Bruzzone et al. 10.1073/pnas.0609379104. |
Fig. 5. (A) Chemical structures of cis, trans ABA (a), trans, trans ABA (b), ABAmethylamide (c), and trans, trans RA (d). The asterisk indicates the chiral carbon, responsible for generation of the (+) and (-) ABA enantiomers. (B) Effect of ABA analogs on the [Ca2+]i of human granulocytes. The arrow indicates addition of 20 mM cis, trans ABA (black circles); trans, trans ABA (black triangles); ABA-methylamide (black squares); or trans, trans RA (white triangles) to Fura-2-AM loaded cells in HBSS. The [Ca2+]i rise triggered by 20 mM trans, trans ABA (black triangles) was due to contaminant cis, trans ABA (0.1 mM, white circles). Traces are from individual experiments, representative of results obtained from at least three experiments. (C) Granulocytes were preincubated for 50 min in HBSS (control, black squares), or in HBSS containing 100 mM 8-Br-cADPR (open squares) and then loaded for further 40 min with Fluo-3-AM. IL-8 (100 nM) was added at 60 sec, and [Ca2+]i was measured in parallel by using a fluorescence plate reader, as described in ref. 1. Representative traces from 3 different experiments, yielding closely comparable results, are shown. Preincubation of granulocytes with ryanodine (50 mM) also did not modify the Ca2+ response to IL-8 (data not shown).
1. Zocchi E, Franco L, Guida L, Benatti U, Bargellesi A, Malavasi F, Lee HC, De Flora A (1993) Biochem Biophys Res Commun 196:1459-1465.
Fig. 6. (A) GDP-ribosyl cyclase was evaluated on control, untreated cells or on cells preincubated with K252a (1 mM for 30 min), with a specific PKA-inhibitor (1 mM for 30 min), with a specific PKC-inhibitor (50 nM for 60 min), or with PTX (2 mg/ml for 1 h). Higher concentrations of the PKC inhibitor did not further increase the percentage of inhibition. GDP-ribosyl cyclase values were calculated at 5 min. (B) CD38 was immunopurified, as described in SI Materials and Methods, from control and ABA-treated (for 10 min with 20 mM ABA) granulocytes. Samples were run in duplicate (control, lanes b and d; ABA-treated, lanes a and c); Western blots were stained with the anti-CD38 antibody (lanes a and b) or with an antiphosphoserine monoclonal antibody (lanes c and d). Results from one of three different experiments, yielding comparable results, are shown. The NetPhos 2.0 server (Center for Biological Sequence Analysis, Technical University of Denmark, Lyngby, Denmark) predicts three serine residues as possible phosphorylation sites in the intracellular domain of human CD38, Ser 19 scoring the highest phosphorylation potential (0.982). The NetPhosK 1.0 (Center for Biological Sequence Analysis) also assigns the highest score (0.68) to the phosphorylation on Ser 19 by PKA.
Fig. 7. Binding of biotinylated ABA to human granulocytes. Granulocytes were incubated with 10 mM biotinylated ABA in the absence (A and C) or in the presence (B and D) of excess unlabelled ABA (1 mM) for 5 min. Cells were then washed and incubated with Alexa-strp for further 5 min. Images are from one representative experiment of six. No fluorescence was detectable on cells incubated with Alexa-strp alone or with 10 mM biotin and then with Alexa-strp (data not shown). Low magnification (A and B), single plane images. High magnification (C and D), Z-stacks of 50 sections for a total thickness of 5.5-6 mm. Insets, corresponding phase contrast light images.
Fig. 8. Binding of [3H]-ABA to human granulocytes. Scatchard plot analysis of high- (A) and low-affinity (B) ABA-binding sites in intact human granulocytes. For experimental details, see SI Materials and Methods.
Fig. 9. ABA-induced phagocytosis, ROS and nitrite generation, chemotaxis and chemokinesis in human granulocytes. (A) Granulocytes were pretreated (open squares, not treated) for 15 min at 25°C with different ABA concentrations (50 nM, closed circles; 1 mM, open circles; 20 mM, closed squares) and then incubated for the time indicated in the presence of fluorescent latex beads. Closed triangles, PTX-pretreated cells, incubated with 20 mM ABA. Results are expressed as fluorescence increase relative to time 0. SD bars, omitted for the sake of clarity, were always £18% of the mean values; n = 4. (B) Granulocytes were preincubated for 1 h at 25°C in the presence or absence of 100 mM 8-BrcADPR or 2 mg/ml PTX. Cells were then loaded with the ROS-specific fluorescent probe H2DCFDA, placed in 96-well plates, and challenged with 20 mM ABA. Fluorescence was monitored as described in the SI Materials and Methods. Results are expressed as fluorescence increase at time 10 min, compared to time = 0. Results are the mean ± SD from at least three determinations for each condition. (C) Granulocytes, isolated from 2 subjects (open bars, subject 1; closed bars, subject 2), were treated for 4 h at 37°C with ABA at the concentrations indicated and nitrite generation was measured. Results are expressed as percentage of control values, measured in unstimulated cells and are the means ± SD of three determinations. The basal concentrations of nitrite were 0.98 ± 0.08 and 1.67 ± 0.17 mM in subject 1 and 2, respectively. (D) Migration of granulocytes isolated from two subjects representative of a high- (open bars) and of a low-responder (closed bars) through 3-mm pore membranes toward a solution containing ABA at the concentrations indicated, was measured in ChemoTx chambers and expressed as chemotaxis index (CI). Results shown are the mean ± SD from three different determinations for each subject (P < 0.001). Granulocytes from both subjects were also preincubated with PTX (2 mg/ml for 1 h, gray bar) or with 8-Br-cADPR (100 mM for 90 min, striped bar) and then challenged with 20 mM ABA. Results shown are the mean ± SD from three experiments for each subject (P < 0.001 compared to chemotaxis index values of untreated granulocytes).
Fig. 10. Detection of ABA by HPLC-MS. A representative sample analysis is shown. (A) Total ion chromatogram (TIC). (B) ion chromatogram of m/z 263.3 extracted from the TIC. (C) Deprotonated ABA molecular ion (m/z 263.3). (D) ABA MS/MS spectrum (m/z 153.1 and 219.2 are two diagnostic ions for cis, trans ABA, obtained from ion 263.3).
Fig. 11. ABA release from granulocytes stimulated with zymosan or latex beads. Granulocytes (5 ´ 107/determination) were incubated in 2.0 ml of HBSS for 30 min at 20°C without (control) or with zymosan (0.37 mg/ml) or latex beads. The ABA content in cells and supernatants was determined by ELISA. Results are expressed as picomoles of ABA detected in the cells (white bars), or in the supernatants (gray bars). Results shown are the mean ± SD from four experiments performed on granulocytes from different subjects. Black bars, sum of mean values of intracellular and released ABA.
Table 1. ABA stimulates A(G)DP-ribosyl cyclase, but not NAD+-ase and cADPR hydrolase, activity in granulocytes
Enzymatic activity | Basal pmol/min/mg | ABA-treated pmol/min/mg |
ADPRC (n = 4; P < 0.05) | 8.17 ± 1.36 | 13.88 ± 2.01 |
NAD+-ase (n = 4) | 750 ± 168 | 794 ± 112 |
cADPR hydrolase (n = 3) | 46 ± 5 | 45 ± 3 |
GDPRC (n = 11; P < 0.005) | 129 ± 41 | 230 ± 52 |
Intact granulocytes were incubated with 20 mM ABA in the presence of the substrates NAD+, cADPR, or NGD+, and production of cADPR, ADPR, ADPR, and cGDPR was evaluated for estimation of ADPRC, NAD+-ase, cADPR hydrolase and GDPRC activities, respectively. The enzymatic activities were measured at 5 min (A/GDPRC and NAD+-ase) or at 60 min (cADPR hydrolase) from the addition of ABA and the substrate.
Table 2. Nitrite production induced by ABA in granulocytes from different subjects
Subject | % of Basal value |
1 | 183 |
2 | 184 |
3 | 220 |
4 | 259 |
5 | 451 |
6 | 750 |
7 | 840 |
8 | 942 |
Median | 355 |
Range | 183-942 |
Granulocytes were isolated from eight different subjects and stimulated in RPMI medium for 4 h at 37°C with 20 mM ABA. Nitrite production is expressed as percentage of the basal production in unstimulated cells from the same subject.
Table 3. Chemotaxis induced by ABA in granulocytes from different subjects
Subject | Chemotaxis index |
1 | 1.51 |
2 | 2.43 |
3 | 2.63 |
4 | 4.75 |
5 | 6.44 |
median | 2.63 |
range | 1.51-6.44 |
Migration of granulocytes (obtained from five different subjects) through 3 mm-pore membranes, towards a solution containing 20 mM ABA was measured in ChemoTx chambers and expressed as chemotaxis index (CI).
SI Materials and Methods
Materials.
The anti-CD38 monoclonal antibody (IB6) was a generous gift of F. Malavasi (University of Torino, Torino, Italy). Fura-2 a.m., protein kinase inhibitors (K252a; protein kinase A inhibitor peptide sequence 14-22, cell-permeant, myristoylated; and bisindolylmaleimide I, a specific protein kinase C inhibitor) and Ro-20-1724 were purchased from Calbiochem (Milan, Italy). Dichlorodihydrofluorescein diacetate (H2DCFDA, D-399) and SYTOX Green were obtained from Molecular Probes (Eugene, Oregon). Ficoll-Paque Plus and [3H](±)-ABA (40 Ci/mmol) were from Amersham Pharmacia Bioscience AB. Chemical synthesis of biotinylated ABA was performed as described in ref. 1, whereas synthesis of C1 methylamide of (±)-abscisic acid (ABA-methylamide) was performed as follows: (±)-cis, trans abscisic acid (10 mg, 38 mmol) was dissolved in 1 ml of N,N dimethylformamide (Biosolve, Valkenswaard, The Netherlands), and O-(7azabenzotriazol-1-yl)-1,1,3,3 tetramethyluroniumhexafluorophosphate (HATU) (14.5 mg, 38 mmol) (Advanced Biotech, Seveso, Italy) was slowly added under stirring in the dark. After a few minutes, a 40% solution of methylamine (6 ml, 75 mmol) was added, and the reaction was stirred at 25°C, protected from light, for 24 h. When reaction was complete, the solvent was evaporated under vacuum, and the residue was dissolved in about 3 ml of deionized water. The pH was adjusted to 9 with addition of NaOH 0.1N, and the solution was extracted with diethyl ether (3 ´ 5 ml). The organic solution was evaporated under vacuum. The residue was purified by reverse phase high performance chromatography on a Shimadzu LC-9A preparative HPLC equipped with a C18 RP Luna column (21.20 ´ 250 mm) (Phenomenex, Torrance, CA). The solvent program was a gradient starting with 100% solvent A for 5 min, linearly increasing to 70% solvent B in 35 min, and up to 100% B in 5 min. Solvent A was 0.1% trifluoroacetic acid (TFA) in water and solvent B was 0.1% TFA in acetonitrile. The molecular weight of ABA-methylamide (m/z = 277.3) was then confirmed by electrospray ion trap mass spectrometry (ESI-TRAP-MS). (±)-cis, transABA, the purified enantiomers (+)- and (-)-ABA and all other chemicals were obtained from Sigma. (±)-trans, transABA was obtained from AG Scientific (San Diego, CA), and HPLC-purified with the same analysis used for ABA purification from cell extracts (see below). [2H6]-ABA was prepared as described in ref. 2.Assays of ADP-ribosyl cyclase, GDP-ribosyl cyclase, NAD+-ase and cADPR-hydrolase activities.
Intact granulocytes were resuspended in HBSS (6 ´ 107/ml). Ectocellular ADP-ribosyl cyclase (ADPRC) and GDP-ribosyl cyclase (GDPRC) activities were measured, respectively, on NAD+ or on NGD+; an NAD+ analogue was used to determine cyclase activity because cGDPR is not a substrate of the hydrolase (3). ADPRC and GDPRC activities were estimated NAD+ or NGD+ (both 0.4 mM) in the absence (control) or in the presence of 20 mM ABA. cADPR-hydrolase and NAD+-ase activities were estimated by adding 0.2 mM cADPR and 0.4 mM NAD+ as substrates, respectively, in the presence or absence of 20 mM ABA. All assays of enzymatic activities were carried out at 37°C. At various times (0, 5, 15, 30, 60, 120 min), 100-ml aliquots were withdrawn and were centrifuged at 5,000 ´ g for 15 sec. Supernatants were recovered and deproteinized with 2.5% (vol/vol) trichloroacetic acid. HPLC analyses of nucleotides to measure the production of cGDPR (for GDP-ribosyl cyclase), cADPR (for ADP-ribosyl cyclase), or ADPR (for cADPR-hydrolase and NAD+ase) were performed as described in ref. 4. Protein determination on aliquots of each incubation saved before TCA addition was performed according to Bradford (5).Immunopurification of CD38 and Western blot analysis.
Freshly drawn granulocytes (1-ml suspension at 5 ´ 107/ml in RPMI medium 1640) were incubated at 25°C for 15 min in the absence (control) or in the presence of 20 mM ABA. Cells were centrifuged at 5,000 ´ g for 15 sec, washed twice with PBS, and resuspended in 200 ml of PBS. After addition of protease inhibitors (0.1 mM aprotinin, 0.1 mM leupeptin, and 10 mM pepstatin) and 1:100 phosphatase inhibitor mixture (Sigma; catalog no. P2850), control and ABA-stimulated cells were lysed by sonication in ice for 1 min at 3W (W380; Heat-System Ultrasonics, New York, NY). Immunopurification of CD38 was carried out, using the anti-CD38 monoclonal antibody IB6 coupled to Sepharose, as described in ref. 6. Elution from the resin was obtained by adding 2´ sample buffer, prepared according to ref. 7 but lacking EDTA and b-mercaptoethano l. Each sample was incubated at 85°C for 10 min. The resin was removed by centrifugation, and the supernatant was analyzed by SDS/PAGE. Eluates from control and ABA-treated samples were loaded in duplicate on a 10% SDS-polyacrylamide gel (7). Prestained molecular weight standards were run in parallel. Western blot was performed on PVDF membranes according to standard procedures (8). Immunodetection was obtained with the anti-human CD38 monoclonal antibody (Sigma) and with a monoclonal antiphosphoserine antibody (clone PSR-45; Sigma catalog no. P3430).Staining of human granulocytes with biotinylated ABA
. Freshly isolated granulocytes (106 per assay) were incubated in 100 ml of HBSS with 10 mM biotinylated ABA in the presence or absence of excess unlabelled ABA (1 mM) for 5 min on ice. Cells were then washed in HBSS by centrifugation and incubated for further 5 min on ice with 5 mg/ml Alexa 488-conjugated streptavidin (Alexa-strp; Molecular Probes, Carlsbad, CA). Controls included cells incubated with Alexa-strp alone and cells incubated first with 10 mM biotin, washed, and then incubated with Alexa-strp. Cells were then seeded on eight-wells chambered coverglasses (Lab-Tek, Nalge Nunc, Naperville, IL) at a cell density of 5 ´ 105 per well.Images were obtained by using a Leica TCS SL confocal microscope equipped with argon/He-Ne laser sources and a HCX PL APO CS 63.0 ´ 1.40 oil objective. During image acquisition, laser energy was set at 25%, emission range was between 500 and 600 nm, and the photomultiplier voltage gain was set to eliminate cell autofluorescence. At low magnification, single-plane images were taken at the center of cell thickness; for the higher magnification images (4´ digital zoom), Z-stacks of 50 sections were taken with a Z-step of 122 nm for a total thickness of 5.5-6 mm. Single images and average projections were processed using Leica LCS software.
Scatchard plot analysis of ABA-binding sites on human granulocytes.
Freshly isolated granulocytes (7 ´ 106 per determination) were incubated in triplicate at 20°C for 60 min in 50 ml of HBSS with or without excess unlabeled ABA (to determine nonspecific binding) in the presence of increasing concentrations of [3H]-ABA. At the end of the incubation time, cells were centrifuged (30 sec at 5,000 ´ g), the supernatant was discarded, cell pellets were washed once in excess HBSS by centrifugation, and the radioactivity of the cells was determined on a Packard b-counter. Specific binding was calculated as the difference between the total binding of [3H]-ABA to the cells and the [3H]-ABA binding in the presence of excess unlabeled ABA (nonspecific binding).Phagocytosis -
Granulocytes (107/ml) were preincubated for 20 min in a total volume of 0.4 ml HBSS without (control) or with 0.05, 0.25, 1, or 20 mM ABA. Fluorescent latex beads (Sigma, catalog no. L5655) were then added (2.5 ´ 105 beads/100 ml): after 0, 10, 15, 30 min, 100 ml of each suspension was centrifuged at 5,000 ´ g for 15 sec. Cell pellets were washed with 1 ml of ice-cold HBSS and resuspended in 350 ml of ice-cold HBSS. The fluorescence of 100-ml aliquots was determined in triplicate by using a fluorescence plate reader (Fluostar Optima; excitation, 485 nm; emission, 590 nm) (BMG Labtechnologies, Offenburg, Germany). Preincubation with PTX (2 mg/ml for 1 h) was performed, when needed, before ABA addition.Determination of ROS and NO production.
Granulocytes were resuspended at 107/ml in HBSS and loaded for 30 min with 10 mM dichlorodihydrofluorescein diacetate (H2DCFDA). Cells were centrifuged at 5,000 ´ g for 15 sec, washed with HBSS, and resuspended at 1 ´ 106/ml in HBSS. After 30 min at 25°C, cells were placed on 96-well plates and ABA was added (or for control, not added). Fluorescence was monitored every 10 sec, over a 10-min period, with a fluorescence plate reader (Fluostar Optima; excitation, 485 nm; emission, 590 nm; BMG Labtechnologies). Granulocytes (6 ´ 107/ml) were incubated for 60 min at 37°C in 24-well plates in RPMI medium 1640, without (control) or with ABA at 0.05, 0.25, 1, 5, or 20 mM, then collected and centrifuged at 5,000 ´ g for 15 sec. Nitrite content in the supernatants was evaluated as described in ref. 9.Chemotaxis and chemokinesis.
Granulocytes were resuspended at 107/ml in chemotaxis buffer (HBSS, PBS, and 5% albumin, 39:16:1). Chemotaxis and chemokinesis assays were performed using 96-well ChemoTx system microplates (Neuro Probe, Gaithersburg, MD) with a 3-mm pore size polycarbonate filter. For chemotaxis assays, ABA was diluted at different concentrations in chemotaxis buffer and added to the bottom wells. For chemokinesis assays, cells were preincubated for 20 min in the presence or absence of 20 mM ABA; chemotaxis buffer was added in the bottom wells. Cell suspensions (25-ml) were placed directly on top of the filter and the plates were incubated for 60 min at 37°C. The transmigrated cells were collected following ChemoTx system instructions, transferred into a 96-well plate, and quantified by adding 60 ml of a solution composed of 0.2% Nonidet P-40 and 1 mM SYTOX Green. After 20 min of incubation at 37°C, fluorescence was recorded (excitation, 485 nm; emission, 520 nm). A standard curve was obtained by placing a serial dilution of the cell suspension in the bottom wells. The results were expressed as chemotaxis index (chemotaxis index = N° of cells migrated toward chemoattractant/ N° of cells migrated toward medium) and chemokinesis index (chemokinesis index = N° of ABA-treated cells migrated toward medium/N° of untreated cells migrated toward medium).Detection of ABA by ELISA and by HPLC-coupled mass spectrometry (HPLC-MS).
Granulocytes (25 ´ 106/ml) were centrifuged at 5,000 ´ g for 15 sec: cell pellets were resuspended in 500 ml of water, 50-ml aliquots were saved for protein determination and 2 ml of methanol were added to the rest of the cell lysates. Trace amounts of [3H]-ABA (3 ´ 103 cpm) were added as internal standard to each sample, and free ABA was extracted as described in ref. 10. For measurement by ELISA, extracts were assayed with a sensitive and specific kit (Agdia) according to the manufacturer's instructions. Conjugated ABA was extracted at pH 11 from the aqueous phase following acid extraction of the free hormone, as described in ref. 11. For measurement by HPLCMS, acid and alkaline extracts were injected into an analytical Atlantis (Waters, Milford, MA) dC18 column (3.9 ´ 150 mm; particle size, 5 mm). Buffer A was water containing 0.01% trifluoroacetic acid (TFA), buffer B was 80% acetonitrile in water containing 0.01% TFA, and the flow rate was 0.8 ml/min. The gradient was from 100% A to 100% B in 30 min. Fractions were collected every 30 sec: the radioactive fraction(s) were dried and redissolved in 11 ml of 90% buffer A and 10% buffer B of the HPLC-MS analysis (see below), containing [2H6]-ABA as internal standard. Two microliters were counted to calculate the extraction yield, and the rest was subjected to capillary chromatography. Samples were analyzed on an Agilent 1100 capillary chromatography system, equipped with a diode array detector and coupled to a mass spectrometer Agilent 1100 series LC/MSD Trap, equipped with an orthogonal geometry electrospray source and ion trap analyser. HPLC separation was performed on a Waters Atlantis dC18 column (150 ´ 1 mm; particle size, 3 mm) at a flow rate of 30 ml/min; buffer A was 1% vol/vol acetic acid in water, buffer B was 90% acetonitrile and 10% buffer A, and the gradient was the following: from 0 to 3 min, 100% A and from 3 to 35 min, linearly increasing to 100% B. Detection wavelength was at 254 nm. MS and MS/MS spectra were acquired in negative ion mode in the m/z range 50-300. Presence of the 153 Da and 219 Da fragments (12) unambiguously identified the eluting peak as cis, trans ABA. The ABA concentration in the extracts was calculated from the area of its HPLC peak and taking into consideration: (i) the percentage of recovery, as assessed with the radioactive tracer; (ii) the efficiency of ionization, as assessed with the internal standard of deuterated ABA, added before HPLC-MS analysis; and (iii) a calibration curve, obtained with known amounts of ABA, injected separately.1. Yamazaki D, Yoshida S, Asami T, Kuchitsu K (2003) Plant J 35:129-139.
2. Gómez-Cadenas A, Pozo OJ, García-Augustín P, Sancho JV (2002) Phytochem Anal 13:228-23.
3. Graeff RM, Walseth TF, Fryxell K, Branton WD, Lee HC (1994) J Biol Chem 269:30260-30267.
4. Guida L, Franco L, Zocchi E, De Flora A (1995) FEBS Lett 368:481-484.
5. Bradford M (1976) Anal Biochem 72:248-252.
6. Zocchi E, Franco L, Guida L, Benatti U, Bargellesi A, Malavasi F, Lee HC, De Flora A (1993) Biochem Biophys Res Commun 196:1459-1465.
7. Laemmli UK (1970) Nature 227:680-685.
8. Towbin H, Staehelin T, Gordon J (1979) Proc Natl Acad Sci USA 76:4350-4354.
9. Bruzzone S, Moreschi I, Guida L, Usai C, Zocchi E, De Flora A (2006) Biochem J 393:697-704.
10. Zocchi E, Carpaneto A, Cerrano C, Bruzzone S, Guida L, Franco L, Usai C (2001) Proc Natl Acad Sci USA 98:14859-14864.
11. Le Page-Degivry MT, Bidard JN, Rouvier E, Bulard C, Lazdunski M (1986) Proc Natl Acad Sci USA 83:1155-1158.
12. Gomez-Cadenas A, Pozo OJ, Garcia-Augustin P, Rancho JV (2002) Phytochem Anal 13:228-234.
13. Moreschi I, Bruzzone S, Nicholas RA, Fruscione F, Sturla L, Benvenuto F, Usai C, Meis S, Kassack MU, Zocchi E, De Flora A (2006) J Biol Chem 281:31419-31429.