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J Immunol. Author manuscript; available in PMC 2010 March 30.
Published in final edited form as: J Immunol. 2010 February 1; 184(3): 1507–1515. doi:10.4049/jimmunol.0901219.

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Diabetes-Induced Oxidative Stress Is Mediated by Ca2+Independent Phospholipase A2 in Neutrophils
Srinivas Ayilavarapu, Alpdogan Kantarci, Gabrielle Fredman, Oya Turkoglu, Kazuhiro Omori1, Hongsheng Liu, Tomoyuki Iwata, Motohiko Yagi2, Hatice Hasturk, and Thomas E. Van Dyke Department of Periodontology and Oral Biology, Goldman School of Dental Medicine, Boston University, Boston, MA 02118

Abstract
Neutrophils from people with poorly controlled diabetes present a primed phenotype and secrete excessive superoxide. Phospholipase A2 (PLA2)-derived arachidonic acid (AA) activates the assembly of NADPH oxidase to generate superoxide anion. There is a gap in the current literature regarding which PLA2 isoform regulates NADPH oxidase activation. The aim of this study was to identify the PLA2 isoform involved in the regulation of superoxide generation in neutrophils and investigate if PLA2 mediates priming in response to pathologic hyperglycemia. Neutrophils were isolated from people with diabetes mellitus and healthy controls, and HL60 neutrophil-like cells were grown in hyperglycemic conditions. Incubating neutrophils with the Ca2+-independent PLA2 (iPLA2) inhibitor bromoenol lactone (BEL) completely suppressed fMLP-induced generation of superoxide. The nonspecific actions of BEL on phosphatidic acid phosphohydrolase-1, p47phox phosphorylation, and apoptosis were ruled out by specific assays. Small interfering RNA knockdown of iPLA2 inhibited superoxide generation by neutrophils. Neutrophils from people with poorly controlled diabetes and in vitro incubation of neutrophils with high glucose and the receptor for advanced glycation end products ligand S100B greatly enhanced superoxide generation compared with controls, and this was significantly inhibited by BEL. A modified iPLA2 assay, Western blotting, and PCR confirmed that there was increased iPLA2 activity and expression in neutrophils from people with diabetes. AA (10 μM) partly rescued the inhibition of superoxide generation mediated by BEL, confirming that NADPH oxidase activity is, in part, regulated by AA. This study provides evidence for the role of iPLA2 in enhanced superoxide generation in neutrophils from people with diabetes mellitus and presents an alternate pathway independent of protein kinase C and phosphatidic acid phosphohydrolase-1 hydrolase signaling. Neutrophil function has been shown to be altered in diabetes; one of the major neutrophil functional changes in diabetes is increased extracellular superoxide generation (1–5). The chronic hyperglycemia of poorly controlled diabetes can prime neutrophils and monocytes, resulting in an exaggerated inflammatory response and tissue damage (1–5). The neutrophil respiratory burst seems to be related directly to glycemic control with an increase in protein kinase C (PKC) and NADPH oxidase activity (5–7). Mechanistically, hyperglycemia results in increased phosphorylation of p47phox, leading to an increase in the generation of superoxide

Copyright © 2010 by The American Association of Immunologists, Inc Address correspondence and reprint requests to Dr. Thomas E. Van Dyke, Department of Periodontology and Oral Biology, Boston University, Goldman School of Dental Medicine, 100 East Newton Street, Suite 107, Boston, MA 02118. tvandyke@bu.edu. 1Current address: Department of Pathophysiology-Periodontal Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan. 2Current address: Department of Periodontology, Nihon University School of Dentistry, Tokyo, Japan. Disclosures The authors have no financial conflicts of interest.

Ayilavarapu et al.

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anion (O2−) (5–7). Recent work has shown that p47phox, a key protein in the assembly of NAPDH oxidase, prematurely translocates to the membrane and associates with p22phox in neutrophils from diabetic subjects (4). This was also observed in cells cultured in high glucose (HG) and the receptor for advanced glycation end products (RAGE) ligand S100B. The premature translocation of p47phox in neutrophils in response to hyperglycemia results in increased superoxide generation. Upon activation, the cytosolic subunits p47phox, p67phox, and p40phox of the NADPH oxidase translocate to the plasma membrane and bind with the cytochrome b558 (gp91phox and p22phox) complex (8). Translocation of the cytoplasmic components to the membrane and their association with cytochrome b558 renders the complex functional; the cytochrome then transfers electrons from NADPH to O2 to create O2− (superoxide anion) (9). The assembly of the subunits of the NADPH oxidase on the membrane is not sufficient; the final activation requires arachidonic acid (AA) (10,11). Results from several studies suggest that AA release catalyzed by phospholipase A2 (PLA2) is necessary for both the activation and the maintenance of O2− generation by the NADPH oxidase (10–12). PLA2 comprises a superfamily of enzymes that catalyze the hydrolysis of membrane phospholipid sn-2 ester bonds, generating free fatty acid and a lysophopholipid (13,14). The PLA2 reaction is the primary pathway through which AA is liberated from membrane phospholipids, providing substrate for enzymatic conversion of the eicosanoids, which include PGs and leukotrienes (LTs) (15). The PLA2 family consists of 15 groups and many subgroups and includes five distinct types of enzymes, namely secreted PLA2 (sPLA2), cytosolic PLA2 (cPLA2), Ca2+-independent PLA2 (iPLA2), platelet-activating factor acetylhydrolases, and lysosomal PLA2 (15). Different isoforms of PLA2 play roles in regulation of inflammation. iPLA2 is associated with the initiation of inflammation, whereas sPLA2 and cPLA2 are involved in the resolution of inflammation (16). The role of PLA2 in the generation of superoxide in neutrophils is not clear. In cPLA2 null mice, it was shown that superoxide generation was not inhibited, suggesting that cPLA2 may not be involved (17). iPLA2, on the other hand, has been shown to be associated with AA mobilization and to be necessary for superoxide generation by neutrophils stimulated with Aroclor 1242, an organochloride compound (18). Because augmentation of an activated neutrophil respiratory burst requires AA generation in response to advanced glycation end products, through which neutrophil NADPH oxidase may be upregulated, enhancing reactive oxygen species output (19), we hypothesize that iPLA2 mediates the hyperglycemia-mediated neutrophil-generated oxidative stress in diabetes. It is also not known if the iPLA2-mediated superoxide generation in neutrophils involves PKC activation. In this study, we examine the role of iPLA2 in the priming of enhanced superoxide generation by neutrophils in diabetes.

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Reagents

Materials and Methods
N-fMLP, S100B, cytochrome C, and propranolol hydrochloride were purchased from SigmaAldrich (St. Louis, MO). A23187, pyrrolidine-1, and bromoenol lactone (BEL) were purchased from EMD Biosciences (San Diego, CA). LTB4 ELISA kits were purchased from R&D Systems (Minneapolis, MN). AA, rabbit polyclonal antiserum to iPLA2, and a cPLA2 assay kit were purchased from Cayman Chemicals (Ann Arbor, MI). HL60 cells, RPMI medium, and Iscove's medium were obtained from American Type Cell Culture (Manassas, VA). Trizol reagent was purchased from Invitrogen (Carlsbad, CA). High-capacity cDNA reverse transcriptase kits, real-time PCR probes for β-actin, iPLA2, and master mix were obtained from Applied Biosystems (Foster City, CA). Amaxa Nucleofector V solution and human monocyte Nucleofector medium were purchased from Amaxa (Gaithersburg, MD). Small interfering RNA (siRNA) for GAPDH, nontargeting, and iPLA2 were obtained from Dharmacon RNAi technologies (Lafayette, CO). Ab to p47phox was purchased from BD Biosciences (San Jose,

J Immunol. Author manuscript; available in PMC 2010 March 30.

and neutrophils were isolated by Ficollhypaque gradient centrifugation as described previously (4). Diabetic subjects were subgrouped according to their glycemic control as poor. and 1.5 mM). The glycemic control of diabetes was assessed by glycated hemoglobin (HbA1c). In order to simulate the hyperglycemic conditions of diabetes-induced hyperglycemia. Because primary neutrophils cannot be cultured for extended periods. available in PMC 2010 March 30. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Subject recruitment.5 × 105) were suspended in PBS (200 μl/well) and stimulated by the addition of fMLP (1 μM) and the absorbance (OD) recorded in a Vmax kinetic microplate reader (SpectraMax 340PC. 37°C for 120 min. Page 3 CA) and PKC phosphospecific substrate Ab was obtained from Cell Signaling Technology (Danvers.5 mM). + glutamine). moderate. neutrophils (25 × 106/ml) were incubated with specific inhibitors for 15 min in prewarmed PBS at 37°C. glucose (5. differentiated HL-60 cells (1 × 106/ml) were incubated in RPMI 1640 culture medium (− glucose. Thermo Fisher Scientific. Waltham. Superoxide assay Superoxide (O2−) release was monitored spectrophotometrically at 37°C by measuring superoxide dismutase-inhibitable reduction of ferricyto-chrome C at 550 nm in 96-well plates as described previously (5).05 min in a thermal cycler (ABI 9700. and written informed consent was obtained from all subjects. cell preparations were routinely 99% neutrophils with ≥95% viability. neutrophil isolation. Cell viability was assessed for all concentrations of the inhibitors by trypan blue exclusion. as determined by trypan blue exclusion. CA) at 550 nm and 37°C. Quantitative real-time PCR (QRT-PCR) was performed using primers and TaqMan probes for GAPDH and iPLA2 (group VI) labeled with FAM dye (Applied Bio-systems.25% dimethyl-sulfoxide (DMSO) for 6 d. Applied Biosystems). Molecular Devices. 5. Quantitative real-time PCR Total RNA was extracted from neutrophils or HL60 neutrophil-like cells with Trizol reagent (Invitrogen) as per the manufacturer's protocol. and culture The study was approved by the Institutional Review Board at Boston University. Sunnyvale. S100B plus HG as described previously (4). Contaminating RBCs were hypotonically lysed. Endogenous J Immunol. 5% CO2 atmosphere for a period of 24 h under the following conditions: normal glucose (NG. MA). S100B (5 μg/ml). Cells were then counted and the cell number adjusted accordingly for further experiments. Individuals with diabetes mellitus were selected and classified according to the criteria of the American Diabetes Association (20) and were recruited at the General Clinical Research Center of Boston University Medical Center. O2− release was measured under conditions of linearity with respect to time and cell number and is expressed as nanomoles of O2−/min/105 neutrophils (5). β-actin was selected as an internal control and amplified using preformulated VIC-TAMRA-labeled TaqMan probes (Applied Biosystems.Ayilavarapu et al. or well-controlled using American Diabetes Association definitions (20). Systemically healthy individuals with no signs of infection or inflammatory disease were recruited as controls. For chemical inhibition experiments. .6. Cells (0. TaqMan gene expression assays). MA). HL-60 cells (American Type Culture Collection) were cultured in flasks containing RPMI 1640 medium supplemented with 10% heat-inactivated FBS (HIFBS). Peripheral venous blood was collected into heparinized tubes. The concentration and purity of RNA was estimated by the A260/A280 ratio spectrophotometrically (Nanodrop. The reaction mixtures were thoroughly mixed and assayed at 25°C for 10 min. The A260/A280 ratio was routinely above 1. Author manuscript. Control neutrophils received an equivalent volume of the diluent. 10% HIFBS at 37°C. in some experiments neutrophil-like differentiated HL-60 cells were used for culture or transfection. A total of 1 μg RNA was converted to cDNA using a high-capacity cDNA reverse transcriptase kit (Applied Biosystems). HG (25 mM). and 85°C for 0.

The blocking buffer was removed. Aliquots of these samples were separated by SDS-PAGE (10–20 μg/lane) on 7. 95°C for 15 s (45 cycles). Author manuscript. 95°C for 10 min (one cycle). and finally 60°C for 1 min. The pellet consisting of unbroken cells and debris was discarded. Protein content in each fraction was then estimated using the Bradford assay (4). and the colorimetric reaction was read at 414 nm in a plate reader.Ayilavarapu et al. the reactions were stopped by ice-cold extraction buffer.6). modified iPLA2 Ca2+-free buffer. 192 mM glycine. The supernatant (whole cell fraction) was further centrifuged at 11. The lysate was then spun at 600 × g for 5 min at 4°C. 10 mM HEPES. The final composition of SDS sample buffer after mixing was 2% (w/v) SDS.3 mM Tris-HC1 (pH 6. Neutrophils (4 × 108) were lysed in 1 ml cold buffer consisting of 50 mM HEPES (pH 7.000 × g for 30 min at 4°C. Neutrophils were lysed and fractionated as mentioned above by adding 40 μl of six times SDS sample buffer to 200 μl of the reaction mixture and then boiling the samples for 5 min. 10 mM EGTA. . 58. a modified Ca2+-free assay buffer was prepared comprising 300 mM NaCl. Ten microliters of the sample was incubated in a cPLA2 assay buffer containing 20 mM CaCl2.5% or 10% (v/v) polyacrylamide slab gels.5% Triton X-100 and 1 mM PMSF by ultrasonication five times at 30 W for 5 s and incubated for 20 min at 4°C. The pellet was suspended in ice-cold extraction buffer with 0. The cell suspension was then sonicated in an ice-cold water bath in two bursts of 15 s each at 1. 1% (v/v) protease inhibitor mixture. In experiments where cells were stimulated with fMLP.5 amplitude at a setting of 30 mA.4)] at 100 V for 1 h at 4°C.4) and 1 mM EDTA. The results were analyzed through a software interface and spreadsheet for the calculation of relative expression (GAPDH/β-actin or iPLA2/β-actin). 6% (v/v) glycerol. 8 mM Triton X-100. The reaction mixtures were kept at 50°C for 2 min (one cycle). and the fractions were prepared as described above. The whole cell lysate was centrifuged at 1000 × g for 5 min at 4°C. Briefly. and the membranes were NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript J Immunol.8). Applied Biosystems). 1% (weight for weight) protease inhibitor mixture] and ultrasonicated five times at 30 W for 5 s.002% (w/v) bromophenol blue. 4 mM EGTA. To determine the activity of iPLA2. 10 mM HEPES. Membranes were blocked for 1 h at room temperature with 5% skim milk in TBS (pH 7. Western blot The protein samples for Western blotting were prepared as described in the literature (4). The authenticity of the cell fractions was verified using the plasma membrane marker Na+/K+ ATPase. The reaction was stopped by the addition of 10 μl 5'. 25 mM NaCl. 60% glycerol. and the supernatant was further centrifuged at 11. neutrophils (25 × 106/ ml) were lysed in ice-cold extraction buffer [20 mM Tris-HCl (pH 7. available in PMC 2010 March 30. The protein concentration from each fraction was determined by Bradford protein assay. 5% (v/v) 2-ME. Page 4 Control). The resulting pellet was discarded.5'-dithio-bis(2nitrobenzoic acid)/EGTA.4) (22). iPLA2 activity was calculated and expressed as nanomoles thiophosphatidylcholine produced/ min/ml.000 × g for 30 min.4). and arachidonylthiophosphatidylcholine for 60 min in a 96-well plate. Cell fractionation Cell fractionation was performed as described previously (4). A cPLA2 assay kit was obtained from Cayman Chemicals. and 2 mg/ml of BSA (pH 7. The separated proteins were immediately transferred electrophoretically to polyvinylidene difluoride (PVDF) membranes utilizing transfer buffer [25 mM Tris. Quantification was performed in an automated thermal cycler (ABI Prism 7000 Sequence Detector. and 1 mM PMSF. 0. 2 mM EDTA. The resulting pellet was the membrane-rich fraction and the supernatant was the cytosol-rich fraction. and 20% (v/v) methanol (pH 8. iPLA2 activity assay A modified iPLA2 activity assay was used as described previously (21) to detect the activity of iPLA2.

002% (w/v) NaN3. and whole cell lysates were collected for Western blotting experiments.Ayilavarapu et al. Superoxide generation was evaluation for siNT and si-iPLA2– transfected neutrophils. cells (4 × 106) were resuspended in 100 μl of Nucleofector solution in Amaxa-certified cuvettes (Amaxa. The blots were then stained with an antiactin Ab (1:2000 dilution) to confirm that equal amounts of protein were present in each lane of the gel.1% (v/v) Tween 20. and 1:500 for Phospho PKC substrate) overnight at 4°C in 20 mM Tris-HCl (pH 7. antihuman 1:200) or propidium iodide (PI) to label apoptotic neutrophils. Statistical analysis All data are presented as the average of at least three experiments with SEM unless otherwise stated. or small interfering iPLA2 (si-iPLA2). The activity of HRP was visualized by incubating the membranes for 5 min at room temperature in an ECL detection system (Pierce Chemical Co. 10.7). Author manuscript. 100 mM 2-ME] for 30 min at 56°C followed by three times washing in TBST. Membranes were washed three times in TBST. . MD).6) containing 150 mM NaCl and 0. Electroporation was set at S-11 program as per the manufacturer's recommendations.5 mM Tris-HCl (pH 6. On day 3. 5. Cells transfected with GFP were analyzed in a fluorescent microscope 24 and 48 h posttransfection to establish the transfection efficiency. available in PMC 2010 March 30. 2% SDS. cells were resuspended in prewarmed Human Monocyte Nucleofector medium (Amaxa) at a concentration of 1 × 106/ml. the Student t test was applied for significance. 1% (w/v) BSA. The device utilizes electroporation to create micropores in the cytoplasmic membrane of the cells and facilitates the entry of siRNA into the cytoplasm. 1:1000 in 3% skim milk for iPLA2. Total RNA was extracted using Trizol re-agent. 0. and neutrophils were resuspended in FACS buffer (PBS with 5% FBS) containing FITC-Annexin V (1:50) and PE-CD16 (mouse. Apoptotic neutrophils were characterized by double-positive staining for Annexin V and PI assessed by CellQuest software (BD Biosciences. 5. and neutrophils were immediately centrifuged at 100 × g for 10 min. and 20 μM).5 mM D-glucose. Membranes were stripped by incubation with reprobing buffer [62. CA). Rockford. The band density was measured using an imaging densitometer (Bio-Rad. Cells were counted after 3 d (72 h posttransfection) using a hemocytometer utilizing the trypan blue exclusion method. IL) followed by autoradiography. Page 5 incubated with the appropriate primary Abs (1:500 dilution for p47phox. 1:5000 dilution) in TBST for 1 h at room temperature. Hercules. Gaithersburg. The cuvettes were then transferred to an Amaxa Nucleofector device (Amaxa). After transfection. and for multiple group analysis. FACS Neutrophils (1 × 106/ml) were incubated with BEL (1.25% DMSO. The membranes were subsequently washed three times in TBST [20 mM Tris-HC1 (pH 7. Incubations were stopped on ice. Transfection experiments HL60 cells (1 × 106/ml) were differentiated for 3 d in RPMI 1640 medium supplemented with 1. The supernatant was removed. CA) and normalized to the actin band. or vehicle (0. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript J Immunol. For comparison between two groups. one-way ANOVA was used with post hoc Bonferroni or least significance difference corrections.6) containing 250 mM NaCl. San Jose.1% (v/v) Tween 20] and then incubated with secondary Ab (goat anti-mouse or rabbit IgG-HRP conjugate. and 0. 15. The cells received 1 μM GFP small interfering nontargeting (si-NT). small interfering GAPDH. and 10% HIFBS..1% DMSO and PBS) for 15 min at 37°C.

HL60 cells were differentiated into neutrophil like cells over a period of 6 d and were then cultured for 24 h with NG (5. if BEL-mediated inhibition of PAP-1 results in the suppression of superoxide generation. there was a significant reduction in superoxide generation in all groups. 1B). 2B).01–20 μM) as a specific inhibitor of cPLA2 and DTT (0. HG (25 mM). The combination of HG with S100B demonstrated significantly increased superoxide generation compared with HG alone (p < 0.01) and S100B+HG (p < 0. To investigate the role of PAP-1 in superoxide generation in neutrophils. BEL completely blocked superoxide generation in neutrophils from healthy and diabetic subjects (Fig. suggesting that the involvement of iPLA2 in superoxide generation is similar in neutrophils from healthy and diabetic subjects. Preincubation with pyrrolidine-1 (0. This suggests a role for glycemic control in neutrophil priming in the patient cohort studied (4–6). Neutrophils were preincubated with the inhibitor and stimulated with fMLP (1 μM). HL-60 cells were used for the knockdown of iPLA2 expression. Neutrophils from people with poorly controlled diabetes demonstrated significantly increased superoxide generation compared with healthy controls (Fig. and there was a 100% reduction in the superoxide generation in response to 20 μM BEL (Fig. 1C).001–1 mM)) for sPLA2 did not inhibit the superoxide generation in healthy neutrophils (data not shown). iPLA2 knockdown inhibits superoxide generation in neutrophils In order to confirm the specificity of iPLA2 in neutrophil super-oxide generation. HG alone did not significantly increase superoxide generation. 2A). Mannitol also demonstrated significantly increased superoxide generation compared with NG alone. 2A). Results from this experiment suggested that propranolol hydrochloride (150 μM) did not significantly inhibit superoxide reduction in neutrophils compared with BEL (Fig. When BEL was added.Ayilavarapu et al. Author manuscript. 5 μg/ml). 1A). BEL was used to investigate the specific involvement of iPLA2 in the generation of superoxide in neutrophils. propranolol hydrochloride. cells were transfected with si-iPLA2 J Immunol. Previous studies have suggested that BEL is also a potent inhibitor of phosphatidic acid phosphohydrolase-1 (PAP-1) (23). Hence. After 3 d. BEL inhibited superoxide generation in a dose-dependent manner (IC50. an in vitro system modeling the neutrophil response to hyperglycemia was established. demonstrating the impact of osmotic pressure changes on neutrophil superoxide generation. The experiment was repeated with neutrophils from people with diabetes. The superoxide response from these experimental groups show that compared with NG alone. RAGE ligand (S100B. Hyperglycemia-mediated increase in superoxide generation is mediated by iPLA2 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript To understand the mechanism of increased superoxide generation and the role of iPLA2 in diabetes. and superoxide generation was evaluated. The mannitol group served as an osmotic control for the hyperglycemia experiments.5 mM). For these experiments. S100B (p < 0.001) demonstrate significantly increased superoxide generation (Fig. a specific inhibitor of PAP-1. HL-60 cells were differentiated with 1. was used. RAGE ligand and HG (S100B+HG) or mannitol (25 mM). then the direct inhibition of PAP-1 would give the same results. PAP-1 catalyzes the conversion of phosphatidic acid into diacylglycerol via the Kennedy pathway. further confirming that the BEL-mediated inhibition of iPLA2 and not PAP-1 regulates superoxide generation in neutrophils.001). suggesting that the increase in superoxide generation is mediated by iPLA2 (Fig. the receptor-mediated stimulation of neutrophil function by the RAGE ligand S100B was more robust. however.25% DMSO for 6 d to create neutrophil-like cells. Page 6 Results Superoxide generation mediated by iPLA2 is increased in neutrophils from people with diabetes Table I compiles the demographic data of the study population. available in PMC 2010 March 30.10 μM). .

we show a representative image of increased iPLA2 protein in the whole cell fraction of neutrophils from people with diabetes. Page 7 using the Amaxa Nucleofector device (Amaxa). The QRT-PCR data shows that neutrophils from people with poorly controlled diabetes (Hb1Ac >8) had significantly increased iPLA2 mRNA compared with healthy controls (Fig. The next experiment was undertaken in the presence of EDTA and without calcium to specifically investigate iPLA2 activity in the membrane fractions of neutrophils from healthy and diabetes donors. Author manuscript. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript J Immunol. 3C). 3A). The data from iPLA2 knockdown experiments confirm the chemical inhibition of iPLA2 by BEL. diverse processes in neutrophils (14). Neutrophils treated with BEL (20 μM) were also counted in a hemocytometer using the trypan blue exclusion method to assess viability. by an increase in iPLA2 activity. 4A). and 20 μM BEL (Fig.Ayilavarapu et al. 5B). available in PMC 2010 March 30. The viability of the neutrophils was not impacted by the inhibition of iPLA2. 4B). Transfection efficiency was confirmed by the uptake of GFP and by the knockdown of GAPDH RNA and protein and was found to be 25%. and si-iPLA2. 3B shows the typical amplicon associated with the reduction of iPLA2 mRNA in the targeted group. 80%. The results of experiments using normal neutrophils show that the basal PLA2 activity in neutrophil cytoplasmic membrane preparations is significantly increased in the presence of EDTA (Fig. siGAPDH. and 57%. iPLA2 activity is increased in neutrophils from people with diabetes To investigate whether increased superoxide generation in neutrophils from people with diabetes is directly related to increased iPLA2 activity. 4B. a modified iPLA2 assay was used. 6B). respectively. To investigate possible increased expression of iPLA2 in neutrophils from diabetic subjects. Neutrophils from people with diabetes showed significantly increased expression of membrane-bound iPLA2 compared with healthy controls (Fig. FACS analysis was used to determine Annexin V expression (apoptotic marker) and PI binding to DNA (necrotic marker). There was no significant difference in the Annexin V-positive neutrophils incubated in PBS. The iPLA2 mRNAwas comparable between the control and nontargeted groups. QRT-PCR and Western blotting experiments were undertaken. Fig. 5A).1%). we wanted to examine the involvement of iPLA2 in apoptosis of neutrophils. This knockdown resulted in 30% inhibition of fMLP-stimulated superoxide generation (Fig. Western blotting reveals that iPLA2 was exclusively expressed in the membrane fraction of the neutrophils with no expression in the cytosol. . suggesting that iPLA2 is the key PLA2 isoform involved in superoxide generation by neutrophils. which is mediated. DMSO (0. The iPLA2 protein migrates at 85 kDa on a 10% SDS-PAGE gel (data not shown). iPLA2 does not regulate cell apoptosis or cell death in neutrophils Because iPLA2 is associated with multiple. 6A). The membrane-bound iPLA2 activity is significantly increased in neutrophils from people with diabetes compared with healthy controls (Fig. Transfection with iPLA2 siRNA resulted in ~50% reduction in mRNA levels of iPLA2 (Fig. The transfected reagents were GFP. The cells were further incubated for 3 d. si-NT. Results also revealed that inhibition of iPLA2 did not induce significant apoptotic or necrotic cell death compared with the vehicle control (Fig. In Fig. suggesting that membrane-bound basal PLA2 activity is calcium-independent and confirming the specific involvement of iPLA2. FACS analysis confirmed that >95% of cells isolated were CD16+ neutrophils. iPLA2 expression is increased in neutrophils from people with diabetes and mediates superoxide generation The hypothesis to be tested is that superoxide generation is increased in neutrophils from people with diabetes. at least in part.

AA rescues neutrophil superoxide inhibition by BEL Our data suggests that inhibition of iPLA2 by BEL abolishes superoxide generation in neutrophils. The data shows that AA (10 μM) partly restores NADPH oxidase activity. Conversely.5). The data reveals that BEL completely inhibits superoxide generation by normal neutrophils. exhibit increased PKC activity. p47phox was not phosphorylated. diglyceride formation. Because BEL has been shown to have nonspecific actions (23). To investigate whether the inhibition of iPLA2 impacts PKC-dependent p47phox phosphorylation.25).Ayilavarapu et al. Author manuscript. and 180 s. and p47phox phosphorylation (5). This study demonstrates a role for iPLA2 in superoxide generation in neutrophils and shows that iPLA2 might at least. the cPLA2 inhibitor pyrrolidine-1 did not have activity on superoxide generation by neutrophils. which was also consistent with findings from in vitro hyperglycemia experiments (4). There was no significant difference in the phosphorylation of p47phox in cells treated with or without the iPLA2 inhibitor at 30. These findings strongly support a primed neutrophil phenotype in poorly controlled diabetes. AA alone did not stimulate the superoxide generation by neutrophils (data not shown). Maximum phosphorylation of p47phox was evident at 30 s with a gradual reduction at 180 s (Fig. A study on a human myeloid cell line PLB-985 expressing antisense cPLA2 mRNA failed to activate NAPDH oxidase activity. available in PMC 2010 March 30. 8). The Ab used for these experiments detects phosphorylated PKC substrates (26. which is consistent with previous reports (18). and in normal neutrophils cultured under hyperglycemic conditions. Therefore. This is the first study to demonstrate a role for iPLA2 in superoxide generation in neutrophils from people with diabetes mellitus. and addition of exogenous AA fully restored this activity J Immunol. in addition to p47phox. pleckstrin is also labeled (27). stimulation of apoptosis. contribute to neutrophil priming in diabetes. confirming previous findings (19). Neutrophils from poorly controlled diabetic subjects generate significant amounts of superoxide compared with healthy controls (4. To identify the role of iPLA2 in superoxide generation. Findings from this study suggest that the fMLP-induced iPLA2-dependent superoxide generation is independent of the PKC and p47phox phosphorylation. In un-stimulated cells. 7). priming and superoxide generation is prevented. normal neutrophils were stimulated with fMLP (1 μM) in the presence or absence of BEL (20 μM) for a period of 180 s because studies have shown that neutrophils stimulated for 180 s with fMLP induced p47phox phosphorylation and translocation (24.27). we used BEL. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Discussion Hyperglycemia-mediated priming of neutrophils results in an enhanced oxidative burst in diabetes mellitus (1–5). a chemical inhibitor known to potently inhibit iPLA2 (28). To prove our hypothesis that iPLA2-derived AA activates NAPDH oxidase. Increased superoxide generation by neutrophils from diabetes is blocked by specific inhibitors of iPLA2. we performed experiments to rule out BEL-mediated inhibition of PAP-1. Pleckstrin phosphorylation was also not changed with the inhibition of iPLA2. and are characterized by a premature translocation of p47phox to the plasma membrane. we added exogeneous AA to neutrophils treated with BEL (20 μM). resulting in an increased oxidative stress. in part. . 60. suggesting that the iPLA2 regulation of superoxide generation in neutrophils is independent of the phosphorylation of these PKC substrates. The data presented in this study extend and further clarify these findings. it is apparent that BEL is only effective at high concentrations in the range of 10 μM for inhibition of superoxide generation in neutrophils (18). resulting in superoxide generation (Fig. Taken together. and p47phox phosphorylation. Page 8 iPLA2 regulates superoxide generation independently of p47phox or pleckstrin phosphorylation The phosphorylation and translocation of p47phox to the cytoplasmic membrane is a key event in superoxide generation catalyzed by NADPH oxidase.

it would be interesting to investigate if the increase in the activity and expression of iPLA2 in neutrophils is associated with a reduction of activity and expression of cPLA2 and sPLA2.31). a novel 60. Our findings (Fig. The resulting data confirm the specificity of the involvement of iPLA2 in superoxide generation by the human neutrophils and further suggest that increased iPLA2 expression and function. we wanted to investigate whether increased expression and activity of iPLA2 was involved in priming. and protein level. LTB4. Because hyperglycemia primes neutrophils for enhanced superoxide generation and iPLA2 directly regulates this process. the expression of iPLA2 at the message. also known as group cPLA2-IVC. Our data suggest that superoxide generation is regulated by the iPLA2 rather than the cPLA2. suppresses bacterial killing by inhibiting NAPDH oxidase in alveolar macrophages (35). chelation of intracellular calcium releases calmodulin from its binding site on iPLA2 and renders it active. The increased cAMP-1 levels inhibited the phosphorylation and translocation p47phox to the cytoplasmic NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript J Immunol. We report that an increase in superoxide generation in neutrophils from people with diabetes is associated with an increase in iPLA2 activity. binding of calmodulin to the iPLA2 renders the enzyme inactive and release of calmodulin in the absence of calcium activates iPLA2 (30. Another PLA2 isoform has been shown to exhibit similar calcium-independent activity (32). a metabolite of AA. To this end. The data from our PLA2 activity experiments suggest that in the presence of EGTA. platelet-activating factor.Ayilavarapu et al. by facilitating the translocation of cytosolic components to the membrane. increasing the affinity of GTP binding sites in the membrane and enhancing the dissociation of Rac G protein from its regulatory molecule. and when neutrophil membrane fractions were treated with 4 mM EGTA in the absence of any exogenous calcium. 8) show that the suppression of O2− by BEL-mediated iPLA2 inhibition can be partly restored by AA. regulation of a proton channel.34). To date however. There is also evidence that PGE2.9-kDa membrane-bound calcium-independent cPLA2-γ. Page 9 (29). and the β form of pro-IL-1 (16). an assay developed previously was used to assess cPLA2 and iPLA2 activity independently. In the presence of intracellular calcium. It was shown that PGE2 increases cAMP-1 levels via activation of G protein coupled E prostanoid (EP) receptors EP2 and EP4. suggesting the activation of NADPH oxidase by AA. Another study in mice reported that disruption of the cPLA2-α gene suppressed neutrophil AA release but had no effect on NAPDH oxidase activity (17). Author manuscript. AA mediates priming of NADPH oxidase and enhances the production of superoxide generation when cells are challenged with a secondary stimulus like fMLP (34). to separate the activity of iPLA2 and cPLA2. iPLA2 activity is enhanced by depleting calcium levels (21). During the resolution phase. there was induction of sPLA2 (type II and V) and cPLA2. . We confirmed these findings with suppression of iPLA2 expression by RNA silencing. one of the mechanisms by which iPLA2 induces O2− release will be AA liberation from membrane phospholipids. AA can act by increasing the affinity of NADPH to the oxidase. suggesting that the predominant PLA2 activity from neutrophil membrane fractions is calcium independent and therefore reflects iPLA2 activity. Studies have shown that AA is necessary for the activation of O2− production and the associated H+ channel-mediated efflux (33. Hence. iPLA2 has also been shown to drive the onset of acute pleurisy-induced inflammation in a male Wistar rat model through the generation of PGE2. this group shows a significant increase in basal PLA2 activity compared with the activity in the presence of 20 mM of exogenous calcium. available in PMC 2010 March 30. which showed that iPLA2 activity was increased in the presence of BAPTA-AM or thapsigargin (calcium chelators) (22). the expression of cPLA2-γ in neutrophils is not known and warrants further investigation. contribute to the neutro-phil priming in people with diabetes. The results are in agreement with data from another study. in part. GDI (34). This calcium-independent PLA2 activity was significantly elevated in neutrophils from diabetes. We used specific cPLA2-α inhibitor pyrrolidine-1 and DTT to rule out the involvement of cPLA2-α and sPLA2 in neutrophil superoxide generation. Because the complications of diabetes are associated with inflammation.

Ayilavarapu et al. enhancing reactive oxygen species output (19). 2A). This work was supported in part by United States Public Health Service Commissioned Corps Grants DE-15566. BEL significantly blocked the hyperglycemia-mediated priming of iPLA2 under these conditions.37). Neutrophils cultured under hyperglycemia-like conditions demonstrated significantly elevated superoxide generation compared with NG alone (Fig. DE-16933. this study demonstrates that increased iPLA2 expression and activity in people with uncontrolled diabetes might. AA is directed to superoxide generation and cPLA2-produced AA is primarily routed toward eicosanoid generation (18). The interpretation of a 30% knockdown in activity with 25% transfection efficiency as significant is consistent with the literature (38). and RR-00533. DE-18917. Our findings confirm the existence of these two routes where inhibition of iPLA2 by BEL inhibited fMLP-induced superoxide generation. The data reveals that the transfection efficiency in HL-60 cells was 25%. it is possible that excess release of AA by iPLA2 in part. and PGE2. and this was not due to suppression of p47phox phosphorylation. we were able to provide evidence that iPLA2-mediated AA release. AA. Like other investigators. Because AA partly rescued the superoxide inhibition by BEL. 2A). We also confirmed the role of iPLA2 in superoxide generation in a differentiated neutrophil-like HL60 model by suppressing iPLA2 mRNA expression.7). The data reported in this study suggest novel pathways of host immune response in the regulation of NADPH oxidase activity by iPLA2. in the case of iPLA2. However. The inhibition of superoxide by BEL in differentiated HL60 cells was 75–80% (Fig. contributes to the priming mechanism in hyperglycemic conditions. Page 10 membrane (35). contributes to NADPH oxidase activity. . We also determined the transfection efficiency using GFP as a marker. The regulation of superoxide generation in neutrophils by iPLA2 involves a mechanism that is independent of PKC and PAP-1 hydrolase signaling. Amanda Blackwood for laboratory assistance. which is similar to findings of Magalhães et al. In conclusion. contribute to the priming of neutrophils for enhanced superoxide generation upon secondary stimulus and confirms the regulatory role of iPLA2 in neutrophil superoxide generation. (38). available in PMC 2010 March 30. in part. In this study. we are unable to achieve 100% transfection efficiency with HL-60 cells. This was pronounced in neutrophils cultured with S100B. Author manuscript. simultaneous activation of both of these pathways is critical in the generation of superoxide anion in neutrophils. at least. suggesting a mechanism that involves iPLA2 activity. at least in part. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Neutrophils prelabeled with [3H] AA display increased [3H] arachidonate release on exposure to advanced glycation endproduct-albumin over exposure to albumin alone (19). Studies have demonstrated that two parallel pathways regulate NADPH oxidase activation: p47phox phosphorylation and PLA2 activation (36. It may be speculated that different pools of AA exist that serve different functions. Abbreviations used in this paper AA BEL cPLA2 arachidonic acid bromoenol lactone cytosolic PLA2 J Immunol. Advanced glycation endproduct augmentation of the activated neutrophil respiratory burst required AA generation. through which neutrophil NADPH oxidase may be upregulated. Acknowledgments We thank Ms. which is known to bind the RAGE receptor and induces significant phosphorylation of p47phox and its premature translocation to the cytoplasmic membrane (4.

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Data are shown as nanomoles of O2− expressed as mean ± SEM for six different donors. Data are represented as percent inhibition and are expressed as mean ± SEM for three different donors. Data is presented as the mean ± SEM in nanomoles of O2− for healthy (n = 27). B. and superoxide generation was evaluated using a superoxide dismutase-inhibitable cytochrome C assay. Neutrophils (25 × 106/ml) isolated from healthy and diabetic subjects were resuspended in PBS and incubated at room temperature for a period of 15 min. moderately controlled diabetes (n = 6). The intensity of the colorimetric reaction was measured in a microplate reader at 550 nm. NIH-PA Author Manuscript NIH-PA Author Manuscript J Immunol. well-controlled diabetes (n = 8). Neutrophils from poorly controlled diabetic subjects generate significantly increased superoxide regulated by iPLA2. Neutrophils were resuspended in PBS and incubated at room temperature for a period of 15 min and were then stimulated with 1 μM fMLP and superoxide generation evaluated. and superoxide generation was evaluated using a superoxide dismutase-inhibitable cytochrome C assay. Cells were then stimulated with 1 μM fMLP. Neutrophils (25 × 106/ml) from healthy and diabetic subjects were incubated with BEL (20 μM) for 15 min at 37°C and washed once with PBS. Page 14 NIH-PA Author Manuscript FIGURE 1. Cells were then stimulated with 1 μM fMLP. and poorly controlled diabetes (n = 13). The intensity of the colorimetric reaction was measured in a microplate reader at 550 nm. . Author manuscript. Neutrophils (25 × 106/ ml) were incubated with the indicated concentrations of BEL for 15 min at 37°C and then washed once with PBS. available in PMC 2010 March 30. C. A. Neutrophils were resuspended in PBS and incubated at room temperature for a period of 15 min.Ayilavarapu et al.

HL-60 cells were differentiated into neutrophils by incubation with 1. #p<0. Results are expressed as mean ± SEM for n=4. NIH-PA Author Manuscript NIH-PA Author Manuscript J Immunol. and superoxide generation was evaluated in nanomoles of O2−. . S100B. ##p<0. Results are expressed as mean ± SEM for n= 3. **p < 0. and 25 mM mannose (MANN) as an osmotic control. Page 15 NIH-PA Author Manuscript FIGURE 2. *p < 0. Author manuscript. and mannitol). S100B+HG. The cells were stimulated with 1 μM fMLP. the cells were washed and resuspended in PBS and were incubated with BEL 20 μM for 15 min at 37°C. available in PMC 2010 March 30. Superoxide release was evaluated by the reduction of cytochrome C read at 550 nM. which is regulated by iPLA2.05. Neutrophils (25 × 106/ml) were incubated with either BEL (20 μM) or propranolol hydrochloride (150 μM) for 15 min at 37°C. HG + S100B. Cells were washed once and resuspended in PBS and were stimulated with 1 μM fMLP. A.25% DMSO for 6 d. After 24 h. S100B (5 μg/ml).01 (oneway ANOVA between stimulated and BEL-treated groups). The cells were then cultured under NG (5 mM). Neutrophils cultured in hyperglycemic medium generate significantly increased superoxide. B. HG (25 mM).Ayilavarapu et al.01 (one-way ANOVA between groups NG.05.

Real-time PCR using probes for actin and iPLA2 was set to 45 cycles to estimate the fold change of iPLA2 normalized to actin. Page 16 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript J Immunol.Ayilavarapu et al. Figure shows a typical amplicon of real-time PCR. HL60 cells (1 × 106 /ml) were differentiated with 1. On day 3. C. and RNA was extracted by Trizol reagent and reverse transcribed to cDNA. cells were collected. Cytochrome C reduction was read in a spectrophotometer at 550 nm for triplicate samples. After 72 h. . Nontransfected differentiated cells served as controls.5 mM) for 3 d. iPLA2 knockdown inhibits super-oxide generation in neutrophils A. available in PMC 2010 March 30. FIGURE 3. B. p < 0. Author manuscript. cells were transfected with nontargeting siRNA (1 μM) and si-iPLA2 (1 μM) by nucleofection and re-suspended in the differentiation media.25% DMSO in RPMI 1640 media supplemented with 10% HIFBS and D-glucose (5. Cells transfected with siiPLA2 and nontargeting siRNA were counted and resuspended in PBS at a concentration of 25 × 106/ml and stimulated with 1 μM fMLP. Data are presented as nanomoles of O2− and expressed as mean ± SEM.05.

FIGURE 4. Author manuscript. iPLA2 membrane fractions and one representative whole cell image was quantified by densitometry and normalized to actin. Neutrophils from people with poorly controlled diabetes mellitus exhibit increased expression of iPLA2.05. One hundred nanograms of cDNA was used for QRT-PCR using probes for actin and iPLA2 to estimate the fold change of iPLA2 normalized to actin for healthy (n = 23). well-controlled diabetes (n = 6). Results are expressed as mean ± SEM. available in PMC 2010 March 30. moderately controlled diabetes (n = 6). A. and 1 μg of RNA was used to prepare cDNA using RTPCR. p < 0. Page 17 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript J Immunol. The membranes were probed overnight with iPLA2 Ab (in 3% skim milk). and poorly controlled diabetes (n = 10). n = 6. Total RNA was extracted from resting neutrophils from healthy and diabetic subjects. Twenty micrograms of whole cell and membrane fraction was run on 10% SDS-PAGE gel and transferred to a PVDF membrane.Ayilavarapu et al. B. .

Data represent percent increase of iPLA2 activity over healthy controls and are expressed as mean ± SEM for n = 4. Calcium-independent PLA2 activity is increased in membrane fractions of neutrophils from diabetic subjects. available in PMC 2010 March 30. Page 18 NIH-PA Author Manuscript FIGURE 5. . The reaction was initiated by the addition of sample to the substrate arachidonyl thiophosphatidyl choline. Results are expressed as mean ± SEM for n = 3. and the plate was read at 415 nm. B. thus activating calcium-independent PLA2. p. The buffers for this assay were calcium free and contained EDTA to chelate all calcium. Author manuscript.5'-dithio-bis(2nitrobenzoic acid)/EGTA.Ayilavarapu et al. Cells were then fractionated to isolate the membrane components.05. The reactions were stopped by the addition of 5'. The membrane fraction of neutrophils from diabetic subjects demonstrated a significant increase in calcium-independent PLA2 activity. Neutrophils (4 × 108/ml) were sonicated in a homogenization buffer supplemented with protease inhibitor. 0. NIH-PA Author Manuscript NIH-PA Author Manuscript J Immunol. A.

Neutrophils (10 ± 106/ml) were incubated with PBS. NIH-PA Author Manuscript NIH-PA Author Manuscript BEL does not stimulate apoptosis in neutrophils.1%). . and data are represented as percent cells positive and as mean ± SEM for n = 3. B. and BEL (1– 20 μM) for 15 min at 37°C. Author manuscript. Cells were labeled with Annexin V or PI to detect apoptotic and necrotic neutrophils. available in PMC 2010 March 30.and PI-positive cells. DMSO (0.Ayilavarapu et al. The upper right quadrant of the dot blot represents Annexin V. Page 19 NIH-PA Author Manuscript FIGURE 6. Annexin V-positive cells were counted and are represented on a bar graph. The histogram represents Annexin Vpositive cells at a threshold setting of 102 FL1-H. J Immunol. A.

FIGURE 7. BEL does not inhibit the phosphorylation of p47phox. . Author manuscript. Results are expressed as mean ± SEM for n = 3. The membranes were stripped and reprobed with anti-p47phox (1:500). and samples were prepared for Western blotting. Page 20 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript J Immunol. or 180 s. 60. Equal amounts of protein (10 μg/lane) were loaded on 10% SDS-PAGE gels and transferred to PVDF membranes. The reaction was stopped by adding ice-cold lysis buffer. available in PMC 2010 March 30. Membranes were then probed with anti-PKC phosphoserine substrate Ab (1:500). Data were obtained by a densitometry. and the results are presented as phospho-p47phox/total p47phox ratio.Ayilavarapu et al. Neutrophils (25 × 106/ml) were incubated with or without BEL (20 μM) and then stimulated with 1 μM fMLP for 30.

Neutrophils (25 × 106/ml) were incubated with BEL (20 μM) for 15 min at 37°C and then washed at 1000 rpm for 10 min at room temperature with PBS. Author manuscript.Ayilavarapu et al. Superoxide generation released was assessed by cytochrome C reduction and was read in a spectrophotometer at 550 nm for triplicate samples. . Cells were then incubated with AA (10 μM) at 37°C for 30 min before being stimulated by 1 μM fMLP. available in PMC 2010 March 30. Data are presented as nanomoles of O2−. Page 21 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript J Immunol. AA partly restores the activity of iPLA2 in superoxide generation in neutrophils. FIGURE 8. Results are expressed as mean ± SEM for n = 3.

93 19 20 9 23 J Immunol. based on HbA1C %)  Poorly controlled (>8% HbA1C)  Moderately controlled (7–8% HbA1C)  Well controlled (<7% HbA1C) Duration of diabetes (mean in y ± SD) Periodontal condition (no.)  Type1  Type 2 Mean HbA1C % (± SD) Glycemic control (no.)  Healthy  Mild  Moderate  Severe There were no significant differences between the age groups..)  Male  Female Ethnicity (no.42 22 19 20 8. Page 22 Table I Demographic data of healthy and diabetic subjects NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Healthy n Age (mean in y ± SD) Gender (no.Ayilavarapu et al.6 43 28 39 27 3 2 33 18 20 12 59 8.)  African-American  Caucasian  Hispanic  Asian Smoking status (no.84 ± 6. 60 3 8 15 47 4 5 43 28 71 36. . HbA1C. available in PMC 2010 March 30.)  Nonsmokers  Smokers  Former smokers Type of diabetes (no. Author manuscript.6 ± 10.0 ± 1.5 ± 9. glycated hemoglobin.3 Diabetic 71 54.