Phosphoinositide and Inositol Phosphate Analysis in Lymphocyte Activation

Karsten Sauer1, Yina Hsing Huang2, Hongying Lin3, Mark Sandberg4, Georg W. Mayr3

1 The Scripps Research Institute, La Jolla, California, 2 Washington University School of Medicine, St. Louis, Missouri, 3 University Medical Center Hamburg‐Eppendorf, Hamburg, Germany, 4 Genomics Institute of the Novartis Research Foundation (GNF), San Diego, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 11.1
DOI:  10.1002/0471142735.im1101s87
Online Posting Date:  November, 2009
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Lymphocyte antigen receptor engagement profoundly changes the cellular content of phosphoinositide lipids and soluble inositol phosphates. Among these, the phosphoinositides phosphatidylinositol 4,5‐bisphosphate (PIP2) and phosphatidylinositol 3,4,5‐trisphosphate (PIP3) play key signaling roles by acting as pleckstrin homology (PH) domain ligands that recruit signaling proteins to the plasma membrane. Moreover, PIP2 acts as a precursor for the second messenger molecules diacylglycerol and soluble inositol 1,4,5‐trisphosphate (IP3), essential mediators of PKC, Ras/Erk, and Ca2+ signaling in lymphocytes. IP3 phosphorylation by IP3 3‐kinases generates inositol 1,3,4,5‐tetrakisphosphate (IP4), an essential soluble regulator of PH domain binding to PIP3 in developing T cells. Besides PIP2, PIP3, IP3, and IP4, lymphocytes produce multiple other phosphoinositides and soluble inositol phosphates that could have important physiological functions. To aid their analysis, detailed protocols that allow one to simultaneously measure the levels of multiple different phosphoinositide or inositol phosphate isomers in lymphocytes are provided here. They are based on thin layer, conventional and high‐performance liquid chromatographic separation methods followed by radiolabeling or non‐radioactive metal‐dye detection. Finally, less broadly applicable non‐chromatographic methods for detection of specific phosphoinositide or inositol phosphate isomers are discussed. Support protocols describe how to obtain pure unstimulated CD4+CD8+ thymocyte populations for analyses of inositol phosphate turnover during positive and negative selection, key steps in T cell development. Curr. Protoc. Immunol. 87:11.1.1‐11.1.46. © 2009 by John Wiley & Sons, Inc.

Keywords: lymphocyte; inositol; phosphoinositide; phospholipid; second messenger; T cell; thymocyte; signal transduction; IP3; IP4; IP5; IP6; PIP2; PIP3; HPLC; MDD‐HPLC

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Myo‐[3H] Inositol Labeling and Stimulation of Primary Murine Thymocytes
  • Alternate Protocol 1: Myo‐[3H] Inositol Labeling and Stimulation of Immortalized T Cells
  • Enriching Unstimulated CD4+CD8+ DP Thymocytes
  • Support Protocol 1: MHCI−MHCII− (MHC−) Mice
  • Support Protocol 2: CD53−CD4+CD8+ DP Thymocyte Enrichment by Magnetic Bead Immunoaffinity Cell Sorting (MACS)
  • Basic Protocol 2: [3H] Inositol Phosphate Resolution by HPLC with In‐Line β‐Detector
  • Alternate Protocol 2: [3H]‐Inositol Phosphate Resolution by HPLC without In‐Line β‐Detector
  • Basic Protocol 3: Soluble Inositol Phosphate Resolution by HPLC with Metal Dye Detection (MDD)
  • Alternate Protocol 3: Inositol Phospholipid Separation and Quantification by HPLC with Metal Dye Detection (MDD)
  • Basic Protocol 4: [3H] Inositol Phosphate Resolution by Dowex Anion‐Exchange Chromatography
  • Basic Protocol 5: [3H] Inositol Phospholipid Resolution by Thin Layer Chromatography
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Myo‐[3H] Inositol Labeling and Stimulation of Primary Murine Thymocytes

  • PBS and Ca2+‐free PBS ( appendix 2A)
  • Fibronectin (lyophilized human plasma fibronectin; Invitrogen, cat. no. 33016‐015 or equivalent; or fibronectin‐like engineered protein polymer‐plus; Sigma, cat. no. F8141)
  • 1% bovine serum albumin (BSA; Fischer, cat. no. AC61191‐0010 or equivalent) in PBS
  • Mice (6‐week‐old C57BL/6 mice) or purified DP cells
  • CO 2 source
  • M199/FCS/HEPES (see recipe)
  • PharmLyse (BD Biosciences, cat. no. 555899), 1× dilution in double distilled water and filter sterilized, optional
  • Inositol‐free DMEM/2.5% FCS (see recipe)
  • myo‐[3H] inositol (∼2.89 TBq/mmol; GE Healthcare, cat. no. TRK883)
  • Recombinant IL‐7 (R&D Systems, cat. no. 407‐ML)
  • 0.5 mM EDTA in Ca2+‐free PBS
  • HBSS/HEPES (see recipe), ice cold
  • Biotinylated anti‐mouse CD3 antibodies, clones 145‐2C11 or 500A2 (Invitrogen; CALTAG; BD Biosciences)
  • Unconjugated streptavidin (SA; Jackson Immunoresearch, cat. no. 016‐000‐113)
  • Concanavalin A type IV (ConA; Sigma‐Aldrich, cat. no. C5275)
  • 3% perchloric acid (PCA) in water
  • 6‐ and 12‐well tissue culture plastic plates, untreated
  • 37°C incubator with and without 5% CO 2
  • Mouse vivarium with appropriate euthanasia equipment in procedure room
  • 60‐mm petri dishes
  • 40‐µm nylon cell strainer
  • 1‐ to 5‐ml syringes
  • 50‐ml conical tubes
  • Hemacytometer or electronic cell counter
  • 2‐ml microcentrifuge tubes
  • 37°C water bath or heating block
  • Additional reagents and equipment for thymi removal (unit 1.9)

Alternate Protocol 1: Myo‐[3H] Inositol Labeling and Stimulation of Immortalized T Cells

  • Human Jurkat αβT cells (ATCC # TIB‐152) or other cells of interest
  • RPMI/10% FCS/PSG (see recipe)

Support Protocol 1: MHCI−MHCII− (MHC−) Mice

  • B6.129‐H2‐Ab1tm1Gru B2mtm1Jae mice [Taconic, cat. no. 004080‐MM‐F (females) or ‐M (males)]
  • PharmLyse (BD Biosciences, cat. no. 555899): dilute to 1× in double distilled water and then filter sterilize
  • PBS containing 2% FCS
  • Fc block: anti‐mouse CD16/32 antibody
  • FACS antibodies: H‐2Kb‐FITC (BD Pharmingen, cat. no. 553569); I‐Ab‐PE (BD Pharmingen, cat. no. 553552); CD4 (L3T4) APC (BD Pharmingen, cat. no. 553051); CD8a (Ly‐2) PE‐Cy7 (BD Pharmingen, cat. no. 552877)
  • Tail digestion buffer (see recipe)
  • 25:24:1 (v/v) phenol/chloroform/isoamyl alcohol (Invitrogen, cat. no. 15593‐031)
  • Genotyping primers: B2m primers [oIMR0160 (mutant): TCTggACgAAgAgCATCAggg; oIMR0184 (common): TATCAgTCTCAgTgggggTg; oIMR0185 (wild type): CTgAgCTCTgTTTTCgTCTg]
  • I‐Ab primers [oIMR5241 (mutant): gTgTTgggTCgTTTgTTCg; oIMR5239 (common): AgggAggTgTgggTCTCC; oIMR5240 (wild type): gTACCAgTTCATgggCgAgT]
  • 96‐well U‐bottom plate
  • Additional reagents and equipment for care and handling of laboratory animals (Chapter 1); flow cytometry (Chapter 5)

Support Protocol 2: CD53−CD4+CD8+ DP Thymocyte Enrichment by Magnetic Bead Immunoaffinity Cell Sorting (MACS)

  • 6‐week‐old C57BL/6 mice
  • MACS staining buffer (see recipe)
  • Anti‐CD53 Ab (OX‐79) (BD Biosciences, cat. no. 559364)
  • Biotinylated anti‐rat IgG (Jackson Immunoresearch, cat. no. 112‐065‐167)
  • Anti‐biotin microbeads (Miltenyi Biotec, cat. no. 130‐042‐401)
  • MidiMACS separation unit (Miltenyi Biotec, cat. no. 130‐042‐302)
  • Additional reagents and equipment for harvesting thymocytes (see protocol 1); flow cytometry (see Chapter 5)

Basic Protocol 2: [3H] Inositol Phosphate Resolution by HPLC with In‐Line β‐Detector

  • HPLC‐grade (NH 4)H 2PO 4
  • Phosphoric acid
  • NaN 3
  • [3H]‐Labeled inositol phosphate standard solutions, e.g.,:
    • D‐myo‐inositol‐1,4,5‐P 3, [inositol‐1‐3H(N)] (Perkin Elmer, cat. no. NET‐911001UC)
    • D‐myo‐inositol‐1,3,4,5‐P 4, [inositol‐1‐3H(N)] (Perkin Elmer, cat. no. NET‐941002UC)
  • Scintillation fluid compatible with high‐salt solutions (Uniscint National Diagnostics, cat. no. LS‐276, or equivalent)
  • HPLC system compatible with aqueous solutions
  • Inline β‐detector (β‐RAM‐RHPLC detector from IN/US with a 500‐µl flow cell or equivalent)
  • Partisphere strong anion exchange (SAX) column (12.5 cm × 4.6–mm; Whatman, cat. no. 4621‐0505 or equivalent) or a 25‐cm column (Whatman, cat. no. 4621‐1507)

Alternate Protocol 2: [3H]‐Inositol Phosphate Resolution by HPLC without In‐Line β‐Detector

  • Thymocytes (see protocol 1) or T cell lines (see protocol 2)
  • PBS ( appendix 2A)
  • Lysis buffer (see recipe), ice cold
  • Water‐saturated diethyl ether: prepared by vigorously mixing 1 vol deionized water with 2 vol diethyl ether for at least 2 min
  • 1 M triethanolamine (TEA, p.a., >99% purity; see recipe)
  • Charcoal suspension (see recipe)
  • 0.1 M NaCl ( appendix 2A)
  • Methanol (LiChrosolv)
  • 10% (w/v) trichloroacetic acid (TCA) solution, 4°C
  • 0.2 M EDTA ( appendix 2A)
  • 0.1 M NaF (see recipe)
  • MDD‐HPLC eluent A (see recipe)
  • MDD‐HPLC eluent B (see recipe)
  • Post‐column reagent C (see recipe)
  • HPLC‐injection solution (see recipe)
  • Degassed, filtered HPLC‐grade water
  • 30% analytical‐grade HCl (suprapure)
  • Phytic acid
  • 10 mM 4‐(2‐pyridylazo)‐resorcinol monosodium salt monohydrate (PAR; see recipe)
  • 18 mM yttrium trichloride (YCl 3; see recipe)
  • 0.5 M sodium acetate (see recipe)
  • Cell scraper
  • 12‐ and 14‐ml polypropylene tubes with caps (Greiner Bio‐One, cat. no. 187262)
  • 35°C water bath
  • 2‐ml microcentrifuge tubes
  • SpeedVac
  • Ultra‐Turrax homogenizer
  • 0.22‐µm pore size membrane filters (Millipore, type GV) in a Pyrex glass filtration device
  • Vacuum pump
  • 2‐ml Pyrex glass vials
  • HPLC auto‐sampler with a 1‐ml injection loop and a 2.5‐ml loading syringe (inert valve made from titanium or PEEK; HPLC auto‐sampler 560, Kontron; loading syringe available from Hamilton)
  • MiniQ PC 3.2/3 column (3‐µm bead diameter, GE Healthcare/Pharmacia Biotech, Uppsala)
  • Hand‐made knitted coil from a 40‐cm 1/16‐in. × 0.5‐mm i.d. PTFE capillary with 7 knots (CS‐Chromatographie Service)
  • Two inert HPLC pumps for gradient elution (with titanium or PEEK pump‐head; Pump 422, Kontron)
  • Pump for post‐column dye reagent addition (Shimadzu, cat. no. LC‐10AD)
  • UV/Vis recorder for absorbance recording (with titanium or PEEK flow cell; Shimadzu, cat. no. SPD‐10Avvp)
  • Chromatography data system for control and data processing (e.g., Galaxie chromatography data system, Varian)
  • Graphing/interpolation software (e.g., GraphPad Prism)

Basic Protocol 3: Soluble Inositol Phosphate Resolution by HPLC with Metal Dye Detection (MDD)

  • PIP 2, PIP 3 standards
  • Methanol (LiChrosolv)
  • Chloroform (CHCl 3; LiChrosolv)
  • 0.1 M HCl
  • 3:48:47 (v/v/v) chloroform/methanol/0.6 M HCl
  • n‐Butanol
  • 33% methylamine in ethanol
  • n‐Propanol
  • 20:4:1 (v/v/v) butanol/petroleum‐ether/ethyl formate
  • 53°C water bath or heating block

Alternate Protocol 3: Inositol Phospholipid Separation and Quantification by HPLC with Metal Dye Detection (MDD)

  • Cells
  • Medium supplemented with 2 to 20 µCi/ml myo‐[3H] inositol
  • Balanced salt solution (see recipe)
  • Stimulus
  • 10% (w/v) trichloroacetic acid (TCA), ice cold
  • Diethyl ether, water‐saturated
  • 1:100 (v/v) dilution of concentrated ammonia
  • 0.5 g/ml Dowex 1‐X8 resin (100 to 200 mesh; formate form; Bio‐Rad, Sigma‐Aldrich, GFS Chemicals, Serva Electrophoresis; formate form may require custom production), slurry in water
  • 60 mM sodium formate/5 mM disodium tetraborate
  • 0.2 M ammonium formate/0.1 M formic acid
  • 0.4 M ammonium formate/0.1 M formic acid
  • 0.8 M ammonium formate/0.1 M formic acid
  • Scintillation fluor cocktail (compatible with aqueous samples)
  • 13 × 100–mm glass tubes
  • Pasteur pipets
  • 0.6‐cm diameter disposable columns
  • Liquid scintillation counter

Basic Protocol 4: [3H] Inositol Phosphate Resolution by Dowex Anion‐Exchange Chromatography

  • 1% potassium oxalate/2 mM EDTA
  • Solvent: 60:20:23:18:12 (v/v/v/v/v) chloroform/methanol/acetone/acetic acid/H 2O
  • Cells (see , protocol 1, and protocol 2)
  • Phosphate‐free medium (e.g., GIBCO/BRL) containing 10% heat‐inactivated fetal calf serum (FCS), dialyzed against HBSS or TBS ( appendix 2A) to remove phosphate
  • Carrier‐free [32P] orthophosphate (296 mBq/ml, 8 mCi/ml)
  • Stimulus (see )
  • 50:100:1 (v/v/v) chloroform/methanol/concentrated HCl
  • 100 mM EDTA, pH 7.4 ( appendix 2A)
  • 100 mM KCl
  • Chloroform
  • Nitrogen source
  • 2:1 (v/v) chloroform/methanol
  • PI, PIP, PIP 2, and PA standards (Sigma)
  • Iodine
  • Silica‐gel plates (Baker Si250, J.T. Baker)
  • TLC tanks
  • 100° and 110°C ovens
  • Filter paper
  • 2‐ml microcentrifuge tubes
  • 37°C water bath or heating block
  • Kodak X‐Omat film
  • EN3HANCE spray surface autoradiography enhancer (Perkin Elmer)
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  •   FigureFigure 11.1.1 Mammalian inositol phosphate metabolism. Simplified scheme of the known inositol phosphate metabolic pathway in mammalian cells. Circled P, phosphate moiety; R, R', fatty acid side chains. The hatched box encloses pathway components for which genetic data suggest relevance in lymphocytes. For more details and discussions of the enzymes involved and of potential cellular inositol phosphate functions, see previously published works (Irvine, , , ; Irvine and Schell, ; Irvine et al., ; Rusten and Stenmark, ; Otto et al., ; Seeds et al., ; Miller et al., ; Alcazar‐Roman and Wente, ; Huang et al., ; Lin et al., ). The membrane phospholipid phosphatidylinositol 4,5‐bisphosphate (PtdIns(4,5)P2/PI(4,5)P2, PIP2) acts as a precursor for the phosphoinositide PI(3,4,5)P3 (PIP3), and for the second messenger molecules diacylglycerol (DAG) and inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3/I(1,4,5)P3/IP3). In mammalian cells, I(1,4,5)P3 acts as a key precursor for multiple higher order, soluble inositol phosphates. An important step in the synthesis of several inositol phosphates is I(1,4,5)P3 phosphorylation into I(1,3,4,5)P4 (IP4) by either one of three IP3 3‐kinases (IP3KA, B or C, also termed ItpkA, B, or C; Pouillon et al., ; Wen et al., ; Huang et al., ) or by IPK2/IPMK (Irvine, ; Irvine et al., ; Otto et al., ). Multiple higher order inositol phosphates have been reported in lymphocytes, including several of those shown here. The levels of some inositol phosphates are modulated after antigen receptor engagement (Imboden and Stobo, ; Stewart et al., , ; Imboden and Pattison, ; Zilberman et al., ; Guse and Emmrich, , ; Guse et al., , ; Pouillon et al., ). Complementing known PIP3, IP3, and DAG functions in lymphocyte development and function (Starr et al., ; Fruman, ; Cante‐Barrett et al., ; Jodi et al., ; Juntilla and Koretzky, ), it has been recently found that IP4 is essential for these processes through novel roles in antigen receptor signaling and myelopoiesis (Pouillon et al., ; Wen et al., ; Huang et al., , ; Jia et al., , ; Marechal et al., ; Miller et al., ). The protocols described here are thus optimized for analyses of IP3 and IP4 isomers.
  •   FigureFigure 11.1.2 Analysis of TCR induced inositol phosphate production in MCHIMHCII thymocytes. (A) HPLC elution profiles of extracts from unstimulated or αCD3‐stimulated MHC murine thymocytes. 2 × 108 cells were labeled overnight with 40 µCi myo‐[3H] inositol, the precursor for all IPs. At 5 min post‐stimulation with medium or 5 µg αCD3 (2C11), cells were lysed in 100 µl of 3% PCA and loaded onto a Whatman cartridge Col SAX PRTSPHR 15‐cm HPLC column. [3H] IP content in the eluates was monitored with an IN/US systems Bram‐4 in‐line β‐detector. IP3 or IP4 retention times were determined by spiking [3H] IP3 or [3H] IP4 into unlabeled cell extracts (not shown). IP3′ represents Ins(1,3,4)P3, an IP3 isomer originating from IP4 metabolism (Pouillon et al., ). IP5 represents a pool of IP5 isomers (Pouillon et al., ). (B) MHC thymocytes contain ≥98% DP cells, shown by FACS analysis of CD4 and CD8 expression on total thymocytes from 6‐week‐old C57BL/6 wild type (wt) or MHCIMHCII (MHC) mice. The two‐dimensional plots indicate CD4 ( y‐axis) or CD8 ( x‐axis) fluorescence intensity for individual cells (dots). The numbers indicate % cells in the respective quadrant.
  •   FigureFigure 11.1.3 Analysis of thymocyte populations pre‐ and post‐anti‐CD53 AB sort. Two‐dimensional plots showing CD4 ( y‐axis) and CD8 ( x‐axis) fluorescence intensities for individual thymocytes (dots) from 6‐week‐old C57BL/6 mice before (pre‐sort) or after (post‐CD53 sort) depletion of CD53+ cells. The numbers indicate percent cells in the respective quadrant.
  •   FigureFigure 11.1.4 A sample trace obtained from Jurkat cells labeled with myo‐[3H] inositol and stimulated for 5 min with OKT3 and αCD28 (1 µg/ml). The inositol phosphate isomers detected are indicated. The peaks corresponding to Ins(1,4,5)P3 and Ins(1,3,4,5)P4 were verified with [3H]‐labeled purified standards (Perkin‐Elmer).
  •   FigureFigure 11.1.5 MDD‐HPLC analysis of phytic acid hydrolysis products. (A) Elution profile. Peak identities were determined by comparison with the retention times for external standards (not shown). Peak 1 (retention time of 6.77 min) contains IP2 isomers, peak 2 (10.9 min) contains I(1,3,4)P3 and I(1,4,5)P3, peak 3 (11.23 min) contains D/L‐I(1,5,6)P3, peak 4 (11.89 min) contains I(4,5,6)P3, peak 5 (13.63 min) contains I(1,2,3,5)P4 and I(1,2,4,6)P4, peak 6 (13.79 min) contains I(1,2,3,4)P4 and I(1,3,4,6)P4, peak 7 (13.91 min) contains I(1,2,4,5)P4 and I(1,3,4,5)P4, peak 8 (14.41 min) contains I(1,2,5,6)P4, peak 9 (14.78 min) contains I(2,4,5,6)P4, peak 10 (15.31 min) contains I(1/3,4,5,6)P4, peak 11 (15.83 min ) contains D/L I(1,2,3,4,6)P5, peak 12 (16.31 min) contains D/L I(1,2,3,4,3)P5, peak 13 (17.04 min) contains D/L I(1,2,4,5,6)P5, peak 14 (17.32 min) contains I(1,3,4,5,6)P5, and peak 15 (18.97 min) contains I(1,2,3,4,5,6)P6 (unhydrolyzed phytic acid). (B) Calibration curve obtained with known amounts of an IP6 external standard.
  •   FigureFigure 11.1.6 MDD‐HPLC analysis of soluble inositol phosphate isomers in Jurkat T cells. The MDD‐HPLC method has been applied in a number of studies to analyze IPs in cells and tissues, including human Jurkat T cells (Guse et al., ). In the example shown here, soluble IPs were extracted from ∼5 × 107 unstimulated or 10 µg/ml OKT3‐stimulated Jurkat T cells. (A) a, separation of an IP standard mixture containing 554 pmol I(1,4,5)P3 (peak 1), 43 pmol I(1,2,3,5)P4 (peak 2), 113 pmol I(1,3,4,6)P4 (peak 3), 217 pmol I(1,3,4,5)P4 (peak 4), 116 pmol I(1,4,5,6)P4 (peak 5), 300 pmol I(1,2,3,4,6)P5 (peak 6), 20 pmol I(1,2,4,5,6)P5 (peak 7), 415 pmol I(1,3,4,5,6)P5 (peak 8), 646 pmol IP6 (peak 9), and 215 pmol PP‐IP5 (peak 10). b‐e, samples from unstimulated (b), 3 (c), 6 (d), or 20 min (e) OKT3‐stimulated Jurkat cells. (B) Quantified amounts of the indicated IPs in the Jurkat cell samples from A. *, p < 0.01, obtained via Student's t‐test.


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