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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 61:1111-1118 (2006)
© 2006 The Gerontological Society of America

Role of Phosphoinositide 3-Kinase and Extracellular Signal-Regulated Kinase Pathways in Granulocyte Macrophage–Colony-Stimulating Factor Failure to Delay Fas-Induced Neutrophil Apoptosis in Elderly Humans

Cosimo Tortorella, Olivia Simone, Giuseppina Piazzolla, Isabella Stella, Valentina Cappiello and Salvatore Antonaci

Department of Internal Medicine, Immunology and Infectious Diseases, University of Bari Medical School, Italy.

Address correspondence to Cosimo Tortorella, MD, Department of Internal Medicine, Immunology and Infectious Diseases, Section of Internal Medicine, Policlinico, 70124 Bari, Italy. E-mail: c.tortorella{at}intmed.uniba.it


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Fas-stimulated neutrophils from elderly individuals show impaired granulocyte macrophage–colony-stimulating factor (GM–CSF)-induced apoptosis cell rescue. Herein, this defect was found to be associated with a significant reduction in GM–CSF-mediated Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Using Akt and ERK1/2 inhibitors, we demonstrated that both kinases were critical for GM–CSF antiapoptotic effects. Whereas Akt inhibition also affected GM–CSF-dependent ERK1/2 phosphorylation, ERK1/2 inhibition did not affect GM–CSF-induced Akt phosphorylation, suggesting that phosphoinositide 3-kinase (PI3-K)/Akt and ERK1/2 are activated in series and that PI3-K is located upstream of ERK1/2 along the GM–CSF-dependent signaling pathway. No age-associated changes in GM–CSF receptor expression were observed. Interestingly, both suppressors of cytokine signaling (SOCS)1 and SOCS3 proteins were significantly higher in unstimulated neutrophils from elderly individuals and, unlike in young individuals, did not further increase following GM–CSF cell triggering. These results indicate that defective PI3-K/Akt/ERK1/2 activation, likely dependent on elevated SOCS1 and SOCS3 levels, may affect the GM–CSF capacity to delay neutrophil apoptosis in elderly persons.

NEUTROPHILS play a significant role in the organism's defense against bacteria and parasites, through the release of toxic oxygen metabolites and proteolytic enzymes. Apoptosis terminates neutrophil functions within the inflammatory areas, inducing the removal of cells without the release of hazardous cell contents. Intact unwanted apoptotic neutrophils are, in fact, recognized and engulfed by phagocytes, thus preventing damage to surrounding tissues (1). Although neutrophils are constitutively committed to apoptosis, their progression through the cell death program can be accelerated or delayed by cytokines and other inflammatory mediators. Granulocyte macrophage–colony-stimulating factor (GM–CSF), interleukin (IL)-8, lipopolysaccharide (LPS), C5a, leukotriene B4, and hypoxia have been reported to delay neutrophil apoptosis (2–6), whereas tumor necrosis factor-{alpha}, Fas ligand (FasL), and UV radiation have the opposite effect (7–9).

It is widely accepted that aging is associated with an increase in the susceptibility to infections, likely as a result of an overall imbalance of the immune response. In this context, a growing body of evidence suggests that a key role is played by impaired neutrophil activity. The neutrophil functions most strongly affected by aging are chemotaxis, phagocytosis, respiratory burst, and killing (10–14). Despite the strict analogies between these alterations and the depressed functional capacities displayed by apoptotic neutrophils (15), we have recently reported, in keeping with findings by other groups, that advancing age does not affect either spontaneous or Fas-induced apoptotic events (16–19). Nevertheless, neutrophils from aged individuals display a diminished rescue capacity when challenged with antiapoptotic molecules, such as GM–CSF, G–CSF, LPS, or IL-2 (16,17). Among these compounds, the pro-inflammatory cytokine GM–CSF appears to have a specialized role in modulating neutrophil functions. Besides the above mentioned capacity to delay apoptosis (2,3,5), its pleiotropic activities include the enhancement of cell adhesion to biological surfaces as well as the priming or activation of the respiratory burst (20,21). Furthermore, GM–CSF is released during septicemia; accordingly, a defective response to this cytokine might have dramatic consequences for neutrophil functions and the host defense against infections.

We have recently reported that neutrophils from elderly individuals show a significant decrease in GM–CSF-induced oxidative burst when assayed on fibronectin-coated plates (12). This effect was strictly related to an age-related impairment in the activation of extracellular signal-regulated kinase 1/2 (ERK1/2), a threonine/tyrosine kinase belonging to the mitogen-activated protein kinase (MAPK) family (22). Interestingly, many reports have demonstrated that ERK is also involved in cell rescue activities from apoptosis induced by various different mediators, including GM–CSF (23–25), and evidence has already been provided of a role of ERK pathway alterations in the age-related inability of GM–CSF to prolong neutrophil survival (19). Another kinase that has been implicated in controlling cell survival in many different cell types, including neutrophils, is phosphoinositide 3-kinase (PI3-K; 25–27). Three classes of PI3-Ks have been identified on the basis of the structure and function of each enzyme. Class IA enzymes comprise three different isoforms, namely PI3-K{alpha}, ß, and {delta}, consisting of a p110 catalytic subunit and a p85 regulatory subunit. Only one Class IB PI3-K enzyme exists, made up of a p110{gamma} catalytic and a p101 regulatory subunit. Whereas PI3-K{gamma} is activated by G protein–coupled receptors, Class IA enzymes are activated in response to GM–CSF and other growth factors (28). A key downstream target of PI3-K in mediating survival signals has been identified as the serine/threonine kinase protein kinase B (PKB), also known as Akt (29). The current study was thus undertaken to assess the relative role of ERK and PI3-K/Akt pathways in the decreased GM–CSF-induced delay of neutrophil apoptosis observed during aging.

We found that GM–CSF-stimulated neutrophils from elderly individuals exhibit a significant reduction of Akt and ERK1/2 phosphorylation, these findings largely accounting for the age-related impaired ability of GM–CSF to prolong neutrophil survival in Fas-triggered cells. Pharmacological inhibition of Akt also affected ERK1/2 activation, demonstrating that PI3-K/Akt and ERK1/2 belong to a single signal transduction pathway and that PI3-K is located upstream of ERK1/2 along the GM–CSF-dependent signaling pathway. Whereas no age-associated changes in GM–CSF receptor expression were observed, significant suppressors of cytokine signaling (SOCS)1 and SOCS3 levels were found in unstimulated neutrophils from elderly individuals. The role of these molecules in the age-related inhibition of the GM–CSF signaling pathway is further discussed.


   MATERIALS AND METHODS
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Donors
Thirty volunteers (16 men and 14 women) older than 65 years (mean age, 79 years; range, 72–94 years), free from diseases affecting the immune system, cancer, or infections and taking no antiinflammatory drugs or corticosteroids, were selected according to the SENIEUR protocol (30). Thirty healthy young blood donors (mean age, 32 years; range, 22–35 years) were enrolled as controls.

Cell Purification
To isolate neutrophils, heparinized peripheral blood, diluted 1:4 (vol/vol) with phosphate-buffered saline (0.02 M; pH 7.2), was fractionated by Ficoll-Hypaque gradient centrifugation (Amersham Biosciences Europe, Milan, Italy) at 170 x g for 45 minutes. Thereafter, the cell pellets were washed twice with phosphate-buffered saline and mixed with a 6% dextran (40,000 m.w.) solution. Following sedimentation for 1 hour at 37°C, the neutrophil-rich supernatants were collected and centrifuged at 400 x g for 10 minutes. Pellets were then subjected to hypotonic lysis of erythrocytes and finally resuspended in RPMI-1640 (Sigma Chemical Co., Milan, Italy) supplemented with penicillin (200 IU/mL), streptomycin (100 µg/mL), L-glutamine (2 mM), and 10% heat-inactivated fetal calf serum (FCS) (complete medium). The preparation contained more than 95% granulocytes, of which more than 90% were neutrophils, as determined by May Grünwald-Giemsa staining of cytocentrifuged samples. All reagents were LPS-free, as assessed by the Limulus amebocyte lysate assay (Sigma Chemical Co.).

Culture Conditions
Neutrophils (106 cells/well in a total volume of 300 µL/well) were cultured at 37°C in humidified air containing 5% CO2 in 24-well cell culture clusters (Corning, Milan, Italy), in the absence or presence of murine monoclonal antibody (mAb) 2R2 (antihuman Fas inducing apoptosis; 300 ng/mL; Alexis Biochemicals, Florence, Italy). After 12 hours of incubation, cells were recovered and washed once in RPMI-1640 before being used for apoptotic assays. To test the GM–CSF capacity to rescue neutrophils from Fas-mediated apoptosis, neutrophils were preincubated with GM–CSF at 20 ng/mL (Sigma Chemical Co.) for 2 hours before the addition of mAb 2R2. In some cases, cells were pretreated with 100 µM MAPK/ERK kinase (MEK) inhibitor PD98059 for 30 minutes (Sigma Chemical Co.) or 25 µM Akt Inhibitor for 2 hours (Calbiochem, La Jolla, CA) before being challenged with GM–CSF.

Assessment of Neutrophil Apoptosis
Neutrophil apoptosis was measured by flow cytometry using the Annexin V-FITC/7-AAD Kit (provided by Beckman Coulter Inc., Miami, FL), following the manufacturer's instructions. Fluorescence intensity was measured on a Coulter Epics Elite cell sorter (Beckman Coulter Inc.) equipped with a 15 mm air-cooled, 488 nm argon-ion laser. Gating on physical parameters was used to exclude cell debris and clumps. Morphological analysis of apoptotic neutrophils and a DNA ladder assay were performed as previously described (17).

GM–CSF Receptor Expression
Aliquots of blood specimens collected into EDTA tubes were incubated with phycoerythrin-conjugated mAbs directed to the {alpha} subunit of the GM–CSF receptor (CDw116; Beckman Coulter Inc.) for 15 minutes at room temperature in the dark. After red blood cell lysis and fixing, samples were analyzed on a Coulter Epics Elite cell sorter (Beckman Coulter Inc.). Neutrophils were gated on the basis of physical parameters. In each experiment, phycoerythrin-labeled isotype-matched control mAbs were used to determine positive and negative staining.

Western Blotting
Human neutrophils (1 x 107/mL) in Hanks' Balanced Salt Solution were prewarmed for 10 minutes at 37°C and then stimulated with GM–CSF at 20 ng/mL for 15 minutes. In some cases, cells were pretreated with PD98059 (100 µM for 30 minutes) or Akt Inhibitor (25 µM for 2 hours) at 37°C before being challenged with GM–CSF. The stimulation reaction was terminated by rapid centrifugation at 4°C. Immediately after removal of supernatants, the cell pellets were resuspended in ice-cold lysis buffer containing 25 mM Tris-HCl (pH 7.5), 1% Tryton X-100, 1% Igepal, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 150 mM sodium chloride, 1 mM EDTA, 1 µM pepstatin, 2 mM phenylmethylsulfonyl fluoride, 1 mM sodium pyrophosphate dibasic, 1 µM aprotinin, and 1 µM leupeptin for 10 minutes at 4°C. After centrifugation, the supernatant was mixed 1:1 (vol/vol) with 2X sample buffer (4% sodium dodecyl sulfate, 20% glycerol, 10% mercaptoethanol, and a trace amount of bromophenol blue dye in 125 mM Tris-HCl, pH 6.8), heated at 100°C for 5 minutes, and then frozen at –80°C until use. Samples were subjected to 10% sodium dodecyl sulfate gel electrophoresis, after which proteins were electrophoretically transferred from the gel onto a nitrocellulose membrane in a buffer containing 25 mM Tris, 192 mM glycine, and 20% methanol at 5.5 mA/cm2 for 30 minutes at room temperature. Residual binding sites on the membrane were blocked by incubating the membrane in blocking buffer (Tris-buffered saline pH 7.6 with 0.1% Tween 20 and 5% nonfat dry milk) for 1 hour at room temperature under gentle agitation. After washing in Tris-buffered saline containing 0.1% Tween 20 (TBST), the membrane was incubated with rabbit polyclonal Abs anti-Thr202/Tyr204–phosphorylated ERK1/2 (1:750), rabbit mAbs anti-Ser473-phosphorylated Akt (1:1000) (both purchased from Cell Signaling Technology, Beverly, MA), rabbit polyclonal Abs anti-SOCS1 and SOCS3 (at 2 µg/mL) (obtained from Abcam Ltd., Cambridge, U.K.) at 4°C overnight, and then with goat antirabbit immunoglobulin G Abs conjugated with horseradish peroxidase (1:10,000 in blocking buffer; Cell Signaling Technology) at room temperature for 90 minutes. The Ab complexes were visualized by ECL Plus Western Blotting Detection Reagents (Amersham Biosciences Europe), following the manufacturer's instructions and, subsequently, densitometric analysis of each band was performed. Values were normalized to ß-actin, assessed by stripping and reprobing the membranes with rabbit polyclonal anti-ß-actin Abs (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA).

To compare values from different gels, one lane of each gel was loaded with a control sample. After transfer and blocking, the membrane was cut and the control lane incubated with rabbit polyclonal Abs against p38 MAPK (1:750; Cell Signaling Technology), arbitrarily chosen as housekeeping protein, before being processed together with the remaining part of the membrane as described above. Therefore, for each protein, values obtained from different experimental points were further normalized to the total p38 MAPK protein to offset any variations due to the different exposure of single membranes.

Statistical Analysis
Student's t test was used to determine statistical significance.


   RESULTS
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Age-Related Effects of GM–CSF on Fas-Induced Neutrophil Apoptosis
To determine the age-related variability of neutrophil cell death in response to Fas activation, purified cells were cultured in vitro in serum-containing medium and challenged with anti-Fas 2R2 mAbs inducing apoptosis up to 12 hours. As depicted in Figure 1, in neutrophils from elderly individuals stimulated with 2R2 mAbs there was a significant increase in the percentage of cells expressing Annexin V (4.3 ± 1.7% at 0 hours vs 73.5 ± 8.7% at 12 hours, p <.0001). Comparable results were obtained for neutrophils from young volunteers, in which 4.8 ± 1.8% and 70.3 ± 6.7% (p <.0001) were apoptotic, as assessed by the expression of Annexin V at 0 hours and 12 hours, respectively. The unaffected tendency of neutrophils from elderly individuals to respond to Fas-induced apoptotic events was further confirmed by morphologic analysis, as well as by a DNA ladder assay (data not illustrated). Such a relatively long culture time is associated with the occurrence of spontaneous apoptotic phenomena in neutrophils, the incidence of which ranges from 20% to 30% according to different studies (16,17). Therefore, it is possible that, in our experimental protocol, the role of the Fas pathway in neutrophil apoptosis might be overestimated. However, this does not seem the case as 12-hour cell incubation with neutralizing anti-FasL mAb resulted in a marked reduction in the percentage of spontaneously occurring Annexin V+ neutrophils both in young (28.4 ± 6.6% without anti-FasL vs 12 ± 3.7% with anti-FasL) and in elderly participants (32.5 ± 5.9% without anti-FasL and 14.2 ± 4.5% with anti-FasL).


Figure 01
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  		Figure 1. Effects of granulocyte macrophage–colony-stimulating factor (GM–CSF) on Fas-induced neutrophil apoptosis in elderly and young humans. Neutrophils (106/well in a total volume of 300 µL/well) were pretreated with GM–CSF (20 ng/mL) for 2 hours before being incubated for 12 hours with antihuman Fas inducing apoptosis monoclonal antibodies (mAbs) (2R2; 300 ng/mL). Neutrophil apoptosis was assessed by flow cytometry using the Annexin V-FITC/7-AAD Kit, and expressed as the percentage of Annexin V+/7-AAD– neutrophils, gated on the basis of physical parameters. Data are representative of 15 similar experiments

 
To test the GM–CSF ability to delay Fas-triggered neutrophil cell death in elderly persons, cell cultures were pretreated with GM–CSF at 20 ng/mL for 2 hours before being incubated for additional 12 hours with mAb 2R2. Under these conditions, whereas neutrophils from young donors exhibited a significant inhibition of apoptotic events, cells from elderly donors underwent only a slight, but not significant delay of neutrophil apoptosis, as assessed by Annexin V expression (36.5 ± 5.2%, p <.0001 and 60.8 ± 8.9%, p = nonsignificant, respectively) (see Figure 1), morphology or DNA ladder analysis (data not shown).

Involvement of ERK1/2 and PI3-K Signaling Pathways in the Age-Related Decrease of GM–CSF-Induced Delay of Neutrophil Apoptosis
Neutrophils from elderly and young participants were incubated with medium alone or GM–CSF at 20 ng/mL for 15 minutes, and tyrosine phosphorylation of ERK1/2 and serine phosphorylation of Akt were assessed by immunoblotting using antibodies against the phosphorylated forms of the kinases. Preliminary experiments on the time course of ERK1/2 and Akt phosphorylation had shown that the interval of stimulation used induced maximal kinase activation in cells from both groups of individuals. As illustrated in Figure 2, higher baseline levels of ERK1/2 and Akt phosphorylated forms but lower GM–CSF-induced phosphorylated ERK1/2 and Akt levels were detected in the neutrophils from the elderly group as compared with those from younger counterparts. As a result, only a 10-fold increase in GM–CSF-induced ERK1/2 or Akt activation over baseline values was found in the cells of the elderly group. These findings were markedly different from those obtained in the neutrophils of the young controls, in which a 25-fold increase in phosphorylated ERK1/2 levels and a 40-fold increase in phosphorylated Akt levels were observed as an effect of the GM–CSF cell challenge (Figure 2).


Figure 02
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  		Figure 2. Extracellular signal-related kinase (ERK)1/2 and Akt phosphorylation in granulocyte macrophage–colony-stimulating factor (GM–CSF)-stimulated neutrophils of elderly and young individuals. Neutrophils (107/mL) were stimulated with GM–CSF at 20 ng/mL for 15 minutes. Phosphorylated ERK1/2 and Akt were then determined by immunoblotting. a, Representative immunoblots with antiphosphorylated ERK1/2 polyclonal antibodies (Abs) or antiphosphorylated Akt monoclonal antibodies (mAbs) are shown. An immunoblot performed by stripping the membrane incubated with anti-pERK1/2 and reprobing it with anti-ß actin Abs is also shown. b, Histograms show the levels of phosphorylated ERK1/2, double-normalized to ß-actin and p38 mitogen-activated protein kinase (MAPK) (see Materials and Methods), and the increase in ERK1/2 phosphorylation over baseline, in young (open columns) and elderly (filled columns) volunteers, using data from densitometric analyses of 15 experiments Values are expressed as mean ± standard deviation. c, Same as (b), with phosphorylated Akt. Significance of results in elderly versus young group: *p <.01, #p <.001

 
Then, we studied the link between the age-related defects in GM–CSF-induced cell signaling and the age-associated inability of GM–CSF to rescue neutrophils from Fas-induced apoptosis using pharmacological inhibitors of ERK1/2 and Akt. We found that both PD98059 and the Akt Inhibitor almost completely abolished the GM–CSF-induced neutrophil survival, with no further inhibition being observed as a result of combined PD98059 and Akt Inhibitor cell treatment (Figure 3). Clearly, this effect reached statistical significance only in the young individuals, but a similar trend was observed in the group of elderly donors (see Figure 3). The inhibitory effect of the two agents was not due to their intrinsic capacity to induce apoptosis commitment. In later experiments we found, in fact, that the percentage of Annexin V+ neutrophils did not increase with respect to the control values after cell culture incubation with ERK1/2 or Akt inhibitors, or coincubation with both compounds, regardless of whether spontaneous or Fas-induced apoptotic cell death was assessed (data not shown). Interestingly, whereas Akt phosphorylation triggered by GM–CSF was affected by cell treatment with the Akt Inhibitor, but not with PD98059, both the Akt Inhibitor and PD98059 significantly reduced GM–CSF-mediated ERK1/2 phosphorylation in neutrophils from both elderly and young participants (Figure 4).


Figure 03
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  		Figure 3. Effects of extracellular signal-related kinase (ERK)1/2 and Akt inhibitors on the granulocyte macrophage–colony-stimulating factor (GM–CSF)-induced delay of neutrophil apoptosis. Neutrophils were pretreated with PD98059 (100 µM) for 30 minutes or with the Akt Inhibitor (25 µM) for 2 hours before being challenged with GM–CSF (20 ng/mL for 2 hours). Antihuman Fas 2R2 monoclonal antibodies (300 ng/mL) were then added, and the cell cultures were incubated for 12 hours. Neutrophil apoptosis was assessed by flow cytometry using the Annexin V-FITC/7-AAD Kit, and determined as the percentage of Annexin V+/7-AAD– neutrophils, after cell gating on physical parameters. a, Representative experiment is shown. As the percentage of 7-AAD cells was never higher than 0.2%, only the expression of Annexin V is reported. b, Histogram shows the percentage of the GM–CSF-induced delay of apoptosis (mean ± standard deviation), either in the absence or in the presence of Akt and/or ERK inhibitors, in young (open columns) and elderly (filled columns) individuals, using data from five experiments. Significance versus homologous cells treated only with GM–CSF: *p <.001

 

Figure 04
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  		Figure 4. Effects of PD98059 and Akt inhibitor on granulocyte macrophage–colony-stimulating factor (GM–CSF)-mediated neutrophil extracellular signal-related kinase (ERK)1/2 and Akt phosphorylation. Cells were pretreated with PD98059 (100 µM) for 30 minutes or with the Akt Inhibitor (25 µM) for 2 hours and then stimulated with GM–CSF (20 ng/mL) for 15 minutes. Phosphorylated ERK1/2 and Akt levels were determined by immunoblotting. Representative immunoblots with antiphosphorylated ERK1/2 polyclonal antibodies or antiphosphorylated Akt monoclonal antibodies are shown. Results are representative of three independent experiments carried out on cells from young and elderly participants

 
Cell Surface Expression of GM–CSF Receptor {alpha} Chain (CD116) in Neutrophils From Elderly and Young Participants
With the aim of identifying which step of the GM–CSF signaling pathway is potentially responsible for both PI3-K and ERK1/2 defective activation in neutrophils of elderly individuals, we first evaluated the possibility of an age-related decrease in GM–CSF receptor cell surface expression. However, as depicted in Figure 5, no differences were found in the mean fluorescence intensity of CD116 expressed on the neutrophil surface as a function of age.


Figure 05
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  		Figure 5. CD116 surface expression in neutrophils from young and elderly individuals. Aliquots of blood specimens collected into EDTA tubes were incubated with phycoerythrin (PE)-conjugated anti-CD116 monoclonal antibodies (mAbs) or with PE-labeled isotype-matched control mAbs for 15 minutes at room temperature in the dark. After red blood cell lysis and fixing, samples were analyzed on a Coulter Epics Elite cell sorter. Neutrophils were gated on the basis of physical parameters. Histograms indicate staining with positive and control mAbs (filled and open histograms, respectively), and are representative of seven similar experiments, the results of which, expressed as mean ± standard deviation of the mean fluorescence intensity (MFI) in control and positive staining, are reported in the insets

 
Role of SOCS Proteins in the Age-Related Impairment of GM–CSF-Dependent Delay of Neutrophil Apoptosis
Members of the SOCS protein family are key regulators of cellular responses to cytokines; their production is induced by the same cytokines they inhibit, in accordance with a classic negative feedback mechanism (31,32). On the basis of recent evidence showing that SOCS1 and SOCS3 are induced by GM–CSF cell stimulation (33,34), it seemed relevant to assess whether changes in the levels of these proteins might occur with age. Interestingly, both SOCS1 and SOCS3 basal expression was significantly higher in the neutrophils of elderly individuals than in those of their younger counterparts (Figure 6). GM–CSF stimulation had no effect on SOCS1 or SOCS3 expression in the neutrophils of elderly individuals, but produced a substantial increase in both SOCS protein levels in the cells of the young controls, the values of which finally reached, but did not exceed, those found in the cells from the elderly group (see Figure 6).


Figure 06
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  		Figure 6. Significant suppressors of cytokine signaling (SOCS)1 and SOCS3 expression in unstimulated and granulocyte macrophage–colony-stimulating factor (GM–CSF)-stimulated neutrophils of elderly and young participants. SOCS1 and SOCS3 expression was determined by immunoblotting performed on both unstimulated neutrophils and cells stimulated with GM–CSF (20 ng/mL for 15 minutes). a, Representative immunoblots with anti-SOCS1 or anti-SOCS3 polyclonal antibodies (Abs) are shown. An immunoblot performed by stripping the membrane incubated with anti-SOCS1 and reprobing it with anti-ß actin Abs is also shown. b, Histograms show levels of SOCS1 and SOCS3 proteins (mean ± standard deviation), double normalized to ß-actin and p38 mitogen-activated protein kinase (MAPK) (see Materials and Methods) in young (open columns) and elderly (filled columns) volunteers, using data from densitometric analyses of seven experiments. Significance versus young: *p <.05

 

   DISCUSSION
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 Materials and Methods
 Results
 Discussion
References
 
The microbicidal activities of mature human neutrophils are strongly influenced by the proinflammatory cytokine GM–CSF. A number of reports indicate that neutrophil responses to GM–CSF are mostly compromised with advancing age, casting doubt on the capacity of senescent cells to mount an effective stand against infectious events (12,16,17). In the current work, we demonstrate that the age-related failure of GM–CSF to rescue neutrophils from Fas-induced apoptotic cell death is strictly related to an impairment of GM–CSF-dependent PI3-K/Akt and ERK1/2 activation. In addition, due to the high baseline levels of phosphorylated forms of the two kinases, an even higher decrease occurred in Akt and ERK1/2 activation over baseline values in the cells of the elderly group as compared with those of the young controls.

The relatively high baseline levels of phosphorylated Akt and ERK1/2 in the cells of the elderly persons are in line with previous studies showing low, yet detectable signs of activation in unstimulated neutrophils from elderly individuals (12,22), likely as a result of continuous cell challenge with proinflammatory mediators in vivo (35). This neutrophil activation state may have a functional counterpart. Besides contributing to the development of the aging process through a pro-oxidative shift in the redox state (36), it may at least partially account for the age-associated defects in neutrophil functions. In the light of the adaptation phenomena commonly observed in homeostatic systems, the magnitude of a physiologic response is seen to be related to the difference between the stimulus-induced conditions and the starting conditions more than to the absolute levels of stimulation. Accordingly, the possibility should be borne in mind that in our experimental system the age-related defect in GM–CSF ability to prolong neutrophil survival might be consequent to reduced PI3-K/Akt or ERK1/2 activation with respect to baseline values rather than to diminished absolute levels of kinase-phosphorylated forms.

Although the role of PI3-K/Akt and ERK1/2 in the antiapoptotic effects of GM–CSF on neutrophils is widely accepted (24–27), other steps in the GM–CSF-triggered signaling leading to neutrophil survival, either downstream or upstream of these kinases, remain to be defined. Uncertainties still exist as to the target of PI3-K/Akt and ERK1/2 activity. Although it was initially reported that Bad, a member of the Bcl-2 family of proteins involved in the modulation of cell death, was serine-phosphorylated in response to PI3-K/Akt- and MAPK-dependent survival signals (25,37–39), further studies did not confirm these findings, suggesting that other survival pathways might be involved (40,41). Another unsolved issue is whether PI3-K/Akt and ERK belong to a single signal transduction pathway, or are activated in parallel following GM–CSF cell stimulation. Our results support the former hypothesis. Cell pretreatment with either Akt or ERK1/2 inhibitors, in fact, gave rise to a similar, almost complete, inhibition of the GM–CSF capacity to delay Fas-induced neutrophil apoptosis. Furthermore, combined treatment with PD98059 and the Akt inhibitor did not induce further suppression of the GM–CSF-induced apoptosis delay as compared with the effect of each agent. Finally, GM–CSF-induced ERK1/2 phosphorylation was significantly affected by neutrophil challenge with either the Akt Inhibitor or PD98059, clearly indicating that PI3-K/Akt and ERK1/2 are activated in series and that ERK1/2 is located downstream of PI3-K along the GM–CSF-dependent signaling pathway. In keeping with our data, several studies have reported that PI3-K is a component of the signal transduction pathway leading to Raf-dependent activation of ERK1/2 (42,43). In addition, inhibition of PI3-K by wortmannin has been found to attenuate both Raf-1 and ERK1/2 activation by GM–CSF (44). In a recent work, Kotone-Miyahara and colleagues (45) reported that the PI3-K inhibitor LY294002 was not able to affect GM–CSF-induced ERK1/2 phosphorylation and that GM-CSF-induced Akt phosphorylation was not affected by PD98059, thus reaching the opposite conclusion that PI3-K and ERK1/2 work along independent, parallel pathways. The only reasonable explanation for these discrepancies is that the PI3-K inhibitor concentration they used was too low to effectively inhibit the signaling induced by the high dosages of GM–CSF (100 ng/mL) used. Moreover, in the same work, at concentrations that have usually proved to be effective in inhibiting neutrophil survival induced by GM–CSF at 10 or 20 ng/mL (25,27,41), LY294002 and PD98059 had no effect on the capacity of GM–CSF at 100 ng/mL to delay Fas-triggered neutrophil apoptosis, unless used in combination.

The GM–CSF receptor is a heterodimer composed of a ligand-specific {alpha} subunit (CD116), and a ß subunit shared with the IL-3 and IL-5 receptors. Binding of GM–CSF induces the aggregation of receptor subunits, which brings Jak2 molecules in close proximity and allows them to phosphorylate and activate each other. After activation, Jak2 phosphorylates receptor subunits on tyrosine residues, allowing the recruitment of proteins endowed with Src homology-2 domains, which in turn mediates the activation of the signaling pathways used by GM–CSF to induce its various biological responses (46). These proteins can also be phosphorylated by Jak2 and, in this regard, evidence has been provided that GM–CSF-induced PI3-K activation may be mediated by Jak2, although the association between Jak2 and the p85 subunit of PI3-K depends on an adaptor protein that has yet to be identified (47). Thus, PI3-K appears to be located at a very upstream position along the GM–CSF signaling pathway, implying that defective activation, as we found in the neutrophils of elderly individuals, must necessarily involve the GM–CSF receptor or the activity of receptor-associated tyrosine kinases. When evaluating the former possibility, however, no age-related change in neutrophil CD116 expression was observed; this finding prompted us to examine whether a functional inhibition of the GM–CSF receptor-associated kinases might occur with aging. Among the several families of proteins involved in down-regulating cytokine signaling, particular interest has recently been paid to the SOCS proteins, a group of cytokine-inducible molecules containing an Src homology-2 domain through which they bind to and inhibit tyrosine-phosphorylated signaling molecules (31,32). Although SOCS were originally discovered, and are best known, as inhibitors of Jak-STAT signaling, they may potentially interfere with any Jak-dependent transduction pathway (48). Of the eight members of this family so far identified, SOCS1 and SOCS3 have been shown to increase in response to GM–CSF cell stimulation (33,34) and were thus investigated in our study. Notably, we found that both SOCS1 and SOCS3 levels were significantly higher in unstimulated neutrophils from elderly individuals than their younger counterparts, and unlike the neutrophils from the young participants, they did not further increase following GM–CSF cell triggering. As a result, a more effective SOCS1 and SOCS3 binding to either the GM–CSF receptor or Jak2, which would largely account for the GM–CSF-dependent defect of PI3-K/Akt/ERK activation, may occur in senescent neutrophils. In agreement with this view, preliminary experiments from our group seem to indicate an impaired GM–CSF-induced Jak2 activation in neutrophils of elderly donors as compared with cells from young volunteers (Tortorella C, Simone O, Piazzolla G, Stella I, Antonaci S, unpublished observations, 2006). The mechanisms leading to the upregulation of neutrophil SOCS1/3 expression with aging are unknown, and their definition may presently be only a matter of speculation. However, on the basis of the recent demonstration of elevated SOCS3 levels also in resting lymphocytes from elderly donors (49), we can speculate that the increase in this class of inhibitory molecules is a general phenomenon associated with aging. In this regard, the high expression of SOCS proteins might be considered as the physiological response to SOCS natural inducers, including the proinflammatory cytokines IL-1, IL-6, and tumor necrosis factor-{alpha}, whose levels have been found to be significantly increased in elderly persons (50).

Taken together, our results are consistent with the conclusion that a defect in PI3-K/Akt and ERK1/2 activation, likely dependent on elevated SOCS1/3 levels, may affect the GM–CSF capacity to delay neutrophil apoptosis in elderly persons. This defect may have an in vivo counterpart in clinical conditions, such as infectious events, in which a prolonged and efficient neutrophil function is required. Controlling SOCS expression might therefore be an important therapeutic target for the restoration of normal immune responses in aging.


   Acknowledgments
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This work was supported by grants (Ricerca Finanziabile con Fondi di Ateneo) from the Università degli Studi di Bari, Bari, Italy.


   Footnotes
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Decision Editor: Huber R. Warner, PhD

Received January 4, 2006

Accepted April 5, 2006


   References
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 Discussion
 References
 

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