Changes in PKC isoforms in human alveolar macrophages compared with blood monocytes

Martha M. Monick, A. Brent Carter, Gunnar Gudmundsson, Lois J. Geist, and Gary W. Hunninghake

Department of Medicine, University of Iowa College of Medicine and Veterans Affairs Medical Center, Iowa City, Iowa 52242

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

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

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

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 (alpha , beta 1, beta 2, and gamma ) and eight that are Ca2+ independent (delta , epsilon , eta , µ, theta , lambda , tau , and zeta ; 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.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

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 alpha 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% beta -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 beta -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 gamma -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.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

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 alpha , beta 1, beta 2, and gamma . PKC-gamma was not detected in either alveolar macrophages or monocytes. PKC-alpha , -beta 1, and -beta 2 were present in greater amounts in monocytes than in alveolar macrophages (Fig. 1A). We next evaluated the Ca2+-independent isoforms (novel and atypical) delta , epsilon , theta , eta , µ, iota , and zeta . The theta - and iota -isoforms were not detected in either alveolar macrophages or monocytes. PKC-delta and -epsilon were present in greater amounts in alveolar macrophages than in monocytes (Fig. 1B). PKC-eta , -µ, and -zeta 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-delta and -epsilon 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.


View larger version (45K):
[in this window]
[in a new window]
 


View larger version (39K):
[in this window]
[in a new window]
 


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 1.   Alveolar macrophages (AM) and blood monocytes (Mono) exhibit differential expression of protein kinase C (PKC) isoforms. Whole cell protein was extracted from cells immediately after isolation. A: Ca2+-dependent isoforms are present in greater amounts in Mono than in AM. B: Ca2+-independent isoforms are present in varying amounts in AM compared with Mono. C: composite graph of monocyte-to-macrophage ratios obtained with relative gray values from densitometry (n = 3).

To determine if differentiation may play a role in the marked differences in Ca2+-dependent isoforms between alveolar macrophages and blood monocytes (especially beta 2), we evaluated monocytes that had been differentiated into macrophages. Whole cell lysates were obtained, and amounts of PKC-beta 2 in alveolar macrophages, undifferentiated monocytes, and differentiated monocytes were determined. As shown in Fig. 2, differentiated monocytes have less PKC-beta 2 than do undifferentiated monocytes (an amount about halfway between undifferentiated monocytes and macrophages). This suggests that the extremely low levels of PKC-beta 2 found in alveolar macrophages may partially be a consequence of the differentiation process.


View larger version (51K):
[in this window]
[in a new window]
 
Fig. 2.   Differentiated Mono contain less PKC-beta 2 than do undifferentiated Mono. Whole cell protein was extracted from cells immediately after isolation (macrophages and undifferentiated Mono) and after a 7-day in vitro differentiation (differentiated Mono). PKC isoform (beta 2) mass was evaluated as described. Figure is representative of 2 studies.

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-beta 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-delta and -epsilon in membranes, whereas PKC-eta , -µ, and -zeta 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.


View larger version (28K):
[in this window]
[in a new window]
 


View larger version (42K):
[in this window]
[in a new window]
 


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 3.   Resting AM contain greater amounts of some PKC isoforms in membranes than do blood Mono. Cytosol and membrane protein fractions were obtained immediately after cell isolation, and PKC isoform mass was evaluated as described in METHODS. For Mono, most of the Ca2+-dependent (A) and Ca2+-independent (B) isoforms were expressed in cytosol. In contrast, AM expressed a substantial portion of some PKC isoforms (beta 1, delta , and epsilon ) in membranes. C: composite graph of %membrane-bound PKC obtained from densitometry data (n = 3).

Alveolar macrophages and blood monocytes respond to short-term PMA stimulation with translocation to the membrane of all PKC isoforms except zeta . 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 (alpha , beta 1, and beta 2) had moved to the membrane. The Ca2+-independent isoforms showed a range of responses (Fig. 4B). Alveolar macrophages showed minimal translocation of PKC-delta , and monocytes translocated only part of the PKC-delta . Alveolar macrophages and monocytes both translocated epsilon , eta , and µ isoforms to the membrane fraction after 120 min. Alveolar macrophages and monocytes exhibited minimal translocation of PKC-zeta . The latter finding is consistent with prior studies showing that PKC-zeta does not contain a PMA-responsive element found in the other isoforms (15).


View larger version (32K):
[in this window]
[in a new window]
 


View larger version (42K):
[in this window]
[in a new window]
 
Fig. 4.   AM and blood Mono respond to short-term phorbol 12-myristate 13-acetate (PMA) stimulation with translocation to membranes of most Ca2+-dependent (A) and Ca2+-independent (B) PKC isoforms. AM and Mono were cultured in RPMI medium with no serum. Cells were untreated (control) or treated with PMA (10 ng/ml) for 5 or 120 min. Cytosol and membrane fractions were isolated and evaluated for expression of specific PKC isoforms. In both AM and Mono, PMA caused translocation of all PKC isoforms, except for zeta , to the membrane. Figure is a representative study of 3 separate experiments.

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-delta was not depleted in alveolar macrophages after 24 h with 100 ng/ml PMA. Monocytes showed a time- and dose-dependent decrease of delta after PMA exposure. PKC-epsilon , -eta , and -µ were all depleted by long-term PMA exposure in both alveolar macrophages and monocytes. PKC-zeta 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.


View larger version (40K):
[in this window]
[in a new window]
 


View larger version (54K):
[in this window]
[in a new window]
 
Fig. 5.   Long-term exposure to PMA causes depletion of most Ca2+-dependent (A) and Ca2+-independent (B) isoforms in AM and blood Mono. AM and Mono were either harvested immediately or were cultured in RPMI medium with no added serum for 24 h and were either untreated (control) or treated with PMA (10 or 100 ng/ml). Whole cell protein was isolated and analyzed for specific PKC isoforms. In both Mono and AM, all of the Ca2+-dependent isoforms (A) were depleted by PMA. Most of the Ca2+-independent isoforms (B) were also depleted by PMA in Mono and AM. The exceptions were PKC-zeta in both Mono and AM and PKC-delta in AM. Figure is a representative study of 3 separate experiments.

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.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 6.   PMA causes activation of mitogen-activated protein (MAP) kinase [extracellular signal-regulated kinase (ERK) 2] in AM and Mono. AM and Mono were cultured with and without PMA (10 ng/ml) for 1-60 min. ERK2 protein was then immunoprecipitated from the cells and incubated with myelin basic protein (MBP) under phosphorylating conditions. Figure is a representative study of 3 separate experiments.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 7.   Blood Mono respond to PMA with greater activation of ERK kinase compared with AM. AM and Mono were cultured with and without PMA (10 ng/ml) for 30 min. ERK2 protein was then immunoprecipitated from the cells and evaluated for ERK2 kinase activity. Densitometry was performed on 3 different experiments, and degree of activation was calculated as PMA values divided by control values. PMA-induced activation of ERK2 in Mono was significantly greater than in AM, P < 0.05.

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.


View larger version (40K):
[in this window]
[in a new window]
 


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 8.   PMA causes activation of activator protein-1 (AP-1) in Mono but not in AM, and this activation is linked to PKC. AM and blood Mono were cultured with and without PMA (10 ng/ml) for 3 h. Nuclear protein was obtained, and electrophoretic mobility shift assays were performed. A: PMA causes greater AP-1 activation in Mono compared with macrophages. B: PMA-induced monocyte activation of AP-1 is inhibited by PKC inhibitors (50 nM bisindolylmaleimide, 1 nM staurosporine). Figure is representative of 3 experiments.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

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, eta , µ, and zeta  were also present in greater amounts in monocytes than in alveolar macrophages. Only the delta and epsilon  isoforms were present in greater amounts in alveolar macrophages. Resting alveolar macrophages had significant amounts of some PKC isoforms in the membrane (beta 1, delta , and epsilon ), 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 delta  in the alveolar macrophages and zeta  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-beta isoform with dihydroxyvitamin D3. Consistent with these observations, Tonetti et al. (30) showed that transfection of PKC-beta 1 and -beta 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 delta  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-delta 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 alpha , beta , and epsilon  isoforms and that this blocked subsequent Fcgamma R-mediated killing of Staphylococcus aureus. Godson et al. (10), by blocking alpha , but not beta , 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 beta 2, epsilon , and zeta  isoforms. Pretreatment with PMA significantly depleted the beta 2 and epsilon  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-beta 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 alpha  and beta  isoforms caused a substantial enhancement (5-fold) of ERK kinase activation by PMA. Overexpression of PKC-epsilon 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 alpha  and beta  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.

    ACKNOWLEDGEMENTS

This manuscript was supported by a Veterans Affairs Merit Review grant and by National Institutes of Health Grants HL-37121 and AI-35018.

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Aihara, H., Y. Asaoka, K. Yoshida, and Y. Nishizuka. Sustained activation of protein kinase C is essential to HL-60 cell differentiation to macrophage. Proc. Natl. Acad. Sci. USA 88: 11062-11066, 1991[Abstract].

2.   Balter, M. S., G. B. Toews, and M. Peters-Golden. Different patterns of arachidonate metabolism in autologous human blood monocytes and alveolar macrophages. J. Immunol. 142: 602-608, 1989[Abstract/Free Full Text].

3.   Davis, R. J. The mitogen-activated protein kinase signal transduction pathway. J. Biol. Chem. 268: 14553-14556, 1993[Free Full Text].

4.   Edwards, D. R. Cell signaling and the control of gene transcription. Trends Pharmacol. Sci. 15: 239-244, 1994[Medline].

5.   Elias, J. A., A. D. Schreiber, K. Gustilo, P. Chien, M. D. Rossman, P. J. Lammie, and R. P. Daniele. Differential interleukin-1 elaboration by unfractionated and density fractionated human alveolar macrophages and blood monocytes: relationship to cell maturity. J. Immunol. 135: 3198-3204, 1985[Abstract/Free Full Text].

6.   Frost, J. A., T. D. Geppert, M. H. Cobb, and J. R. Feramisco. A requirement for extracellular signal-regulated kinase (ERK) function in the activation of AP-1 by Ha-Ras, phorbol 12-myristate 13-acetate, and serum. Proc. Natl. Acad. Sci. USA 91: 3844-3848, 1994[Abstract].

7.   Fujihara, M., N. Connolly, N. Ito, and T. Suzuki. Properties of protein kinase C isoforms (beta II, epsilon , and zeta ) in a macrophage cell line (J774) and their roles in LPS-induced nitric-oxide production. J. Immunol. 152: 1898-1906, 1994[Abstract/Free Full Text].

8.   Gaynor, C. D., F. X. McCormack, D. R. Voelker, S. E. McGowan, and L. S. Schlesinger. Pulmonary surfactant protein A mediates enhanced phagocytosis of Mycobacterium tuberculosis by a direct interaction with human macrophages. J. Immunol. 155: 5343-5351, 1995[Abstract].

9.   Genot, E. M., P. J. Parker, and D. A. Cantrell. Analysis of the role of protein kinase C-alpha, -epsilon, and -zeta in T cell activation. J. Biol. Chem. 270: 9833-9839, 1995[Abstract/Free Full Text].

10.   Godson, C., K. S. Bell, and P. A. Insel. Inhibition of expression of protein kinase Calpha by antisense cDNA inhibits phorbol ester-mediated arachidonate release. J. Biol. Chem. 268: 11946-11950, 1993[Abstract/Free Full Text].

11.   Hanks, S. K., and T. Hunter. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J. 9: 576-596, 1995[Abstract/Free Full Text].

12.   Hempel, S. L., M. M. Monick, B. He, T. Yano, and G. W. Hunninghake. Synthesis of prostaglandin H synthase-2 by human alveolar macrophages in response to lipopolysaccharide is inhibited by decreased cell oxidant tone. J. Biol. Chem. 269: 32979-32984, 1994[Abstract/Free Full Text].

13.   Hempel, S. L., M. M. Monick, and G. W. Hunninghake. Lipopolysaccharide induces greater amounts of prostaglandin H synthase-2 protein and mRNA in human alveolar macrophages compared to blood monocytes. J. Clin. Invest. 93: 391-396, 1994[Medline].

14.   Huberman, E., and M. F. Callaham. Induction of terminal differentiation in human promyelocytic leukemia cells by tumor-promoting agents. Proc. Natl. Acad. Sci. USA 76: 1293-1297, 1979[Abstract].

15.   Hug, H., and T. F. Sarre. Protein kinase C isoenzymes: divergence in signal transduction? Biochem. J. 291: 329-343, 1993[Medline].

16.   Hunninghake, G. W. Immunoregulatory functions of human alveolar macrophages. Am. Rev. Respir. Dis. 136: 253-254, 1987[Medline].

17.   Karin, M. The regulation of AP-1 activity by mitogen-activated protein kinases. J. Biol. Chem. 270: 16483-16486, 1995[Free Full Text].

18.   Kharbanda, S., A. Saleem, Y. Emoto, R. Stone, U. Rapp, and D. Kufe. Activation of raf-1 and mitogen-activated protein kinases during monocytic differentiation of human myeloid leukemia cells. J. Biol. Chem. 269: 872-878, 1994[Abstract/Free Full Text].

19.   Kline, J. N., D. A. Schwartz, M. M. Monick, C. S. Floerchinger, and G. W. Hunninghake. Relative release of IL-1beta and IL-1RA by alveolar macrophages in asbestos-induced lung disease, sarcoidosis, and idiopathic pulmonary fibrosis. Chest 104: 47-53, 1993[Abstract].

20.   Macfarlane, D. E., and L. Manzel. Activation of beta -isozyme of protein kinase C (PKCbeta ) is necessary and sufficient for phorbol ester-induced differentiation of HL-60 promyelocytes. J. Biol. Chem. 269: 4327-4331, 1994[Abstract/Free Full Text].

21.   Marquardt, B., D. Frith, and S. Stabel. Signaling from TPA to MAP kinase requires protein kinase C, raf and MEK: reconstitution of the signaling pathway in vitro. Oncogene 9: 3213-3218, 1994[Medline].

22.   Mischak, H., J. H. Pierce, J. Goodnight, M. G. Kazanietz, P. M. Blumberg, and J. F. Mushinski. Phorbol ester-induced myeloid differentiation is mediated by protein kinase C-alpha and -delta and not by protein kinase C-beta II, -epsilon , -zeta , and -eta . J. Biol. Chem. 268: 20110-20115, 1993[Abstract/Free Full Text].

23.   Newman, S. L., J. E. Henson, and P. M. Henson. Phagocytosis of senescent neutrophils by human monocyte-derived macrophages and rabbit inflammatory macrophages. J. Exp. Med. 156: 430-442, 1982[Abstract].

24.   Peters-Golden, M., R. W. McNish, J. K. Brieland, and J. C. Fantone. Diminished protein kinase C-activated arachidonate metabolism accompanies rat macrophage differentiation in the lung. J. Immunol. 144: 4320-4326, 1990[Abstract/Free Full Text].

25.   Peters-Golden, M., R. W. McNish, P. H. S. Sporn, and K. Balazovich. Basal activation of protein kinase C in rat alveolar macrophages: implications for arachidonate metabolism. Am. J. Physiol. 261 (Lung Cell. Mol. Physiol. 5): L462-L471, 1991[Abstract/Free Full Text].

26.   Qui, Z., and C. C. Leslie. Protein kinase C-dependent and -independent pathways of mitogen-activated protein kinase activation in macrophages by stimuli that activate phospholipase A2. J. Biol. Chem. 269: 19480-19487, 1994[Abstract/Free Full Text].

27.   Rovera, G., D. Santol, and C. Damsky. Human promyelocytic leukemia cells in culture differentiate into macrophage-like cells when treated with a phorbol diester. Proc. Natl. Acad. Sci. USA 76: 2779-2783, 1979[Abstract].

28.   Seger, R., and E. G. Krebs. The MAPK signaling cascade. FASEB J. 9: 726-735, 1995[Abstract/Free Full Text].

29.   Strieter, R. M., S. W. Chensue, M. A. Basha, T. J. Standiford, J. P. Lynch, M. Baggiolini, and S. L. Kunkel. Human alveolar macrophage gene expression of interleukin 8 by tumor necrosis factor-alpha, lipopolysaccharide, and interleukin-1 beta. Am. J. Respir. Cell Mol. Biol. 2: 321-326, 1990[Medline].

30.   Tonetti, D. A., C. Henning-Chubb, D. T. Yamanishi, and E. Huberman. Protein kinase C-beta is required for macrophage differentiation of human HL-60 leukemia cells. J. Biol. Chem. 269: 23230-23235, 1994[Abstract/Free Full Text].

31.   Trejo, J., T. Massamiri, T. Deng, N. N. Dewji, R. M. Bayney, and J. H. Brown. A direct role for protein kinase C and the transcription factor Jun/AP-1 in the regulation of the Alzheimer's beta-amyloid precursor gene. J. Biol. Chem. 269: 21682-90, 1994[Abstract/Free Full Text].

32.   Wang, Q. J., P. Acs, J. Goodnight, T. Giese, P. M. Blumberg, H. Mischak, and J. F. Mushinski. The catalytic domain of protein kinase C-delta in reciprocal delta  and epsilon  chimeras mediates phorbol ester-induced macrophage differentiation of mouse promyelocytes. J. Biol. Chem. 272: 76-82, 1997[Abstract/Free Full Text].

33.   Whitmarsh, A. J., P. Shore, A. D. Sharrocks, and R. J. Davis. Integration of MAP kinase signal transduction pathways at the serum response element. Science 269: 403-407, 1995[Medline].

34.   Winitz, S., M. Russell, N. Qian, A. Gardner, L. Dwyer, and G. L. Johnson. Involvement of Ras and Raf in the Gi-coupled acetylcholine muscarinic m2 receptor activation of mitogen-activated protein (MAP) kinase kinase and MAP kinase. J. Biol. Chem. 268: 19196-19199, 1993[Abstract/Free Full Text].

35.   Xing, M., B. L. Firestein, G. H. Shen, and P. A. Insel. Dual role of protein kinase C in the regulation of cPLA2-mediated arachidonic acid release by P2U receptors in MDCK-D1 cells: involvement of MAP kinase-dependent and -independent pathways. J. Clin. Invest. 99: 805-814, 1997[Abstract/Free Full Text].

36.   Young, S. W., M. Dickens, and J. M. Tavare. Activation of mitogen-activated protein kinase by protein kinase C isotypes alpha , beta 1 and gamma , but not epsilon . FEBS Lett. 384: 181-184, 1996[Medline].

37.   Zavala, D., and G. W. Hunninghake. Lung lavage. In: Recent Advances in Respiratory Medicine, edited by D. C. Glenley, and T. L. Petty. Edinburgh, UK: Churchill Livingstone, 1983, p. 21-23.

38.   Zheng, L., T. P. L. Zomerdijk, C. Aarnoudse, R. van Furth, and P. H. Nibberling. Role of PKC isozymes in Fcgamma receptor-mediated intracellular killing of Staphylococcus aureus by human monocytes. J. Immunol. 155: 776-784, 1995[Abstract].


Am J Physiol Lung Cell Mol Physiol 275(2):L389-L397
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society