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INTRODUCTION |
Alveolar macrophages are critical cells that are important for
pulmonary host defense and the development of inflammation in the lung
(1-12). Alveolar macrophages are derived from monocytes after
migration of the cells into the lung and differentiation into more
mature macrophages (13, 14). During the process of differentiation,
there are a number of phenotypic changes that result in an increased
capacity to adhere to various surfaces, an increased phagocytic
ability, a difference in morphology, and a changed ability to secrete
cytokines (15, 16).
Although these differences in phenotype are well described, there is
little information about changes in signal transduction pathways that
might mediate the different functions of these two cell types. The only
relevant studies are those of Peters-Golden et al. (14) and
a study by Monick et al. (11). Both of these studies noted
that normal alveolar macrophages had a decreased capacity to express
protein kinase C activity, compared with monocytes or other
macrophages. In the study by Monick et al. (11), we also
found that normal human alveolar macrophages had a decreased capacity
to express protein kinase C-induced AP-1 DNA binding.
There are a number of FOS- and JUN-like proteins that can form
the heterodimers or homodimers that make up the AP-1 complexes (17-20). The classic complex is comprised of c-FOS and c-JUN, and these proteins are found in the nucleus of cells. AP-1 activity can be
regulated at many levels, including transcription of genes that code
for AP-1 proteins, message stability, and translation of the mRNAs.
AP-1 activity is also regulated by the composition of the AP-1
complexes, phosphorylation of the proteins, and redox regulation of
cysteine residues in the AP-1 proteins (17-21). The redox status of
the AP-1 protein complex determines the binding of AP-1 to DNA. A dual
function nuclear protein called REF-1 (or apurinic/apyrimidinic
endonuclease DNA repair enzyme) regulates the binding activity of AP-1
(22-25). This protein has two distinct and separate functions that
involve different parts of the protein. REF-1 repairs
apurinic/apyrimidinic sites in DNA, and it is activated by thioredoxin
to reduce cysteine residues on both FOS and JUN enabling DNA binding by
AP-1 (22-25).
This study analyzed possible mechanisms for the lack of AP-1 DNA
binding in alveolar macrophages. We found no differences in expression
of c-FOS or c-JUN proteins in alveolar macrophages compared with
monocytes at base line or after protein kinase C stimulation. We did
find that alveolar macrophages are deficient in REF-1. Reconstituting
alveolar macrophage nuclear proteins with monocyte-derived REF-1
reconstituted AP-1 binding activity. In addition, when monocytes were
allowed to differentiate into macrophages, in vitro, they
lost REF-1 and the ability to respond to
PMA1 with increased AP-1
binding. We conclude that the process of differentiation of monocytes
into alveolar macrophages is accompanied by a loss of REF-1, affecting
AP-1 binding and subsequent expression of AP-1-driven genes.
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MATERIALS AND METHODS |
Isolation of Human Alveolar Macrophages--
Alveolar
macrophages were obtained from bronchoalveolar lavage as described
previously (5). Briefly, normal volunteers with a lifetime non-smoking
history, no acute or chronic illness, and no current medications,
underwent bronchoalveolar lavage. The lavage procedure used five 25-ml
aliquots of sterile, warmed saline in each of three segments of the
lung. The lavage fluid was filtered through two layers of gauze and
centrifuged at 1500 × g for 5 min. The cell pellet was
washed twice in Hanks' balanced salt solution without Ca2+
and Mg2+ and suspended in complete medium, Roswell Park
Memorial Institute (RPMI) tissue culture medium (Life Technologies,
Inc.), with 5% fetal calf serum (HyClone, Logan, UT) and added
gentamycin (80 µg/ml). Differential cell counts were determined using
a Wright-Giemsa-stained cytocentrifuge preparation. All cell
preparations had between 90 and 100% alveolar macrophages. This study
was approved by the Committee for Investigations Involving Human
Subjects at the University of Iowa.
Isolation of Human Blood Monocytes--
180 ml of heparinized
blood was obtained by venipuncture of the same volunteers who underwent
bronchoscopy. Monocytes were isolated using a Ficoll-Paque gradient
(Sigma). After harvesting the mononuclear cell layer, cells were washed
four times in phosphate-buffered saline and then resuspended in RPMI
medium. Additional purification was obtained by a 1-h adherence at
37 oC. Non-adherent cells were then washed off, and RPMI
medium was added back to the adherent cells. In some instances the
monocytes were allowed to differentiate into more macrophage-like
cells. In order to do this, adherent monocytes were cultured in RPMI with 10% added human AB serum for a period of 7-10 days. At the end
of that period, floating cells were washed off, and the remaining adherent cells had spread out and obtained the pancake-like appearance of macrophages.
Isolation of Nuclear Extracts and Electrophoretic Mobility Shift
Assays--
Alveolar macrophages and monocytes were cultured for
3 h with or without 100 ng/ml PMA. The nuclear pellets were
prepared by resuspending cells in 0.4 ml of lysis buffer (10 mM HEPES, pH 7.8, 10 mM KCl, 2 mM
MgCl2, 0.1 mM EDTA), placing them on ice for 15 min, and then vigorously mixing after the addition of 25 µl of 10%
Nonidet P-40. After a 30-s centrifugation (16,000 × g,
4 °C), the pelleted nuclei were resuspended in 50 µl of extraction buffer (50 mM HEPES, pH 7.8, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 10% glycerol) and
incubated on ice for 20 min. Nuclear extracts were stored at 70 °C.
The DNA binding reaction (electrophoretic mobility shift assay) was
done at room temperature in a mixture containing 5 µg of nuclear
proteins, 1 µg of poly(dI-dC), and 15,000 cpm of
32P-labeled double-stranded oligonucleotide probe for 30 min. The samples were fractionated through a 5% polyacrylamide gel in
1× TBE (Tris base 6.05 g/liter, boric acid 3.06 g/liter,
EDTA-Na2·H2O 0.37 g/liter). Sequence of the
nucleotide was 5'-CGCTTGATGAGTCAGCCGGAA-3' (AP-1). Experiments were
repeated 3 times. Supershift assays were performed by incubating the
nuclear protein with antibodies specific for c-FOS and c-JUN (Santa
Cruz Biotechnology, Santa Cruz, CA) for 30 min at room temperature
before the addition of the labeled DNA probe. Specific c-FOS and c-JUN
proteins are indicated by a higher band on the gel and by an overall
decrease in binding.
Isolation of RNA and Northern Analysis--
Whole-cell RNA was
isolated using RNA Stat-60 according to the manufacturer's
instructions (Tel-Test "B", Friendswood, TX). The isolated RNA was
fractionated in a 1.5% denaturing agarose gel containing 2.2 M formaldehyde. An RNA ladder (0.24-9.5 kilobase pairs;
Life Technologies, Inc.) was included as a molecular size standard. RNA
loading was confirmed by equivalent ethidium bromide staining in each
lane. The RNAs were transferred to GeneScreen Plus (NEN Life Science
Products) as suggested by the manufacturer. c-FOS and c-JUN cDNA
probes (generated by polymerase chain reaction with primers obtained
from CLONTECH, Palo Alto, CA) were labeled with
[
-32P]CTP (NEN Life Science Products) by the random
primer method. Blots were prehybridized for 3 h at 42 °C
(formamide 10 ml, NaCl 5 mM, 50% dextran 4 ml, 10% SDS,
Tris, pH 7.0, 1 M, and 0.4 ml 50× Denhardt's solution)
and then hybridized with the labeled probe overnight at 42 °C. The
filters were washed twice with 1× SSC at 25 °C, twice with 1× SSC
plus 1% SDS at 65 °C, and then once with 0.1× SSC at 25 °C. The
filters were exposed to autoradiographic film at
70 °C.
Western Analysis--
For these studies, alveolar macrophages
and monocytes were cultured for 3 h with or without PMA (10 or 100 ng/ml). At the end of the culture period, either whole-cell protein
extracts or nuclear protein extracts (see gel shift protocol) were
obtained as described previously (11). The cell material was sonicated 15 s on ice, allowed to sit for 20 min, and then centrifuged at 15,000 × g for 10 min. An aliquot of the supernatant
was used to determine protein concentration by the Coomassie Blue
method. Equal amounts of protein (100 µg for whole cell extracts and
20 µg for nuclear extracts) were mixed 1:1 with 2× sample buffer and
loaded onto a 10% SDS-polyacrylamide gel and run at 80 V for 2 h.
Cell proteins were transferred to nitrocellulose (ECL, Amersham Pharmacia Biotech) overnight at 30 V and visualized using c-FOS, c-JUN,
or REF-1-specific antibodies (Santa Cruz Biotechnology, Santa Cruz,
CA). Immunoreactive bands were developed using a chemiluminescent substrate (ECL, Amersham Pharmacia Biotech).
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RESULTS |
PMA Induces Increased AP-1 DNA Binding in Monocytes but Not
Alveolar Macrophages--
In order to evaluate the effect of PMA (a
model of protein kinase C-driven activation) on AP-1 DNA binding in
monocytes and alveolar macrophages from the same individual, cells were
cultured for 3 h with and without PMA (10 ng/ml) and LPS (1 µg/ml) for 3 h. Nuclear proteins were isolated, and DNA binding
was evaluated in a standard gel shift assay. In Fig.
1A, we show that PMA caused an
increase in protein binding to the AP-1 consensus sequence with nuclear
protein from monocytes and but not with nuclear protein from alveolar
macrophages. LPS did not increase AP-1 binding in monocytes or alveolar
macrophages. In Fig. 1B, we use cold competition to show
that it is the upper band (seen only with monocyte samples) that can be
competed off with cold AP-1 oligonucleotide. In order to confirm that
the alveolar macrophages were functional, we also evaluated binding to
the NF-
B sequence. We showed that LPS increases both monocyte and
alveolar macrophage nuclear protein binding to the NF-
B consensus
sequence (data not shown).

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Fig. 1.
PMA induces increased AP-1 DNA binding in
monocytes but not alveolar macrophages. Monocytes and alveolar
macrophages were cultured for 3 h with LPS (1 µg/ml) or PMA (100 ng/ml). Nuclear protein was isolated; 5 µg of protein was mixed with
32P-labeled DNA, under the appropriate conditions, and the
protein-bound DNA separated on a 5% polyacrylamide gel. A
shows PMA-induced nuclear protein binding to the AP-1 site in monocytes
but not alveolar macrophages. LPS did not induce AP-1 binding in
monocytes or alveolar macrophages. B shows that the upper
band in the gel shift assay is specific for AP-1. A gel shift similar
to A was performed using monocyte-derived nuclear proteins
with a 100-fold excess of unlabeled DNA oligonucleotide added to the
reaction mixture. The higher molecular weight band is completely
eliminated by an excess of unlabeled probe.
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In Fig. 2, we show that PMA causes an
increase in AP-1 binding in monocytes over a prolonged time course
(0.5-6 h). At none of these time points was there the appearance of
AP-1 binding activity in alveolar macrophages. These data show that the
difference in AP-1 binding activity between monocytes and macrophages
is not simply a function of different response times.

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Fig. 2.
PMA induces increased AP-1 DNA binding in
monocytes but not in alveolar macrophages over an extended time
period. Monocytes and alveolar macrophages were cultured for
differing periods (0.5 to 6 h) with 100 ng/ml of PMA. Nuclear
protein was isolated, and a gel shift assay was performed as described
for Fig. 1.
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PMA Induces c-FOS and c-JUN mRNA and Protein in Both Monocytes
and Alveolar Macrophages--
One possible explanation for the lack of
AP-1 DNA binding in alveolar macrophages is that alveolar macrophages
do not make the same amounts of AP-1 proteins as monocytes. We
initially evaluated the composition of the PMA-induced AP-1-binding
proteins in monocytes with a supershift assay. Both the upward shift of
the band and the decrease in binding shown in Fig.
3 demonstrate that the monocyte AP-1
complex includes both c-FOS and c-JUN proteins. We next analyzed both
cell types for the production of specific c-FOS and c-JUN mRNAs.
Cells were cultured for 3 h, as in the gel shift experiments, and
then harvested- and whole-cell RNA was extracted (Fig.
4A). Both monocytes and
alveolar macrophages responded to PMA with increased amounts of c-FOS
and c-JUN message. Compared with monocytes, the alveolar macrophages
show increased amounts of both c-FOS and c-JUN mRNA. We next
evaluated c-FOS and c-JUN proteins in PMA-treated cells. Both alveolar
macrophages and monocytes responded to PMA with increases in amounts of
c-FOS and c-JUN proteins (Fig. 4B). These experiments
suggest that the decreased AP-1 binding to DNA observed in alveolar
macrophages is not due to a lack of the appropriate AP-1 proteins.

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Fig. 3.
The monocyte AP-1 complex contains both c-FOS
and c-JUN. Monocytes were cultured for 3 h with and without
100 ng/ml PMA. Nuclear protein was isolated, and a supershift assay was
performed. Nuclear extracts were incubated for 30 min with nothing or
with c-FOS- or c-JUN-specific antibodies before the addition of labeled
probe. The position of the arrows denotes the position of
the AP-1-specific band and the band caused by an antibody-specific
shift.
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Fig. 4.
PMA induces increased c-FOS and c-JUN
mRNA and protein in both monocytes and alveolar macrophages.
Monocytes and alveolar macrophages were cultured with LPS (1 µg/ml)
or PMA (100 ng/ml) for 3 h. A, whole cell RNA was
obtained and run out on a 1.5% formaldehyde gel. A Northern blot was
obtained and probed with 32P-labeled c-DNA, specific for
c-FOS and c-JUN. This is an autoradiogram of the labeled blot.
B, whole-cell protein from cells treated identically to the
mRNA cells was isolated and run out on a 10% polyacrylamide-SDS
gel. Western analysis was performed, and c-FOS and c-JUN were
visualized using c-FOS- and c-JUN-specific antibodies and
chemiluminescence. These are autoradiograms of the immunoreactive
bands.
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Monocytes Contain More of the Nuclear Protein, REF-1, Than Do
Alveolar Macrophages--
Binding of AP-1 proteins to the DNA is
determined not only by presence of the transcription factor but also by
its redox state that is regulated by REF-1. In order to determine if
the observed differences in AP-1 binding between monocytes and alveolar
macrophages could be explained by differences in amounts of the protein
REF-1, we obtained nuclear protein from both alveolar macrophages and monocytes from three different individuals. Western analysis for REF-1
was performed, and the results are shown in Fig.
5, A and B. In all
three individuals there was significantly more REF-1 in the nuclei of
monocytes than in the nuclei of alveolar macrophages. This experiment
provided a possible explanation for the lack of AP-1 binding observed
in PMA-treated alveolar macrophages. In order to make sure that
treatment of the cells is not changing the amounts of REF-1, we
evaluated REF-1 protein levels in both alveolar macrophages and
monocytes that were treated with either LPS or PMA for 3 h. As
shown in Fig. 6, neither LPS nor PMA
altered the amounts of REF-1 in the cells.

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Fig. 5.
Monocytes contain increased amounts of the
nuclear protein, REF-1, compared with alveolar macrophages.
Nuclear protein was isolated from monocytes and alveolar macrophages
immediately after isolation as described under "Materials and
Methods." Western analysis was performed on 20 µg/sample of nuclear
protein from three individuals. REF-1 was visualized using an
REF-1-specific antibody and chemiluminescence. A is an
autoradiogram of the immunoreactive bands. 1, 2, and
3 refer to the three individuals whose matching monocytes
and alveolar macrophages were used for protein isolation. B
shows a quantitation of the densitometry performed on the Western
analysis, and statistical significance was evaluated using the mean
gray level values and the Student's t test.
p < 0.001 for monocytes compared with alveolar
macrophages.
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Fig. 6.
LPS and PMA do not change the amounts of
REF-1 in either alveolar macrophages or monocytes. Nuclear protein
was isolated from monocytes and alveolar macrophages after 3 h of
treatment with either LPS (1 µg/ml) or PMA (100 ng/ml). Western
analysis was performed on 20 µg/sample of nuclear protein. REF-1 was
visualized using an REF-1-specific antibody and chemiluminescence. This
is an autoradiogram of the immunoreactive bands.
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Monocytes Have a Decreased Amount of REF-1 Following
Differentiation into Macrophages--
We next performed an experiment
that links differentiation of monocytes to the defect in AP-1 DNA
binding seen in alveolar macrophages. As an in vitro
correlate of the in vivo differentiation of monocytes into
alveolar macrophages, we evaluated the amount of REF-1 in the nucleus
of differentiated monocytes and the ability of PMA to induce AP-1
binding in differentiated monocytes. In order to do this, we obtained
blood monocytes and harvested nuclear protein from half the cells
immediately after isolation. The remaining cells were put into culture
with 10% human AB serum for 7 days. At the end of this time period,
the cells morphologically resembled macrophages and nuclear protein was
isolated. Fig. 7 shows that the process
of differentiation into macrophage-like cells is associated with
decreased amounts of REF-1. Fig. 8 is an
AP-1 gel shift with PMA-treated nuclear proteins from monocytes
compared with differentiated monocytes. The differentiated monocytes
have lost their ability to increase AP-1 binding with PMA. There is
more AP-1 binding in the control differentiated monocytes than in the
control undifferentiated monocytes. This could just be a function of
the difference between in vitro differentiation and in
vivo differentiation. As a composite these two experiments show
that with differentiation monocytes have reduced amounts of REF-1 and
do not respond to PMA with increased AP-1 DNA binding.

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Fig. 7.
In vitro differentiation of
monocytes results in decreased levels of nuclear REF-1. Monocytes
were harvested and either processed immediately for nuclear protein or
cultured for 7 days on plastic before isolating nuclear protein.
Western analysis for REF-1 protein in the nucleus was performed as
described in Fig. 5. This is an autoradiogram of the immunoreactive
bands.
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Fig. 8.
In vitro differentiation of
monocytes results in a decreased amount of PMA-induced AP-1
binding. Monocytes were harvested and either treated immediately
with PMA (100 ng/ml) and nuclear protein isolated or cultured for 7 days on plastic, followed by 3 h incubation with PMA (100 ng/ml),
followed by isolation of nuclear protein. A gel shift assay, as
described for Fig. 1, was then performed.
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REF-1 from Monocytes Can Increase AP-1 Binding in Nuclear Extracts
from Alveolar Macrophages--
The second experiment to link a defect
in AP-1 binding to REF-1 was performed in alveolar macrophages. Both
monocytes and alveolar macrophages were cultured with and without PMA,
and nuclear protein was isolated. We next immunoprecipitated REF-1 from
20 µg of monocyte nuclear extract and added it to the macrophage nuclear proteins 15 min before addition of the labeled probe and allowed to sit at room temperature. Fig.
9 shows that PMA-treated macrophages
alone do not exhibit significant amounts of AP-1 DNA binding
(2nd lane) and that the addition of monocyte
REF-1 results in AP-1 binding (3rd lane). The
amount of AP-1 binding in the control alveolar macrophage is a function
of the variation found between individuals. Some people showed no AP-1
binding, and others showed a small amount of base-line binding. None of
the individuals studied showed any increase in binding with the
addition of PMA. We also performed a number of controls as follows: the
4th lane contains macrophage nuclear extract with
monocyte protein precipitated with an isotype control antibody (rabbit
IgG), the 5th lane contains macrophage nuclear
protein with monocyte protein precipitated with an irrelevant antibody
(
p65, Santa Cruz Biotechnology), and the 6th
lane contains immunoprecipitated monocyte REF-1 with no
macrophage nuclear protein. Also shown on this blot is an AP-1 gel
shift of the monocyte nuclear protein used for the
immunoprecipitations. This figure shows that AP-1 proteins from
PMA-treated alveolar macrophages can bind to DNA after monocyte REF-1
has been added.

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Fig. 9.
The addition of monocyte-derived REF-1 to
alveolar macrophage nuclear protein results in increased AP-1 DNA
binding. Blood monocytes were cultured with PMA for 3 h;
nuclear protein was isolated, and REF-1 or control proteins were
immunoprecipitated using antibody-coated Sepharose. An AP-1 gel shift
was then performed with alveolar macrophage nuclear protein alone
(Control and PMA) and with alveolar macrophage
nuclear protein (PMA) with immunoprecipitated proteins from
monocytes. This figure shows no AP-1 binding by alveolar macrophages,
AP-1 binding by PMA-treated alveolar macrophages after the addition of
monocyte REF-1 and a series of controls: Control A,
immunoprecipitation with an isotype control instead of REF-1;
Control B, immunoprecipitation with an irrelevant antibody
(p65) instead of REF-1; and Control C, immunoprecipitated
REF-1 without alveolar macrophage nuclear protein. Shown also is an
AP-1 gel shift with the monocyte nuclear protein used for the
immunoprecipitations. This is an autoradiogram showing binding to AP-1
DNA by macrophage nuclear protein with the addition of monocyte
REF-1.
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DISCUSSION |
This study shows that the defect in protein kinase C-induced AP-1
DNA binding activity seen in normal alveolar macrophages, compared with
monocytes, is due to a relative lack of the redox protein, REF-1, and
is not due to a lack of AP-1 proteins. We were able to reconstitute
normal AP-1 DNA binding in alveolar macrophage nuclear extracts by
adding REF-1 from monocytes. This was specific for AP-1, since the DNA
binding activity of NF-
B was similar in alveolar macrophages and
monocytes. This loss of REF-1 in alveolar macrophages may be due to the
process of macrophage differentiation since monocytes lost REF-1 as
they differentiated in vitro, into macrophage-like cells. To
our knowledge, this study is the first description of normal cells with
a defect in AP-1 binding caused by a lack of REF-1. AP-1 is an
important regulator of expression of many genes. The relative lack of
REF-1 and AP-1 DNA binding activity may explain, in part, the
functional differences between alveolar macrophages and their
precursors, monocytes.
REF-1 is a ubiquitous nuclear protein found in mammalian nuclear
extracts. It was initially studied for its DNA repair function, which
is found at the C-terminal portion of the molecule. The redox
regulator, located close to the N terminus, is structurally and
functionally distinct from the DNA portion of the molecule (35, 36).
REF-1, itself, is controlled by redox modification of cysteines by the
protein, thioredoxin (25). REF-1 controls the DNA binding capacity of
FOS and JUN proteins through conserved cysteine residues flanked by
basic amino acids found in the DNA binding domain of all FOS- and
JUN-related proteins (37, 38).
There are only a few previously described instances of modulation of
the expression of REF-1. Fung et al. (26) showed that asbestos caused an increase in REF-1 gene expression in
mesothelial cells. Asai et al. (27) showed that thyrotropin
increased REF-1 mRNA and protein in rat thyroid FRTL-5 cells, and
Suzuki et al. (28) showed that human chorionic gonadotropin
increased REF-1 in murine Leydig cells. In an animal model, Gillardon
et al. (29) showed that global ischemia induced by cardiac
arrest increased expression of REF-1 in the rat hippocampus. All of
these studies are cases in which an intervention results in an increase
in REF-1 expression.
In contrast to these studies, Robertson et al. (30) showed
that induction of apoptosis in HL-60 cells results in down-regulation of REF-1. Walton et al. (31) evaluated rat brains following ischemia and found that apoptotic cells had a significant decrease in
amounts of REF-1. One possible explanation for our data is that the
alveolar macrophages are undergoing apoptosis. This is not likely since
all studies of normal human alveolar macrophages reported to date
showed that the cells must be exposed to a toxic stimulus to trigger
apoptosis. We showed in a number of studies that alveolar macrophages
are not activated, unless they are exposed to endotoxin or another
stimulus in vitro (5, 9-11, 32, 33). Our studies evaluated
REF-1 immediately after the cells were obtained from normal volunteers.
These observations are supported by observations of Bingisser et
al. (34) who showed that unstimulated normal human alveolar
macrophages do not undergo apoptosis but that apoptosis could be
induced in human alveolar macrophages by high levels of endotoxin.
These studies are the first to show that a normal cell can be deficient
in REF-1 and that this can result in a defect in AP-1 DNA binding.
These studies also suggest that it is the process of differentiation
that results in the decreased levels of REF-1 in alveolar macrophages.
The findings that we describe in this study may explain some of the
functional differences between alveolar macrophages and their
precursors blood monocytes.