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Originally published In Press as doi:10.1074/jbc.M508997200 on December 21, 2005

J. Biol. Chem., Vol. 281, Issue 8, 5058-5064, February 24, 2006
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Human Alveolar Macrophages Are Deficient in PTEN

THE ROLE OF ENDOGENOUS OXIDANTS*

Dawn M. Flaherty1, Martha M. Monick, and Sara L. Hinde

From the Department of Internal Medicine, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, Iowa 52242

Received for publication, August 15, 2005 , and in revised form, December 15, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Human alveolar macrophages play a critical role in host defense and in the development of inflammation and fibrosis in the lung. Unlike their precursor cells, blood monocytes, alveolar macrophages are long-lived and tend to be resistant to apoptotic stimuli. In this study, we examined the role of differentiation in altering baseline phosphatidylinositol (PI) 3-kinase/Akt activity. We found that differentiation increased activity of pro-survival PI 3-kinase/Akt while decreasing amounts of the negative PI 3-kinase regulator, PTEN. PTEN is a lipid phosphatase with activity against phosphatidylinositol 3,4,5-trisphosphate (PI3,4,5P3), the major bioactive product of PI 3-kinase. Examining in vivo differentiation of alveolar macrophages (by comparing blood monocytes to alveolar macrophages from single donors), we found that differentiation resulted in increased baseline reactive oxygen species (ROS) in the alveolar macrophages. This led to a deficiency in PTEN, increased activity of Akt, and prolonged survival of alveolar macrophages. These data support the hypothesis that alterations in ROS levels contribute to macrophage homeostasis by altering the balance between PI 3-kinase/Akt and the phosphatase, PTEN.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Alveolar macrophages are critical effector cells in immunity and inflammation in the lung. They are derived from peripheral blood monocytes that migrate to the lung and differentiate into alveolar macrophages. Unlike their precursor cells, blood monocytes, which undergo rapid apoptosis, alveolar macrophages are long-lived and tend to be resistant to apoptotic stimuli (1). An important cell survival pathway (and the focus of this study) is the phosphatidylinositol (PI)2 3-kinase/Akt pathway. PI 3-kinase phosphorylates membrane phospholipids, generating phosphatidylinositol 3,4,5-trisphosphate (PI3,4,5P3). PH domain proteins, including Akt, are recruited to PI3,4,5P3 in the plasma membrane, leading to their activation. Akt modulates cell survival by both up-regulating pro-survival pathways and down-regulating pro-apoptotic pathways (2, 3). Akt activity is negatively regulated by the lipid phosphatase, PTEN. PTEN dephosphorylates PI3,4,5P3, reducing the pool of phosphoinositides required for Akt activity (4).

PTEN was originally described as a tumor suppressor protein and is deleted or mutated in a number of malignancies; however, it has become clear in numerous studies that PTEN also serves an important role in the regulation of normal cellular functions, including development, immunity, and cell growth (for review, see Ref. 5). Regulation of PTEN has been shown to occur at several levels. Phosphorylation of PTEN results in a conformational change in the protein and masking of the membrane binding surface, thus preventing interaction of PTEN with its membrane-localized substrate, PI3,4,5P3 (6-8). Oxidant-mediated negative regulation of PTEN function has also been shown to occur (9), resulting in reversible inactivation of PTEN phosphatase activity. PTEN has also been shown to be regulated transcriptionally (10-13) and via alterations in protein stability (7, 14). Our studies demonstrate that alveolar macrophages are deficient in PTEN and that this is linked to constitutive activation of Akt. Differentiation-induced increases in baseline ROS contribute to the decreased PTEN protein and increased Akt observed in alveolar macrophages.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Chemicals were obtained from Sigma-Aldrich. The PI 3-kinase inhibitor LY294002 was obtained from Calbiochem. Protease inhibitors were obtained from Roche Diagnostics (Mannheim, Germany). Antibodies were from various sources: anti-PTEN antibodies were from Cascade Biosciences (Winchester, MA), antibodies to phosphorylated Akt (serine 473) and cleaved caspase 7 were from Cell Signaling Technology (Beverly, MA), the antibody to beta-actin was from Sigma-Aldrich, and antibodies to total Akt were from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary antibodies for Western analysis (horseradish peroxidase-conjugated anti-rabbit or -mouse Ig) were also from Santa Cruz Biotechnology.

Isolation of Human Alveolar Macrophages—Alveolar macrophages were obtained from bronchoalveolar lavage as previously described (15). Briefly, normal volunteers with a lifetime non-smoking history, no acute or chronic illness, and no current medications, with the exception of oral contraceptives, underwent bronchoalveolar lavage. The lavage procedure used five 20-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 x g for 5 min. The cell pellet was washed twice in saline and suspended in complete medium, Rosewell Park Memorial Institute tissue culture medium (Invitrogen) with gentamicin. Differential cell counts were determined using a Wright-Giemsa-stained cytocentrifuge preparation. All cell preparations had between 90 and 100% alveolar macrophages. All protocols were approved by the University of Iowa Institutional Review Board.

Isolation of Human Blood Monocytes—A volume of 180 ml of heparinized whole blood was obtained by venipuncture of the same volunteers that underwent bronchoscopy. Peripheral blood mononuclear cells were isolated over a Ficoll-Hypaque gradient (Sigma-Aldrich), and monocytes were isolated by negative selection using magnetic labeling and separation techniques (Miltenyi Biotech, Auburn, CA). Monocyte purity was evaluated using Wright-Giemsa staining and was >98%. Monocytes were differentiated in 10% human AB serum for 8-10 days at which time cells were harvested for either total protein or RNA.

Cell Survival Assay—Freshly isolated alveolar macrophages were plated in serum-free media plus gentamicin alone or with the PI 3-kinase inhibitor, LY294002 (50 µM) for 24 h. Samples were analyzed for cell death using two different methods: trypan blue exclusion and ethidium homodimer nuclear staining (Molecular Probes, Eugene, OR). The percentage of dead cells was calculated by counting at least 300 cells per field in a blinded fashion. Triplicate cultures were performed on each of two separate experiments. These cells were assessed for apoptosis using Western analysis and an antibody directed against cleaved caspase 7. In some experiments, cell viability was also assessed using a commercially available kit to quantitate ATP amounts (CellTiter-Glo®, Promega, Madison, WI). ATP amounts have been shown to correlate with metabolically active (viable) cells (16).

Adenovirus Vectors—First generation recombinant adenovirus was generated by the University of Iowa Gene Transfer Vector Core (17). The particle titers of the adenoviral stocks were typically 1012 DNA particles/ml; functional titers were ~4 x 1010 particle forming units/ml. Adenovirus vectors expressing the transgene for green fluorescent protein (AdGFP) driven by the cytomegalovirus promoter, PTEN (AdPTEN, kindly provided by Dr. J. Engelhardt at our institution), or an empty vector containing no transgene (AdVector), were used to infect the cells at a multiplicity of infection of 500. These vectors were free of wild-type virus contamination as determined by plaque assay and PCR. Freshly isolated human alveolar macrophages were plated, and virus was added in serum-free media. The cells were incubated at 37 °C for 24 h and harvested for total cellular protein. In parallel studies, cell survival was determined using either ethidium homodimer staining or the ATP viability assay (see "Cell Survival Assay" above). Efficiency of infection was determined in each experiment by examining green fluorescence of the AdGFP-infected cells using a Leica DMIRB inverted fluorescence microscope (Wetzlar, Germany). Similar infection efficiencies were assumed with the AdVector and AdPTEN viruses.

Western Analysis—Western analysis for the presence of specific proteins was performed on freshly isolated alveolar macrophages or blood monocytes using total cellular protein. The protein concentrations of the lysates were measured by the Bradford assay. Equal amounts of protein (10 µg of nuclear extracts and 30 µg of total cellular protein) were mixed 1:1 with 2x 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), loaded onto a 10% SDS-PAGE gel, and run at 100 volts for 2 h. Cell proteins were transferred to nitrocellulose (ECL, Amersham Biosciences, Arlington Heights, IL), 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 for 1 h at room temperature or overnight at 4 °C. The blots were washed and incubated with a horseradish peroxidase-conjugated secondary antibody and developed with a chemiluminescent substrate, ECL Plus (Amersham Biosciences). Following development of the primary antibodies, bound immunoglobulins were removed from the membranes by washing 2 times for 15 min each at room temperature in Restore Western blot Stripping Buffer (Pierce), and the membranes were re-probed for beta-actin. An autoradiograph was obtained, and protein levels were quantitated using a FluorS scanner and Quantity One software for analysis (Bio-Rad). The data were analyzed, and statistics were performed using GraphPad Prism software (San Diego, CA).

Real-time RT-PCR Detection of PTEN mRNA—Total RNA was prepared from freshly isolated human alveolar macrophages or blood monocytes using RNA Stat-60 (Tel-Test B, Friendwood, TX). One microgram of RNA was reverse-transcribed to cDNA using a RETRO-script RT-PCR kit (Ambion, Austin, TX) and was quantitated using the RiboGreen kit (Molecular Probes, Eugene, OR). The resulting cDNA was subjected to PCR as follows. In a 0.2-ml PCR tube (Bio-Rad), 2 µlof cDNA was added to 48 µl of PCR mixture containing 160 µM of each dNTP (Invitrogen), 3.0 mM MgCl2 (Invitrogen), 1:15,000 SYBR Green I DNA dye (Molecular Probes), 0.2 µM of each sense and antisense primer (IDT, Coralville, IA), and 2.5 units of Platinum TaqDNA Polymerase (Invitrogen). Amplification was then performed in an iCycler iQ fluorescence thermocycler (Bio-Rad). Fluorescence data were captured during the 10-s dwell to ensure that primer dimers were not contributing to the fluorescence signal generated with SYBR Green I DNA dye. Specificity of the amplification was confirmed using melting curve analysis. Data were collected and recorded by iCycler iQ software (Bio-Rad) and expressed as a function of threshold cycle (Ct), the cycle at which the fluorescence intensity in a given reaction tube rises above background (calculated as 10x mean ± S.D. of fluorescence in all wells over the baseline cycles). Specific primer sets used for human PTEN and HPRT genes are as follows (5' to 3'): PTEN (sense, TAA AGG CAC AAG AGG CCC TA; antisense, AGG TAA CGG CTG AGG GAA CT); HPRT (sense, CCT CAT GGA CTG ATT ATG GAC; antisense, CAG ATT CAA CTT GCG CTCATC). Primers were selected based on nucleotide sequences downloaded from the National Center for Biotechnology Information data bank and designed with software by S. Rozen and H. J. Skaletsky.3

Quantitation of PTEN mRNA—Relative quantitative gene expression was calculated as follows: for each sample assayed, the Ct values for reactions amplifying PTEN and HPRT were determined. Abundance of PTEN mRNA, relative to abundance of HPRT mRNA, in each cell type was calculated by the formula 2-{Delta}{Delta}Ct. The validity of this approach was confirmed by using serial 10-fold dilutions of template of template containing PTEN and HPRT genes. Using the 10-fold dilutions, the amplification efficiencies for PTEN and HPRT amplimers were found to be identical.

Electron Spin Resonance—Freshly isolated blood monocytes and alveolar macrophages were placed in chelated phosphate-buffered saline after culture in serum-free media or in media plus 15 mM N-acetyl cysteine. The spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was added to the samples at a final concentration of 50 mM. ESR spectra for HO· were recorded using a Bruker EMX ESR spectrometer with the following settings: receiver gain, 1 x 106; modulation amplitude, 1.0 G; modulation frequency, 100.0 kHz; sweep width, 80.0 G; microwave power, 39.8 milliwatt; and frequency of 9.854 GHz. Spectra were the result of eight signal-averaged scans collected over 15 min. The analyses of the ESR spectra were based on the hyperfine splitting values characteristic of the spin adduct (18, 19).

Statistical Analysis—Statistical analysis was performed on the densitometry data, the survival assays, and real-time PCR data. Significance was determined by the Students t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Alveolar Macrophages Have High Constitutive Akt Activation and Are PTEN-deficient as Compared with Blood Monocytes—Previous studies in our laboratory showed that resting alveolar macrophages have constitutive activation of Akt (20) which is thought to play an important role in survival of these cells (21). Given that alveolar macrophages have a much longer in vivo survival time than their precursor cells, blood monocytes (22), we hypothesized that monocytes would have less Akt activation. Also, because PTEN is a negative regulator of the PI 3-kinase pathway, we further speculated that alveolar macrophages would have low amounts of PTEN. We obtained matching sets of freshly isolated peripheral blood monocytes and alveolar macrophages from a series of normal human volunteers, isolated total cellular protein, and performed Western analysis using antibodies directed against active Akt (phosphorylated on serine 473) and total PTEN. As shown in Fig. 1, human alveolar macrophages have higher constitutive Akt activity compared with blood monocytes with a reciprocal decrease in PTEN. Conversely, blood monocytes have very little constitutive Akt activation and high amounts of PTEN.


Figure 1
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  		FIGURE 1.
Alveolar macrophages have constitutive Akt activation and are deficient in PTEN. Matching sets of human alveolar macrophages and peripheral blood monocytes were obtained from normal volunteers and total protein was isolated. Western analysis was performed using antibodies against phospho-Akt (serine 473) and PTEN. The same blot was probed with total Akt to demonstrate equal loading. Densitometry was obtained on the film, and the graph represents the three separate experiments.

 
PI 3-Kinase Activation Is Important for Survival in Alveolar Macrophages—To lend physiologic relevance to our observations, we examined in vitro survival of human alveolar macrophages in culture in the presence of the PI 3-kinase inhibitor, LY294002. In these experiments, macrophages were cultured under serum-free conditions with and without inhibitor, and cell death was measured using trypan blue exclusion and ethidium homodimer staining after 24 h. Fig. 2 shows these results and illustrates the importance of PI 3-kinase activation and downstream phosphorylated Akt expression on survival of these cells. We also examined survival by harvesting cells for total protein and performing a Western analysis for cleaved caspase 7 expression. Caspase 7 is a major contributor in the execution of apoptosis. It is a major effector caspase that cleaves intracellular substrates and serves as a marker for apoptosis (23-25). In the Western blot shown as an insert to the survival graph (Fig. 2), we demonstrate that inhibiting PI 3-kinase activity induces significant caspase 7 cleavage, consistent with the increase in cell death demonstrated by trypan blue exclusion and ethidium homodimer assays. We also show that blood monocytes, which express very low levels of active Akt, are hypersusceptible to cell death and apoptosis via inhibition of PI 3-kinase. This also supports the idea that Akt activation is critical for cell survival.

Overexpression of PTEN Alters Akt Activation and Alveolar Macrophage Survival—To evaluate the effect of PTEN expression on alveolar macrophage survival, we infected alveolar macrophages with an adenovirus vector containing the PTEN transgene. These data are shown in Fig. 2B. The upper left panel shows that alveolar macrophages can be infected with 75% efficiency using an adenovirus vector containing GFP. The upper right panel is a Western analysis of PTEN and phospho Akt showing that PTEN is overexpressed in cells infected with the PTEN vector but is unaffected by the empty vector. Importantly, overexpression of PTEN results in loss of Akt activation (phospho Akt) and enhanced cell death, shown by ethidium homodimer staining (middle panel) and by ATP viability assay (Fig. 2C).

Endogenous Oxidant Production by Alveolar Macrophages Affects PTEN Expression—In these experiments, we demonstrate that differentiation of blood monocytes to alveolar macrophages leads to an increase in baseline oxidant tone. Using ESR and the spin trap DMPO to trap hydroxyl radicals, we found that alveolar macrophages, compared with blood monocytes, spontaneously produce large amounts of reactive oxygen species in the unstimulated state, whereas blood monocytes produce little or none. This is consistent with a recent study demonstrating that alveolar macrophages contain increased superoxide compared with blood monocytes (26). Observations in the literature led us to ask if the alterations in oxidant tone are linked to the changes in PTEN protein amounts. Vasudevan and colleagues have shown that PTEN transcription can be down regulated indirectly by NF{kappa}B p65 via sequestration of the transcriptional co-activator proteins, CBP/p300 (13), and we have observed that alveolar macrophages have high constitutive expression of NF{kappa}B p65 (data not shown). Furthermore, it is known that NF{kappa}B activation is oxidant-mediated (27, 28) and that N-acetylcysteine (NAC) can inhibit this activation (29-34). Given these observations, we hypothesized that the loss of PTEN might be modulated by oxidant tone in macrophages. To test this hypothesis, we used the antioxidant, NAC or the addition of exogenous glutathione, to inhibit endogenous oxidant production of alveolar macrophages and examined the effect on PTEN in macrophages. These results are shown in Fig. 3. Using ESR, we determined generation of HO· in blood monocytes and alveolar macrophages at baseline and after treatment with NAC. Fig. 3A shows that alveolar macrophages, unlike monocytes, generate large amounts of HO· under control conditions and that this is inhibited by NAC. To examine the effect of the high oxidant levels on PTEN expression, we evaluated whole cell lysates for PTEN after treatment with NAC (3 h) or NAC and glutathione (24 h). Fig. 3B shows that PTEN expression is increased in alveolar macrophages after treatment with NAC or glutathione, suggesting that endogenous oxidant production by alveolar macrophages is important in down-regulation of PTEN. When we performed a similar experiment in blood monocytes (which exhibit no spontaneous oxidant production), we found no changes in PTEN protein amounts after treatment with NAC (Fig. 3C).

PTEN Is Down-regulated during in Vitro Monocyte Differentiation—Given that human alveolar macrophages are derived from blood monocytes via a lung-specific differentiation process and that these cells have very different expression levels of PTEN and Akt activity, we asked if PTEN amounts were consistently altered during in vitro differentiation. Peripheral blood monocytes obtained from normal human volunteers were differentiated in culture with human serum for 7-10 days, and RNA or total protein was isolated from the cells. Fig. 4 demonstrates that monocyte-derived macrophages have decreased PTEN mRNA compared with the housekeeping gene, HPRT, after the differentiation process. This is similar to the differences noted in baseline PTEN mRNA seen when comparing blood monocytes to human alveolar macrophages (Fig. 4A). It is important to note that levels of HPRT mRNA between monocytes and alveolar macrophages are comparable. Examining protein levels of PTEN during the in vitro differentiation process, we found a consistent decrease over time of relative amounts of PTEN (Fig. 4B). When we examined Akt activation during in vitro differentiation, we found that active Akt increased (Fig. 4C). Consistent with our primary cell data, we found that monocyte differentiation leads to an increase in ROS production (Fig. 4D). These data suggest that the differences in PTEN levels and Akt activity between blood monocytes and alveolar macrophages are the result of a differentiation process that can be replicated in vitro. We also examined the effect of inhibition of oxidant production on survival of monocytes differentiated in vitro. Fig. 4E demonstrates that monocytes differentiated in the presence of NAC have diminished survival and fail to differentiate into macrophage-like cells. This suggests that spontaneous production of oxidants during the process of differentiation is important for acquiring the alveolar Macrophage survival phenotype.


Figure 2
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  		FIGURE 2.
PI 3-kinase activation is important for survival in alveolar macrophages. Human alveolar macrophages or blood monocytes were cultured with and without the PI 3-kinase inhibitor, LY294002, in serum-free media for 24 h. A, photomicrographs of cells stained with ethidium homodimer (a marker of cell death). The graphs show percent cell death as determined by trypan blue exclusion. The graph inset shows the immunoblot analysis for cleaved caspase 7, an indicator of apoptosis. B, alveolar macrophages were infected with replication-deficient adenovirus vectors containing the transgene for GFP (AdGFP), PTEN (AdPTEN), or no transgene (AdVector). The panel to the right shows 75% infection efficiency with AdGFP. To the left is the Western analysis showing PTEN and phospho Akt expression in AdVector- and AdPTEN-infected macrophages. The center panel shows ethidium homodimer staining (cell death). C, ATP viability assay in the Ad-infected cells (EV = AdVector).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
One of the most intriguing characteristics of alveolar macrophages is their ability to survive for prolonged periods of time in vivo. In fact, the estimated turnover time of alveolar macrophages in humans is 81 days (22). This is in contrast to their precursor cells, blood monocytes, which have a much shorter life span and undergo apoptosis within 1-2 days (1). Macrophages are derived from peripheral blood monocytes that migrate to the lung where they undergo differentiation. Few studies have addressed the survival differences between alveolar macrophages and their precursor cells (21, 35-38), and none have examined the role of differentiation in this process. We have shown for the first time that human alveolar macrophages are deficient in PTEN and that this correlates with high constitutive expression of activated Akt. The decrease in PTEN can be reversed by blocking the alveolar macrophage specific high levels of reactive oxygen species. Furthermore, differentiation plays a role in the process since PTEN is down regulated with in vitro differentiation of monocytes.


Figure 3
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  		FIGURE 3.
Endogenous oxidant production by alveolar macrophages affects PTEN expression. Matching sets of human alveolar macrophages and peripheral blood monocytes were obtained from normal volunteers and cells were treated with and without N-acetylcysteine (NAC) for 3 h. Electron spin resonance was performed using the spin trap, DMPO (panel A). In a parallel set of experiments, freshly isolated alveolar macrophages or blood monocytes were treated with and without NAC or reduced glutathione (macrophages only) for the specified time points, and cells were harvested for total protein. Western analysis was performed using an anti-PTEN antibody (panels B and C). The graphs below show corresponding densitometry.

 
PTEN, originally described as a tumor suppressor protein (39, 40), clearly plays a role in normal cellular development and function, primarily through its action as a lipid phosphatase for PI3,4,5P3 (for reviews see Refs. 4 and 8). The reduction of PI3,4,5P3 levels suppresses activation of Akt allowing apoptosis to occur (41, 42). In mice, homozygous PTEN deletion is embryonic lethal, whereas Pten+/- heterozygotes survive to adulthood but develop multiple tumors and immune system abnormalities (41-44). Studies of immortalized embryonic fibroblasts from PTEN null mice show that these cells have increased amounts of PI3,4,5P3, high constitutive expression of activated Akt, and enhanced survival in response to pro-apoptotic stimuli (42) illustrating the importance of PTEN in modulating Akt activation through its action as a lipid phosphatase. These genetic deletion studies underscore the importance of PTEN in modulating embryonic development, immune function, and tumor suppression. Our studies show that alterations in amounts of PTEN may play a role in regulation of normal cell survival via differential expression in blood monocytes and alveolar macrophages. Whereas blood monocytes, which express high levels of PTEN and low active Akt, undergo rapid apoptosis, alveolar macrophages, expressing high active Akt and low PTEN, have prolonged survival in vivo and in vitro.

Very little is known about PTEN regulation in normal cells. In early embryonic development in mice, PTEN levels are low until ~day 11 when expression increases in a variety of tissues (44). PTEN appears to be constitutively expressed in many cells and regulated by transcription and by post-translational phosphorylation-dependent effects on protein stability (7, 12, 45, 46). PTEN has been shown to be up-regulated by Egr-1 (12), p53 (11), and PPAR{gamma} (10), but little is known about negative regulation of PTEN in normal cells. The only relevant study was done by Vasudevan et al. (13) in which they examined the role of NF{kappa}B on inhibition of PTEN expression in mouse embryonic fibroblasts and found that PTEN is indirectly down regulated by NF{kappa}B p65 via sequestration of the transcriptional co-activator proteins CBP/p300. They noted a correlation between high NF{kappa}B p65 and low PTEN amounts in several tumor cell lines but did not examine normal cells. We have found a similar association in human alveolar macrophages (high levels of p65 and low PTEN). Our data shows that there is less PTEN mRNA in alveolar macrophages compared with blood monocytes suggesting that either PTEN transcription is down-regulated in macrophages or that PTEN mRNA is less stable.

In examining a mechanism for the decreased PTEN mRNA and protein in alveolar macrophages, we found that differentiation-induced oxidant production by alveolar macrophages (absent in monocytes) contributed to the differential expression of PTEN. Although oxidant-mediated regulation of PTEN phosphatase function has been previously described (9, 47, 48), our study is the first to show oxidant-mediated regulation of PTEN protein amounts. Further studies to examine this mechanism are ongoing.


Figure 4
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  		FIGURE 4.
PTEN is down-regulated during in vitro monocyte differentiation. Matching sets of human alveolar macrophages and peripheral blood monocytes were obtained from normal volunteers and total RNA was obtained. Additional blood monocytes were cultured in the presence of 10% human AB serum to induce differentiation into macrophage-like cells. The cells were harvested for total RNA or cellular protein at the indicated time points, and real-time PCR and Western analysis was performed. In separate experiments, oxidant production was measured in freshly isolated cells or in differentiated monocytes at day 8. A, real-time RT-PCR graph showing PTEN mRNA relative to the housekeeping gene, HPRT. B, immunoblot for PTEN with corresponding densitometry. C, immunoblot for phosphorylated (active) Akt with corresponding densitometry. D, electron spin resonance in alveolar macrophages, blood monocytes, and differentiated monocytes. E, ATP viability assay in the presence of 10 mM NAC during differentiation of blood monocytes.

 
Few studies have examined the mechanism resulting in prolonged survival of macrophages and the role of differentiation in the development of this phenotype. The most relevant studies come from Pope and colleagues and have shown that monocyte-derived macrophages require constitutive expression of phosphorylated Akt for survival and that constitutive activation of Akt and STAT3 are important for expression of the downstream anti-apoptotic Bcl family member, Mcl-1 (21, 36). They have also shown that inhibition of constitutively active NF{kappa}B in a macrophage cell line and in monocyte-derived macrophages induces apoptosis of these cells in a caspase 3-independent manner presumably related to loss of the anti-apoptotic Bcl family member, A1 (35). Others have shown that differentiation of bone marrow macrophages results in up-regulation of the anti-apoptotic proteins, XIAP, Bcl-2, and Bcl-xL (49, 50), although this has not been studied in alveolar macrophages. Our laboratory has recently shown that activation of ERK and Akt is required for survival of alveolar macrophages and that sphingosine contributes to survival via an effect on the ERK and Akt pathways (38). Our current study further examines upstream effectors of constitutive Akt activation and demonstrates that a deficiency in PTEN likely contributes to and is functionally important for macrophage survival. We show that the down-regulation of PTEN seen in alveolar Macrophage correlates with alterations of PTEN that occur during differentiation. Only one other study has reported PTEN amounts during differentiation of blood monocytes (10). These authors examined the role of the ligand-activated nuclear receptor peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) on regulation of PTEN amounts. They found that monocytes that were differentiated in the presence of the PPAR{gamma} agonist, rosiglitazone, had a marked up-regulation in PTEN with a corresponding loss of activated Akt and postulated that PPAR{gamma} may have a role in regulation of the PI 3-kinase pathway in inflammatory cells. In contrast to our study, they found an induction of PTEN mRNA with differentiation, although they did not examine total PTEN protein in the monocytes relative to the macrophages. These contrasting observations might be due to different methods of in vitro differentiation and to time points at which PTEN mRNA was quantitated. These authors obtained RNA at day 4 of differentiation, whereas in our studies, differentiated monocytes were not harvested until day 8.

It is not known whether these alterations in PTEN are specific for alveolar macrophages, because we have not examined other primary tissue macrophages. It would be interesting to know if there are environmental factors unique to the lung that have an affect on PTEN regulation in macrophages. Studies to examine this possibility are underway. As a composite, these data present the novel finding that differentiation of monocytes results in macrophages with a phenotype characterized by high oxidant tone, low levels of PTEN, high Akt activity, and prolonged survival times.


   FOOTNOTES
 
* This work was supported by a Veterans Affairs Merit Review grant, National Institutes of Health Grants HL-60316, HL-077431, and HL-04428, and National Center for Research Resources, National Institutes of Health Grant RR00059 from the General Clinical Research Centers Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Division of Pulmonary, Critical Care, and Occupational Medicine, 100 Eckstein Medical Research Bldg., Iowa City, IA 52242. Tel.: 319-384-7123; Fax: 319-335-6530; E-mail: dawn-flaherty{at}uiowa.edu.

2 The abbreviations used are: PI, phosphatidylinositol; ROS, reactive oxygen species; PI3,4,5P3, phosphatidylinositol 3,4,5-trisphosphate; DMPO, 5,5-dimethyl-1-pyrroline N-oxide; NAC, N-acetylcysteine; XIAP, X-chromosome-linked inhibitor of apoptosis; ERK, extracellular signal-regulated kinase; PPAR{gamma}, peroxisome proliferator-activated receptor {gamma}; GFP, green fluorescent protein; PTEN, phosphatase and tensin homologue deleted on chromosome 10; Ad, adenovirus; HPRT, hypoxanthine-guanine phosphoribosyltransferase. Back

3 S. Rozen and H. J. Skaletsky (2000) Primer3, source code available at fokker.wi.mit.edu/primer3/. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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