Pannexin-1 Is Required for ATP Release during Apoptosis but Not ...

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Activationduring Apoptosis but Not for Inflammasome Pannexin-1 Is Required for ATP Release

David Hackos and Vishva M. DixitGilmour, Salina Louie, James E. Cupp, George R. Dubyak, Yan Qu, Shahram Misaghi, Kim Newton, Laurie L.

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The Journal of Immunology

Pannexin-1 Is Required for ATP Release during Apoptosis butNot for Inflammasome Activation

Yan Qu,* Shahram Misaghi,† Kim Newton,* Laurie L. Gilmour,‡ Salina Louie,*

James E. Cupp,‡ George R. Dubyak,x David Hackos,{ and Vishva M. Dixit*

Apoptotic cell death is important for embryonic development, immune cell homeostasis, and pathogen elimination. Innate immune

cells also undergo a very rapid form of cell death termed pyroptosis after activating the protease caspase-1. The hemichannel

pannexin-1 has been implicated in both processes. In this study, we describe the characterization of pannexin-1–deficient mice.

LPS-primed bone marrow-derived macrophages lacking pannexin-1 activated caspase-1 and secreted its substrates IL-1b and IL-

18 normally after stimulation with ATP, nigericin, alum, silica, flagellin, or cytoplasmic DNA, indicating that pannexin-1 is

dispensable for assembly of caspase-1–activating inflammasome complexes. Instead, thymocytes lacking pannexin-1, but not

the P2X7R purinergic receptor, were defective in their uptake of the nucleic acid dye YO-PRO-1 during early apoptosis. Cell

death was not delayed but, unlike their wild-type counterparts, Panx12/2 thymocytes failed to recruit wild-type peritoneal

macrophages in a Transwell migration assay. These data are consistent with pannexin-1 liberating ATP and other yet to be

defined “find me” signals necessary for macrophage recruitment to apoptotic cells. The Journal of Immunology, 2011, 186: 000–

000.

Several members of the NOD-like receptor (NLR) family ofintracellular adapter proteins expressed in innate immunecells assemble inflammasome complexes for caspase-1 ac-

tivation upon detecting pathogen-associated molecular patterns ordamage-associated molecular patterns (DAMPs) (1, 2). NLRC4(also known as Ipaf) activates caspase-1 in response to bacterialflagellin or infection with bacteria such as Salmonella typhi-murium, Legionella pneumophila, and Shigella flexneri (3–6).NLRP3 (also called cryopyrin and NALP3) activates caspase-1 inresponse to diverse stimuli, which include the DAMPs ATP anduric acid (7, 8), but the mechanism of NLRP3 activation remainsunclear. The hemichannel protein pannexin-1 was suggested toform large, nonselective pores after ATP stimulation of P2X7R,thereby allowing DAMPs and pathogen-associated molecular pat-terns access to cytosolic NLRP3 (9, 10). Pannexin-1–dependentpore formation also has been suggested as a mechanism for IL-1bsecretion after cleavage of pro–IL-1b by caspase-1 (11–17), as IL-1b lacks signal sequences for export via the classical endoplasmicreticulum/Golgi-dependent secretory pathway (18, 19).Pannexin-1 is one of three vertebrate pannexins that show ho-

mology to gap junction-forming invertebrate innexins (20, 21).

Pannexin-1 is expressed broadly, whereas pannexin-2 is expressedlargely in brain, and pannexin-3 is in skin and connective tissues(22). Pannexins and connexins have four transmembrane domains,cytosolic N and C termini, and two extracellular loops (22). De-spite their structural similarities, these two protein families seemto have distinct biological functions. Gap junction channelsformed by connexins mediate metabolic and electrical coupling ofadjacent cells, whereas the sugar moiety on pannexin-1 preventsalignment and docking of adjacent pannexin hemichannels to formgap junctions (23). Thus, evidence for pannexin-1 functioning asa physiologically relevant gap junction channel in vivo is lacking(24). Recent studies suggest that pannexin-1 in brain may con-duct small molecules up to ∼1 kDa, such as ions, ATP, inositoltriphosphate, and amino acids (25, 26). In cultured astrocytes,pannexin-1 was reported to mediate ATP release, and in RBCs, ithas been implicated in the secretion of arachidonic acid andits metabolites (26, 27). A recent study targeting pannexin-1 withpharmacological inhibitors or small interfering RNAs identifiedpannexin-1 as the conduit for ATP release from apoptotic cells(28). Cleavage of pannexin-1 by the executioner caspases 3 and 7appeared to trigger its channel activity, and this was necessary forthe recruitment of phagocytic cells (28, 29). Pannexin-1 was notrequired, however, in macrophages migrating in response to ATP(30).We generated mice lacking pannexin-1 by gene targeting to

explore the contribution of pannexin-1 to inflammasome function.We found that neither caspase-1 activation nor IL-1b secretionmediated by either NLRC4 or NLRP3 was compromised bypannexin-1 loss. Instead, Panx12/2 cells were less permeable thantheir wild-type (WT) counterparts to the nucleic acid dye YO-PRO-1 after apoptosis induction, and the dying Panx12/2 cellswere less effective at recruiting macrophages in a Transwellassay. Our data support previous studies (28, 30) indicating thatpannexin-1 releases “find me” signals from apoptotic cells in needof clearance by phagocytic cells but is dispensable in the mi-grating phagocyte itself. Contrary to previous studies (9, 10), weprovide genetic evidence that pannexin-1 and P2X7R function indistinct signaling pathways.

*Physiological Chemistry Department, Genentech, Inc., South San Francisco, CA94080; †Early Stage Cell Culture Department, Genentech, Inc., South San Francisco,CA 94080; ‡Immunology Department, Genentech, Inc., South San Francisco, CA94080; xDepartment of Physiology and Biophysics, Case Western Reserve UniversitySchool of Medicine, Cleveland, OH 44120; and {Neuroscience Department, Genen-tech, Inc., South San Francisco, CA 94080

Received for publication February 14, 2011. Accepted for publication March 25,2011.

Address correspondence and reprint requests to Dr. Vishva M. Dixit or Dr. DavidHackos, Physiological Chemistry Department, Genentech, Inc., 1 DNA Way, SouthSan Francisco, CA 94080 (V.M.D.) or Neuroscience Department, Genentech, Inc., 1DNA Way, South San Francisco, CA 94080 (D.H.). E-mail addresses: [email protected] (V.M.D.) and [email protected] (D.H.)

Abbreviations used in this article: BMDM, bone marrow-derived macrophage; CHX,cycloheximide; DAMP, damage-associated molecular pattern; NLR, NOD-like re-ceptor; PI, propidium iodide; WT, wild-type.

Copyright� 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100478

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Materials and MethodsReagents

Key reagents and their sources were as follows: Escherichia coli LPSserotype O1101:B4 (List Biological Laboratories); ATP, cycloheximide(CHX), dexamethasone, and nigericin (Sigma); z-VAD-fmk (MBL); Imjectalum (Thermo Scientific); silica (US Silica); S. typhimurium flagellin(InvivoGen); Lipofectamine 2000, pcDNA3.1(+), Pacific blue annexin V,and YO-PRO-1 (Invitrogen); complete protease inhibitor mixture andDOTAP (Roche); Jo2 hamster anti-mouse CD95 and propidium iodide (PI;BD); cleaved (Asp175) and total (8G10) caspase-3 Abs (Cell Signaling);GAPDH, caspase-1 p10, and actin Abs (Santa Cruz); 4B4 caspase-1 p20Ab (Genentech); and 3ZD anti–IL-1b and pro–IL-1b Ab (BiologicalResources Branch, National Cancer Institute). S. enerica serovar typhi-murium strain SL1344 was from D. Monack (Stanford University).

Mice

The strategy used to generate Panx12/2 mice is depicted in Fig. 1A. Ho-mologous recombinant C57BL/6 C2 embryonic stem cells were deletedof the neomycin selection cassette with adenovirus-expressed FLP re-combinase. Deletion of exon 2, which generates a frameshift and pre-mature stop codon, was achieved by breeding to the C57BL/6-Gt(ROSA)26Sortm16(Cre)Arte Cre deleter strain (TaconicArtemis). Mice were geno-typed with PCR primers (59-TGA CCA CAG ACA GCA CTTAAG-39 and59-CGT CTG AGA GCT CCC TGG CG-39) that yield a 651-bp WT DNAfragment and a 335-bp knockout DNA fragment (Fig. 1B). P2rx72/2 micewere generated by Lexicon Pharmaceuticals. The Genentech InstitutionalAnimal Care and Use Committee approved all experiments.

Cells

Bone marrow and thioglycolate-elicited peritoneal macrophages werecultured in DMEM supplemented with 20% L cell-conditioned medium,

10% heat-inactivated FBS, 100 U/ml penicillin, 10 mM L-glutamine, and100 mg/ml streptomycin. Adherent bone marrow-derived macrophages(BMDMs) were obtained after 5–7 d and replated in 6- or 24-well platesfor experiments. Thymocytes were cultured in RPMI 1640 media sup-plemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 mg/mlstreptomycin, and 10 mM L-glutamine.

Western blots and ELISAs

BMDMs were routinely seeded in 6-well plates (2 3 106 cells/well) forWestern blot analysis and in 24-well plates for ELISA. Cell supernatantswere concentrated by TCA precipitation for Western blotting (31), andcells were lysed in 150 ml RIPA lysis buffer (1% Nonidet P-40, 0.5%sodium deoxycholate, 0.1% SDS, pH 7.4, in PBS) containing a proteaseinhibitor mixture. Mouse tissues were homogenized in KINEXUS buffer(20 mM HEPES pH 7.2, 2 mM EGTA, 5 mM EDTA, 30 mM NaF, 60 mMb-glycerolphosphate, 20 mM sodium pyrophosphate, 1 mM Na3VO4, and1% Triton X-100) supplemented with protease and Halt Phosphataseinhibitor cocktails (Thermo Scientific Pierce). Total protein concentra-tion was determined by bicinchoninic acid protein assay (Thermo Scien-tific Pierce). IL-1b and IL-18 in culture supernatants were measured byBioplex-23 assay (Bio-Rad).

ATP measurements

Culture supernatants were heated at 98˚C for 5 min and then assayed witha CellTiter-Glo kit (Promega) in an EnVison Multilabel Reader (Perkin-Elmer).

FACS analysis

Thymocytes were washed twice with cold PBS and then stained with Pacificblue-conjugated annexin V, YO-PRO-1, and PI for 30 min at room tem-perature. Cells were analyzed in an LSR-II cytometer (BD).

FIGURE 1. Generation of Panx12/2 mice. A, Strategy for deleting exon 2 of Panx1 in C57BL/6 C2 embryonic stem cells. B, Identification of Panx12/2

mice by PCR. C, Western blot analysis of pannexin-1 protein expression in mouse tissues (70 mg total protein/lane). Variable pannexin-1 migration likely

represents variable glycosylation of the protein. GAPDH is presented to indicate protein loading.

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Migration assay

Thymocytes (2 3 106/ml) were plated on 12-well dishes and left untreatedor stimulated with 0.5 mM dexamethasone in the absence or presence of 25mM zVAD for 4 h. Culture supernatants were collected by centrifugationtwice at 500 3 g for 5 min. Transwell migration assays were performedwith 500 ml apoptotic cell supernatant in the lower chamber and 250 ml ofWT or Panx12/2 thioglycolate-elicited peritoneal macrophages (2 3 106/ml) in the upper chamber/insert of a 24-well plate with 3-mM pore-sizemembrane (Corning) for 90 min at 37˚C. Cells that migrated into the lowerchamber were counted with a hemocytometer. Where indicated, culturesupernatants were supplemented with 1 U/ml apyrase for 5 min at roomtemperature. Separately, 500 ml of the supernatants from these migrationassays were boiled at 98˚C for 5 min prior to ATP measurement.

Cell viability assay

Thymocytes (1 3 106/ml) were plated on 96-well dishes and left untreatedor stimulated with dexamethasone in the absence or presence of zVADfor 4, 24, 30, and 48 h. Cell viability was measured with a CellTiter-GloLuminescent Cell Viability Assay Kit (Promega).

YO-PRO-1 influx

BMDMs (1 3 106/ml) plated on PDL-coated, black 96-well plates werewashed once with PBS and cultured in the following media: 135 mM

NaCl, 5 mM KCl, 10 mM glucose, 10 mM HEPES pH 7.4, 1 mM CaCl2,and 1 mM MgCl2. After addition of YO-PRO-1 (5 mM) and ATP (3 mM),cells were imaged using a 320 Plan Fluor ELWD objective lens andimages collected every 60 s for 80 min. Fluorescence intensity wasrecorded and calculated using Nikon’s NIS Elements software, whichenables regions-of-interest selection and automatic tracking of 20–25 nu-clei per image at each time point throughout the recording time.

ResultsPannexin-1 is dispensable for caspase-1 activation by ATP ornigericin

Panx12/2 mice generated by gene targeting (Fig. 1A, 1B) wereconfirmed to lack pannexin-1 with a polyclonal Ab against theprotein’s C terminus (23). Western blotting of WT tissues revealedseveral pannexin-1 bands between ∼41 and 48 kDa, but thesewere absent in Panx12/2 tissues (Fig. 1C). Pannexin-1 was de-tected readily in kidney, lung, muscle, spleen, brain, and thymus.BMDMs also expressed pannexin-1 but at a much lower level.Prolonged stimulation of the purinergic receptor P2X7R with

ATP is proposed to trigger pannexin-1 pore formation for extra-cellular bacterial products to enter the cell and promote NLRP3-

FIGURE 2. Pannexin-1 is not required for P2X7R-dependent activation of the NLRP3 inflammasome. WT (Panx1+/+), Panx12/2, or P2rx72/2 BMDMs

were primed with 0.5 mg/ml LPS for 4 h and then treated with 5 mM ATP or 10 mM nigericin for the time indicated. Culture supernatants were Western

blotted for IL-1b and the processed caspase-1 subunits p10 and p20. Cell lysates were Western blotted for p45 procaspase-1 (A–C). IL-1b (D) and IL-18 (E)

secreted into the culture supernatant are presented as the mean 6 SD of triplicate samples. Results are representative of three independent experiments.

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dependent activation of caspase-1 (10, 14–17). We tested thisnotion with WT, Panx12/2, and P2rx72/2 BMDMs (Fig. 2). Thecells were primed with LPS for 4 h and then stimulated with eitherATP or the ionophore nigericin to activate caspase-1. Both WTand Panx12/2 BMDMs cleaved procaspase-1 (p45) into its p10and p20 subunits after 15 min of ATP stimulation (Fig. 2A, 2B),whereas P2rx72/2 BMDMs did not cleave procaspase-1 even after1 h of ATP stimulation (Fig. 2C). Panx12/2 BMDMs also pro-

cessed procaspase-1 in response to nigericin, which triggerscaspase-1 cleavage independently of the P2X7R (Fig. 2A–C).Further confirmation of caspase-1 activation in Panx12/2 BMDMswas the normal processing (Fig. 2A–C) and secretion (Fig. 2D) ofbiologically active IL-1b (p17). Caspase-1–dependent secretionof IL-18 also occurred normally in Panx12/2 BMDMs (Fig. 2E).These data prove that pannexin-1 is dispensable for NLRP3-dependent activation of caspase-1 and secretion of the proin-

FIGURE 3. Pannexin-1 is not required for NLRP3-, NLRC4-, or AIM2-dependent caspase-1 activation and cytokine release. WT (Panx1+/+) (A, C) and

Panx12/2 (B, D) BMDMs were primed with 0.5 mg/ml LPS for 2 h and then treated with 5 mM ATP for 30 min, 500 ng/ml alum, 125 ng/ml silica, 2.5 mg/

ml flagellin, 1 mg/ml pcDNA3.1, or Salmonella (multiplicity of infection = 20) for 6 h. Culture supernatants were Western blotted for the processed caspase-

1 subunits p10 and p20. Cell lysates were Western blotted for p45 procaspase-1 (A–D). IL-1b (E), and IL-18 (F) secreted into the culture supernatant are

shown as the mean 6 SD of triplicate samples. Results are representative of three independent experiments.

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flammatory cytokines IL-1b and IL-18 in response to LPS/ATP orLPS/nigericin.

Pannexin-1 is not required for NLRP3-, NLRC4-, orAIM2-dependent caspase-1 activation

Next, we determined whether pannexin-1 might contribute tocaspase-1 activation by other stimuli. NLRP3-dependent caspase-1activation in response to alum or silica (32, 33), NLRC4-dependentcaspase-1 activation in response to S. typhimurium or transfectedflagellin (3, 6, 32), and AIM2-dependent caspase-1 activation inresponse to transfected plasmid DNA (34–36) occurred normallyin Panx12/2 BMDMs (Fig. 3A–D). Consistent with these data,WT and Panx12/2 BMDMs secreted equivalent amounts of IL-1band IL-18 (Fig. 3E, 3F). Our results indicate that pannexin-1 is

dispensable for caspase-1 activation by distinct inflammasomecomplexes and the subsequent secretion of IL-1b and IL-18.

P2X7R pore formation in BMDMs does not require pannexin-1

The P2X7R forms large pores permeable to ions up to 900 Da inmolecular mass (37, 38), so we considered whether pannexin-1might be required for P2X7R pore formation in BMDMs dis-tinct from ATP-induced caspase-1 activation. WT and Panx12/2

BMDMs, but not P2rx72/2 BMDMs, took up the 375 Da nucleicacid dye YO-PRO-1 after stimulation with ATP for 30 min (Fig. 4A).To exclude differences in the kinetics of dye uptake, we monitoredthe rate of YO-PRO-1 uptake by live imaging (Fig. 4B). YO-PRO-1entered WT and Panx12/2 BMDMs with similar kinetics, indicatingthat pannexin-1 is not essential for P2X7R channel formation.

FIGURE 4. Pannexin-1 is dispensable for increased membrane permeability in response to ATP. A, WT (Panx1+/+), Panx12/2, and P2rx72/2 BMDMs

cultured with YO-PRO-1 in 96-well plate format were stimulated with 3 mM ATP for 30 min at 37˚C. The cells were imaged using a 320 Plan Fluor

ELWD objective lens. The image has a scale of 0.33 mm per pixel and is 1280 pixels 3 1024 pixels. B, Kinetics of YO-PRO-1 dye uptake by cells in A.

Fluorescence intensity is shown as the average of six images containing 20–25 cells per image at each time point (n = 6)6 SEM. Results are representative

of three independent experiments.

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Pannexin-1 mediates YO-PRO-1 uptake and ATP release inapoptotic cells

Pannexin-1 is reported to release nucleotide “find me” signals fromapoptotic cells for the recruitment of phagocytic cells (28). Wedetermined whether primary Panx12/2 cells recapitulated resultsobtained with pharmacological or small interfering RNA in-hibition of pannexin-1. WT and Panx12/2 thymocytes exhibitedequivalent spontaneous apoptosis in culture (Fig. 5A, 5B). In ad-dition, both genotypes showed comparable killing by dexameth-asone or Fas Abs in combination with CHX (Fig. 5). Of note,Panx12/2 cells in the early stages of apoptosis (annexin V+ PI2)(Fig. 5B) were less permeable than WT or P2rx72/2 cells to YO-PRO-1 (Fig. 6A). These data indicate that pannexin-1 increases cellpermeability in early apoptotic cells independent of P2X7R sig-naling. Consistent with pannexin-1 being a caspase-3 substrate (28),endogenous pannexin-1 was reduced in dexamethasone-treated WTthymocytes, and this decrease along with caspase-3 processing wasblocked by the pancaspase inhibitor zVAD (Fig. 6B).Next, we determined the ability of culture supernatants from

apoptotic thymocytes to induce macrophage chemotaxis. WT

thioglycolate-elicited peritoneal macrophages migrated efficiently

in a Transwell assay into media taken from apoptotic WT but not

Panx12/2 cells (Fig. 7A). This migration was reduced markedly

when thymocyte apoptosis was inhibited with zVAD, supporting

a role for pannexin-1 in the release of chemoattractants from

apoptotic cells. It is worth noting that Panx12/2 macrophage

migration into apoptotic cell culture supernatants was not signif-

icantly different from WT macrophage migration (Fig. 7B). These

data agree with the report by Kronlage et al. (30) that pannexin-1

is dispensable in macrophages for chemotaxis toward apoptotic

cells.Because nucleotides such as ATP and UTP are released from

apoptotic cells and function as chemoattractants (28, 39), we

measured ATP release from WT and Panx12/2 apoptotic thymo-

cytes. WT but not Panx12/2 cells released detectable amounts of

ATP after culture with dexamethasone for 4 h (Fig. 7C). ATP

release from WT cells correlated with apoptosis induction because

it was blocked by zVAD (Fig. 7C). These results indicate that

pannexin-1 is essential for ATP release from apoptotic cells. ATP

likely is a major chemoattractant released from apoptotic cells

FIGURE 5. Panx12/2 thymocytes are not resistant to apoptotic stimuli. A, Panx1+/+ and Panx12/2 thymocytes were cultured in medium alone or 0.5 mM

dexamethasone. Cell viability was determined with a CellTiter-Glo Kit. Data represent the mean 6 SD of triplicate samples. Results are representative of

two independent experiments. B, Thymocytes were cultured in medium alone (control), 5 mg/ml Jo2 Fas Ab plus 20 mg/ml CHX, or 0.1 mM dexamethasone

for 8 h. Where indicated, cells were also treated with 25 mM zVAD. Cell viability was determined by flow cytometry after staining with annexin V and PI.

Results are representative of three independent experiments. Dex, dexamethasone.

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because ATP depletion from apoptotic thymocyte culture super-natants with apyrase reduced macrophage migration to that seen incontrol supernatants (Fig. 7A, 7B).

DiscussionPannexin-1 in immune cells has been linked to inflammasome as-sembly and caspase-1 activation (9, 10), secretion of proinflam-matory cytokines (16, 40), P2X7R-dependent dye uptake, and che-

moattractant release during apoptosis (17, 28, 39). Our analyses of

Panx12/2 BMDMs indicate that pannexin-1 is not required for

NLRP3-, NLRC4-, or AIM2-dependent caspase-1 activation, nor is

it needed for caspase-1–dependent secretion of the proinflammatory

cytokines IL-1b and IL-18 (Figs. 2, 3). We also demonstrate that

ATP engagement of the P2X7R increases BMDM membrane per-

meability independent of pannexin-1 (Fig. 4). Consistent with

Chekeni et al. (28), our data reveal that pannexin-1 regulates

FIGURE 6. Pannexin-1 is required for YO-PRO-1 dye uptake during early apoptosis. A, WT, Panx12/2, and P2rx72/2 thymocytes were cultured with 5

mg/ml Jo2 Fas Ab plus 20 mg/ml CHX or 0.5 mM dexamethasone for 8 h. YO-PRO-1 uptake by annexin V+, PI2 apoptotic cells was determined by flow

cytometry. White histograms represent cells cultured with DMSO carrier, whereas gray histograms represent cells cultured with 25 mM zVAD. B, WT

(Panx1+/+) and Panx12/2 thymocytes were treated with dexamethasone and/or 25 mM zVAD for 18 h. Dex, dexamethasone.

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membrane permeability in early apoptosis (Fig. 6) and the releaseof macrophage chemoattractants such as ATP (Fig. 7). Therefore,P2X7R and pannexin-1 function in distinct signaling pathways.Prior studies concluding pannexin-1 involvement in inflamma-

some activation and cytokine release in BMDMs (9, 10, 16, 17)relied in large part on mimetic blocking peptides, pharmacologicalinhibitors, and/or overexpression approaches in transformed celllines. Possible off-target activities (24) associated with these ex-perimental approaches and the use of transformed cells may ac-

count for the discrepancy with our genetic evidence. Our data thatpannexin-1 regulates the release of chemoattractants for phago-cytic cells during apoptosis while being dispensable for inflam-masome activation is consistent with the notion that apoptosisis an immunologically silent, anti-inflammatory mode of celldeath (41). Panx12/2 mice are viable and fertile indicating thatpannexin-1 is not essential for mouse development. MonitoringPanx12/2 mice from birth to adulthood for 12 mo did not revealany gross defects. Histological and pathology examinations of

FIGURE 7. Release of chemoattractants from apoptotic thymocytes requires pannexin-1. WT (Panx1+/+) and Panx12/2 thymocytes were treated with 0.5

mM dexamethasone in the absence or presence of 25 mM zVAD for 4 h. Culture supernatants were used in Transwell migration assays with thioglycolate-

elicited peritoneal macrophages from WT (A) or Panx12/2 (B) mice. Where indicated, 1 U/ml apyrase was added to culture supernatants for 5 min before

the migration assay. The ATP content in the culture supernatants is shown in C. Data represent the mean 6 SD of triplicate samples. Results are rep-

resentative of three independent experiments. All p values were determined by t test using independent variants.

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major organs were also normal. Whether clearance of apoptoticcell populations during development or pathogen challenge isdelayed remains to be determined.

DisclosuresY.Q., S.M., K.N., L.L.G., S.L., J.E.C., D.H., and V.M.D. are employees of

Genentech, Inc.

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