and 2) 5% CO2 and 5% O2 using N2 to flush excess oxygen (“low
oxygen”). Using an Orion Model 830A dissolved oxygen meter, we determined the oxygen partial pressure (PO2) of RPMI with 10% FBS and compared
it with known values. Error bars, SE of 3 independent readings. BMC, bone
marrow-derived macrophage.
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Animals. BALB/c mice were obtained from Jackson Laboratories
(Bar Harbor, ME), and DO11.10 [BALB/c background with transgene
for ovalbumin (OVA)-specific T cell receptor (TCR)] breeding pairs
were kindly provided by Corixa (Hamilton, MT). Euthanasia was
performed by intraperitoneal injection of a lethal dose of pentobarbital
sodium (for AM) or CO2 asphyxiation, which are both consistent with
the recommendations of the Panel on Euthanasia of the American
Veterinary Medical Association. The animal room was set on 12-h
light/dark cycles at ⬃18–26°C, with mouse feed and deionized water
provided ad libitum. All protocols for the use of animals in experiments have been approved by the University of Montana Institutional
Animal Care and Use Committee. The mice were maintained in
microisolation containers in the Laboratory Animal Resources animal
facility, in accordance with the “Guide for the Care and Use of
Laboratory Animals” prepared by the Institute of Laboratory Animal
Resources, National Research Council.
Collection and culture of BMC. The femurs of euthanized BALB/c
mice were exposed, excised, and placed into a dish containing sterile
PBS. In a sterile hood, the bone marrow cells were aspirated via a
1-ml syringe filled with culture media (RPMI 1640; Mediatech,
Herndon, VA; 20% FBS; GIBCO, Grand Island, NY; 1% penicillin/
streptomycin, GIBCO). Aspirated material was centrifuged and resuspended in fresh medium, counted, and seeded to tissue culture flasks
or plates for experiments. After stromal cell elimination by adherence
overnight, nonadherent cells were transferred to new flasks, and
macrophage colony stimulating factor (M-CSF; R&D Systems, Minneapolis, MN) was added to give 20 ng/ml. The medium, with CSF,
was replaced after 3–4 days. By 7–10 days the cells were fully
differentiated and stained positive for F4-80 macrophage marker, as
well as an antiaminopeptidase antibody (clone ER-BMDM1; Cedarlane, Hornby, Ontario, Canada) that is used for monitoring macrophage differentiation in culture. For challenge experiments, 10 ␮g/ml
lipopolysaccharide (LPS, Salmonella typhimurium; Sigma, St. Louis,
MO) was added for indicated times. Viability was determined to be
⬎90% by trypan blue staining. Cell counts were performed on test
cultures following differentiation and treatment with LPS to show that
the cultures in the different incubators yielded similar numbers of
cells.
Harvest of AM and PM. The lungs from euthanized BALB/c mice
were surgically removed and thoroughly lavaged with five repeated
1-ml instillations of sterile PBS. We harvested peritoneal cells following CO2 asphyxiation by injecting 8 ml of sterile PBS into the
peritoneal cavity following death and withdrawing the fluid into a
sterile 10-ml syringe via an 18-gauge needle. The cells were kept on
ice until centrifuged, resuspended, and counted on a Coulter counter
(Z1 particle counter; Beckman Coulter, Miami, FL). To purify AM
and PM by adherence, we plated the cells in RPMI 1640 with 10%
FBS or mouse serum (preincubated in high or low oxygen for AM and
PM, respectively) supplemented with antibiotics and placed them in
the high- or low-oxygen 37°C cell culture incubator for ⬃1 h. More
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TNF-␣ measurement from BMC. BMC or AM/PM were cultured in
respective incubators in 24-well plates and challenged for 4 h with
either media alone or media with 10 ␮g/ml LPS. Culture supernatants
were collected and assayed for TNF-␣ by ELISA kit (Pharmingen),
against a standard curve according to the manufacturer’s instructions.
To ensure that differences seen were not due to unequal cell numbers
after treatment, we repeated the experiments in eight-well chamber
slides, and, after removal of supernatants for TNF-␣ assay, the cells
were stained with PI (Molecular Probes). Identical-size scan areas
were counted for multiple replicates of each treatment with the LSC.
Cell numbers were shown not to vary significantly between different
treatments (data not shown).
Statistical analysis. The statistical significance of differences between the BMC from two different oxygen tensions was determined
by an unpaired two-tailed t-test with Prism statistical software. For
statistical analysis of ratios, P values were calculated by the MannWhitney nonparametric t-test, with Prism software. We analyzed flow
cytometric data using Kolmogorov-Smirnov statistics with the
CellQuest software (Becton-Dickinson). A P value of ⱕ0.05 was
considered significant and is represented as an asterisk. Error bars
represent SE of a minimum of three replicate samples. Where data are
presented in the text, the symbol ⫾ indicates SE. Experiments were
repeated at least twice with similar results, and representative data are
shown.
RESULTS
Effect of oxygen tension on phagocytosis and APC activity.
To demonstrate functional differences between the bone marrow cells differentiated in high- vs. low-oxygen conditions,
phagocytosis and antigen presentation were assayed for BMC
as well as AM and PM. The phagocytic capacity of PM
compared with AM for E. coli uptake is shown in Fig. 2A. The
fluorescence of PM following incubation with fluorescent bacterial particles was significantly higher than that of AM.
Phagocytosis by BMClow was similarly increased over that of
BMChigh in this assay system (Fig. 2B).
To measure antigen presentation activity by AM and PM,
and similarly by BMChigh and BMClow, we cocultured the
macrophages with T cells from the spleens of DO11.10 mice in
the presence of OVA and assayed APC-induced T cell proliferation by BrdU uptake. DO11.10 BALB/c mice express a
transgenic TCR that is specific for OVA presented on MHC II
so that the T cells will respond to APC presenting OVA
peptides, by proliferation and production of cytokines. As
expected, PM induced significantly more T cell proliferation
than AM (Fig. 3A). Figure 3B shows that BMClow were also
significantly better APC in terms of inducing T cell proliferation than the BMChigh. As another measure of the macrophages’ ability to activate the T cells, the supernatants from
these experiments were assayed for IFN-␥ by ELISA. There
was significantly more IFN-␥ released from T cells cultured
with BMClow (14.9 ␮g/ml ⫾ 0.63, n ⫽ 4) compared with those
cultured with BMChigh (7.98 ␮g/ml ⫾ 0.49, P ⬍ 0.05 compared with BMClow). Together, these results demonstrate that
in functional assays, BMChigh performed similarly to AM, and
BMClow behaved more like PM.
Effect of oxygen tension on intracellular oxidation conditions. To assess how the cultures’ oxygen tensions affected
intracellular redox state, dihydroxy-dichloro-fluorescein diacetate was added to cultures. After incubation with the reduced
diacetate form, H2DCFDA, the fluorescence intensity of oxidized dye was measured in BMC from high- vs. low-oxygen
environments. Figure 4 shows that AM and BMChigh had a
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2-vinylpyridine before the above assay. Values were derived from
standard curves.
Phagocytosis assay. To measure phagocytosis, we added fluorescent Escherichia coli particles (Phagocytosis Assay Kit, Molecular
Probes) to the cultures that were plated at 5 ⫻ 105 cells/ml in
eight-well chamber slides (Nalge-Nunc, Naperville, IL). The cells
were allowed 1 h in their respective incubators for particle uptake,
then were washed and analyzed by laser scanning cytometer (LSC;
CompuCyte, Cambridge, MA) in which identical numbers of cells
were counted (600 per well), and the mean/median integral fluorescence was determined.
APC assay. For APC activity, macrophages were cultured as above,
treated with mitomycin C (Calbiochem, San Diego, CA) to prevent
replication, washed, and then plated to 96-well tissue culture plates at
1 ⫻ 105 cells per well in 100 ␮l of RPMI with 20% FBS and
antibiotics. OVA (Sigma) was added to give 8 mg/ml. T cells were
obtained from DO11.10 mice that express an OVA-specific ␣␤ TCR;
their CD4⫹ T cells recognize an epitope of OVA presented on APC.
Stimulation of DO11.10 T cells with antigen on APC induces proliferation and cytokine production without prior immunization. Spleens
were removed from the mice, macerated between glass slides, and
mixed well in a volume of PBS by pipetting to break up clumps. The
cells were pelleted at 1,500 rpm for 5 min, and red blood cells were
lysed in 10 ml of 0.83% ammonium chloride. The cells were then
washed in PBS 3⫻ to remove debris. T cells were enriched through a
CD3 T cell enrichment column, according to the manufacturer’s
protocol (R&D Systems). The T cells were suspended in RPMI as
above at 4 ⫻ 106 cells per ml and added to the wells containing
macrophages in 100 ␮l per well to give 4 ⫻ 105 T cells/well. The
plates were incubated for 24 h in their respective incubators, and then
a bromodeoxyuridine (BrdU) label was added for the proliferation
assay. After another 24 h, the plates were centrifuged (5 min at 1,500
rpm), and supernatants were removed for analysis of cytokine production by ELISA (Pharmingen, San Diego, CA). The protocol for
fixing and staining the DNA was followed according to kit instructions (Oncogene, Boston, MA), and the plate was developed and read
on a colorimetric plate reader at 450 nm.
NF-␬B translocation by fluorescence microscopy/LSC. Cells to be
analyzed were seeded to multiwell glass slides (Cel-Line) at equal
density (5 ⫻ 104 cells/ml) and treated with media or media containing
10 ␮g/ml LPS for 1 h. The cells were fixed with 1% paraformaldehyde
and then permeabilized with 0.2% Triton X-100. After blocking steps,
the cells were stained with primary antibody, anti-NF-␬B p65 subunit
(Santa Cruz Biotechnology, Santa Cruz, CA), followed by AlexaFluor
488-labeled goat anti-rabbit IgG (Molecular Probes). The cells were
counterstained with 5% propidium iodide (PI, Molecular Probes) with
100 ␮g/ml RNase to localize the nucleus. This staining protocol
allowed visualization of the cellular location of NF-␬B by confocal
fluorescence microscopy with the oil-immersion ⫻60 objective, as
well as quantitation by LSC (CompuCyte) (6). To show translocation,
we programmed the LSC to measure the green fluorescence intensity
in two separate cell compartments: 1) within the nucleus, as defined
by the PI staining (integration contour), and 2) in the cytoplasm,
defined outside the nucleus by a specified number of pixels (peripheral
contour). Background fluorescence (background contour, outside cell
membrane) was subtracted from both regions. A minimum of 2,000
cells were counted per condition, based on preliminary experiments
showing statistical differences of defined scan areas containing
⬃5,000 cells.
NF-␬B activity ELISA. We determined the binding activity of
NF-␬B by a consensus oligonucleotide binding ELISA, using EZDetect Transcription Factor Kits for NF-␬B p65 (Pierce), according to
the manufacturer’s instructions. Equal numbers of cells were cultured
in flasks, treated with media or media containing 10 ␮g/ml LPS for
1 h, and then washed and lysed with NE-PER nuclear and cytoplasmic
extraction reagents (Pierce), following the manufacturer’s recommended protocol. The extracts were frozen at ⫺80°C until use.
OXYGEN TENSION AFFECTS MACROPHAGE PHENOTYPE
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Fig. 2. Effect of oxygen tension on phagocytosis. Fluorescent particles were
added to cell cultures that were plated at 5 ⫻ 105 cells/ml in 8-well chamber
slides. After 1 h in their respective incubators for particle uptake, the cells were
washed and analyzed by laser scanning cytometer (LSC), in which identical
numbers of cells were counted (600 per well), and the median integral
fluorescence was determined. A: alveolar (AM) and peritoneal (PM) macrophages. B: BMC differentiated in high or low oxygen. Error bars ⫽ SE; n ⫽
4 independent wells for each bar; *P ⬍ 0.05.
significantly higher overall mean fluorescence intensity than
PM and BMClow, respectively, suggesting the presence of a
more oxidative state. Despite the apparently higher oxidative
condition in BMChigh, measurement of intracellular GSH levels demonstrated an upregulation of GSH in BMChigh (2.3 ⫾
0.16 nmol/106 cells vs. 1.3 ⫾ 0.1 nmol/106 cells in BMClow,
n ⫽ 6 separate samples for each condition, P ⬍ 0.05 compared
with BMChigh). This was shown to be consistent with higher
GSH levels in AM compared with PM (1.6 ⫾ 0.4 nmol/106
cells vs. 1.0 ⫾ 0.1 nmol/106 cells, n ⫽ 6, P ⬍ 0.05) and
suggests a possible compensation for the more oxidative environment. However, when the ratios of GSSG/GSH were calculated, AM and BMChigh both showed higher ratios (Fig. 5, A
and B), indicating a more oxidized intracellular environment,
consistent with the DCFDA data. These data suggest that the
BMC model system induced similar redox conditions in cells
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Fig. 3. Effect of oxygen tension on antigen presenting cell (APC) activity.
Macrophages were cocultured with spleen T cells from DO11.10 mice in the
presence of ovalbumin, and APC-induced T cell activation was assayed by
bromodeoxyuridine (BrdU) uptake. A: AM and PM. B: BMC differentiated in
high or low oxygen. Error bars ⫽ SE; n ⫽ 4 independent wells for each bar;
*P ⬍ 0.05.
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as those measured in AM and PM. However, unlike AM and
PM, the functional differences between BMChigh and BMClow
can be attributed to the redox environment, since otherwise
their culture conditions were identical. Consequently, the data
strongly suggest that the single variable of the redox environment is responsible for these differences in cell function of
the BMC.
Effect of oxygen tension on NF-␬B translocation and TNF-␣
production. Intracellular redox state can affect cell function
through modification of the activity of redox-sensitive transcription factors. To determine whether the redox environment
was affecting NF-␬B activity, we measured LPS-induced
translocation of NF-␬B. We performed immunofluorescent
labeling of the NF-␬B p65 subunit using antibodies to p65 in
BMC cultured in the high- and low-oxygen incubators. Its
translocation to the nucleus following LPS challenge (10
␮g/ml for 1 h) was quantified by an LSC. This instrument
allowed visualization of the localization of fluorescent staining,
as well as quantification of the fluorescence in the nucleus vs.
the cytoplasm (6). Figure 6A shows fluorescent images of the
BMC with and without LPS challenge. In both BMChigh
(panels 1 and 2, from the left) and BMClow (panels 3 and 4),
the green staining around the red nuclei shows cytoplasmic
localization of NF-␬B in the resting cells (panels 1 and 3).
After LPS challenge, the nuclei for both cell types appear more
orange/yellow, demonstrating overlap of the green and red
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OXYGEN TENSION AFFECTS MACROPHAGE PHENOTYPE
ing with LPS treatment. These results further support increased
NF-␬B activity in the cells from lower oxygen environments.
Consistent with this activation of NF-␬B, BMClow also
produced significantly more TNF-␣ (a NF-␬B-regulated proinflammatory cytokine) in response to LPS than BMChigh (Fig.
7B). Figure 7A shows a similar experiment using AM vs. PM,
in which LPS-stimulated TNF-␣ production was significantly
higher from PM than from AM.
DISCUSSION
staining and suggesting translocation of NF-␬B to the nuclei
(panels 2 and 4). However, the translocation is more striking
for BMClow in which nearly all of the NF-␬B appears in the
nuclei (panel 4). Figure 6B shows the LSC fluorescence data
from replicate wells of the images shown, as ratios of nuclearto-cytoplasmic staining. The data confirm that the translocation
of NF-␬B into the nucleus is greater in BMC cultured in low
vs. high oxygen following LPS challenge and suggest that the
NF-␬B pathway is more active in cells grown in low oxygen.
Similar to these results with BMC, there was a significantly
higher ratio of nuclear to cytoplasmic staining following LPS
challenge of PM than with AM (Fig. 6B), confirming more
NF-␬B translocation in cells from lower oxygen environments.
In a separate experiment, the mean nuclear staining of NF-␬B
(by LSC) in LPS-treated cells increased 39% after 4 h in BMC
from the low oxygen condition but was not significantly
increased in the BMC from high oxygen (not shown).
To test the binding of NF-␬B to its target DNA sequence, we
added nuclear extracts of the treated and untreated cells to
microtiter plates coated with NF-␬B consensus oligonucleotides. The extent of binding to the wells by the NF-␬B in the
cell lysates was determined as luminescence using a fluorescence plate reader after addition of enzyme conjugated antiNF-␬B antibodies and enzyme substrate. Figure 6C shows
increased NF-␬B activity in the BMClow following treatment
with LPS, whereas the BMChigh did not show increased bindAJP-Lung Cell Mol Physiol • VOL
Fig. 5. Effect of oxygen (Ox) tension on intracellular glutathione disulfideglutathione (GSSG/GSH) ratios. GSSG and GSH were measured per 106 cells
by a microassay as described in METHODS. Ratios were calculated, and P values
were determined by the Mann-Whitney nonparametric t-test. A: AM and PM;
B: BMC from high vs. low Ox. Error bars ⫽ SE; n ⫽ 6 independent wells;
*P ⬍ 0.05.
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Fig. 4. Effect of oxygen tension on oxidation of intracellular redox dye
dichloro-dihydro-fluorescein diacetate (DCFDA). Macrophages were stained
with the redox-sensitive dye DCFDA (10 ␮M) in 6-well plates. After 1 h, the
cells were washed, and fluorescence was measured after an additional hour of
incubation, by flow cytometry for AM and PM (P value calculated by
Kolmogorov-Smirnov analysis of 104 cells, A) or on a microtiter plate reader
at 485-nm excitation, 530-nm emission for BMC differentiated in high or low
oxygen (B). Error bars ⫽ SE; n ⫽ 6 independent wells for each bar; *P ⬍ 0.05.
The overall hypothesis for this study was that the normal
phenotype of AM is in part due to the redox environment of the
lung, which is relatively hyperoxic compared with other tissues. To test this hypothesis, BMC were differentiated under
conditions where the oxygen environment could be altered. An
oxygen environment similar to alveolar PO2 not only altered
phenotypic parameters in a predictable fashion, but the resulting macrophages were similar to AM in terms of the functions
tested. Both AM and BMChigh showed reduced phagocytosis of
fluorescent particles compared with PM or BMClow, respectively. Reports are inconsistent regarding relative phagocytic
capacity of AM compared with other macrophages, and this
may be due to length of culture time, culture conditions, type
of particle and opsonization (13, 26). Under the conditions
used for this study, the macrophages from low-oxygen environments phagocytosed more bacterial particles, based on
fluorescence intensity, than those from high oxygen. However,
microscopic visualization of the cells showed that AM had
taken up large numbers of particles, consistent with their
function in clearing particles from the lung. Nevertheless, the
functional similarities between the cells from different oxygen
tensions in this specific assay were consistent with the hypothesis that oxygen tension could affect phenotype. The BMChigh
were also less active as APCs than BMClow, similar to the
OXYGEN TENSION AFFECTS MACROPHAGE PHENOTYPE
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Fig. 6. Effect of oxygen tension on NF-␬B translocation to nucleus. Macrophages were plated to cell culture-treated multiwell
slides, treated with media or media containing 10 ␮g/ml LPS for 1 h, fixed, permeabilized, and stained with rabbit anti-NF-␬B p65
antibodies followed by anti-rabbit IgG conjugated with AlexaFluor 488. The cells were counterstained with 5% propidium iodide
with 100 ␮g/ml RNase to localize the nucleus vs. cytoplasmic regions and quantify fluorescence in each compartment by LSC.
Ratios of nuclear to cytoplasmic staining were calculated, and P values were determined by the Mann-Whitney nonparametric t-test.
A: images taken by confocal microscopy of representative wells. B: LSC quantitation for AM vs. PM and BMC. Error bars ⫽ SE;
n ⫽ 6 independently treated wells for each bar; *P ⬍ 0.05. C: NF-␬B activity in BMC by oligonucleotide-binding ELISA. One
representative of 4 separate experiments with similar results is shown.
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relatively low APC activity of AM. These results suggest that
BMC differentiated in the high oxygen incubator are similar to
AM in terms of phagocytic capacity and antigen presentation,
whereas BMC differentiated in low oxygen are more active
phagocytes and APCs, similar to PM. Although the environments of AM and PM have differences besides oxygen tension,
many of which could affect cell function, the BMC system
allowed the examination of the effects of oxygen tension alone.
The effects of different redox environments on these macrophage functions provide compelling evidence that redox regulation may be very important in contributing to the AM
phenotype.
To understand the intracellular events regulating signal
transduction in these conditions, it was important to determine
how the extracellular oxygen environment affected the cells
intracellularly and to demonstrate similarities between the two
models, BMC and AM/PM. Earlier studies have shown that
AM and PM have different levels of glycolytic and oxidative
phosphorylation activities and that these metabolic differences
not only are attributed to the different oxygen tensions but are
also adaptive in that AM are more efficient in oxidative
metabolism (22). BMC grown at 5 and 20% oxygen, similar to
our model, developed metabolic characteristics that matched
PM and AM, respectively (1). These studies suggest that
environmental oxygen tension does modify the intracellular
redox state and that this can lead to altered cell function. The
experiments reported here provide additional details about the
specific changes occurring and how those changes relate to
other cell functions.
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Fig. 7. Effect of oxygen tension on TNF-␣ production. BMC from high vs.
low oxygen conditions or AM/PM were plated in 24-well plates and challenged with media or media containing 10 ␮g/ml LPS for 4 h. TNF-␣ in the
culture supernatants was quantified by ELISA against a standard curve. B: AM
vs. PM. A: BMC from high or low oxygen. Error bars ⫽ SE; n ⫽ 3
independently treated wells for each bar; *P ⬍ 0.05.
Despite higher levels of total GSH, both the DCFDA assay
and measurement of GSSG/GSH ratios suggested that BMChigh
and AM are both more oxidative intracellularly than their
counterparts from low oxygen, possibly due to an inability of
the cells to completely buffer against their environment. Murata et al. (18) have demonstrated that intracellular redox status
can modify cytokine production by macrophages when challenged by IFN-␥ and that cells that are more oxidized (less
GSH) tend to produce less of the proinflammatory T helper
(Th) 1 cytokine IL-12 than cells with more reduced GSH. This
is consistent with our model in which cells with more reduced
GSH appear more active in several macrophage activities.
Because GSH can affect signal transduction through redoxsensitive transcription factors (7, 11), these data suggested that
the redox environment might affect phenotype through NF-␬B,
a redox-sensitive transcription factor involved in many proinflammatory activities.
To explore a possible mechanism of the redox regulation of
macrophage phenotypes, LPS-induced NF-␬B translocation
was analyzed. The LSC allowed visualization of the localization of the NF-␬B p65 subunit by fluorescent staining, as well
as quantification of the fluorescence in the nucleus vs. the
cytoplasm (6). The translocation of NF-␬B into the nucleus
was much more evident in BMC cultured in low vs. high
oxygen following LPS challenge, suggesting that the NF-␬B
pathway is more active in cells grown in low oxygen. The
activity of NF-␬B also appeared greater in the BMClow, based
on a consensus oligonucleotide binding assay. To support these
data with an effect on protein expression, we measured TNF-␣
as an important macrophage proinflammatory cytokine that is,
in part, regulated by NF-␬B. Consistent with the NF-␬B
results, BMClow produced more TNF-␣ following LPS challenge than BMChigh, suggesting that the redox regulation of
NF-␬B translocation led to downstream effects on proinflammatory cytokine production. Because NF-␬B has been shown
to coordinately regulate many functions of bone marrowderived DC (27), this regulation of NF-␬B could significantly
impact overall phenotype of macrophages as well.
Overall, this study demonstrates a clear phenotypic difference between BMC differentiated in environments of different
oxygen tensions, suggesting that at least some macrophage
activities may be significantly regulated by redox mechanisms
in vivo. GSH is a critical cellular redox buffer that has also
been shown to regulate signal transduction through reduction
of thiol groups in active sites of signaling components (7, 11,
20). Cells residing in the lung, where the PO2 is relatively high,
would be expected to upregulate levels of GSH to provide
sufficient buffering. Nevertheless, these cells would be challenged by their environment to maintain the GSH in a reduced
form. The data presented here show that modifying extracellular oxygen tension does lead to more oxidized glutathione
(GSSG), represented as increased ratios of GSSG to GSH,
despite higher overall levels of total GSH. Because oxidative
stress has been implicated as an activator of NF-␬B (16), our
data would appear to contradict this classic picture of NF-␬B in
that it was more active in cells with more reduced GSH.
However, recent studies have challenged the validity of a
causal relationship between oxidative stress and NF-␬B activation (4). Oxidative conditions have actually been shown to
inhibit cytokine-induced activation of NF-␬B through oxidative inactivation I␬B kinase (12). In the study reported here, the
OXYGEN TENSION AFFECTS MACROPHAGE PHENOTYPE
ACKNOWLEDGMENTS
We gratefully acknowledge Dr. Terrance Kavanagh, University of Washington, and Dr. Jay Patel, University of Florida School of Medicine, for review
and advice regarding this project.
GRANTS
This work was supported by Environmental Protection Agency Center
Grant R828602 and National Institute of Environmental Health Sciences
National Research Service Award Post-Doctoral Fellowship Grant ES-11249.
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mildly oxidative conditions appear to have inhibited LPSinduced NF-␬B activation. The mechanism of this unique
redox setting is not yet clear, but other important redox buffers
may be involved, such as thioredoxin, which can also affect
NF-␬B activation (8). In the lung environment, a mechanism
must exist to protect the cells from oxidative stress without the
often concurrent upregulation of proinflammatory responses,
since that would lead to chronic lung inflammation. The data
presented here support the hypothesis that AM have a unique
redox setting that allows adequate oxygen buffering while
downregulating proinflammatory activities. Disruption of this
balance occurs following environmental exposures, often leading to chronic inflammatory conditions. A clearer understanding of the redox regulation of the AM phenotype could lead to
improvements in the prevention and treatment of chronic
inflammatory conditions.
Several interesting studies have recently addressed the possibility that intracellular redox conditions may play a role in
skewing the Th1/Th2 immune responses (7, 17, 18). This study
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due to its ability to downregulate inflammatory responses. One
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more Th2 response. The ability of redox signals to skew the T
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immune suppression. An understanding of this tendency in the
normal lung could provide important clues to controlling specific pathological changes following exposures that disrupt this
balance and explain the unique immune regulation of these
critical players in lung immune homeostasis.
Although it has become evident in recent years that redox
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a clear understanding of how moderate changes in redox
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control of AM phenotypes are profound, so that studies in this
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normal homeostasis but also the mechanisms that prevent a
return to homeostasis in diseases manifested by chronic inflammation.
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