Introduction

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Monocytes are produced in the bone marrow and, in the absence of specific survival signals, are programmed to undergo apoptosis in 24-48 h (1, 2). In the presence of these survival factors, monocytes can differentiate into tissue macrophages, which have life spans of up to 3 mo (2). In a number of cardiopulmonary diseases, like atherosclerosis, and interstitial lung diseases like pulmonary fibrosis and sarcoidosis, monocyte accumulation and the presence of MCP-1 and/or M-CSF in involved tissues correlates with disease pathogenesis and progression (3). A number of inflammatory cytokines that promote monocyte survival, such as interleukin (IL)-1 and tumor necrosis factor (TNF)-, are also found in biologic fluid or pathologic samples from these affected areas (9, 10). We speculate that these factors may promote disease progression by stimulating the survival and accumulation of these recruited monocytes. Thus, understanding the biochemical pathways involved in monocyte survival pathways may allow targeting of key intracellular events in these cells to treat patients with these diseases more effectively.

Previous investigators identified the inflammatory cytokines IL-1 and TNF-, the bacterial cell wall antigen lipopolysaccharide (LPS), and growth factors M-CSF and granulocyte macrophage-colony-stimulating factor (GM-CSF) (9) as factors that promote monocyte survival (10). In addition, we hypothesized that the IL-1 family member IL-18 (11) may also promote monocyte survival. To promote survival, these factors should repress monocyte apoptotic pathways. In apoptosis induced by serum deprivation of normal human monocytes, activation of caspase-3 appears to be a critical execution event (12). However, the intracellular events responsible for promoting monocyte survival in response to these factors are not well understood. To begin to address this issue, we reported that monocytes stimulated with M-CSF required the activation of PI 3-kinase and suppression of caspase-9 and caspase-3 for survival (13). Using in vitro kinase assays, we found that Akt was activated in human monocytes stimulated with M-CSF, and that Akt activation was suppressed by PI 3-kinase inhibitors (13). Finding that Akt activation correlated to monocyte survival is in agreement with other investigators, who found that Akt activation appeared to be a central survival factor in growth factor-stimulated cells (14). Akt is a cytosolic protein whose translocation is induced by the binding to PI 3-kinase products via the pleckstrin homology domain of Akt (17). Once Akt translocates to the cell membrane, a separate kinase, PDK1, phosphorylates Akt on threonine residue 308 and serine residue 473, inducing the kinase activity of Akt (17). In addition to in vitro kinase assays, antibodies to serine (ser⁴⁷³) or threonine 308 (thr³⁰⁸) phosphorylated Akt have been used to demonstrate Akt activation.

Once phosphorylated, Akt is released from the cell membrane and migrates to intracellular locations where Akt can activate other proteins, resulting in the suppression of cellular apoptosis. Activated Akt leads to cellular survival by several mechanisms, including suppressing caspase-9 and caspase-3 activation (13), promoting production of the survival factor Bcl-x_L, repressing the activity of the proapoptotic factor BAD, suppressing the production of Fas ligand production, and augmentation of the endothelial cell isoform of nitric oxide synthase (14, 16, 20, 21). In previous studies from our laboratory, we found that monocyte survival, Akt activation, and the suppression of caspase-3 activity in M-CSF-stimulated monocytes were reversed by PI 3-kinase inhibitors (13), suggesting that PI 3-kinase may function upstream of Akt and caspase-3. Based on these findings, we hypothesized that factors tested in this study may activate PI 3-kinase and Akt to suppress caspase-3 activation.

In support of this hypothesis, we found that monocyte survival factors appeared to induce the tyrosine phosphorylation of p85 PI 3-kinase, an activating step in PI 3-kinase activity. PI 3-kinase inhibitors reversed the suppression of DNA fragmentation and caspase-3 activation induced by M-CSF in normal human monocytes. Moreover, PI 3-kinase inhibitors also reduced the serine and threonine phosphorylation of Akt in M-CSF-stimulated monocytes. Using in vitro kinase assays, M-CSF appeared to induce more Akt kinase activity than other monocyte survival factors in human monocytes. We speculate that activation of PI 3-kinase and Akt by these monocyte survival factors may promote the suppression of caspase-3, potentially representing a common biochemical pathway for monocyte survival.

	Materials and Methods

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Materials

Recombinant human M-CSF, GM-CSF, IL-1, and IL-18 were purchased from R&D Systems (Minneapolis, MN). TNF- was purchased from Sigma (St. Louis, MO). LY294002 and wortmannin were from Calbiochem (La Jolla, CA). Protein G-agarose was purchased from Gibco Life Technologies, Inc (Rockville, MD). RPMI 1640 medium was obtained from BioWhittaker, Inc. (Walkersville, MD). Fetal calf serum was obtained from Hyclone Laboratories (Logan, UT). Anti-Akt and Anti-PhosphoAkt antibodies were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). All other reagents were from Sigma unless otherwise specified.

Isolation of Peripheral Blood Monocytes and Cell Culturing

Monocytes (66 + 2.1% CD14+) were isolated from the heparinized blood of normal volunteers as described previously (22, 23). For DNA fragmentation analysis, monocytes were cultured under the indicated conditions immediately after isolation from blood. For signaling experiments, monocytes were subsequently grown in RPMI 1640 medium supplemented with 10% fetal calf serum and 30 ng/ml recombinant human M-CSF for 16 h at 37°C. Samples were serum-starved on ice in RPMI 1640 medium alone for 2 h before being subjected to stimulation with one of the cytokines or growth factors as indicated.

Cytosolic DNA Fragmentation Analysis

Monocytes (3 × 10⁶/condition) were preincubated for 30 min in RPMI 1640 medium supplemented with 5% fetal calf serum and 10 µg/ml polymyxin B (added to all samples except those stimulated with LPS) at 37°C and 5% CO₂ with the PI 3-kinase inhibitor LY294002 (50 µM) or DMSO solvent control. Monocytes were then stimulated with IL-1 (100 ng/ml), TNF- (1 µg/ml), GM-CSF (100 ng/ml), IL-18 (100 ng/ml), or LPS (100 ng/ml) for 18 h before DNA fragmentation analysis. The rate of monocyte apoptosis was compared with untreated time-matched controlled monocytes using DNA fragmentation assays as previously described (13).

LDH Assay

Lactate dehydrogenase (LDH) release was determined using an in vitro assay kit (Sigma). Readings were obtained using a spectrophotometer at 690 nm with the background medium (RPMI-1640) subtracted. As a positive control cell lysates from serum-starved monocytes were used.

Immunoprecipitation and Immunoblotting

Monocytes (10 × 10⁶/condition) in 2 ml 1640 RPMI medium supplemented with 10% heat inactivated fetal bovine serum were stimulated with either IL-1 (100 ng/ml), TNF- (1 µg/ml), GM-CSF (100 ng/ml), IL-18 (100 ng/ml), or LPS (100 ng/ml) for the indicated times and then lysed by the addition of 1,000 µl of ice-cold lysis buffer (50 mM Tris-Hcl [pH 7.5], 0.1% [wt/vol] Triton X-100, 1 mM EGTA, 50 mM NaF, 10 mM sodium glycerophosphate, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 0.1% 2-mercaptoethanol) and incubated on ice for 60 min. Nuclei were removed by centrifugation, and samples were subjected to immunoprecipitation with anti-Akt or isogenic control IgG antibodies (1 µg/ml) overnight at 4°C as described (13).

In Vitro Kinase Assays

In vitro kinase assays for Akt using histone 2B as a substrate were performed as previously described (13).

Preparation of Lysates and Detection of Caspase Activity

Enzymatic caspase activity measured with amino trifluoromethyl coumarin (AFC) as described for caspase-9 and caspase-3 (13).

Statistical Evaluation

For comparisons between groups, ANOVA with Fisher's post-hoc testing was performed. Statistical significance was defined by P  0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

PI 3-Kinase Inhibitors Reverse Monocyte Survival Induced by Survival Factors

Because we previously observed that PI 3-kinase inhibitors appeared to reverse the suppression of DNA fragmentation and caspase-3 activation induced by M-CSF in human monocytes, we hypothesized that this same biochemical pathway may be operative for other monocyte survival factors. Consistent with an important role for PI 3-kinase in monocyte survival, we found that the PI 3-kinase inhibitor LY294002 promoted DNA fragmentation in monocytes stimulated with the tested monocyte survival factors (Figures 1A and 1B). Incubating monocytes in the presence of LY294002 (10 µM) or Wortmannin (10 nM) in the absence of M-CSF did not appear to have independent effects on cellular survival (data not shown). In contrast, monocytes stimulated with M-CSF alone or with M-CSF with DMSO did not demonstrate evidence of DNA fragmentation. To ensure that the effects of LY294002 were not from nonspecific cellular cytotoxicity, we found that cells treated with or without LY294002 had no statistical difference in LDH release (P = 0.38 between M-CSF + LY294002 and M-CSF + DMSO) (Figure 2).

View larger version (63K): [in this window] [in a new window]   Figure 1.   PI 3-kinase inhibitor LY294002 activates DNA fragmentation in monocytes stimulated with survival factors. Monocytes (3 × 106/ml) were preincubated with the PI 3-kinase inhibitor LY294002 (50 µM) or DMSO (control) for 30 min. The cells were left not stimulated (NS) or stimulated with TNF- (1 µg/ml), IL-1 (100 ng/ml), LPS (100 ng/ml), IL-18 (100 ng/ml) or GM-CSF (100 ng/ml) for 18 h. The presence of cytosolic low molecular weight DNA fragments indicates apoptosis. This figure is representative of three independent experiments. Note at the left is a 123-bp DNA ladder.

View larger version (17K): [in this window] [in a new window]   Figure 2.   There is no increase in LDH release in cells incubated with the PI 3-kinase inhibitor LY 294002. Human monocytes (3 × 106) were extracted, preincubated with the PI 3-kinase inhibitor LY294002 (50 µM or 10 µM) or DMSO (solvent control) for 30 min and then the cells were left not stimulated (NS) or stimulated with M-CSF (100 ng/ml) for 18 h. As a positive control for the release of lactate dehydrogenase (LDH), cells were lysed after overnight incubation to release intracellular LDH. LDH release was determined using an in vitro assay kit (Sigma). Readings were obtained using a spectrophotometer at 690 nm with the background from media without cells (RPMI-1640) subtracted. These data represent two independent experiments. (P = 0.38 between LY294002 + M-CSF and DMSO + M-CSF-treated cells).

Monocyte Survival Factors Suppress the Activation of Caspase-3 and Caspase-9

Because the activation of caspase-3 appears to be an important execution event in the apoptosis program of human monocytes, we next hypothesized that monocyte survival factors may suppress caspase-3 activation. We found that all tested monocyte survival factors appeared to suppress the activation of caspase-3 (P < 0.05 versus apoptotic cells) (Figure 3). Interestingly, the addition of PI 3-kinase inhibitor LY294002 or wortmannin, but not the solvent control DMSO, resulted in significant activation of caspase-3- like activity in the presence of IL-1, TNF, IL-18, LPS, GM-CSF, and M-CSF (P < 0.05 versus cells incubated in the survival factors with DMSO) (Figures 3A and 3B). Because we previously demonstrated that M-CSF also appeared to suppress caspase-9 activity, we tested monocyte survival factors for their ability to suppress caspase-9. When compared with monocytes left not stimulated for 18 h, these factors also reduced caspase-9 activity (P < 0.05 for all factors by ANOVA with post-hoc analysis) (Figure 3C).

View larger version (40K): [in this window] [in a new window]   View larger version (13K): [in this window] [in a new window]   Figure 3.   Monocyte survival factors suppress caspase-3 and caspase-9 activation, which is reversed by the PI 3-kinase inhibitors LY294002 and wortmannin. Monocytes (5 × 106) were preincubated with the PI -3-kinase inhibitor LY294002 (black bars) or wortmannin (white bars) or the DMSO solvent control for 30 min. The cells were then incubated with one of the indicated survival factors or left not stimulated (NS) for 18 h. Lysates were prepared from monocytes harvested fresh or after 18 h in culture. The presence of caspase-3- or caspase-9-like activity was determined in lysates of monocytes treated as indicated using fluorogenic substrate DEVD-AFC for caspase-3 (A and B) or LEHD-AFC for caspase-9 (C) and defined by fluorescence emission. Both LY294002 and wortmannin induced more caspase-3-like activity in monocytes treated with survival factors compared with cells treated with solvent control DMSO and the survival factors (P < 0.05). In addition, these survival factors reduced caspase-9-like activity in monocytes compared with monocytes left not stimulated for 18 h (P < 0.05). Experiments for caspase-3-like activity represents three experiments and those for caspase-9-like activity represents two studies.

Monocyte Survival Factors Induce the Activation of p85 PI 3-Kinase and Akt

Because Akt activation is important in facilitating M-CSF- induced cellular survival and is dependent on the activity of PI 3-kinase, we next wanted to determine if other monocyte survival factors induced the activation of p85 PI 3-kinase and Akt activation. We found that each of the monocyte survival factors evaluated in this study induced the tyrosine phosphorylation of p85 PI 3-kinase (Figure 4). The monocyte survival factors also induced the serine (Figure 5A) and threonine (Figure 6A) phosphorylation of Akt, which was reversed by the PI 3-kinase inhibitor LY294002. These data were quantitated using densitometry (Figures 5B and 6B). To ensure that an equal amount of Akt was assayed in each of the samples, the blots were stripped and reprobed for Akt (Figures 5C and 6C). Moreover, in vitro kinase assays for Akt using the substrate histone 2B revealed that M-CSF appeared to be the most potent inducer of Akt kinase activity (Figure 7).

View larger version (34K): [in this window] [in a new window]   Figure 4.   p85 is phosphorylated after stimulation with monocyte survival factors. Human monocytes (10 × 106/ml) were serum-starved and then left not stimulated (NS) or stimulated with IL-1 (100 ng/ml), TNF- (1 µg/ml), GM-CSF (100 ng/ml), IL-18 (100 ng/ml), or LPS (100 ng/ml) for 5 min. The samples were then lysed, immunoprecipitated with anti-p85 or isogenic control antibodies, and separated using SDS-PAGE. The membranes containing the samples were blotted with antiphosphotyrosine antibodies to detect tyrosine-phosphorylated proteins and developed using autoradiography (top panel ). The membranes were stripped and reprobed for the adapter p85 PI 3-kinase protein (bottom panel ). This figure is illustrative of two independent studies.

View larger version (19K): [in this window] [in a new window]   Figure 5.   Akt is serine phosphorylated after stimulation by monocyte survival factors and is suppressed by LY294002. Human monocytes (10 × 106/ ml) were serum-starved for 2 h. The cells were then left not stimulated (NS) or stimulated with GM-CSF (100 ng/ ml), IL-1 (100 ng/ml), IL-18 (100 ng/ml), LPS (100 ng/ml), or TNF- (1 µg/ml) for the 5 min. Immunoprecipitation was performed with or anti-Akt1 antibodies. After separation by SDS-PAGE, immunoblotting was performed with anti-phosphoserine473 Akt antibodies. Membranes from monocytes stimulated as above were stained with (A) anti-phosphoserine473 Akt antibodies, (B) densitometry measurements of the bands in (A) in relative densitometric units, or (C) the membrane was stripped and reblotted with anti-Akt1 antibodies to ensure equal loading of all samples. This figure is representative of three independent studies.

View larger version (20K): [in this window] [in a new window]   Figure 6.   Akt is threonine phosphorylated after stimulation by monocyte survival factors and is suppressed by LY294002. Human monocytes (10 × 106/ml) were serum-starved for 2 h. The cells were then left not stimulated (NS) or stimulated with GM-CSF (100 ng/ml), IL-1 (100 ng/ml), IL-18 (100 ng/ml), TNF- (1 µg/ml), or LPS (100 ng/ml) for 5 min. Immunoprecipitation was performed with anti-Akt1 antibodies. After separation by SDS-PAGE, immunoblotting was performed with anti-phosphothreonine308 Akt antibodies. Membranes from monocytes stimulated as above were stained with (A) anti-phosphothreonine308 Akt antibodies, (B) densitometry measurements of the bands in (A) in relative densitometric units, or (C) the membrane was stripped and reblotted with anti-Akt1 antibodies to ensure equal loading of all samples. This figure is representative of three independent studies.

View larger version (33K): [in this window] [in a new window]   Figure 7.   Monocyte survival factors induce Akt kinase activity. Human monocytes (10 × 106/condition) were left not stimulated (NS) or treated with monocyte survival factors M-CSF (100 ng/ml), GM-CSF (100 ng/ml), IL-1 (100 ng/ml), TNF- (1 µg/ml), IL-18 (100 ng/ml), or LPS (100 ng/ml) for 3 min. Akt in vitro kinase assays were performed using histone 2B as the substrate and (A) developed using autoradiography as previously described (13), (B) densitometric analysis of bands in (A) in relative densitometric units, (C) the membranes were reprobed for total Akt1 to ensure equal loading. This figure is representative of three independent studies.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

These studies evaluated the hypothesis that monocyte survival factors may each promote Akt activation and suppress caspase-3 activation in human monocytes. To test this hypothesis, we assessed factors previously described as being able to promote monocyte survival, and included the cytokine IL-18 in our analysis. We found that these factors appeared to reduce cytosolic DNA fragmentation and suppressed the activation of caspase-3 and caspase-9 in human monocytes. Moreover, these survival factors appeared to induce the tyrosine phosphorylation of p85 PI 3-kinase. Monocyte survival events appeared to be reversed by the addition of PI 3-kinase inhibitors, suggesting a potentially important role for PI 3-kinase in this survival pathway. Because PI 3-kinase appeared important in this survival pathway, we next hypothesized that these survival factors may activate the phosphorylation of Akt on serine⁴⁷³ and/or threonine³⁰⁸ residues. Indeed, we found that Akt was phosphorylated on each of these residues by the monocyte survival factors evaluated in this study. Moreover, these phosphorylation events were suppressed by the addition of PI 3-kinase inhibitors. To further explore the ability of these monocyte survival factors to activate Akt, we next performed in vitro kinase assays using histone 2B as the substrate. Interestingly, M-CSF appeared to be a more potent activator of Akt kinase activity than the other monocyte survival factors, a finding consistent with the known critical role of M-CSF in monocyte and macrophage production and survival.

Of the monocyte survival factors tested in this study, the factors appear to fall into two general categories: inflammatory cytokines and growth factors (9, 10). It is interesting to speculate that these factors may be important in facilitating host immune responsiveness through their abilities to extend monocyte survival. These activated monocytes may then act as direct responder cells, which are able to phagocytose opsonized particles, or may direct trafficking and activation of other inflammatory cells through the release of cytokines and chemokines (24). This important role for monocyte recruitment and survival in host immunity and inflammation is underscored by findings in transgenic animals deficient in monocyte recruitment factors. For example, MCP-1^/ mice, which have reduced monocyte recruitment, have increased morbidity and mortality to experimental infection challenge (27). However, because MCP-1 does not support monocyte survival and to promote effective immune surveillance, these recruited monocytes also require survival factors, such as those evaluated in this study.

In addition, monocytes may participate in tissue inflammation and injury in clinical disease, such as in apolipoprotein E^/ or LDL receptor^/ mice, which are hypercholesterolemic and die of precocious coronary artery disease. Crossbreeding these apolipoprotein E^/ or LDL receptor^/ mice with animals deficient in the ability to recruit monocytes (MCP-1^/ or CCR-2^/, the receptor for MCP-1) or deficient in monocyte survival factors (M-CSF^/) protect the resulting homozygous deficient offspring from developing atherosclerosis (4, 5, 28). Thus, it appears that the regulated recruitment of monocytes may have important immune benefits, but if not carefully controlled, these same biologic processes may lead to the genesis or progression of inflammatory diseases. Thus, we speculate that by defining molecular targets and biochemical pathways important in the monocyte survival program, we may be able to better regulate tissue inflammation. Based on previous work from our laboratory and others, we further speculated that an activation of the PI 3-kinase and Akt might play a role in the survival of human monocytes.

PI 3-kinase is an enzyme complex that is important in a variety of cellular biochemical processes, including cellular survival (29). In cellular survival, PI 3-kinase has been found to be an important target of growth factor receptors, through its ability to stimulate the activation of Akt/protein kinase B (32). Once activated, Akt has several important molecular targets that may serve to allow Akt to promote cellular survival, including suppressing caspase-3 activation, reducing nuclear translocation of Forkhead transcription factors, inducing the production of prosurvival proteins and promoting NF-B nuclear translocation (14, 16, 20, 21, 40, 41).

In this report, we provide evidence to support the hypothesis that the monocyte survival factors tested in this study appeared to facilitate monocyte survival through a biochemical pathway requiring PI 3-kinase activity that resulted in suppressing caspase-9 and caspase-3 activation. Similarly, these monocyte survival factors also appeared to induce the serine and threonine phosphorylation of Akt in a PI 3-kinase-dependent manner. These intermediate pathways may serve as molecular targets to suppress unwanted monocyte survival in inflammatory disease states.