Department of Medicine, University of Iowa College of Medicine and Veterans Affairs Medical Center, Iowa City, Iowa 52242
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ABSTRACT |
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Alveolar macrophages play an important role in host defense and in other types of inflammatory processes in the lung. These cells exhibit many alterations in function compared with their precursor cells, blood monocytes. To evaluate a potential mechanism for these differences in function, we evaluated expression of protein kinase C (PKC) isoforms. We found an increase in Ca2+-dependent PKC isoforms in monocytes compared with alveolar macrophages. We also found differential expression of the Ca2+-independent isoforms in alveolar macrophages compared with monocytes. One consequence of the activation of PKC can be increased expression of mitogen-activated protein (MAP) kinase pathways. Therefore, we also evaluated activation of the MAP kinase extracellular signal-regulated kinase (ERK) 2 by the phorbol ester phorbol 12-myristate 13-acetate (PMA). PMA activated ERK2 kinase in both alveolar macrophages and monocytes; however, monocytes consistently showed a significantly greater activation of ERK2 kinase by PMA compared with alveolar macrophages. Another known consequence of the activation of PKC and subsequent activation of ERK kinase is activation of the transcription factor activator protein-1 (AP-1). We evaluated the activation of AP-1 by PMA in both monocytes and macrophages. We found very little detectable activation of AP-1, as assessed in a gel shift assay, in alveolar macrophages, whereas monocytes showed a substantial activation of AP-1 by PMA. These studies show that the differential expression of PKC isoforms in alveolar macrophages and blood monocytes is associated with important functional alterations in the cells.
protein kinase C; mitogen-activated protein kinase; differentiation; inflammation
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INTRODUCTION |
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ALVEOLAR MACROPHAGES play an important role in host defense and in other types of inflammatory processes in the lung (8, 12, 16, 19, 29). They are derived from blood monocytes after migration of the cells into the lung and differentiation into more mature macrophages (13, 24). As a result of this process of differentiation into macrophages, the phenotype of the cells is altered, including changes in morphology and cytokine production, increased capacity to adhere to various surfaces, and increased phagocytic activity (2, 5, 23). Although these changes in cell phenotype are well defined, very little is known about how cellular differentiation alters various signal transduction pathways in alveolar macrophages. Various studies in myelomonocytic cell lines have identified protein kinase C (PKC) as an important pathway that regulates the differentiation of immature cells into more mature monocytes/macrophages (1, 22, 30). In fact, exposure to phorbol 12-myristate 13-acetate (PMA) is often used to stimulate differentiation of immature cells into more mature macrophages (14, 20). Thus far, however, no studies have determined if the normal process of differentiation of monocytes into alveolar macrophages (or other mature tissue macrophages) is associated with alterations in PKC.
At least 12 different isoforms of PKC have been described, four of
which are Ca2+ dependent (,
1,
2, and
) and eight that are
Ca2+ independent (
,
,
,
µ,
,
,
, and
; see Ref. 11). One important consequence of
activation of various PKC isoforms is a downstream activation of one of
the mitogen-activated protein (MAP) kinase pathways, the extracellular
signal-regulated kinase (ERK) kinase pathway (26, 36). A
well-described pathway for ERK kinase activation is a PKC-mediated
effect on Raf-1 kinase (28). Activation of Raf-1 is followed by
sequential activation of MAP kinase kinase (MEK) and one or more of the
ERK kinases (21, 34). Activation of ERK kinases is an important means by which various growth factors and other signals activate cell proliferation and expression of various genes (3, 33). Another important effect of PKC activation is an increase in the
amount of the transcription factor activator protein-1 (AP-1; see Refs. 6 and 9). Activation of AP-1 is an important means by which cells
regulate the activity of various genes (4). This study shows that there
are important differences in the amounts of some PKC isoforms in
alveolar macrophages compared with monocytes and that these alterations
in PKC isoforms are reflected in alterations in ERK kinase activation
and activation of AP-1.
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METHODS |
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Isolation of human alveolar macrophages. Alveolar macrophages were obtained from bronchoalveolar lavage as previously described (37). Briefly, normal volunteers with a lifetime nonsmoking 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 1,500 g for 5 min. The cell pellet was washed two times in Hanks' balanced salt solution without Ca2+ and Mg2+ and suspended in RPMI tissue culture medium (GIBCO-BRL, Gaithersburg, MD) with added gentamicin (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. Heparinized blood (180 ml) was obtained by venipuncture of the same volunteers who underwent bronchoscopy. Monocytes were isolated using a Ficoll-Hypaque gradient (Sigma Chemical, St. Louis, MO). After the mononuclear cell layer was harvested, cells were washed four times in phosphate-buffered saline (PBS) and then resuspended in RPMI medium. Additional purification was obtained by a 1-h adherence at 37°C. Nonadherent cells were then washed off, and RPMI medium was added back to the adherent cells. To assess potential effects of adherence on PKC isoforms, alveolar macrophages were analyzed both before and after a 1-h adherence to mimic the monocyte isolation protocol. There were no changes in the PKC isoforms after the 1-h adherence step.
Differentiation of monocytes. Monocytes were isolated as described above. After the 1-h adherence step in 100-mm tissue culture plates, nonadherent cells were washed off with PBS. Adherent cells were cultured in RPMI with 10% human AB serum for 7-14 days. At the end of the incubation period, the cells had undergone morphological changes consistent with a macrophage-like cell type. Viability was >95% as assessed by trypan blue exclusion.
Western analysis of whole cell PKC
isoforms. For these studies, whole cell lysates from
alveolar macrophages and monocytes were either evaluated immediately or
cultured for 24 h with or without PMA (10 or 100 ng/ml). Cell pellets
were lysed in 400 µl of lysis buffer (1% Tween 20, 50 mM Tris, pH 8, 10 mM EDTA, 20 µg/ml (4-amidinophenyl)-methanesulfonyl fluoride
(APMSF), 10 µg/ml aprotinin, 10 µg/ml leupeptin, 170 µg/ml diethyldithiocarbamic acid, 200 µg/ml
2-macroglobulin, 0.4 mM sodium
orthovanadate, and 10 mM sodium fluoride; all reagents were from
Boehringer Mannheim, Indianapolis, IN). The cell material was sonicated
for 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 (30-60 µg) were
mixed 1:1 with 2× sample buffer (20% glycerol, 4% SDS, 10%
-mercaptoethanol, 0.05% bromphenol blue, and 1.25 M Tris, pH 6.8;
all chemicals were from Sigma Chemical), loaded onto a 10% SDS-PAGE
gel, and run at 80 volts for 2 h. Cell proteins were transferred to
nitrocellulose (ECL; Amersham, Arlington Heights, IL) overnight at 30 volts. The nitrocellulose was then blocked with 5% milk in TTBS
(Tris-buffered saline with 0.1% Tween 20) for 1 h, washed, and then
incubated with the primary antibody (1:250-1:1,000 dilution; all
PKC isoform- specific antibodies were obtained from Santa Cruz
Biotechnology, Santa Cruz, CA) for 1 h. The blots were washed four
times with TTBS and incubated for 1 h with horseradish
peroxidase-conjugated anti-rabbit IgG antibody (at 1:5,000 dilution;
Amersham). Immunoreactive bands were developed using a chemiluminescent
substrate (ECL; Amersham). Antibody specificity was verified by adding
an excess of PKC- specific peptides that were used to develop the
antibodies (also from Santa Cruz Biotechnology).
Isolation of cytoplasmic and membrane PKC
isoforms. Alveolar macrophages and monocytes were
cultured for 5 or 120 min with or without 10 ng/ml PMA. Cell pellets,
either before or after culture, were suspended in lysis buffer without
Tween 20 (400 µl), sonicated for 10 s on ice, and then spun at
100,000 g (55,000 rpm) for 10 min. The
supernatant (cytoplasmic fraction) was saved at 70°C. The
membrane pellet was resuspended in 200 µl of lysis buffer with 1%
Tween 20 and sonicated for 5 s on ice. After the resuspension was
allowed to sit for 20 min, cell debris was removed (14,000 rpm for 10 min), and the supernatant was saved. Western analysis was performed as
described above.
Depletion of PKC isoforms with PMA. Alveolar macrophages and monocytes were cultured for 24 h with or without PMA (10 or 100 ng/ml). At the end of the incubation period, cells were harvested, and whole cell protein was prepared as described above. PKC isoform mass was determined by Western analysis.
Immunoprecipitation of ERK2 kinase. Alveolar macrophages and monocytes were cultured in RPMI medium with and without PMA (10 ng/ml). After culture, cells were lysed on ice for 20 min in 500 µl of kinase lysis buffer (0.05 M Tris, pH 7.4, 0.15 M NaCl, 1% Nonidet P-40, 0.5 M phenylmethylsulfonyl fluoride, 50 µg/ml aprotinin, 10 µg/ml leupeptin, 50 µg/ml pepstatin, 0.4 mM sodium orthovanadate, 10 mM sodium fluoride, and 10 mM sodium pyrophosphate; all from Boehringer Mannheim). The lysates were then spun at 15,000 g for 10 min, and the supernatant was saved. Protein levels were measured, and 600 µg from each sample were removed for immunoprecipitation. The samples were cleared by incubating for 2 h with 1 µg/sample of rabbit IgG and 10 µl/sample of GammaBind Sepharose (Pharmacia, Piscataway, NJ). After centrifugation, the supernatants were transferred to a tube containing 3 µg/sample of rabbit anti-ERK2 antibody (no. sc-154; Santa Cruz) bound to GammaBind Sepharose and rotated at 4°C overnight. The beads were subsequently washed three times with high-salt buffer (0.05 M Tris, pH 7.4, 0.48 M NaCl, and 1% Nonidet P-40) and three times with kinase lysis buffer without the protease inhibitors. The ERK2 complexes were either released with 2× sample buffer for Western analysis or used to determine kinase activity.
Analysis of ERK2 kinase activity.
After immunoprecipitation of ERK2 from alveolar macrophages and
monocytes, the ERK2 pellet was washed two times with kinase buffer (20 mM MgCl2, 25 mM HEPES, 20 mM
-glycerophosphate, 20 mM
p-nitrophenyl phosphate, 20 mM sodium
orthovanadate, and 2 mM dithiothreitol). The pellet was then suspended
in 20 µl of kinase buffer, and the following were added: 20 µM ATP,
5 µCi
-ATP (no. BLU 002Z; NEN, Boston, MA), and 10 µg myelin basic protein (MBP; Sigma Chemical). The reaction was
continued for 15 min at 25°C and then stopped by the addition of 40 µl/sample of 2× sample buffer. The samples were boiled for 5 min and run on a 10% SDS-PAGE gel. The gel was dried, and
autoradiography was performed to visualize the
32P-labeled MBP. Exposure
times of 10-30 min were used. Densitometry was performed on films,
and the degree of increase was calculated as experimental sample
divided by control sample.
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. In some instances, PKC inhibitors, bisindolylmaleimide (50 nM) or staurosporine (1 nM; Boehringer Mannheim), were added 15 min before the PMA. The nuclear pellets were prepared by resuspending cells in 0.4 ml lysis buffer (10 mM HEPES, pH 7.8, 10 mM KCl, 2 mM MgCl2, and 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, and 10% glycerol) and incubated on ice for 20 min. Nuclear extracts were stored at 70°C.
The DNA binding reaction was done at room temperature in a mixture containing 5 µg of nuclear proteins, 1 µg poly[d(I-C)], and 15,000 counts/min of 32P-labeled double-stranded oligonucleotide probe for 30 min. The samples were fractionated through a 5% polyacrylamide gel in 1× 6.05 g/l Tris base, 3.06 g/l boric acid, and 0.37 g/l EDTA-Na2 · H2O. Sequence of the nucleotide was 5'-CGCTTGATGAGTCAGCCGGAA-3' (AP-1). Experiments were repeated three times.
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RESULTS |
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Alveolar macrophages and blood monocytes exhibit
differential expression of PKC isoforms. To determine
the relative amounts of PKC isoforms in alveolar macrophages and
monocytes, both cell types were isolated from the same donors and
analyzed for the presence of various PKC isoforms. Initially, we
evaluated the Ca2+-dependent
isoforms ,
1,
2, and
. PKC-
was not detected in either
alveolar macrophages or monocytes. PKC-
, -
1, and -
2 were
present in greater amounts in monocytes than in alveolar macrophages
(Fig.
1A).
We next evaluated the
Ca2+-independent isoforms (novel
and atypical)
,
,
,
, µ,
, and
. The
- and
-isoforms were not detected in either alveolar macrophages or
monocytes. PKC-
and -
were present in greater amounts in alveolar
macrophages than in monocytes (Fig.
1B). PKC-
, -µ, and -
were
present in greater amounts in monocytes than alveolar macrophages. An
excess of specific peptide, added with the antibody (2:1; data not
shown), could block the detection of specific PKC isoforms. In Fig.
1C, we show the composite data from
three separate experiments, expressed as monocyte-to-macrophage ratios
of relative gray values, obtained from densitometry. It is of interest
that PKC-
and -
are the only isoforms present in a greater
abundance in macrophages compared with monocytes. For the majority,
monocytes contain relatively greater amounts of PKC isoforms. These
observations show that alveolar macrophages contain less of the
Ca2+-dependent PKC isoforms
compared with monocytes.
Ca2+-independent PKC isoforms were
well represented in both alveolar macrophages and monocytes; however,
different amounts of specific isoforms were present in alveolar
macrophages compared with monocytes.
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To determine if differentiation may play a role in the marked
differences in Ca2+-dependent
isoforms between alveolar macrophages and blood monocytes (especially
2), we evaluated monocytes that had been differentiated into
macrophages. Whole cell lysates were obtained, and amounts of PKC-
2
in alveolar macrophages, undifferentiated monocytes, and differentiated
monocytes were determined. As shown in Fig. 2, differentiated monocytes have less
PKC-
2 than do undifferentiated monocytes (an amount about halfway
between undifferentiated monocytes and macrophages). This suggests that
the extremely low levels of PKC-
2 found in alveolar macrophages may
partially be a consequence of the differentiation process.
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Resting alveolar macrophages contain greater amounts
of some PKC isoforms in membranes than do blood
monocytes. To determine the relative presence of PKC
isoforms in membranes versus the cytosol of resting cells, alveolar
macrophages and monocytes were analyzed immediately after isolation. In
both monocytes and macrophages, Ca2+-dependent isoforms were
primarily in the cytosol (Fig.
3). The exception was
PKC-1 in alveolar macrophages, which, depending on the individual,
had up to 50% of the isoform in membrane fractions (Fig.
3A). In monocytes, the
Ca2+-independent isoforms were
mostly in the cytosol (Fig. 3B). In alveolar macrophages, there was a significant amount of PKC-
and
-
in membranes, whereas PKC-
, -µ, and -
were primarily in
the cytosol (Fig. 3B). In Fig.
3C, we show the composite data from
densitometry values of three experiments. In the two
Ca2+-dependent and two
Ca2+-independent isoforms shown,
significantly more of the macrophage PKC protein is in the membrane
compared with monocytes. These observations suggest that there may be
ongoing activation of some PKC isoforms in resting alveolar
macrophages. The observations are consistent with those of
Peters-Golden et al. (25), whose study also suggested ongoing PKC
activation in resting rat alveolar macrophages compared with rat
peritoneal macrophages. PKC isoforms were not evaluated in this study.
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Alveolar macrophages and blood monocytes respond to
short-term PMA stimulation with translocation to the membrane of all
PKC isoforms except . Both alveolar macrophages
and monocytes responded to PMA by translocating all of the
Ca2+-dependent isoforms to the
membrane (Fig.
4A).
There was little activation at 5 min, but by 120 min most of the PKC
(
,
1, and
2) had moved to the membrane. The
Ca2+-independent isoforms showed a
range of responses (Fig. 4B).
Alveolar macrophages showed minimal translocation of PKC-
, and
monocytes translocated only part of the PKC-
. Alveolar macrophages
and monocytes both translocated
,
, and µ isoforms to the
membrane fraction after 120 min. Alveolar macrophages and monocytes
exhibited minimal translocation of PKC-
. The latter finding is
consistent with prior studies showing that PKC-
does not contain a
PMA-responsive element found in the other isoforms (15).
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Long-term exposure to PMA causes depletion of most PKC
isoforms in alveolar macrophages and blood monocytes.
To study the effect of long-term exposure to PMA in alveolar
macrophages and monocytes, cells were cultured with and without PMA for
24 h. Alveolar macrophages and monocytes both exhibited a
dose-dependent depletion of
Ca2+-dependent isoforms after 24 h
in culture with PMA (Fig.
5A). Interestingly, there also was a spontaneous decrease in the amount of
Ca2+-dependent isoforms in
alveolar macrophages after 24 h in culture. In contrast, unstimulated
monocytes showed little change in the amount of
Ca2+-dependent PKC isoforms over
24 h in culture. With the
Ca2+-independent isoforms,
alveolar macrophages and monocytes showed a variety of responses to
long-term PMA exposure (Fig. 5B).
PKC- was not depleted in alveolar macrophages after 24 h with 100 ng/ml PMA. Monocytes showed a time- and dose-dependent decrease of
after PMA exposure. PKC-
, -
, and -µ were all depleted by
long-term PMA exposure in both alveolar macrophages and monocytes.
PKC-
was unaffected by PMA in alveolar macrophages and only slightly depleted in monocytes. In alveolar macrophages, there was a decrease in
the total amounts of
Ca2+-independent isoforms over
time in culture (24 h). This was not true of monocytes. These
observations show that both time in culture and PMA have different
effects on PKC isoforms in alveolar macrophages compared with
monocytes.
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Blood monocytes respond to PMA with greater activation of ERK kinase compared with alveolar macrophages. To assess the effect of PMA on ERK2 activation in alveolar macrophages and monocytes, an initial time course of PMA activation was performed (Fig. 6). Both cells showed a peak activation at 30 min. All subsequent experiments were done at that time. Monocytes showed an increased activation of ERK2 by PMA over alveolar macrophages (Fig. 7). Using densitometry, we determined the degree of activation (PMA/control) in three different experiments, all of which used alveolar macrophages and monocytes from identical donors. PMA activation of monocytes caused a greater increase in ERK2 activation than did PMA activation of alveolar macrophages (16 ± 2 compared with 4.4 ± 1, P < 0.05). These observations show that changes in PKC isoforms in alveolar macrophages compared with monocytes are associated with important effects on activation of some downstream second messenger pathways.
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Blood monocytes respond to PMA with greater activation of AP-1 than alveolar macrophages. AP-1 exists in resting cells as a Jun/Jun homodimer, and activation creates a Fos/Jun heterodimer (17). In these studies, we looked for both a greater amount of AP-1 and a shift upward, consistent with the heavier Fos/Jun heterodimer as an indication of activation. In Fig. 8A, we show that there is a significant difference between alveolar macrophages and blood monocytes in terms of AP-1 activation by PMA. Alveolar macrophages had only a minimal Jun/Jun band, and PMA caused only a very slight increase. In contrast, monocytes showed a significant AP-1 activation by PMA. To link AP-1 activation to activation of PKC, we used two inhibitors of PKC, bisindolylmaleimide and staurosporine. Figure 8B shows that both inhibitors significantly inhibited the PMA-induced AP-1 activation in monocytes. These experiments show that AP-1 is activated by PMA in monocytes and not in macrophages and that this activation is linked to PKC activation.
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DISCUSSION |
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This study demonstrates that there are important differences in many
PKC isoforms in blood monocytes compared with alveolar macrophages.
These differences in PKC isoform mass were associated with significant
differences in the ability of the two cell types to generate ERK kinase
and AP-1 activity. With the use of peptide-specific isoform antibodies,
the data show relatively greater amounts of Ca2+-dependent isoforms in
monocytes than in alveolar macrophages. Of the other isoforms,
, µ, and
were also present in greater amounts in
monocytes than in alveolar macrophages. Only the
and
isoforms
were present in greater amounts in alveolar macrophages. Resting
alveolar macrophages had significant amounts of some PKC isoforms in
the membrane (
1,
, and
), whereas minimal amounts of these PKC
isoforms were present in the membranes of monocytes. PMA caused a
translocation to the membrane and depletion of all of the PKC isoforms,
with the exception of
in the alveolar macrophages and
in both
cell types. Thus there are important quantitative and qualitative
differences in PKC isoforms in alveolar macrophages compared with
monocytes.
Alveolar macrophages are often used to study tissue macrophages, since they can be obtained in sufficient numbers from the lung for various studies. There have been no studies that have evaluated specific PKC isoforms in human or animal alveolar macrophages. Peters-Golden et al. (25) evaluated differences in PMA responsiveness between rat alveolar macrophages and peritoneal macrophages. They showed that PMA caused release of arachidonic acid in a PKC-dependent manner in peritoneal macrophages but not in alveolar macrophages. This suggested that alveolar macrophages had a deficiency in their ability to activate PKC. Their study also suggested that there was ongoing activation of PKC in these cells. Our studies are consistent with these observations. The finding that human alveolar macrophages have a membrane distribution of some isoforms of PKC suggests ongoing activation of PKC in the cells. Activation of phospholipase A2 and release of arachidonic acid is often regulated by ERK kinase activity (35). Our finding of a decrease in ERK kinase activation in PMA-stimulated alveolar macrophages is consistent with the PKC-mediated defect in release of arachidonic acid in the study by Peters-Golden et al. (25).
There is very little information about the normal differentiation of
blood monocytes into alveolar macrophages. It is known, however, that
PKC plays an important role in cell differentiation. In 1979, Huberman
and Callaham (14) and Rovera et al. (27) reported the differentiation
of HL-60 cells into mature myeloid cells by PMA treatment. Later,
Aihara et al. (1) showed that sustained PKC activation was necessary
for HL-60 differentiation. Several studies have evaluated the
contribution of certain PKC isoforms to this differentiation process.
Macfarlane and Manzel (20) showed that a PMA nonresponsive HL-60 mutant
could be converted to PMA responsiveness (and the ability to
differentiate) by the induction of the PKC- isoform with
dihydroxyvitamin D3. Consistent with these observations, Tonetti et al. (30) showed that transfection of PKC-
1 and -
2 into his cells converted a nonresponding cell line to a PMA-responsive line (30). Two studies in a mouse cell line
(32D) showed that overexpression of the
isoform of PKC is necessary
for differentiation into macrophage-like cells (22, 32). This later
observation is consistent with our findings of a relatively large
amount of PKC-
in alveolar macrophages. These studies, as an
aggregate, suggest that changes in PKC isoform mass are associated with
and may also be necessary for macrophage differentiation. Although our
studies do not show that macrophage differentiation is related to the
changes in PKC that we observed in this study, they are consistent with
this hypothesis.
PKC is known to play an important role in signal transduction. A number
of studies have evaluated the importance of various isoforms of PKC in
mediating cellular responses. Zheng et al. (38) showed that PMA
stimulation of monocytes translocated ,
, and
isoforms and
that this blocked subsequent Fc
R-mediated killing of
Staphylococcus aureus. Godson et al.
(10), by blocking
, but not
, isoform with antisense constructs,
was able to inhibit PMA-induced arachidonic acid release in Madin-Darby
canine kidney cells. Fujihara et al. (7), using a mouse macrophage cell
line, showed the presence of
2,
, and
isoforms. Pretreatment
with PMA significantly depleted the
2 and
isoforms and led
to a reduction of lipopolysaccharide-induced nitric oxide
production (7). These studies suggest that the changes in PKC isoforms in alveolar macrophages compared with monocytes may have important consequences in terms of cell function.
PKC activation is one pathway leading to the activation of ERK kinases.
Marquardt et al. (21) showed, in an in vitro system, that
induction of ERK kinase activation by PMA requires the presence of PKC, c-Raf, and MEK. Kharbanda et al. (18) evaluated PMA-induced activation of ERK kinase in HL-60 cells and showed that an upregulation of PKC- could convert a nonresponsive cell line to one that
exhibited MAP kinase activation. In mouse peritoneal macrophages, Qui
and Leslie (26) showed that PMA activated PKC, leading to
activation of ERK kinase, and that depleting PKC would block this
response. Our studies showed a greater PMA-induced activation of
MAP kinase in monocytes than in alveolar macrophages. This is possibly
explained by the differences in PKC isoform mass between the two cell
types. The study by Young et al. (36) in CHO-T cells showed that
overexpression of
and
isoforms caused a substantial enhancement
(5-fold) of ERK kinase activation by PMA. Overexpression of
PKC-
had no effect on the ERK kinase response. These findings match
the changes that we found in our study in monocytes and
alveolar macrophages. Monocytes have greater amounts of
and
isoforms than alveolar macrophages, and this could explain the
greater degree of ERK kinase activation.
Several studies have linked PKC activation and ERK kinase activation to activation of the transcription factor AP-1 (6, 9, 17, 31). This observation could explain some of the functional differences in monocytes compared with alveolar macrophages. The difference in AP-1 activation in monocytes compared with alveolar macrophages was striking. PMA induced substantial amounts of AP-1 in monocytes, whereas it had little effect in alveolar macrophages. Two distinct inhibitors of PKC, bisindolylmaleimide and staurosporine, could prevent the PMA-induced AP-1 activity. These studies suggest that AP-1 activity that is linked to PKC activity may be compromised in alveolar macrophages compared with monocytes.
In summary, these studies show important differences in the relative abundance of various PKC isoforms in alveolar macrophages compared with monocytes. Although a number of studies have evaluated the importance of specific PKC isoforms in the differentiation of cell lines, it is not known if changes in PKC isoforms accompany the in vivo maturation of blood monocytes to tissue macrophages. To our knowledge, this is the first study to evaluate changes in PKC isoforms that occur in blood monocytes as they differentiate into macrophages in human subjects. The observation that there are differences in PKC isoform mass between alveolar macrophages and monocytes is consistent with prior studies which showed that alterations in PKC isoform mass play an important role in monocyte differentiation. Similar findings have been observed for macrophage differentiation in cell lines. The increased baseline PKC activity seen in alveolar macrophages is consistent with a prior study by Batter et al. (2) using rat alveolar macrophages. It is not clear if this ongoing PKC activation is necessary to maintain macrophage differentiation or if it results from ongoing exposure of these cells to inhaled environmental agents or higher ambient O2 tensions in the lung. The changes in PKC isoforms in alveolar macrophages were also associated with alterations in important downstream second messenger activity. These findings may also explain, in part, some of the known functional differences in blood monocytes compared with alveolar macrophages.
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ACKNOWLEDGEMENTS |
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This manuscript was supported by a Veterans Affairs Merit Review grant and by National Institutes of Health Grants HL-37121 and AI-35018.
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FOOTNOTES |
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Address for reprint requests: G. W. Hunninghake, Pulmonary Division, Rm. C33-G GH, The Univ. of Iowa Hospitals & Clinics, 200 Hawkins Dr., Iowa City, IA 52242.
Received 24 November 1997; accepted in final form 23 April 1998.
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