* Department of Pediatrics, Shinshu University School of Medicine, Matsumoto, Japan; and
Department of Microbiology and Immunology, Yamaguchi University School of Medicine, Japan
Correspondence: Kozo Yasui, M.D., Ph.D., Department of Pediatrics, Shinshu University School of Medicine, Asahi 3-1-1, Matsumoto 390-8621, Japan. E-mail: k-yasui{at}hsp.md.shinshu-u.ac.jp
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Gene-disrupted mice were used to help accurately identify the physiological role of the PI3K isoform in PMN activation in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF). PMN from the p85-/- mice showed normal cellular motility, and the quantity of superoxide anion (O2-) produced by PMN upon stimulation with formyl-Met-Leu-Phe did not significantly differ between p85-/- and wild-type mice under controlled conditions. In p85-/- mice, the O2- production by PMN was enhanced (primed) by GM-CSF when stimulated with the chemotactic peptide but to a significantly lesser extent than in wild-type mice. In addition, no major GM-CSF-dependent delay in apoptosis or activation of Akt protein phosphorylation by GM-CSF was observed in the p85-/- mice. In terms of targeting strategy, however, the mutation actually expressed a small amount of Ia-type (p85-regulated) PI3K activity (partially abrogated) in the mice. These results demonstrate that Ia-type PI3K plays a critical role in the enhancement of the GM-CSF-modulated function of PMN and in the PI3K/Akt pathway-dependent delay of PMN apoptosis.
Key Words: rodent • knockout • signal transduction • cytokines
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Phosphoinositide 3-kinases (PI3Ks) constitute a family of lipid kinases that are thought to regulate an array of fundamental cellular responses, including cellular migration, superoxide production, priming effect, and protection from apoptosis in myeloid cells [13
14
15
]. PMN express all four known PI3K isoforms (PI3K, ß, 
, and 
). The , ß, and isoforms are composed of a dimer containing a p85 regulatory subunit and a p110 catalytic subunit (type Ia PI3Ks). PI3K appears to be composed of a p110 catalytic subunit and a p101 regulatory subunit (type Ib PI3K) [16
] and in knockout mouse studies, has been proven to be linked to heterotrimeric guanosine 5'-triphosphate (GTP)-binding protein-coupled receptor signaling for cellular migration and superoxide production [17
18
19
20
]. Specific seven-transmembrane domain receptor agonists, such as C5a, formyl-Met-Leu-Phe (fMLP), and interleukin-8, trigger the formation of phosphatidylinositol 3,4,5-triphosphate. In addition, some pharmacological studies have indicated that GM-CSF also activates PI3K in human neutrophils and that this process is mediated by tyrosine kinases, linked partly to the priming of PMN functions and delay of PMN apoptosis [21
22
23
24
25
26
]. PI3Ks are known to be activated by a number of growth factor receptor systems and to play essential roles in proliferative and nonproliferative cellular functions. However, it is not clear at present which PI3K isoform enzyme(s) relay signals for these effects induced by GM-CSF.
To clarify the physiological role of the PI3K isoform in PMN activation in the presence of GM-CSF, we used p85 gene-disrupted mice and their PMN [27
, 28
] to examine the effects of GM-CSF on PMN functions and survival.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gene could lead to a lethal phenotype, the first exon of the gene was disrupted, resulting in an abrogated p85 but intact p55 and p50 (p85-/-) [27
, 28
].
Purification of mouse PMN
To obtain enriched populations of PMN, the peritoneal cavity was washed with heparinized phosphate-buffered saline (PBS) 20 h after a peritoneal injection of 2% thioglycollate (2 ml). The PMN population was >90% pure, as assessed by light microscopy of May-Grunwald-Giemsa (Merck Ltd., Darmstadt, Germany)-stained cytospun preparations. The PMN were resuspended in Hanks’ balanced saline solution (HBSS) buffered with 10 mM HEPES, pH 7.35 (Ca2+- and Mg2+-free).
Drugs and analytical reagents
Dextran T500 was obtained from Pharmacia (Uppsala, Sweden). Endotoxin-free Histopaque, fMLP, cytochrome C (type VI), dimethyl sulfoxide (DMSO), N-ethylmaleimide, wortmannin (a PI3K inhibitor), superoxide dismutase (SOD), and mouse tumor necrosis factor (TNF-) were purchased from Sigma Chemical Co. (St. Louis, MO), and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002; a specific PI3K inhibitor) was from Calbiochem (La Jolla, CA). fMLP and PI3K inhibitors were stocked in DMSO and further diluted with the appropriate medium. The final DMSO concentration did not exceed 0.1% by volume.
Measurement of superoxide anion (O2-) production
Superoxide production in mouse PMN (5x106 cells/ml) was determined at 37°C, as a change in absorbance at 550 nm from the baseline using a Hitachi spectrophotometer U2000 [31
]. The reaction lasted for 2 min with fMLP (1 µM) in a standard assay mixture with 1.6 mM Ca2+ and 1.0 mM Mg2+. The reaction was stopped by adding 0.5 mM N-ethylmalemide. Superoxide generation was calculated by subtracting the absorbance change in the presence of SOD (1 mM) from that in its absence and then dividing the value for the molar extinction coefficient. In kinetic studies, changes in the absorbance of light at 550 nm were continuously assayed using a cuvette holder maintained at a constant temperature by a thermostat. Recombinant mouse GM-CSF was supplied by Kirin Brewery (Tokyo, Japan). In several experiments, PMN were incubated with 100 ng/ml TNF- or 10 ng/ml (45 pM) GM-CSF for 45 min prior to fMLP stimulation.
Cell motility (chemotaxis)
PMN motility was measured using the agarose method [31
]. Briefly, 5 ml 1.2% agarose dissolved in HBSS containing Ca2+ and Mg2+ and 10% fetal calf serum was placed in 60 x 15-mm Petri dishes. Cell suspension (10 µl) containing 5 x 105 cells was placed in the center well (3-mm diameter) of the plate, and equal volumes of the chemoattractant (2x10-7 M fMLP) and HBSS were placed in the outer and inner wells, respectively. The outer and inner wells were located 7 mm from the center well. After incubation at 37°C in 5% CO2 for 2 h, the plates were fixed with ethanol and formalin, and the cells were stained with Wright’s stain (Sigma Chemical Co.). Cell motility was defined as the linear distance that the cells moved from the center well in the direction of that containing the chemoattractant (chemotaxis). The amount of migration was compared with that of controls. All results represent averages of duplicate measurements.
Cell culture and assessment of apoptosis
A mouse agonistic anti-Fas monoclonal antibody (mAb), apoptosis-inducing RK-8, was purchased from MBL (Nagoya, Japan). The PMN (1x106/ml) were incubated in Ca2+- and Mg2+-free HBSS buffered with 10 mM HEPES, pH 7.35, and containing 2.5% fetal bovine serum, settled with inclination to prevent clotting under a humidified 5% CO2 atmosphere for 8 h. Incubation proceeded in the absence or presence of anti-Fas Ab (RK-8; 500 ng/ml). The percentage of apoptotic cells was analyzed by flow cytometry (FACScan; Becton Dickinson, San Jose, CA) using propidium iodide (PI), and 1 x 104 cells were counted [32
, 33
]. The PI fluorescence of individual nuclei with an acquisition of FL3 (DNA content) was plotted, and the data registered on a logarithmic scale correlated well with the morphological determination (characteristic chromatin condensation and formation of apoptotic bodies).
DNA fragmentation
PMN (2x106) were lysed by incubation for 15 min in 400 µl of a cold mixture of 10 mM Tris-HCl, pH 7.4, 0.2 mM EDTA, and 0.2% Triton X-100. The lysate was separated by centrifugation, the supernatant was extracted with chloroform/isoamyl alcohol/phenol, and the aqueous phase was collected. The DNA was precipitated with 50% 2-propranol and 0.5 M sodium acetate and placed at -80°C overnight. Following digestion with 50 µg/ml RNase A (Boehringer Mannheim, Mannheim, Germany) for 1 h at 37°C, the samples were resolved by electrophoresis through a 1.2% agarose gel and stained with 0.5 µg/ml ethidium bromide [33
].
Immunoprecipitations and immunoblotting
Anti-p85 polyclonal Ab against a full-length p85-glutathione-S-transferase fusion protein containing the N-terminal SH2 domain of p85 and anti-p110 Ab against the carboxy-terminal region was purchased from Upstate Biotechnology (Lake Placid, NY). Phosphorylated Akt was identified with a polyclonal Ab directed against phosphorylated Ser473 (New England Biolabs, Cambridge, MA). Immunoblotting was performed as described [25
]. Briefly, PMN (2x107 cells/ml) were incubated with GM-CSF (10 ng/ml) for 3 min, and then cells were pelleted and resuspended in lysis buffer containing Tris-HCl, NaCl, Triton X-100, Nonidet P-40, EDTA, EGTA, Na2VO4, NaF, leupeptin, and phenylmethylsulfonyl fluoride. Protein samples were resolved by electrophoresis and transferred to nitrocellulose membranes, which were then incubated overnight in primary Ab (1:150 dilution) in 5% bovine serum albumin/PBS. Ab bound to the membrane was detected with 125I-labeled protein A. Blots were developed on Fuji X Omat films (Tokyo, Japan) overnight at -70°C.
Statistical analysis
Data are presented as means ± SE. Data were analyzed by Student’s t-test or one-way ANOVA among more than three groups. A P value of <0.05 was considered significant.
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as well as the p50 subunit. p85 and p50 were precipitated in wild-type and hetero mice. In p85-/- mice, however, p50 was detected only in the major PI3K regulatory subunit (Fig. 1
), which can also bind to the p110 catalytic subunit and act as a regulatory unit for p110 [28
]. Thus, despite the complete elimination of full-length p85, little PI3K activity could be detected in several cells (including PMN) in the knockout mice (p85-/- mice) [27
, 28
].
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Figure 1. Expression patterns of subunits of PI3K in PMN from p85-/-, hetero, and wild-type (WT) mice. Purified neutrophils were lysed and immunoprecipitated with the p85 Ab (that cross-reacts with p50). In addition, the anti-p110 Ab cross-reacted with the conjugated p85 subunit of PI3K but not with p50 in PMN from WT mice (C).
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-/- and wild-type mice. The time-course study of peritoneal PMN diluted in medium containing 100 nM wortmannin (a PI3K inhibitor) for 30 min showed a dramatic inhibition of O2- production (Fig. 2
). PMN were finally stimulated by a chemotactic factor (1 µM fMLP) that interacts with GTP-binding protein-coupled receptors [17
18
19
20
]. Wortmannin also strongly inhibited the GM-CSF-induced augmentation of O2- production in PMN. Similar inhibitions of O2- production were observed in the presence of 10 µM LY294002 (data not shown). The amounts of O2- produced in the PMN of p85-/- and wild-type mice stimulated with fMLP did not differ under control conditions (Fig. 3A
). Prior exposure to TNF and GM-CSF can accelerate O2- production stimulated with fMLP (priming effect; P<0.002 by ANOVA). PMN were incubated with 100 ng/ml TNF- or 10 ng/ml (45 pM) GM-CSF for 45 min prior to fMLP stimulation. As shown in Figure 3B
, the response of PMN from p85-/- mice to fMLP was significantly lower than that of PMN from the wild-type mice preincubated with GM-CSF (10 ng/ml: 45 pM for 45 min; P<0.01 by ANOVA; n=3). The enhancement of O2- production by GM-CSF in response to fMLP in p85-/- mice was impaired but not completely eliminated (P<0.05 by ANOVA, n=3). Meanwhile, preincubation with TNF (100 ng/ml for 45 min) did not result in any observable difference between the two types of mice.
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Figure 2. Effects of wortmannin on superoxide production in mouse PMN (5x106 cells/ml) stimulated with 1 µM fMLP. PMN were preincubated with 100 nM wortmannin for 30 min at 37°C before stimulation with fMLP (solid marks). Changes in absorbance at 550 nm were monitored. Representative results are shown. The experiment was performed three times with similar results. Cells were incubated with 10 ng/ml (45 pM) GM-CSF for 45 min prior to fMLP stimulation (open squares).
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Figure 3. (A) Time course of O2- production in PMN (5x106 cells/ml) from p85-/- and wild-type (WT) mice stimulated with 1 µM fMLP. Representative changes in absorbance at 550 nm from three separate experiments are shown. Cells were incubated with 100 ng/ml TNF- or 10 ng/ml (45 pM) GM-CSF for 45 min prior to fMLP stimulation. There was a significant difference in response between p85-/- and WT mice (**, P<0.01). (B) Comparison of total amount of O2- production in PMN from p85-/- and WT mice determined with a cytochrome C reduction assay. Cells were incubated with 100 ng/ml TNF- or 10 ng/ml (45 pM) GM-CSF for 45 min prior to fMLP stimulation. Reaction lasted for 2 min. Data represent means ± SE of three experiments, each conducted in duplicate. There was a significant difference in response from that without cytokines (*, P<0.05; **P<0.01).
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-/- mice did not differ from that of wild-type mice cells in terms of fMLP-induced chemotactic activities. Preincubation of control PMN with wortmannin (100 nM) or LY294002 (10 µM) for 30 min reduced cell migration of both types of PMN (Fig. 4
) in a similar manner.
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Figure 4. Chemotaxis of mouse PMN under agarose. PMN motility is expressed as means ± SE on a microscopic scale; the actual distance at the front is the number shown multiplied by 0.375 mm. Chemotaxis (solid bars) is defined as the distance cells moved in response to 2 x 10-7 M fMLP in three determinations; spontaneous migration (open bars) is also shown. PMN from WT mice were preincubated with the PI3K inhibitor wortmannin (100 nM) or LY294002 (10 µM) for 30 min. The response differs significantly from that without inhibitors (**, P<0.01).
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-/- mice. The percentages of apoptotic cells were almost the same for p85-/- and wild-type mice without GM-CSF (64±6–68±3%). Figure 5A
and 5B
, shows that apoptosis was not completely inhibited by GM-CSF in PMN purified from p85-/- mice. Similar effects of GM-CSF were observed on spontaneous apoptosis of PMN (without anti-Fas Ab; data not shown). Cellular apoptosis was further confirmed by electrophoresis (Fig. 6
). Agarose gel electrophoresis of DNA from PMN showed characteristic ladder-like apoptotic patterns (DNA degradation). Ladder formation in DNA extracts was augmented by anti-Fas mAb and inhibited by GM-CSF (Fig. 6)
in PMN from wild-type mice (p85+/+). PMN apoptosis was not delayed by GM-CSF treatment of PMN from p85-/- mice. Preincubating PMN with wortmannin (100 nM) or LY294002 (10 µM) markedly reversed the antiapoptotic effect of GM-CSF as described previously (ref. [25
]; data not shown). One mechanism of GM-CSF that prevents apoptosis is thought to be the activation of the protein kinase B (PKB)/Akt pathway [25
, 34
]. We examined the role of this pathway in GM-CSF-induced apoptosis delay. As shown in Figure 7
, GM-CSF caused phosphorylation of Akt in PMN from wild-type mice (p85+/+), but this phenomenon was not clearly observed in PMN from p85-/- mice.
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Figure 5. Loss of DNA in PMN undergoing apoptosis as measured by flow cytometry of PI-stained PMN (A). Wild-type (WT) or p85-/- knockout mouse PMN were incubated with anti-Fas Ab (RK-8; 500 ng/ml). After 8 h incubation with/without 10 ng/ml GM-CSF, apoptotic cells as a percentage of the total number of PMN were determined. Results are representative data of three independent experiments. Comparison of percentages of apoptotic cells in PMN from p85-/- and WT mice (B) from three independent experiments. The response significantly differs from that of control (**, P<0.01).
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Figure 6. Agarose gel electrophoresis of DNA extracted from p85-/- or wild-type (p85+/+) mouse PMN after culture for 8 h under various conditions. M, Molecular weight markers. Cells were incubated with/without 10 ng/ml GM-CSF (GM) and anti-Fas Ab (RK-8; 500 nM).
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Figure 7. Effect of GM-CSF on phosphorylation of Akt in PMN. PMN were incubated with 10 ng/ml GM-CSF, and cell aliquots were obtained after 45 min incubation at 37°C. Phosphorylation of Akt was determined by immunoblotting. The representative data are shown.
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(Ia-type PI3K). It is possible that GM-CSF augments O2- production and also increases the life span of PMN through PI3K, but the isoform or subunit involved has not been clearly identified [25
, 26
]. Although its participation is beyond doubt, there was no direct evidence that GM-CSF relays signals via PI3K activation during functional augmentation or delaying apoptosis. Therefore, we studied the functions of PMN and cellular survival in p85 gene-disrupted mice to clarify the role of PI3K activation.
In control experiments (without cytokines), the quantity of O2- produced and the extent of chemotactic migration (chemotaxis) as a result of stimulation with fMLP in the PMN of p85-/- and wild-type mice showed no significant differences. PMN purified from thioglycollate-treated p85-/- mice did not differ from that of wild-type cells in terms of fMLP-induced cellular activation. The chemotaxis and O2- production of PMN from PI3K-deficient mice were impaired in response to fMLP [17
18
19
], and preincubation of PMN with wortmannin or LY294002 (nonspecific PI3K inhibitors) for 30 min also diminished cellular functions. These results indicate that only PI3K plays an essential role in chemoattractant-induced PMN activation. These findings also indicate that PI3K activity continues in PMN from p85-/- mice.
The enhancement of O2- production by GM-CSF in response to fMLP in p85-/- mice was poor but not completely eliminated. The remaining PI3K activity with the p55 and p50 isoforms may have little effect, or it is comprehensible that PI3K may have an important but not an essential role in the priming effect of GM-CSF. The activation of mitogen-activated protein kinase (MAPK) subtypes, including extracellular signal-regulated kinases (ERKs; p38 MAPK, ERK1, and ERK2), may be another process that enhances neutrophil function. PMN from p85-/- mice and wild-type mice preincubated with TNF (100 ng/ml for 45 min) had a similar priming effect on O2- production. Previous studies revealed that GM-CSF and TNF activate different pathways, which are related to the activation of MAPK subtype cascades of ERK1/ERK2 and p38 MAPK, respectively [35
36
37
]. Our findings also suggested distinct pathways of PMN activation for these cytokines. Thus, the MAPK subtype cascades may participate in TNF and GM-CSF-mediated priming effects, and this can be interpreted as meaning that the PI3K-dependent pathway is mainly involved in the GM-CSF-mediated priming effect.
Previous studies found significant Akt phosphorylation [25
] and reversal of the GM-CSF-dependent delay of PMN apoptosis as a result of inhibition of PI3K with wortmannin or LY294002 [25
, 26
]. Our results showed that Akt was not phosphorylated in PMN purified from p85-/- mice after incubation with GM-CSF. PMN from PI3K knockout mice, however, can activate PKB and phosphorylate Akt in response to GM-CSF but not to fMLP [17
]. These findings and our results indicate that the PI3K/Akt pathway plays a role in initiation of the GM-CSF-stimulated delay of PMN apoptosis. Several investigators have reported that Janus kinase/signal transducer and activator of transcription and MAPKs also play key roles in regulating neutrophil apoptosis [26
, 37
] and others, that PI3K is an alternative element in the signal transduction pathway leading to ERK activation [38
, 39
]. Further studies are obviously required to clarify the involvement of ERKs and the precise linkage between ERKs and PI3Ks in GM-CSF-regulating PMN apoptosis.
We identified two important differences in GM-CSF-modulated PMN functions between p85-/- and wild-type mice. In the p85-/- mice, O2- production of PMN was enhanced by GM-CSF stimulated with fMLP but to a significantly lesser extent than in wild-type mice. In addition, the GM-CSF-dependent delay in apoptosis was considerably less evident in PMN from p85-/- mice, and GM-CSF failed to activate the PI3K/Akt pathway as determined by Akt phosphorylation. As our targeting strategy left the expression of the p55 and p50 isoforms intact [27
, 28
], the mutant continued to express a small amount of PI3K activity [27
]. Therefore, it is possible that the effects of GM-CSF on PMN functions or cellular survival in the knockout mice were not completely eliminated.
Our study presents genetic evidence for the critical role of PI3K p85 gene products in the functioning of GM-CSF-modulated PMN and Akt-dependent prevention of cellular apoptosis. The adapter isoforms encoded by the p85 gene appear to be critical for PI3K catalytic function, as p85-deficient cells greatly reduced p110 expression and kinase activity [27
]. To summarize, our results demonstrated that PI3K plays a key role in GM-CSF-modulated functions and in the delayed apoptosis of mature, peripheral PMN. The blockade of the PI3K isoform may thus exert an anti-inflammatory effect by promoting cellular apoptosis and sedation of functional priming of PMN. Further studies will be needed to determine whether this pharmacological mechanism is also effective in vivo.
-/- mice. Received April 10, 2002; revised August 12, 2002; accepted August 21, 2002.
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in thymocyte development, T cell activation, and neutrophil migration Science 287,1040-1046 in chemoattractant-mediated signal transduction Science 287,1046-1049 in inflammation Science 287,1049-1053 J. Biol. Chem. 275,23636-23641 subunit of phosphoinositide 3-kinase Science 283,390-392 subunit of phosphoinositide 3-kinase Nature Genet 21,230-235[Medline]-mediated regulation of apoptosis of human neutrophils via caspase-3 Blood 93,3106-3115 and GM-CSF J. Leukoc. Biol. 64,537-545[Abstract] Blood 93,341-349-mediated mitogen-activated protein kinase signaling pathway J. Biol. Chem. 271,12133-12136This article has been cited by other articles:
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