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Originally published online as doi:10.1189/jlb.0805481 on February 24, 2006

Published online before print February 24, 2006
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(Journal of Leukocyte Biology. 2006;79:1061-1072.)
© 2006 by Society for Leukocyte Biology

Impairment of SHP-1 down-regulation in the lipid rafts of human neutrophils under GM-CSF stimulation contributes to their age-related, altered functions

Carl F. Fortin*,{dagger}, Anis Larbi*,{dagger}, Olivier Lesur{dagger},{ddagger}, Nadine Douziech* and Tamas Fulop, Jr.*,{dagger},§,1

* Laboratory of Immunology, Research Center on Aging, and
{dagger} Immunology Graduate Program, Clinical Research Center, and
{ddagger} Pneumology and
§ Geriatrics Divisions, Department of Medicine, Faculty of Medicine, University of Sherbrooke, Quebec, Canada

1Correspondence: Laboratory of Immunology, Research Center on Aging, Immunology Graduate Program, Clinical Research Center, and Department of Medicine, Geriatrics Division, Faculty of Medicine, University of Sherbrooke, 1036 rue Belvédère Sud, Sherbrooke, Québec, J1H 4C4, Canada. E-mail: tamas.fulop{at}usherbrooke.ca


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ABSTRACT
 
It has been shown that the functions and the rescue from apoptosis by proinflammatory mediators of polymorphonuclear leukocytes (PMN) tend to diminish with aging. Here, we investigated the role of protein tyrosine phosphatases (PTP), especially Src homology domain-containing protein tyrosine phosphatase-1 (SHP-1), in the age-related, altered PMN functions under granulocyte macrophage-colony stimulating factor (GM-CSF) stimulation. The inhibition of PTP suggested a differential effect of GM-CSF on phosphatase activity in modulating PMN functions with aging. The down-regulation of phosphatase activity of immunopurified SHP-1 from lipid rafts of PMN of young donors was found significantly altered at 1 min of stimulation with aging. In young donors, SHP-1 is displaced from lipid rafts at 1 min of stimulation, whereas in the elderly, SHP-1 is constantly present. We assessed in PMN lipid rafts the phosphorylation of tyrosine and serine residues of SHP-1, which regulates its activity. We observed an alteration in the phosphorylation of tyrosine and serine residues of SHP-1 in PMN of elderly subjects, suggesting that GM-CSF was unable to inhibit SHP-1 activity by serine phosphorylation. GM-CSF activates Lyn rapidly, and we found alterations in its activation and translocation to the lipid rafts with aging. We also demonstrate that SHP-1 in the PMN of elderly is constantly recruited to Lyn, which cannot be relieved by GM-CSF. In contrast, in the young, the resting recruitment could be relieved by GM-CSF. Our results suggest an alteration of the SHP-1 modulation by GM-CSF in lipid rafts of PMN with aging. These alterations could contribute to the decreased GM-CSF effects on PMN.

Key Words: phosphatase • aging • Lyn • chemotaxis • ROS • apoptosis


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INTRODUCTION
 
Protein tyrosine phosphatases (PTP) act as the counterpart of protein tyrosine kinases (PTK) to maintain the homeostasis in the levels of tyrosine phosphorylation. Signal transduction elicited by a ligand in the cell can be viewed as a shift in this balance, which activates cellular functions. Src homology (SH) domain-containing protein tyrosine phosphatase-1 (SHP-1) was first described in the early 1990s by screening of a cDNA library of human breast carcinoma [1 ]. When it is recruited to the plasma membrane and activated, SHP-1 dephosphorylates proteins activated by receptors, hence, inhibiting cell activation [2 ], as it was shown for the B cell receptor (BCR) signaling [3 ]. Jiao and co-workers [4] showed that the main mechanism for Janus tyrosine kinase 2 (Jak2) dephosphorylation by SHP-1 involves a direct, SH2-independent interaction with Jak2. It is of note that the association was found to be strong in resting cells and was reduced during interferon-{alpha} (IFN-{alpha}) stimulation of a T cell line [5]. Given the wide range of its ligand, it is likely that SHP-1 plays various roles in cells besides inhibition of cell activation, including maturation of receptors [6 ] and of cells such as polymorphonuclear leukocytes (PMN) or platelets [7 8 9 10 11 ], and that specific adaptor proteins mediate each of these roles. Moreover, it was shown that SHP-1 might be the phosphatase responsible for down-regulation of mitogen-activated protein kinase (MAPK) activation by granulocyte-colony stimulating factor (G-CSF) during the course of PMN maturation [10 ].

PMN represent the first line of defense against aggressions, as they are the first cells to arrive at the site of the aggression, where they can directly eliminate the invading organisms. Granulocyte macrophage (GM)-CSF is not only a modulator of granulopoiesis but is also responsible for the priming of mature PMN to a second stimulation such as lipopolysaccharide (LPS) or formyl-Met-Leu-Phe (fMLP). GM-CSF elicits three important pathways in various cells and also in PMN, where these pathways are responsible for cellular activation: Jak/signal transducer and activator of transcription (STAT), MAPK, and phosphatidylinositol-3 kinase (PI-3K) [12 13 14 15 ]. GM-CSF has been shown to rescue human granulocytes from spontaneous apoptosis via an increase in tyrosine phosphorylation of the well-known survival signaling pathways, including Jak/STAT, PI-3K/Akt, and Lyn [14 , 16 , 17 ]. Moreover, SHP-1 has been shown to be of critical importance for negative regulation of Src kinases elicited by GM-CSF, such as Jak2 in PMN or Lyn in others immune cells [2 , 4 , 18 19 20 ]. It was demonstrated that low enzymatic activity of SHP-1 was associated with increased neutrophil survival and that death receptor stimulation blocks the antiapoptotic activity of GM-CSF, G-CSF, or IFN-{gamma} and leads to the recruitment of SHP-1 to the Fas death receptor [19 ]. Furthermore, the inhibition of phosphatase activity, by phenylarsine oxide (PAO), led to an increase in protein tyrosine phosphorylation level in eosinophils and in PMN [21 ]. Recent data show that 20–30% of total SHP-1 is located in the lipid rafts of unstimulated PC12 or A431 cells [22 , 23 ] and in BYDP cells or primary thymocytes [24 ]. Lipid rafts are relatively ordered membrane domains, which float in the disordered glycerophospholipid bilayer, and their central feature is that they allow the lateral segregation of proteins within the plasma membrane [25 ]. Upon cross-linking of signaling receptors associated with lipid rafts, they become a larger and more stable structure, often attached to the cytoskeleton, a phenomenon called coalescence. Lipid rafts serve to spatially segregate signaling components in the plasma membrane so to regulate the initiation and prolongation of signaling [25 ].

Aging causes multiple defects in the functions of PMN, notably for the production of reactive oxygen species (ROS) [26 ], chemotaxis, and in the rescue from apoptosis by proinflammatory mediators [12 ]. Furthermore, the PMN signal transduction is also altered with aging [27 28 29 30 31 32 33 34 35 36 ], and over the past few years, it has been demonstrated that these PMN-specific, receptor-driven functions are altered with aging [12 , 27 , 32 ]. The importance of negative regulators of PMN receptors, such as the GM-CSF receptor, in the alteration of the PMN functions has not yet been studied extensively. In this study, we tested the possibility that altered regulation of GM-CSF signaling by the PTP SHP-1, which has been shown to be important in the regulation of GM-CSF signal transduction [3 , 4 , 18 19 20 ], is also a factor in the impairment of effector functions in the elderly.

We provide evidences for the altered regulation of SHP-1 activity in the PMN of the elderly. SHP-1 was activated and recruited continuously to Lyn in the PMN of elderly donors. Moreover, we show that these alterations in the elderly are in part mediated by the impaired up-regulation of SHP-1 serine phosphorylation and down-regulation of tyrosine phosphorylation.


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MATERIALS AND METHODS
 
Reagents and antibodies
Antiphosphotyrosine, Clone 4G10, was from Upstate (Charlottesville, VA). Anti-SHP-1, anti-Lyn, and apoptosis detection kit (containing annexin V and PI) were from Santa Cruz Biotechnology (CA). Phospho-Lyn was from Cell Signaling Technologies (Beverly, MA). Recombinant human GM-CSF, PTP inhibitor cocktail II, and bisindolylmaleimide I [protein kinase C (PKC) inhibitor] were from Calbiochem (San Diego, CA). Antiphosphoserine was from Sigma-Aldrich (St. Louis, MO). 2',7'-Dichlorofluorescein diacetate (DCFDA) was from Molecular Probes (Burlington, Ontario, Canada). Bradford assay reagent was from Bio-Rad (Hercules, CA). Secondary antibodies were from Chemicon International (Temecula, CA). All other reagents were from Sigma-Aldrich unless otherwise stated.

Subjects and PMN separation
Thirty-five elderly volunteers, aged 65–78 years (mean age 73 years), and 35 young subjects, aged 19–25 years (mean age 22 years), participated in the study. The lipid profile was determined by routine biochemical analysis, and all the subjects were in good health, normolipidemic, and satisfied the inclusion criteria of the SENIEUR protocol for immune investigations of human elderly subjects [37 ]. Citrated blood was obtained by venipuncture, and neutrophils were isolated by Ficoll-Hypaque density sedimentation as already described [38 ]. Activation of neutrophils by the separation method was assessed by the measure of ROS production by cytochrome oxidation [38 ]. Cell viability was greater than 95%, as measured by Trypan blue exclusion.

SHP-1 immunoprecipitation (IP) and phosphatase assay
Neutrophils (5x106 cells) were treated with or without stimuli, sedimented rapidly, left on ice for 5 min, and resuspended in ice-cold lysis buffer [150 mM NaCl, 10 mM EGTA, 5 mM EDTA, 100 mM NaF, 2 mM NaVO4, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM sodium polyphosphate, 1% Triton, antiproteases cocktail in 50 mM HEPES, pH 7.4]. Lysates were cleared by centrifugation, and protein concentration was determined by the Bradford assay. SHP-1 (2 µg) antibodies were added to 250 µg proteins in 250 µl wash buffer [150 mM NaCl, 1 mM EDTA, 1 mM NaVO4, 0.1% Nonidet P-40 (NP-40) in 5 mM Tris, pH 7.5], followed by an incubation overnight at 4°C while rotating end-over-end. A mixture of protein A/G sepharose (25 µl) was added, and incubation was continued for 1.5 h as already described [39 ]. The immunoprecipitates were washed with wash buffer (see above) and subjected to phosphatase assay, which was carried out essentially as described [40 ]. Briefly, immunoprecipitates were washed once with assay buffer (0.5 mM EGTA in 25 mM HEPES, pH 7.0) and then incubated with 200 µl assay buffer containing 10 mM p-nitrophenyl phosphate at 37°C for 6 h while shaking. Reactions were stopped by addition of 800 µl 0.2 M NaOH, beads were sedimented by brief centrifugation, and phosphatase activity was assessed by measuring the absorbance at 420 nm of the supernatant. Results are shown as relative to SHP-1 enzymatic activity in unstimulated cells.

Isolation of lipid rafts
Neutrophils (2x107 cells) were treated as above, except they were resuspended in 300 µl ice-cold lipid rafts lysis buffer (100 mM NaCl, 2 mM EDTA, 2 mM NaVO4, 1 mM PMSF, 0.5% Triton, 10 mg/ml aprotinin, 10 mg/ml leupeptin in 25 mM HEPES, pH 6.9). An 85% (w/v) solution of sucrose (300 µl) in Hepes-buffered saline was added to a final concentration of 42.5%, and the solution was transferred to 2 ml ultracentrifuge tubes. The lysates were gently overlaid with 1 ml sucrose 35% and 300 µl sucrose 5%. Centrifugation were performed at 4°C for 16 h at 200,000 g in a Beckman TLA-100.4 rotor (Beckman Instruments, Montreal, Quebec, Canada). Nine fractions of 200 µl each were collected from the top of the gradient. For the experiments, lipid raft fractions used correspond to Fractions 1–3, and Fractions 5–8 were used as nonraft fractions, as already described [41 , 42 ].

Measurement of ROS production by DCFDA cleavage
Neutrophils (1x106) in 500 µl complete RPMI were primed with 20 ng mL–1 GM-CSF for 90 min at 37°C. If required, neutrophils were preincubated 30 min at 37°C with 50 µM PTP inhibitor II before priming and stimulation. These concentrations were determined as optimal in our experimental settings (data not shown). After priming, cells were loaded with 20 µM DCFDA in phosphate-buffered saline at 37°C for 15 min, then cells were stimulated, and fluorescence in the FL-1 channel was read with a FACSCalibur from Becton Dickinson (Franklin Lakes, NJ).

IP and immunoblotting
For all IP other than SHP-1, they were done as already described earlier. In some experiments, PMN were preincubated for 1 h with PTP inhibitor cocktail (50 µM) or PKC (1 µM) inhibitor prior to GM-CSF stimulation. After appropriate stimulation, PMN were resuspended in lysis buffer as described above, and protein concentration was determined by the Bradford assay. Appropriate antibodies (2 µg) were added to 300 µg lysates proteins in 250 µl IP washing buffer (150 mM NaCl, 1 mM EDTA, 1 mM NaVO4, 0.1% NP-40 in 5 mM Tris, pH 7.5) and incubated for 2 h at 4°C while rotating end-over-end. A mixture of protein A/G sepharose (25 µl) was added, and incubation was continued for 1.5 h. After the last wash of the IP, proteins were incubated for 5 min at 95°C with 25 µl 1x sodium dodecyl sulfate (SDS) gel-loading buffer. The samples were resolved by 7.5% SDS-polyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane. The strips were blocked with 5% milk or 3% bovine serum albumin for antiphospho antibodies for 1.5 h at room temperature. Following the blocking, the strips were probed and analyzed using the enhanced chemiluminescence detection system as already described [43 ]. Densiotometric analyses were performed using an image analyzer, Chemigenius2 bio-imaging system (Syngene, Frederick, MD), as already described [42 ].

Chemotaxis assay
Chemotaxis of freshly isolated neutrophils was assessed by the modified Boyden chamber [38 , 43 ]. Briefly, neutrophils were primed with 20 ng mL–1 GM-CSF with 50 µM PTP inhibitor cocktail (GM-CSF+PTP inhibitor) or without (GM-CSF), and chemotaxis assay toward 20 ng mL–1 GM-CSF was carried for 2 h at 37°C. For the purpose of clarity, the results for negative control (chemotaxis toward complete RPMI) and priming of PMN with PTP inhibitor alone are not shown, as they were low and of similar level. These conditions were determined as optimal in our experimental settings (data not shown).

Apoptosis assay
Freshly isolated neutrophils (1x106) were incubated rapidly with GM-CSF (20 ng mL–1), PTP inhibitor (1 µM) in complete RPMI, or with a combination of both, as indicated in text for 18 h. These concentrations were determined as optimal in our experimental settings (data not shown). Not shown in Results is the rate of PMN apoptosis after 18 h of culture in complete RPMI. They were consistently 60–70% of apoptosis. Staining of cells was done with the apoptosis detection kit according to the manufacturer’s instructions. For the results, we considered as apoptotic cells that stained for annexin V only (early apoptotic) and cells that stained for PI and annexin V (late apoptotic) as already described [44 ].

Statistical analysis
All the statistical calculations were done by GraphPad PRISM (San Diego, CA). Data were analyzed by one-way ANOVA using the Bonferroni correction. P < 0.05 was considered statically significant.


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RESULTS
 
Determination of the role of SHP-1 in PMN functions
GM-CSF is a well-known modulator of PMN functions, which were found previously altered with aging [12 , 26 27 28 , 31 , 33 , 35 , 44 ] and reconfirmed here (Fig. 1A 1B 1C , solid bars). For the PMN of young and elderly donors, incubations with the PTP inhibitor cocktail followed by GM-CSF stimulation resulted in a significant increase in ROS production (P<0.05) and chemotaxis (P<0.01; Fig. 1 A and B ). It is of note that the PTP inhibitor cocktail induced a significant increase in ROS production in PMN of elderly (P<0.001) compared with that of GM-CSF alone. This modulation by the PTP inhibitor was not observed for chemotaxis (data not shown). Nevertheless, this could suggest a deregulation of phosphatase activity, including SHP-1, in PMN with aging. This was further confirmed when we measured PMN apoptosis after 18 h of culture. In PMN of young subjects, the PTP inhibitor or PTP inhibitor and GM-CSF treatment blunted the GM-CSF apoptosis-rescuing effect. In contrast, the preincubation of PMN of elderly with the PTP inhibitor before the 18-h culture with GM-CSF resulted in the recovery of the lost GM-CSF-induced rescue from apoptosis (Fig. 1C) . Altogether, these results suggest a differential effect of GM-CSF on phosphatase activity in modulating PMN functions with aging.


Figure 1
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  		Figure 1. Determination of the role of SHP-1 in PMN functions, where functions of human PMN from young and elderly donors were studied after incubation with PTP inhibitor cocktail or after priming with 20 ng mL–1 GM-CSF. (A) Measurement of ROS production with DCFDA, as described in Materials and Methods, was done by FACScan analysis. Data are shown as a mean ± SEM of stimulation index (mean fluorescence intensities of the stimulated cells relative to mean fluorescence intensities of loaded quiescent cells) for PMN from four different young (open bars) and four different elderly donors (solid bars). (B) The primed PMN were put in the upper wells of Boyden chambers, and chemotaxis was evaluated toward 20 ng mL–1 GM-CSF as a chemoattractant in the lower wells. After 2 h of migration at 37°C, the filter was stained. Ten to 15 fields were counted for each condition at a 400x original magnification. The data are shown as the mean ± SEM of a number of PMN, which migrated from three different young (open bars) and three different elderly donors (solid bars). For clarity, the value obtained with the PTP inhibitor alone is not indicated, as it was not different from zero. (C) Measurement of PMN apoptosis. Staining of PMN was done with the apoptosis detection kit by FACScan according to the manufacturer’s instructions described in Materials and Methods. The data are shown as the mean ± SEM of PMN rescued from apoptosis (relative to PMN cultured 18 h with medium alone) from six different young (open bars) and six different elderly donors (solid bars). Significant differences between young and elderly donors are depicted as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Measurement of SHP-1 activity under GM-CSF stimulation in PMN of young and elderly subjects
As we have shown altered functions of PMN under GM-CSF stimulation as well as a deregulation of PTP activities with aging, we reasoned that PTP, especially SHP-1, as it is the major negative regulator of the pathway elicited by GM-CSF, might play a role in that phenomenon. We immunoprecipitated SHP-1 from GM-CSF-stimulated PMN of young and elderly donors and measured its enzymatic activity using p-nitrophenylphosphate (pNPP) as a substrate. At the quiescent status, the SHP-1 activity (OD at 420 nm) in PMN of elderly subjects was found significantly higher compared with that of young subjects (0.274±0.012 vs. 0.154±0.014, respectively; P<0.05) in the whole cell lysates. Stimulation of PMN from young donors causes a significant reduction (P<0.001) in the activity of SHP-1 at 1 min isolated from whole cell lysates compared with the basal status (Fig. 2A ). This inhibition by GM-CSF seems to be biphasic, as it occurs again after 30 min of stimulation. To the contrary, GM-CSF stimulation on PMN of elderly donors could not inhibit the activity of SHP-1 (Fig. 2B , solid bars) at any of the stimulation time used. Adding 2 mM PTP inhibitor sodium orthovanadate in the assay mixture totally abrogated phosphatase activity of immunopurified SHP-1 in both age groups (data not shown). To assess the specificity of the SHP-1 down-regulation to GM-CSF stimulation, we verified if LPS stimulation of PMN could have an influence on SHP-1 activity by using 1 µg mL–1 LPS and the same time-points of stimulation, which with LPS, did not induce any changes in SHP-1 activity from basal status in the young or in the elderly donors (data not shown).


Figure 2
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  		Figure 2. Measurement of SHP-1 activity under GM-CSF stimulation in PMN of young and elderly subjects. The SHP-1 immunoprecipitates obtained from whole cell lysates (A) or from membrane lipid rafts (B) of GM-CSF-stimulated human PMN were incubated with 20 mM pNPP in assay buffer. Results shown are phosphatase activities of SHP-1 of stimulated PMN relative to unstimulated PMN. Activity without any treatment was set to 100%. (A) Data represent the mean values ± SEM of eight different young (open bars) and eight different elderly donors (solid bars). (B) Data represent the mean values ± SEM of three different young (open bars) and three different elderly donors (solid bars). Significant differences between young and elderly donors are depicted as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

As membrane lipid rafts are now known as a platform with a purpose in amplifying signal transduction [45 ] and that 20–30% of SHP-1 has been shown to be in lipid rafts with no data in PMN, we tested if SHP-1 activity modulation was also altered in membrane lipid rafts of the elderly. SHP-1 activity was lower in lipid rafts at the quiescent status, but there was no statistical difference between young and elderly donors(DO420 nm: 0.038±0.005 vs. 0.030±0.008, respectively). Similarly to the whole cell lysates, there was a significant inhibition (P<0.05) of SHP-1 activity in lipid rafts of the young donors following 1 min of stimulation with GM-CSF (Fig. 2B) . However, longer stimulation with GM-CSF did not cause a sustained inhibition of SHP-1 activity in lipid rafts, in contrast to the whole cell lysates (30 min in Fig. 2B vs. 30 min in Fig. 2A ). When PMN were isolated from elderly donors and stimulated with GM-CSF, there were no changes whatsoever in the levels of SHP-1 activity with regard to times of stimulation (Fig. 2B , solid bars). We also evaluated SHP-1 activity in the nonraft fractions, where the activity stayed around the basal value for all the GM-CSF stimulation time used, and no differences could be demonstrated with aging (data not shown). Again, the use of sodium orthovanadate in the assay mixture reduced to the background value the level of SHP-1 activity in both age groups (data not shown).

Expression of SHP-1 in lipid rafts and in whole cell lysates in human PMN
One possible explanation for the increased SHP-1 activity observed in the elderly could be an overexpression of this enzyme in the cytoplasm and/or in lipid rafts relative to the young donors. Taking into account the results of Figure 2 and the lack of data, whether SHP-1 could be associated to lipid rafts in PMN, we determined the expression of SHP-1 in lipid rafts and whole cell lysates with an anti-SHP-1 antibody. Figure 3A clearly shows that the expression of SHP-1 in the whole cell lysates is the same in the PMN of young and elderly donors and rules out this factor as a possible explanation for the differences seen in its enzymatic activity with aging at a basal and GM-CSF-stimulated status. We next studied the expression of immunopurified SHP-1 in the lipid rafts of PMN of young and elderly donors. Using Fractions 1–3, corresponding to the fractions highly expressing the lipid raft marker GM1, no difference was found in SHP-1 expression in lipid rafts of quiescent PMN of the studied age groups (Fig. 3B) . Stimulation with GM-CSF causes a rapid loss at 1 min of SHP-1 from the lipid rafts in the PMN of young donors compared with the basal status (Fig. 3B , Young, and Fig. 3D , open bars, P<0.05). However, SHP-1 is recruited again to lipid rafts with longer stimulation and returned to the basal level (Fig. 3D , open bars). Probing all the lipid raft fractions with the SHP-1 antibody revealed that it is found in greater abundance in the nonraft fractions (Numbers 5–8, Fig. 3C ) compared with the raft fractions in both age groups (Numbers 1–3, Fig. 3C ), as already found for unstimulated PC12 or A431 cells [22 , 23 ] and in BYDP cells or primary thymocytes [24 ]. However, SHP-1 expression in these fractions did not change with the time of GM-CSF stimulations used, and there were no notable differences with aging (data not shown). In contrast, this effect could not be seen with aging, as it is shown in Figure 3B (Elderly) and Figure 3D (solid bars) that there is no dissociation of SHP-1 from the lipid rafts at 1 min of stimulation. Moreover, SHP-1 association to lipid rafts in PMN of elderly donors remains strong at any time of stimulation with GM-CSF. These results indicate that SHP-1 should be displaced by GM-CSF from lipid rafts for being inhibited in PMN of young subjects, and this is not the case in PMN of elderly, suggesting that the mechanisms responsible for the regulation of phosphatase activity and localization to lipid rafts are altered.


Figure 3
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  		Figure 3. Expression of SHP-1 in lipid rafts and in whole cell lysates in human PMN, which from young and elderly donors were stimulated for the indicated times with 20 ng mL–1 GM-CSF. The cell lysates and isolated lipid rafts from Fractions 1–3 or from Fractions 1–9, as shown by the GM1 staining, were probed directly with anti-SHP-1 antibody (A–C). Immunoglobulin (Ig) bands are shown as loading control. Densiometric analysis is shown in D from the immunoblot (WB) in B. Intensities are relative to an unstimulated state. Experiments were performed from five different donors of each age group (young, open bars; elderly, solid bars) with similar results. Significant differences between young and elderly donors are depicted as follows: *, P < 0.05; **, P < 0.01.

Phosphorylation of tyrosine and serine residues of SHP-1 at a basal and GM-CSF-stimulated status in whole cell lysates and lipid rafts
After having seen altered phosphatase activity and an impairment of lipid rafts dissociation, we next sought to study the phosphorylation state of SHP-1 following GM-CSF stimulation. SHP-1 can be phosphorylated on serine and tyrosine residues in its C-terminal domain. Tyrosine phosphorylation has an enhancing effect on SHP-1 activity [46 47 48 ], whereas serine phosphorylation has a strong, inhibiting effect on SHP-1 phosphatase activity [40 , 49 ]. We were able to study the phosphorylation status of SHP-1 on these critical residues in whole cell lysates of PMN. Figure 4A (Young) shows a gradual decrease in tyrosine phosphorylation of SHP-1 in the PMN of young donors, and serine phosphorylation remained unchanged throughout stimulation. For the elderly donors, Figure 4A (Elderly) also shows no changes in the level of serine and tyrosine phosphorylation in GM-CSF-stimulated PMN. This correlates well with the lack of modulation of SHP-1 activity by GM-CSF in whole cell lysates of PMN from elderly. Our findings in whole cell lysates indicate some discrepancies between the attributed roles of C-terminal phosphorylation and the decreased SHP-1 activity measured for 1 min of GM-CSF stimulation, as neither tyrosine nor serine phosphorylation was found to be modulated by GM-CSF (Figs. 2A and 4A) . In contrast, there is a significant decrease (P<0.001) of tyrosine phosphorylation at 30 min relative to an unstimulated state, and serine phosphorylation is maintained at 30 min (Fig. 4A) in PMN of young subjects, in accordance with the significant decrease of SHP-1 activity measured at 30 min under GM-CSF stimulation (Fig. 2A , P<0.01).


Figure 4
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  		Figure 4. Phosphorylation of tyrosine and serine residues of SHP-1 at basal and GM-CSF-stimulated status in whole cell lysates and lipid rafts. PMN from young and elderly donors were stimulated with 20 ng mL–1 GM-CSF, and whole cell lysates (A) or lipid rafts (B) were subjected to IP with 2 µg antiphosphotyrosine antibody (pTYR) or antiphosphoserine antibody (pSER). Immunodectection was carried on with an anti-SHP-1 antibody. Ig bands are shown as loading control. Densiometric analyses corresponding to the blots are shown in the lower panels. Experiments were performed from four different donors of each age group (young, open bars; elderly, solid bars) with similar results. Significant differences between young and elderly donors are depicted as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

After the slight variations seen in serine and tyrosine phosphorylation levels of SHP-1 in whole cell lysates, we studied the possible differences in lipid rafts. At basal status (Fig. 4B , Young), the tyrosine phosphorylation of SHP-1 is much stronger than that of serine phosphorylation. This suggests an activation of SHP-1 at basal level in lipid rafts in accordance with its inhibitory role in PMN activation. In contrast, in PMN of elderly (Fig. 4B , Elderly) at basal status, the SHP-1 phosphorylation is in favor of serine phosphorylation, indicating a shift toward the inhibition of SHP-1 activity in accordance with the basal-activated status of PMN found in elderly subjects. Moreover, we can see in Figure 4B (Young) that tyrosine phosphorylation of SHP-1 in young donors is decreased significantly at 1 min (P<0.05) of GM-CSF stimulation in comparison with the basal status. Longer stimulation with GM-CSF restores tyrosine phosphorylation to a level of resting state. For serine phosphorylation, there is a significant (P<0.05) increase compared with the unstimulated level at 1 min and 30 min of stimulation. These also indicate that the most important modulation of SHP-1 activity is occurring in relation to lipid rafts in PMN of young subjects. For the elderly, however, the pattern of SHP-1 tyrosine and serine phosphorylation is strongly altered. First, instead of a strong tyrosine phosphorylation of SHP-1, as seen in the young, there is practically no tyrosine phosphorylation in the unstimulated state (Fig. 4B , Young vs. Elderly). Upon stimulation with GM-CSF, there is a high increase of tyrosine phosphorylation (P<0.001) at 5 min and 30 min in elderly donors, indicating that SHP-1 activity is stimulated instead of being inhibited. For serine phosphorylation, there are no significant changes in time with GM-CSF stimulation in the elderly. As an overall result, there is a relatively higher serine phosphorylation in the elderly compared with tyrosine phosphorylation for all the times of the GM-CSF stimulation used. These results suggest that the alterations in the GM-CSF-induced regulation of SHP-1 activity with aging are in part mediated by altered phosphorylation of residues critical for SHP-1 regulation.

Alterations in the activation and translocation to the lipid rafts of the Src kinase Lyn with aging
Lyn is one of the first molecules activated via tyrosine phosphorylation by GM-CSF, and we found earlier an alteration in Lyn phosphorylation in PMN with aging [50 ]. Thus, we next investigated the expression and activation of Lyn in whole cell lysates and in lipid rafts using PTP and PKC inhibitors to assess the eventual role of SHP-1 in this phenomenon. The Src kinase Lyn is one of the important tyrosine kinases for GM-CSF signal transduction with Jak2 [17 , 51 ]. When PMN were treated with GM-CSF, there is a strong, significant phosphorylation of Lyn in the young donors (Fig. 5A , Young and open bars, P<0.001) over the unstimulated state. If the PMN are preincubated with a PTP inhibitor cocktail without further stimulant (no GM-CSF), we see a level of Lyn activation that is the same as that occurring under stimulation with GM-CSF alone (Fig. 5A , Young and open bars). It is surprising that if PMN are preincubated with the PTP inhibitor cocktail and then stimulated again with GM-CSF, there is no further increase in the phosphorylation of Lyn compared with GM-CSF alone. These results show that GM-CSF stimulation of PMN or relief of SHP-1 inhibition by PTP inhibitor cocktail is able to induce a strong Lyn activation but does not act in synergy to increase it. As it is possible to inhibit SHP-1 activity by using a PTP inhibitor, it is also feasible to stimulate SHP-1 activity by using a PKC inhibitor (bisindolylmaleimide I). The inhibition of PKCs will reduce serine phosphorylation in the C-terminal domain of SHP-1 and thus stimulate its activity, as evidences point to an inhibitory role for this residue [40 , 49 ]. Preincubation of PMN with the PKC inhibitor followed by stimulation with GM-CSF did not result in a phosphorylation of Lyn (Fig. 5A , Young and open bars). These results suggest that the GM-CSF could not overcome the anti-PKC-induced activation of SHP-1. For the elderly donors, Figure 5A (Elderly and solid bars) confirms the peculiar, increased phosphorylation in resting PMN. Despite a strong basal phosphorylation in resting PMN, stimulation with GM-CSF did not result in a significant increase in Lyn phosphorylation (Fig. 5A , Elderly and solid bars). As it was already shown, this is characteristic of the PMN from elderly donors, as they are unable to mount an appropriate response following stimulation [12 , 50 ]. This also correlates with the lack of modulation of the SHP-1 phosphorylation in serine by GM-CSF in PMN of elderly subjects (Fig. 4 A and B , Elderly).


Figure 5
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  		Figure 5. Alterations in the activation and translocation to the lipid rafts of the Src kinase Lyn with aging. PMN from young and elderly donors were stimulated with 20 ng mL–1 GM-CSF, PTP, PKC inhibitor, or with a combination of GM-CSF and inhibitor as described in Materials and Methods. (A) Whole cell lysates (50 µg) from GM-CSF or PTP inhibitor- or PKC inhibitor-treated cells were immunoblotted with antiphospho-Lyn (pLyn) antibody. (B) Isolated lipid raft fractions were probed with an anti-Lyn antibody. (C) Isolated lipid raft fractions were probed with an antiphospho-Lyn antibody. Densiometric analyses corresponding to the blots are shown in the lower panel. Experiments were performed from three different donors of each age group (young, open bars; elderly, solid bars) with similar results. Significant differences between young and elderly donors are depicted as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

As Lyn was shown to be present in lipid rafts in mast cells and B cells [52 , 53 ], we wondered whether differences in Lyn lipid raft expression of human PMN exist. Indeed, in resting PMN from young donors, only a small amount of Lyn is present in the lipid rafts and is further recruited upon stimulation with GM-CSF (Fig. 5B , Young and open bars, P<0.05). The eventual role of PTPs and SHP-1 in Lyn lipid rafts mobility was again studied by using PTP inhibitor cocktail and PKC inhibitor. It is interesting that PTPs (including SHP-1) maintain the low level of recruitment of Lyn to lipid rafts, as a vigorous recruitment of Lyn is seen when PTPs are inhibited (Fig. 5B , Young and open bars, P<0.001), which is not increased further after GM-CSF stimulation. In contrast, PKCs are involved in the recruitment of Lyn to lipid rafts, as their inhibition abrogates Lyn recruitment even under GM-CSF stimulation (Fig. 5B , Young and open bars). For the elderly donors, we see a stronger presence of the two isoforms of Lyn in the lipid rafts for resting and GM-CSF-stimulated PMN (Fig. 5B , Elderly and solid bars). Inhibition of PTPs did not cause more Lyn recruitment in the lipid rafts of the elderly compared with the basal and GM-CSF-activated status. PKC inhibitors have the opposite effect of that seen in the young donors, as there is actually more Lyn recruited to lipid rafts, especially under GM-CSF stimulation (P<0.001).

As for the activation by phosphorylation of Lyn in lipid rafts, there is a strong tyrosine phosphorylation of Lyn in lipid rafts of PMN from young subjects following GM-CSF stimulation (Fig. 5C , Young and open bars, P<0.01) compared with the basal status. In contrast, there is no phosphorylation of Lyn with GM-CSF stimulation in PMN of elderly donors compared with the higher phosphorylated basal status (Fig. 5C , Elderly and solid bars). The use of PTP inhibitors influenced the phosphorylation status of Lyn compared with the basal status in the young (Fig. 5C , Young and open bars) in accordance with the strong recruitment of Lyn in lipid rafts. No significant modulation of antiphospho-Lyn in rafts compared with basal status could be observed in PMN of elderly (Fig. 5C , Elderly and solid bars).

Interactions of SHP-1 with Src Lyn are altered with aging
Considering the altered SHP-1 activity with aging under GM-CSF stimulation and our data on SHP-1 lipid rafts association in this present study as well as the previous data concerning antiphospho-Lyn in lipid rafts (Fig. 5C) , we next studied the physical interactions of SHP-1 with the Src kinase family member Lyn. We performed IP experiments in lipid rafts with Lyn antibody to study the interactions between SHP-1 and Lyn. In the young donors, we demonstrate an association of SHP-1 and Lyn in resting PMN. This association in the resting PMN is lost at 1 min of stimulation (Fig. 6A and 6B ) with GM-CSF. SHP-1 is then recruited back to Lyn at 5 min to a level similar to basal state. Then, at 30 min of stimulation with GM-CSF, there is again a strong dissociation between SHP-1 and Lyn. We also observe the same pattern in the whole cell lysates of young donors (data not shown). In striking contrast for the lipid rafts of elderly donors, Figure 6A shows a faint association in unstimulated PMN, suggesting that the increase in tyrosine phosphorylation of Lyn with aging [50 ] is possibly induced by altered recruitment of SHP-1 to Lyn. It is remarkable that instead of the physical dissociation seen in the young under GM-CSF stimulation, there is a constant and strong recruitment of SHP-1 to Lyn for up to 30 min of stimulation in the lipid rafts of PMN of the elderly donors (Fig. 6) . These data indicate that SHP-1 is physically linked to Lyn at the basal status in PMN of young in contrast to PMN of elderly subjects. After GM-CSF stimulation, there is a rapid dissociation in young subjects, and in PMN of elderly, there is a strong recruitment. Moreover, this mobility in and out of lipid rafts also determines the SHP-1 activity (Figs. 2B and 3B) .


Figure 6
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  		Figure 6. Interactions of SHP-1 with Src Lyn are altered with aging. PMN from young and elderly donors were stimulated with 20 ng mL–1 GM-CSF for the indicated times. (A) Isolated lipid raft fractions were subjected to IP with anti-Lyn antibody and revealed with anti-SHP-1 antibody. Ig bands are shown as loading control. (B) Densiometric analysis of A is shown, where the intensities are indicated as relative to the unstimulated state. Experiments were performed from three different donors of each age group (young, open bars; elderly, solid bars) with similar results. Significant differences between young and elderly donors are depicted as follows: *, P < 0.05; ***, P < 0.001.


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DISCUSSION
 
The PMN and receptor-driven functions have been demonstrated to be altered with aging [12 , 27 28 29 30 31 32 33 34 35 36 ]. The alterations of the signal transduction manifested by altered tyrosine kinase phosphorylation during various PMN receptor stimulation including GM-CSF and fMLP could partly explain this impairment with aging. However, the importance of negative regulators of PMN receptors, including the GM-CSF receptor, in the alteration of the effector functions has not been studied extensively. In this study, we have undertaken to test the possibility that altered regulation of cytokine signaling by PTP is also a factor in the impairment of effector functions in the elderly.

Aging causes multiple defects in the functions of PMN, notably in the production of ROS [26 ] in chemotaxis and in their rescue from spontaneous apoptosis by proinflammatory mediators [12 , 54 ]. Our results revealed for both age groups that relieving the cells from the inhibiting effects of phosphatase, including SHP-1, by the use of a PTP inhibitor cocktail prior to GM-CSF stimulation resulted in an increase in ROS production and chemotaxis. It is of note that for young donors, the PTP inhibitor cocktail alone was not able to cause ROS production, whereas for elderly donors, the inhibition of PTP was enough to induce ROS production. In the case of chemotaxis, incubating the PMN with the PTP inhibitor alone was not able to cause chemotaxis in any age group. This suggests that PTPs may not be an inhibiting factor for chemotaxis. The suspected age-related alterations in the effects of GM-CSF on phosphatase activity became mostly evident when PMN from young and elderly donors were cultured during 18 h in the presence of a PTP inhibitor and GM-CSF. In PMN of young subjects, the PTP inhibitor or PTP inhibitor followed by GM-CSF stimulation blunted the GM-CSF apoptosis-rescuing effect. In a similar manner, it was shown that 1 µM of the PTP inhibitor PAO accelerated apoptosis compared with medium alone in eosinophils and in PMN [21 ]. In contrast, in the elderly, PTP inhibition before the 18-h culture resulted in the recovery of the lost GM-CSF-induced rescue from apoptosis. These data about the modulation of PTP activities in relation to GM-CSF-induced PMN functions revealed a differential regulation of PTPases in PMN with aging.

SHP-1 is well-known to be a negative regulator of signal transduction [2 , 18 19 20 , 55 , 56 ], and it has been shown to be of critical importance for negative regulation of Src kinases, such as the Jak or Lyn kinase, elicited by GM-CSF in PMN or other immune cells [2 , 4 ]. Our present study demonstrates that SHP-1 phosphatase activity cannot be down-regulated following a 1-min simulation with GM-CSF in the PMN of the elderly donors in contrast to PMN of young donors. A similar lack of down-regulation was observed during the epidermal growth factor receptor stimulation in fibroblasts, as the aging-caused attenuation of signaling could be mediated in part by this increased PTPase activity [57 ]. Recently, lipid rafts have been shown to be an important platform for cellular signal transduction in various cells including PMN [24 , 25 , 58 , 59 ]. We have also shown the presence of lipid rafts in PMN of young and elderly donors [12 ]. Thus, we mainly focused our present study on SHP-1 modulation in relation to lipid rafts. In contrast to the whole cell lysates in the lipid rafts from PMN of elderly, the SHP-1 is continuously present, whereas in the PMN of young donors, SHP-1 is dissociated at 1 min of stimulation by GM-CSF and is recruited back for a longer period of stimulation (Fig. 3B and 3D) . These studies show two alterations in the elderly compared with the young donors: SHP-1 activity is not down-regulated and is steadfastly present in lipid rafts during GM-CSF stimulation. This pattern of association in the lipid rafts of the PMN of the young correlates well with the measurement of SHP-1 activity (Fig. 2A vs. Fig. 3B ); i.e., there is a rapid dissociation from the lipid rafts associated with the decrease in phosphatase activity. Longer times of stimulation restore recruitment to lipid rafts and phosphatase activity.

SHP-1 can be phosphorylated on serine and tyrosine residues in its C-terminal domain. Tyrosine phosphorylation has an enhancing effect on SHP-1 activity, whereas serine phosphorylation has a strong, inhibiting effect on SHP-1 activity [40 , 46 47 48 49 ]. Our results demonstrated some contradictory data between the measured SHP-1 activity (Fig. 1A) and the tyrosine and serine phosphorylation (Fig. 4A) in whole cell lysates of both age groups. We do not have an explanation for these discrepancies. However, the possibility has been raised that another layer of regulation exists in the C-terminal domain of SHP-1 for its in vivo phosphatase activity modulation [60 ]. In contrast, in the lipid rafts, the serine and tyrosine phosphorylation correlated well with the observed SHP-1 activity alterations with aging. In the lipid rafts, we see several alterations in PMN with aging in tyrosine and serine phosphorylation. As an overall result, there is a relatively higher serine phosphorylation in the elderly compared with tyrosine phosphorylation at any time. As a higher serine phosphorylation status of SHP-1 has an inhibiting effect on its activity, this is in accordance with the fact that PMN were found to have an altered increase of Lyn and MAPK activation at the basal status and that no further activation was possible [50 ]. In contrast, in young subjects, the balanced changes in tyrosine and serine phosphorylation of SHP-1 in lipid rafts of the PMN reflect the GM-CSF-induced changes in SHP-1 activity.

Lyn is one of the first molecules activated via tyrosine phosphorylation by GM-CSF [17 ], and we found earlier an alteration in Lyn phosphorylation in PMN with aging [50 ]. When PMN are treated with GM-CSF, there is a strong phosphorylation of Lyn in the young donors over the unstimulated state, whereas it is only weak for the elderly donors, despite a stronger basal phosphorylation in whole cell lysates. The stronger basal phosphorylation seen in the elderly suggests that PMN were already primed for action and is characteristic for the PMN of the elderly. This is the consequence of a low-grade, chronic inflammation occurring with aging and is called the "Inflamm-aging" theory. This states that this increased basal, age-related, chronic inflammatory activity, resulting from a life-long antigenic load, is detrimental for longevity [61 ].

To our best knowledge, we demonstrate for the first time in this paper that the Src kinase Lyn can be recruited to the lipid rafts in human PMN, as it was already shown for other Src kinases during TCR signaling [24 ] and Fc{gamma} ligation in PMN [62 ]. In resting PMN from young donors, only a small amount of Lyn is present in the lipid rafts and is further recruited upon stimulation with GM-CSF. It is interesting that PTP, including SHP-1, maintain the low level of recruitment of Lyn to lipid rafts, as a vigorous recruitment of Lyn is seen when PTP are inhibited, which is not increased further after GM-CSF stimulation. In contrast, PKC are involved in the recruitment of Lyn to lipid rafts, as their inhibition abrogates Lyn recruitment even under GM-CSF stimulation. For the elderly donors, we see a stronger presence of the two isoforms of Lyn in the lipid rafts for resting and GM-CSF-stimulated PMN, possibly as that SHP-1 activity is reduced in lipid rafts of elderly.

We found a strong tyrosine phosphorylation of Lyn in lipid rafts of PMN from young subjects following GM-CSF stimulation compared with the almost nonphosphorylated basal status. In contrast, there is no phosphorylation of Lyn with GM-CSF stimulation in PMN of elderly donors compared with the higher phosphorylated basal status. A similar situation has been demonstrated already for MAPKs in PMN of elderly [44 , 50 ]. The use of PTP inhibitors influenced the phosphorylation status of Lyn compared with the basal status in the young in accordance with the strong recruitment of Lyn in lipid rafts. This indicates that the inhibition of phosphatase activity, including SHP-1, is necessary to maintain antiphospho-Lyn in rafts, as it is also suggested by the use of the PKC inhibitor, which stimulates SHP-1 activity. No significant modulation of antiphospho-Lyn in rafts compared with basal status could be observed in PMN of elderly. Altogether, these results suggest that SHP-1 not only participates in the negative regulation of Lyn but also in the maintenance in its inactive form in lipid rafts.

Our present data confirmed the interactions between SHP-1 and Lyn, as it was already shown by Daigle et al. [19 ]. We found this interaction in lipid rafts of resting PMN of the young donors, which was lost with stimulation, whereas there was a faint interaction in resting PMN of the elderly and a constant recruitment of SHP-1 to Lyn following stimulation. Putting in parallel the measured SHP-1 activity and Lyn phosphorylation as well as their interactions, we can suggest that the inhibition of SHP-1 activity at 1 min GM-CSF stimulation is concomitant with the dissociation of SHP-1 from Lyn. This indicates that in the lipid rafts, as long as SHP-1 is present, Lyn is not tyrosine-phosphorylated and is excluded. At 1 min GM-CSF stimulation, SHP-1 is displaced from lipid rafts, and Lyn is then recruited to lipid rafts and tyrosine-phosphorylated (Fig. 7 ). These results suggest that the positive or negative regulation of GM-CSF signal transduction is mediated in part by a regulated interaction of SHP-1 with the Src kinase Lyn. Similarly, SHP-1 was shown to bind to caspase-8 constitutively, and LPS stimulation disrupted this association, permitting the tyrosine phosphorylation of caspase-8 in PMN [63 ]. This mechanism also resembles the activation of Lck in T cells by CD45, a receptor-like PTP. In resting T cells, an adaptor protein, PAG, recruits Csk, a PTK, to the lipid rafts, where it maintains Lck, inactive by tyrosine phosphorylation of an inhibiting residue. In TCR-stimulated T cells, CD45 dephosphorylates PAG, thereby resulting in dissociation of Csk from lipid rafts and activation of Lck [2 , 56 ]. In striking contrast for the lipid rafts of elderly donors, there is a faint association in unstimulated PMN, and instead of the physical dissociation seen in the young under GM-CSF stimulation, there is a constant and strong recruitment of SHP-1 to Lyn. These data further indicate that SHP-1 is physically linked to Lyn at the basal status in PMN of young in contrast to PMN of elderly subjects.


Figure 7
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  		Figure 7. Proposed mechanism for the regulation of Lyn activation by SHP-1 in lipid rafts of PMN from young donors. (A) In a resting state, PTP, including SHP-1, maintain Lyn inactivated and outside of lipid rafts. (B) Stimulation by GM-CSF causes inhibition of SHP-1 by displacement from lipid rafts and recruitment of Lyn to lipid rafts, where it is activated by tyrosine phosphorylation. (C) The stimulation of PMN of elderly subjects by GM-CSF is unable to modulate the dissociation of SHP-1 from lipid rafts, explaining the altered Lyn activation. See text for further details. GM-CSFR, GM-CSF receptor; P, phosphorylation.

In an attempt to explain the discrepancies of the regulation of SHP-1 observed between young and elderly donors, we looked for possible alterations in the physical characteristics of PMN. Membrane cholesterol staining with filipin and measurement of the resultant fluorescence intensities by a flow cytometer revealed no differences whatsoever between the PMN of young and elderly donors (data not shown). This is in sharp contrast to what is found in T cells with aging [42 ]. Assessing the membrane fluidity of PMN by anisotropy with a fluorescent probe (diphenylhexatriene) yielded again no differences between young and elderly (data not shown). In contrast, the measurement of protein oxidation revealed that there was more oxidation of proteins in the whole cell lysates of PMN of elderly than in young donors at resting and GM-CSF-stimulated states (data not shown). Furthermore, significantly increased levels of acute-phase proteins such as C-reactive protein, fibrinogens, haptoglobin, and the inflammatory cytokine interleukin-6 in the plasma of elderly, healthy subjects have been reported [64 ]. This is likely to contribute to the low-grade inflammation in the elderly and could negatively affect the immune response. However, this was not the case for our own donors. Other factors not studied in our present work, for example, myristoylation or N-glycosylation, could also act to regulate SHP-1 activity. Further experiments are needed to study the alterations of the regulatory mechanisms that target SHP-1 to lipid rafts in PMN with aging.

In conclusion, the alterations of SHP-1 modulation by GM-CSF in lipid rafts in PMN with aging could contribute to the age-related impairment of PMN functions. These alterations of signaling with aging are likely to contribute to the decrease of PMN functions, leading to increased susceptibility to infections, cancers, autoimmune disorders, and inflammation-based pathologies. The involvement of phosphatase in cellular function alterations with aging was underestimated. However, more studies are needed to determine to which extent they contribute to immunosenescence.


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ACKNOWLEDGEMENTS
 
This work was supported by a grant-in-aid from the Canadian Institute of Health Research (No. 63149) and the Research Center on Aging of Sherbrooke. We thank the Clinical Research Center for assistance and support.

Received August 25, 2005; accepted January 8, 2006.


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