Mature human eosinophils express functional Notch ligands mediating eosinophil autocrine regulation

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doi:10.1182/blood-2008-05-155937Prepublished online January 26, 2009;2009 113: 3092-3101

SpencerAmy L. Radke, Lauren E. Reynolds, Rossana C. N. Melo, Ann M. Dvorak, Peter F. Weller and Lisa A. eosinophil autocrine regulationMature human eosinophils express functional Notch ligands mediating

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PHAGOCYTES, GRANULOCYTES, AND MYELOPOIESIS

Mature human eosinophils express functional Notch ligands mediating eosinophilautocrine regulationAmy L. Radke,1 Lauren E. Reynolds,1 Rossana C. N. Melo,1,2 Ann M. Dvorak,3 Peter F. Weller,1 and Lisa A. Spencer1

1Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA; 2Department of Biology, Federal University of Juiz deFora, Juiz de Fora, Brazil; and 3Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA

Eosinophil chemotaxis and survival withintissues are key components in the develop-ment of tissue eosinophilia and subsequenteffector responses. In this study, we demon-strate a novel mechanism of eosinophil au-toregulation affecting migration and sur-vival mediated through Notch signaling. Weshow for the first time that human bloodeosinophils express Notch receptors andNotch ligands, expressions of which areinfluenced by the presence of eosinophil-

activating granulocyte-macrophage colony-stimulating factor (GM-CSF). Evidence ofNotch receptor activation and subsequenttranscription of the Notch-responsive geneHES1 were observed in GM-CSF–stimulatedeosinophils, confirming functionality ofeosinophil-expressed Notch-signaling com-ponents. Moreover, by inhibiting Notch sig-naling with !-secretase inhibitors or Notchreceptor–specificneutralizingantibodies,wedemonstrate that autocrine Notch signaling

enhances stimulus-mediated actin rear-rangement and eosinophil chemokinesis,and impairs eosinophil viability. Taken to-gether, these data suggest autocrine Notchsignaling, enhanced in response to tissue-or inflammatory-derived signals, influenceseosinophil activity and longevity, which mayultimately contribute to the development oftissue eosinophilia and exacerbation or re-mediation of eosinophil effector functions.(Blood. 2009;113:3092-3101)

Introduction

Eosinophils are innate immune leukocytes implicated in thepathogeneses of multiple inflammatory responses, includingparasitic helminth infections, RNA viral infections, and allergicdiseases (reviewed in Rothenberg and Hogan1). Eosinophils arerecruited from the circulation by chemotactic factors, includinginterleukin (IL)–5 and eotaxin-1 (CCL11), and activated withintissues in response to inflammatory-derived mediators.2,3 Inchronic airway inflammatory conditions, such as asthma, acti-vated eosinophils within tissues modulate immune responsesand elicit effector functions through differential secretion ofcytokine, lipid, and cationic protein mediators.1 In contrast,tissue eosinophilia may be protective in the face of someparasitic helminth infections,4,5 and eosinophil-derived RNasesare effective combatants against infection with RNA viruses, such asrespiratory syncytial virus (RSV).6,7 Delineation of mechanismsmediating chemotaxis, activation, and survival of eosinophils isthus an attractive goal for development of therapies both toalleviate eosinophil-mediated tissue destruction and, conversely, topromote protective functions of eosinophils.

Eosinophil chemotaxis and survival within tissues are enhancedthrough exposure to specific cytokines, chemokines, and otherproinflammatory molecules. Included among the eosinophilopoi-etins are IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF), which play critical roles in promot-ing the generation of eosinophils in the bone marrow, enhancingchemotaxis into tissues, and delaying eosinophil apoptosis withintissues.8 Of note, IL-3, IL-5, and GM-CSF are cytokines alsosynthesized and released from eosinophils, providing for potentialautocrine regulation.1

Notch signaling is a strongly evolutionarily conserved pathwayfirst noted for its principal role in cell-to-cell communications,dictating critical cell fate decisions during development. Signalingis mediated through 4 mammalian Notch receptors (1, 2, 3, and 4)and at least 5 identified Notch ligands, subdivided into 2 families:Jagged 1 (J1) and 2 (J2), and Delta-like 1 (DL1), DL3, and DL4.Binding of Notch ligands to Notch receptors on neighboring cellsinitiates sequential !- and "-secretase–mediated proteolytic cleav-age events, releasing the intracellular portion of the Notch receptor(NICD) from the plasma membrane of target cells, initiatingdownstream effects of Notch activation (reviewed in Maillard etal9). In addition to critical roles throughout development, recentstudies have revealed novel effects of Notch signaling in maturecells, including T and B lymphocytes.9-12 Moreover, Notch signal-ing is implicated in multiple diverse processes from immunemodulation to fibrosis13,14 and oncogenesis.15-17 Currently, inhibi-tion of "-secretase, which also cleaves the amyloid # precursorprotein to produce amyloid # peptide, is in trials as a treatment forAlzheimer disease.18

Notch signaling inhibits eosinophilopoiesis, as demonstrated byenhanced in vitro eosinophil development in the presence of a"-secretase inhibitor.19 Our studies show, for the first time, thatmature human blood eosinophils express Notch receptors andNotch ligands. Eosinophil-expressed Notch ligands are functional,and their expression is enhanced by the eosinophil-activatingcytokine GM-CSF. Notch signaling enhances GM-CSF–inducedeosinophil polarization and chemokinesis, and diminishes eosino-phil viability, demonstrating autoregulatory functions for eosinophil-expressed Notch ligands. This work reveals Notch ligand and

Submitted May 6, 2008; accepted January 10, 2009. Prepublished online as BloodFirst Edition paper, January 26, 2009; DOI 10.1182/blood-2008-05-155937.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 USC section 1734.

3092 BLOOD, 26 MARCH 2009 ! VOLUME 113, NUMBER 13

receptor expression by circulating human eosinophils, and identi-fies Notch signaling–mediated autoregulatory mechanisms in eosin-ophils influencing eosinophil activities and viability in response totissue- or inflammatory-derived signals. Thus, Notch signalingpathways may provide novel therapeutic targets for treatment ofeosinophil-associated pathologies.

Methods

Cell isolation and stimulation

Eosinophils were purified from donor blood by negative selection, aspreviously described,20 with the exception that hypotonic red blood cell(RBC) lysis was omitted to avoid any potential for RBC lysis to affecteosinophil function. Written informed consent was obtained from donors inaccordance with the Declaration of Helsinki, and Institutional ReviewBoard (IRB) approval was obtained from the Beth Israel DeaconessMedical Center. Both mildly atopic and healthy donors were included, withtotal eosinophils recovered from 320 mL of blood ranging from 8 to50 million. Briefly, venous blood was collected into a 6% dextran salinesolution (Baxter, Deerfield, IL), and RBCs were allowed to sediment. Buffycoat was centrifuged over Ficoll to separate eosinophil-containing granulo-cyte pellets from peripheral blood mononuclear cells (PBMCs). Eosino-phils were isolated from granulocyte pellets by incubation with a depletionantibody (Ab) cocktail (containing Abs against CD2, CD14, CD16, CD19,CD56, and glycophorin A, StemSep; StemCell Technologies, Vancouver,BC), followed by passage over columns (Miltenyi Biotec, Auburn, CA)under magnetic force. Most RBCs present in the granulocyte pellet wereretained in the cell separation column during the magnetic negativeselection process, as the depletion Ab cocktail includes Abs to glycophorin.Purity of isolated eosinophils was greater than 99% of nucleated cells withno more than 3% contaminating RBCs and viability greater than 99%, asdetermined by microscopic analysis and trypan blue exclusion, respec-tively. Human T cells were isolated from the PBMC layer by negativeselection using the human CD4$ T-cell enrichment kit (StemSep; StemCellTechnologies), per manufacturer’s instructions. For overnight incubations,cells were resuspended at 106 cells/mL in RPMI supplemented with 10%FBS (Life Technologies, Grand Island, NY) and 1% penicillin/streptomycin(Life Technologies) with or without GM-CSF (R&D Systems, Minneapolis,MN) at the indicated concentrations and in the presence or absence ofinhibitors or dimethylsulfoxide (DMSO) vehicle control, and cultured at 37°C.

Reagents and Abs

Anti-J1 Ab (clone 188331; R&D Systems) was used for immunoprecipita-tion and Western blotting of J1. Abs specific for extracellular (ab10580;Abcam, Cambridge, MA) and intracellular (clone H-114; Santa CruzBiotechnology, Santa Cruz, CA) domains of J1 were used for flowcytometry and microscopy. Abs against J2 (clone H-143), Notch 1 (cloneH-131), and Notch 2 (clone 25-255), used for flow cytometry, were fromSanta Cruz Biotechnology. Anti-Notch 1 (clone A-6; Thermo Scientific,Fremont, CA) was used for immunoprecipitation and Western blotting ofNotch 1. Anti-Notch 1 (ab8925; Abcam) was used for detection of thecleaved intracellular domain of Notch 1. Abs against Aph-1 used for flowcytometry and immuno–electron microscopy (EM) were from Abcam.Secondary Abs for immuno-EM were affinity-purified Fab conjugated to1.4 nm gold (1:100; Nanogold; Nanoprobes, Stony Brook, NY). "-Secretaseinhibitors and their predetermined optimal concentrations included thefollowing: "-secretase inhibitor II (GSI; Calbiochem, San Diego, CA;10 %M); N-[N-(3,5-difluorophenacetyl)-L-alanyl)-S-phenylglycine t-butylester (DAPT; Calbiochem; 0.2 %M); and L-685,458 (Sigma-Aldrich,St Louis, MO; 0.05 %M). For neutralization, Abs against Notch 1 (cloneA-6; Thermo Scientific) were included at a final concentration of 10 %g/mL.

Lysate preparation and Western blotting

Pelleted cells were lysed in lysis buffer (1% Triton X-100, 50 mM Tris-HCl,300 mM NaCl, 5 mM EDTA (ethylenediaminetetraacetic acid), 0.02%

sodium azide, and 1/100 diluted protease inhibitor cocktail (Sigma-Aldrich). Lysates were preincubated with protein A beads conjugated toisotype control Abs. Unbound supernatants were immunoprecipitated withAbs against J1 or Notch 1. Recovered beads were washed well andresuspended in 30 %L reducing buffer. Bound protein was eluted by boilingfor 5 minutes. Samples were run on 4% to 12% bis-Tris gels (Invitrogen,Carlsbad, CA) under denaturing conditions, transferred to polyvinylidenedifluoride (PVDF) membranes, and blocked overnight with 5% milk beforeprobing with anti-J1 or anti–Notch 1 Abs (2 %g/mL), followed by anti–mouse horseradish peroxidase (HRP)–conjugated secondary Abs (JacksonImmunoResearch Laboratories, West Grove, PA). Membranes were devel-oped (WestPico chemiluminescence kit; Pierce, Rockford, IL) per themanufacturer’s instructions.

Immuno-EM

Pre-embedding immunonanogold EM was performed in frozen 10-%m sections,as previously described.21 Pre-embedding immuno-EM optimizes antigen preser-vation and is more sensitive to detect small molecules than postembeddinglabeling done after conventional EM processing. Moreover, to reach antigens atmembrane microdomains such as small vesicles, we used labeling with verysmall (1.4 nm) gold particles (Nanogold). For controls, primary Ab was replacedby an irrelevant isotype control Ab, and primary Ab was omitted. Specimenswere examined on a transmission electron microscope (CM 10; Philips,Eindhoven, The Netherlands) at 60 KV.

Flow cytometry and immunofluorescence

For extracellular detection, nonpermeabilized eosinophils were stained withrelevant primary Abs or appropriate isotype controls for 30 minutes at 4°C,followed by incubation with Alexa 488 (Invitrogen)– or fluoresceinisothiocyanate (FITC; Jackson ImmunoResearch Laboratories)–conjugatedsecondary Abs (diluted 1/100), for 15 minutes at 4°C in the dark. Cells werewashed well and stored in fluorescence-activated cell sorter (FACS) buffer(HBSS&/& $ 0.5% BSA) supplemented with 0.5% paraformaldehyde(PFO) at 4°C until analysis. For staining with intracellularly targeted Abs,eosinophils were fixed for 5 minutes at room temperature in 2% PFO,followed by permeabilization for 5 minutes at 4°C in 0.1% saponin-containing FACS buffer supplemented with 2.5% normal goat serum and2.5% normal human serum. Permeabilized cells were incubated withprimary relevant Abs or appropriate isotype controls for 30 minutes at 4°C,followed by incubation with FITC-conjugated secondary Ab (diluted 1/100;Jackson ImmunoResearch Laboratories) in the continued presence of 0.1%saponin. Cells were washed well, resuspended in FACS buffer, and stored at4°C until analysis.

Data were acquired using a FACScan with CellQuest acquisition andanalysis software (BD Biosciences, Franklin Lakes, NJ). Data are presentedas percentage of increase in mean fluorescence intensity (MFI), calculatedby the following equation: [(geometric MFI of relevant Ab & geometricmean MFI of isotype control Ab) / (geometric MFI of isotype controlAb)] ' 100.

Detection of apoptotic cells

Cells were incubated with FITC-conjugated annexin V (1:100; Biovision,Mountain View, CA) and propidium iodide (PI; 1 %g/mL final concentra-tion; Invitrogen) for 30 minutes on ice before analysis by flow cytometry.

RNA preparation and real-time reversetranscription–polymerase chain reaction

Cell pellets were resuspended in 500 %L RNALater (Ambion, Austin, TX)and incubated at 4°C for at least 15 minutes before extraction of total RNA.RNA was extracted from cell pellets using the RNeasy system (QIAGEN,Valencia, CA), per the manufacturer’s instructions. cDNA was synthesizedusing iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA). Real-timereverse transcription–polymerase chain reaction (RT-PCR) was performedwith the 7300 thermocycler (Applied Biosystems, Austin, TX) using theiTaq system with carboxy-X-rhodamine (ROX; Bio-Rad). Primers andTaqMan-specific probes for all Notch ligands and Notch receptors, and for

NOTCH SIGNALING IN HUMAN EOSINOPHILS 3093BLOOD, 26 MARCH 2009 ! VOLUME 113, NUMBER 13

18S rRNA, were from Applied Biosystems. All reactions were performed intriplicate wells, and 18S rRNA levels were used as an internal endogenouscontrol. Results are presented as log 10 (RQ), in which RQ is the relativequantification (or fold-change), as determined by the equation RQ ( 2&))Ct,where )Ct and ))Ct (Ct indicates threshold cycle) are defined by thefollowing equations:

)Ct ! Ct target gene " Ct 18S endogenous control

))Ct ! )Ct sample " )Ct baseline.

Measurement of eosinophil shape change using flow cytometry

Cell pellets were fixed for 5 minutes in 4% PFO at room temperature,washed, and resuspended in FACS buffer until analysis of forward scatter(FSC) parameter by flow cytometry. Fold increase in FSC percentage wascalculated as FSC % ( (FSC from stimulated eosinophils/FSC from un-stimulated eosinophils) ' 100. Mean changes in FSC were calculated fromtriplicate wells per condition.

F-actin staining of eosinophils

Cells were fixed for 5 minutes in 4% PFO at room temperature, permeabil-ized on ice for 30 minutes in FACS buffer supplemented with 0.5% TritonX-100 and 5% normal goat serum, and incubated for 1 hour with Alexa488–conjugated phalloidin (1:70; Molecular Probes, Eugene, OR) andHoescht stain (1:10 000; Molecular Probes) diluted in permeabilizationbuffer.

Chemokinesis assay

Eosinophils (106/mL) were primed for 5 hours at 37°C with 1 pM GM-CSFor medium alone in the presence or absence of "-secretase inhibitors orneutralizing Abs. After priming, cells were washed in RPMI supplementedwith 0.1% ovalbumin (OVA), resuspended in RPMI plus OVA containingfresh inhibitors, and added (2 ' 105 cells in 100 %L) in triplicate to theupper chambers of 24-well transwell plates (Corning Glass, Corning, MA),separated from medium-containing lower wells by polycarbonate inserts(5 %M pore size). Plates were incubated for an additional hour at 37°Cbefore recovering, and migrated cells from the lower compartment werecounted. Cells were quantitated from triplicate wells by averaging totalevents counted in 15 seconds using a flow cytometer.

Statistical analysis

Levels of significance between individual groups or dose-response curveswere analyzed by unpaired Student t tests or 2-way ANOVA withBonferroni posttests, respectively. P values less than .05 were consideredstatistically significant.

Results

Mature human blood eosinophils express Notch receptors

Notch signaling influences eosinophilopoeisis, which may suggestNotch receptor expression by immature eosinophils.19 To deter-mine whether circulating eosinophils might express Notch recep-tors, RNA was extracted from human blood eosinophils and probedfor Notch receptors 1, 2, 3, and 4 mRNA. mRNAs encoding Notchreceptors 1 and 2 (Figure 1A), but not 3 or 4 (data not shown), werereadily detected in freshly isolated eosinophils.

Notch 1 protein expression was confirmed by immunoprecipi-tation from eosinophil whole-cell lysates with anti-N1 Abs(Figure S1A, available on the Blood website; see the Supplemen-tal Materials link at the top of the online article). To furtherinvestigate localization of eosinophil-expressed Notch receptorproteins, freshly isolated blood eosinophils were incubated,before and after plasma membrane permeabilization, with Abs

against extracellular domains of Notch receptors 1 and 2. Notch1 was detected on the cell surface of eosinophils from 4 of5 donors (Figure 1B), and within permeabilized cells from all5 donors (Figure 1C). (See Figure S1B for comparison ofrelative Notch 1 expression between eosinophils and CD4$

T cells.) Likewise, cell-surface Notch 2 expression was detectedon eosinophils from only 2 of 5 donors (Figure 1B), whereas

100 101 102 0 101 10210

250 200

Notch 1 Notch 2

N1 (95 bp) N2 (73 bp)

Notch 1 Notch 2

Donor ID

100 101 102 100 101 102

Notch 1 Notch 2

Surface expression

Total expression

D Di Di

20.87 23.13 13.46 34

22.76 25.10 13.78 33

22.36 24.58 13.94 31

N2 N1 18S Donor #

Ave. Ct values

Figure 1. Human blood eosinophils express Notch receptors. (A) Total RNAextracted from freshly isolated human eosinophils from 3 separate donors wasreverse transcribed, and cDNA was probed by real-time PCR for Notch receptor1 and 2 mRNA expression. neg, no RNA-negative control. Table includes average Ctvalues for N1 and N2 for comparison with 18S housekeeping controls. Intact (B) orsaponin-permeabilized (C) eosinophils were analyzed by flow cytometry for expres-sion of surface or intracellular Notch receptors, respectively. Shaded histogramrepresents isotype control. (D) Freshly isolated eosinophils were processed forimmuno-EM with Abs against Notch receptor 1. Notch 1 expression was observed atthe plasma membrane (Di) and within granules (Dii). Bars represent 1 %m (D) and480 nm (Di,ii). Nu indicates nucleus.

3094 RADKE et al BLOOD, 26 MARCH 2009 ! VOLUME 113, NUMBER 13

eosinophils from all 5 donors exhibited intracellular Notch 2receptor expression (Figure 1C).

In full corroboration, immuno-EM localization revealed Notch 1expression at the cell periphery (Figure 1Di), with a greater densityof staining observed intracellularly (Figure 1D). Although cytoplas-mic vesicular compartments were labeled, the most robust signallocalized to secretory granules (Figure 1Dii).

Eosinophil expression of Aph-1

Notch receptor activation requires sequential cleavage eventsmediated through !- and "-secretase activities. Functional"-secretase complexes comprising 4 proteins: presenilin, nicas-trin, Pen-2, and Aph-1.22,23 To confirm the potential for classicalNotch receptor activation in human eosinophils, we analyzedthe expression and cellular localization of "-secretase com-plexes as represented by Aph-1 expression. Aph-1, a keycomponent of the functional "-secretase complex, is thought tobe involved in the early stages of "-secretase complex formationwithin the endoplasmic reticulum (ER),24-26 and is present as acomponent of active "-secretase within the Golgi.27 Freshlyisolated human eosinophils stained positively with Abs againstAph-1 (Figure 2A). As anticipated, Aph-1 was expressed withineosinophil Golgi (Figure 2B,Bi). Importantly, Aph-1 was alsostrongly expressed in association with the plasma membrane(Figure 2C,Ci) and granules (Figure 2B,Bii), reminiscent of thepattern of Notch receptor 1 expression.

Mature human blood eosinophils express Jagged and Deltafamily Notch ligands

To determine whether circulating eosinophils might express Notchligands in addition to Notch receptors, freshly isolated bloodeosinophils were evaluated by real-time RT-PCR for expression ofthe 5 known Notch ligands: J1, J2, DL17, DL3, and DL4. J1mRNA was identified in eosinophils from 21 of 22 donors analyzed(95%), whereas J2, DL1, and DL4 mRNAs were detected in cellsfrom 6 of 13 (46%), 3 of 7 (43%), and 5 of 7 (71%) donors,respectively. DL3 mRNA was not detected in any donor tested. Gelanalyses of recovered PCR products revealed amplicons of theexpected sizes for J1, J2, DL1, and DL4, further confirmingspecificity of results (data not shown). Table 1 shows results from5 donors probed simultaneously for mRNA expression of all Notchligands, and results from all donors analyzed are included in Table S1.

In light of constitutive J1 mRNA expression by blood eosino-phils, we investigated protein expression of Jagged family mol-ecules. Western blotting of anti-J1-Ab–immunoprecipitated eosino-phil lysates revealed J1 protein expression (Figure 3A). (See FigureS1C for comparison of eosinophil and T cell J1 expression levels.)Using a different anti-J1 Ab to confirm these results by flowcytometry, J1 protein was detected in eosinophils from 11 of12 donors evaluated (Figures 3B and 4A), extending the findings ofconstitutive J1 mRNA expression in eosinophils (Table 1) todocument eosinophil expression of J1 protein. Total J2 proteinexpression by eosinophils was detected by flow cytometry in 7 of12 donors (Figures 3C and 4A), approximating the percentage ofdonors found to express detectable J2 mRNA.

Notably, detection of cell-surface J1, using an extracellularlytargeted anti-J1 Ab by flow cytometry or immunofluorescencemicroscopy, revealed J1 surface expression in only 2 of 6 experi-ments (Figure S1D), suggesting J1, like Notch receptors, might besequestered within circulating eosinophils. In full support, im-

muno-EM of freshly isolated eosinophils revealed detectable J1(Figure 3D,Di) and J2 (data not shown) expression at the cellperiphery, with more dense expression of J1 and J2 proteinlocalized to specific granules (Figure 3D,Dii, and data not shown).

J1 and J2 protein expression is influenced by GM-CSF

Observed levels of J1 and J2 proteins between donors ranged from6 to 150% of isotype control fluorescence intensities (Figure 4A).Expressions of J1 and J2 were not statistically correlated (Pearsoncoefficient ( 0.34, R2 ( 0.1125); however, J2 was not detected incells with relatively low (* 50-fold increase over isotype control)levels of J1 protein. It is unclear whether the varying levels of J1and J2 expression by eosinophils singularly represent in vivoexpression levels and/or reflect ex vivo down-regulation of theirexpression. That J1 and J2 expression may be transitory issupported by the finding that J1 mRNA expression decreased upon

100 101 102

Figure 2. Human eosinophils express Aph-1. Freshly isolated human bloodeosinophils were analyzed for intracellular Aph-1 expression by flow cytometry ofsaponin-permeabilized cells (A) and immuno-EM (B,C). Aph-1 expression wasobserved within the Golgi (Bi), within granules (Bii), and at the cell periphery (Ci). Bars,870 nm (B,C); 440 nm (Bi,ii and Ci). Nu indicates nucleus. Scale bar equals 1.2 cm.

culture in the absence of exogenous stimuli and remained higher inthe presence of GM-CSF (Figure 4B). Likewise, detection of bothJ1 and J2 proteins is decreased, often to undetectable levels, afterovernight culture in the absence of exogenous stimuli (Figure 4C).

Protein levels of both J1 and J2 were at least partially maintainedupon inclusion of 100 pM of the eosinophil-activating cytokineGM-CSF in the culture medium (Figure 4C), suggesting environ-mental signals may be integral to maintaining Notch ligandexpression by eosinophils in vivo. Despite GM-CSF–inducedenhancement of total Notch ligand expression by eosinophils, wefailed to detect an increase in surface expression by flow cytometryof J1, Notch 1, or Notch 2 after overnight incubation with 100 pMGM-CSF, despite evidence by electron microscopy of significanteosinophil activation and granule mobilization at this dose (datanot shown; n ( 3).

Eosinophil-expressed Notch ligands are functional

Dual expression by mature eosinophils of Notch ligands andreceptors enabled us to investigate the functional potential ofeosinophil-expressed Notch ligands through autocrine assays.Upon Notch receptor activation, sequential specific cleavageevents result in the release of a portion of the Notch intracellulardomain (NICD), which subsequently translocates into the nucleus,where it binds CBF1, suppressor of hairless, Lag-1 (CSL),converting this complex into a transcriptional activator, turning onspecific gene transcription.28 To monitor activation of Notchreceptors, purified eosinophils were stimulated with GM-CSF inthe presence of either the Notch signaling inhibitor GSI or vehiclecontrol, and assessed by flow cytometry at 30, 60, and 120 minutesfor the presence of NICD using an Ab recognizing the cleaved, butnot the intact, intracellular domain of Notch receptor 1. Althoughprecise kinetics of Notch receptor activation between individualdonors varied from 30 to 120 minutes of stimulation, detection ofactivated NICD in GM-CSF–stimulated eosinophils was dimin-ished in the presence of GSI in 5 of 7 experiments (Figure 5A).

The basic helix-loop-helix transcription factor, hairy/enhancerof split homologue-1 (HES1), is an early downstream target ofNotch signaling in many cell types.9 To confirm the functionalpotential of eosinophil-expressed Notch ligands using a down-stream target, HES1 mRNA expression was compared in GM-CSF–stimulated eosinophil cultures in the presence or absence of GSI.As demonstrated in Figure 5B, HES1 mRNA levels were effec-tively suppressed in the absence of Notch signaling. These datademonstrate that Notch ligands expressed by human eosinophilsare functional, that the GSI is successfully inhibiting Notchreceptor signaling, and that autoregulatory Notch signaling occursin human eosinophils.

Treatment with GSI promotes eosinophil viability

To determine whether Notch signaling might regulate eosinophilactivities, eosinophil longevity was evaluated in the presence orabsence of GSI. In the absence of exogenously added growthfactors, 70% to 75% of eosinophils remained viable after overnightincubation in serum-containing medium. However, when GSI wasincluded in the culture medium, eosinophil viability remained at85%-90% after 24-hour culture. Inhibition of Notch signaling

Table 1. Human blood eosinophils constitutively express Jagged 1 and differentially express Jagged 2, Delta 1, and Delta 4

Donor no. Sex Eos count*, "106 Jagged 1 Jagged 2 Delta 1 Delta 3 Delta 4

4 F 27.9 $ $ $ & $

11 M 39.5 $ & & & &

16 F 8.3 $ & & & $

18 F 21.2 $ $ & & $

24 F 15.5 $ & & & &

*Eosinophil (Eos) count is total number of eosinophils isolated from 320 mL whole blood.

Jagged 1

Jagged 2

100 101 102

T Cells EOS

IgG IgG J1 J1

Figure 3. Human blood eosinophils express Notch ligands J1 and J2 protein.(A) Freshly isolated blood eosinophils or CD4$ T cells (positive control) were lysed andimmunoprecipitated with anti-J1 or isotype control Abs. Immunoprecipitated proteins wereimmunoblotted with anti-J1Abs. n ( 3. Saponin-permeabilized eosinophils were analyzedby flow cytometry for expression of J1 (B) or J2 (C). Shaded histograms ( isotype control.(D) Freshly isolated eosinophils were processed for immuno-EM and stained with Absagainst J1. J1 expression was observed at the plasma membrane (Di) and withineosinophil-specific granules (Dii). Bars represent 1.1 %m (D) and 650 nm (Di,ii).

improved cell viability at 24, 48, or 72 hours by approximately20% (Figure 6A; see Figure S2 for GSI dose response). Similarresults were obtained using either uptake of PI or trypan blue asindicators of cell death (data not shown).

To confirm that increased cell viability in the presence of GSIreflected a diminished rate of apoptosis, eosinophils recovered after24 hours of culture were simultaneously stained with PI andannexin V to detect dead and apoptotic cells, respectively. Thepresence of increased numbers of apoptotic cells (annexin V$, PI&)in the control, vehicle-treated wells indicates that GSI treatmentincreases eosinophil viability through inhibition of apoptosis(Figure 6B).

Treatment with GSI diminishes activation-induced actinrearrangement in human blood eosinophils

To explore additional potential Notch signaling–mediated auto-regulatory effects on eosinophils, activation-induced shapechanges were monitored in eosinophils after stimulation withGM-CSF in the presence or absence of GSI. As demonstrated inFigure 7A, Ai and Aii, eosinophils stimulated with GM-CSFundergo a dose-dependent shape change defined by a rearrange-ment of the actin cytoskeleton that can be objectively quantifiedthrough comparison of forward side scatter (FSC) propertiesusing flow cytometry, as previously described.29 Briefly, theFSC parameter of flow cytometry is a measurement of thelight-scattering profile of individual cells. Thus, a round cellwill elicit a decreased FSC value accordant with its minimallight-scattering potential in comparison with an elongated cell.

(Total cellular events are included as histograms in Figure S3A.)Notch signaling did not appear to influence rapid changes inshape occurring within 1 hour of GM-CSF stimulation. How-ever, at time points greater than 3 hours, a dose-dependentimpairment in GM-CSF–induced eosinophil shape change wasobserved in the presence of increasing concentrations of GSI(Figure 7C; see Figure S3B for GSI dose response), furtherdefined by a lack of polarized actin rearrangement (Figure 7Aii).Eosinophil viability after 24-hour incubations in the presence ofGM-CSF was greater than 98% in both vehicle- and GSI-treatedgroups, as determined by trypan blue exclusion and uptake of PI(data not shown), suggesting cell death does not contribute toGSI-inhibitable shape changes. Cultures of eosinophils alone,without exogenous stimuli, revealed a basal level of Notch-mediated shape change (compare 0-pM data points in thepresence or absence of GSI; Figure 7A). Thus, to determinewhether the apparent inhibition of GM-CSF–induced shapechange was solely a reflection of decreased basal eosinophilactivation or indicative of synergistic effects between Notch andGM-CSF signaling, FSC values were normalized to the nonstimu-lated (NS) values of the corresponding condition. By thisreanalysis, GM-CSF–induced shape change (fold increase inFSC above nonstimulated baseline) remains significantly im-paired in the presence of GSI (Figure 7B), suggestive of synergybetween GM-CSF and Notch signaling pathways. Notably,impairment of GM-CSF–induced eosinophil polarization in thepresence of GSI did not reflect an overall defect in eosinophilactivation, as GM-CSF–induced surface expression of CD69

100 101 102 100 101 102 100 101 102

Jagged 1

Jagged 2

200 200 200

Fresh Overnight CultureMedium alone +GM-CSF

Fresh 0 100

pMGM-CSF

25 26 27 28 29 30 31 32

Donor ID

Figure 4. J1 and J2 protein expression is influenced byGM-CSF. (A) Saponin-permeabilized eosinophils were ana-lyzed by flow cytometry for expression of J1 and J2. Data arepresented as percentage of increase in MFI over isotypecontrol. (B) RNA extracted from eosinophils immediatelyafter isolation (Fresh) or after overnight culture in the pres-ence or absence of 100 pM GM-CSF was evaluated byreal-time RT-PCR for J1 mRNA expression. Data are pre-sented as the log 10 of the relative quantification, usingovernight culture in medium alone as the baseline value.Data are from 1 experiment representative of 3 separateexperiments. (C) Total J1 (top panels) or J2 (bottom panels)protein was detected in saponin-permeabilized eosinophilsimmediately after isolation (Fresh) or after overnight culturein the presence or absence of 100 pM GM-CSF. Shadedhistograms ( isotype control.

was not impaired on eosinophils in the presence of GSI after24 hours of stimulation (Figure 7D).

GM-CSF–primed chemokinesis is mediated through Notchreceptor 1

Actin-mediated cell shape change is considered a prerequisite forcell migration and chemotaxis. GM-CSF priming induces random,chemokinetic migration of eosinophils30 and enhances eosinophilchemotactic responses to subsequent stimulation (ie, with IL-8 orleukotriene B4 [LTB4]) in vitro.31 To determine whether Notchsignaling might also be required for optimal GM-CSF–inducedchemokinetic responses, eosinophils were primed with GM-CSFfor 5 hours in the presence or absence of GSI before subsequentassessment of cell migration in a chemokinesis assay. As shown inFigure 7E, inclusion of GSI was sufficient to counteract theGM-CSF–priming effect on cell migration. To validate our find-ings, 2 additional "-secretase inhibitors with distinct compositions(DAPT and L-685,458) were used in parallel and revealed a similarrequirement for intact Notch signaling for GM-CSF–primed chemo-kinesis (Figure 7E).

Although the "-secretase inhibitors used in this study effec-tively depress Notch activation in human eosinophils (as confirmedin Figure 5), and are used at concentrations designed to favorselective Notch signaling inhibition, it is plausible that Notch-independent side effects of the inhibitors may be responsible for theobserved effects. To directly link the Notch pathway to GM-CSF–

primed effects on eosinophils, chemokinesis assays were per-formed in the presence or absence of neutralizing Abs againstNotch receptor 1. Compared with cells treated with irrelevantisotype control Abs, inclusion of anti-Notch 1 Abs during primingeffectively inhibited GM-CSF–induced eosinophil chemokinesis(Figure 7F).

Discussion

Enhanced eosinophil chemotaxis, activation, and survival withininflamed tissues are key components in the development of tissueeosinophilia and subsequent eosinophil effector responses. Delin-eating the mechanistic bases for these critical functions is requisitefor the development of more specifically targeted therapeuticapproaches. Our study is the first to demonstrate that mature humanblood eosinophils express functional Notch receptors and Notchligands and are capable of Notch-mediated autocrine signaling.Inhibition of Notch signaling resulted in enhanced viability,decreased actin polarization, and diminished chemokinesis of

100 101 102

Activated N1

+vehicle

HES1 mRNAFigure 5. Eosinophil-expressed Notch ligands are functional. (A) Eosinophilswere stimulated for 60 minutes with 100 pM GM-CSF in the presence of 10 %M GSI(dotted line) or vehicle alone (heavy line) before permeabilization and detection byflow cytometry of the cleaved, intracellular domain of activated Notch receptor 1.Shaded histogram represents isotype control. (B) RNA was extracted from eosino-phils after overnight stimulation with 100 pM GM-CSF in the presence of 10 %M GSIor vehicle control, and analyzed by real-time RT-PCR for the presence of HES1mRNA. Data are presented as the log 10 of the relative quantification using theGSI-treated sample as the baseline value. Data are from a representative of3 separate experiments.

100vehicleGSI

24 48 72Time (hours)

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6.97% 27.21%

V3.83% 7.34%

vehicle

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Figure 6. Treatment with !-secretase inhibitor promotes eosinophil viability.(A) Eosinophils incubated for 24, 48, or 72 hours in the presence of 10 %M GSI orvehicle alone were recovered and incubated on ice with PI (1 %g/mL) for 30 minutesbefore analysis of PI incorporation by flow cytometry. Data shown are means (+ SD)from a representative of 3 independent experiments. (B) Eosinophils recovered after24 hours of incubation in the presence of 10 %M GSI or vehicle alone were analyzedfor exposed annexin V and uptake of PI to differentiate dead from apoptotic cells.Percentages are calculated from total events and represent apoptotic (annexin V$,PI&) and dead (annexin V$, PI$) populations. Data shown are from 1 representativeof 3 independent experiments.

eosinophils, identifying autoregulatory roles for Notch signaling inmature human eosinophils, which may provide novel therapeutictargets for intervention in eosinophil-associated diseases.

Previous work by Kang et al demonstrated a role for Notchsignaling in eosinophil maturation.19 In this study, we have

demonstrated that mature human eosinophils within the bloodcirculation maintain protein expression of Notch receptors andligands. Parallel intra- and extracellular staining revealed Notchligands and receptors to be more robustly expressed withineosinophils as opposed to on the cell surface, indicating Notch

0 1 10 100

*+ vehicle

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11.051.1

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1 3 5 8 260270280290300310320330340350360370 DMSO

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0 101 102 103

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500 600 700 800 900 1000

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anti-N1

mIgG2b

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Figure 7. Notch signaling is required for GM-CSF–induced eosinophil shape change and chemokinesis. (A) Eosinophils were cultured overnight with increasing dosesof GM-CSF in the presence of 10 %M GSI or vehicle alone, fixed with 4% PFO, and analyzed using the FSC parameter of flow cytometry to assess cell shape changes. Datarepresent average FSC geometric means (+ SD) for 1 experiment representative of 8 independent experiments. (i) Live cells were imaged using phase microscopy with aninverted microscope before fixation (imaged with a Nikon TE-300 inverted microscope coupled to a Retiga Exi cooled digital camera; images acquired using QImagingsoftware). (ii) Fixed and permeabilized cells were stained with Alexa 488–conjugated phalloidin to detect F-actin filaments and Hoescht for nuclear staining, as described in“Methods” (imaged with a BX62 Olympus upright microscope, 60' PlanApo objective with numerical aperture of 1.42, coupled to a Hamamatsu Orca AG fire-wire cooled digitalcamera; images acquired using IPLab). (B) Data from panel A were normalized to the relevant nonstimulated values and replotted. (A,B) Statistical significances betweencurves were confirmed using a 2-way ANOVA test, followed by Bonferroni posttests to determine significances at individual doses. (C) Eosinophils were stimulated with 100 pMGM-CSF in the presence of 10 %M GSI or vehicle alone for 1, 3, 5, or 8 hours. (D) Eosinophils were cultured overnight in medium alone (top panel) or with 100 pM GM-CSF(bottom panel) in the presence of 10 %M GSI (dotted line) or vehicle alone (solid line) before staining for CD69 surface expression. Data shown are from 1 experimentrepresentative of 3 independent experiments. Shaded histogram represents isotype control. (E,F) Eosinophils were primed for 5 hours with 1 pM GM-CSF in the presence orabsence of "-secretase inhibitors (E) or neutralizing Abs against Notch 1 (F). After pretreatment, cells were added to upper wells of chemotaxis chambers, and chemokinesiswithin 1 hour was determined by quantifying cells migrating to the lower wells. *P * .05 versus vehicle alone or isotype control–treated samples.

receptors and ligands may be sequestered within the cell. In fullsupport, immuno-EM demonstrated strong association of Notch recep-tors and ligands with eosinophil-specific granules. Intracellular granulesmay serve as a sequestered repository for Notch receptors and ligands,preventing unwarranted cell-autonomous Notch activation while main-taining a rapidly mobilizable cache of signaling components. Alterna-tively, localization of Notch receptors, ligands, and "-secretase com-plexes all to eosinophil granules might hint at an as yet undefinedintracellular signaling mechanism focally centered at the granules.Functional responses presented in this work, however, are most likelyresultant from canonical, cell-surface expression, as evidenced by theability of exogenously added anti-Notch 1–neutralizing Abs to inhibitGM-CSF–primed chemokinesis (Figure 7F).

Constitutive J1 expression and apparent donor-dependent variabilityin expression of J2, DL1, and DL4 indicate plasticity in the potentialeosinophil Notch ligand repertoire, and suggest that eosinophils maymaintain the capacity to initiate varied effects dependent on specificligand expression. That Notch ligand expression might be influenced byenvironmental cues is evidenced by the ability of GM-CSF stimulationto enhance J1 and J2 protein expression (Figure 4B,C). Thus, it isreasonable to speculate that activating signals within inflamed tissuespromote Notch activation in eosinophils through regulation of specificNotch ligand expression, and may provide a mechanism for inflamma-tion-induced eosinophil autoregulation.

Eosinophil-expressed Notch receptors and ligands are func-tional, as evidenced by autocrine, GSI-inhibitable induction of boththe cleaved fragment of activated Notch receptor 1 and downstreamtranscription of the Notch-responsive gene HES1 (Figure 5). Inaddition to demonstrating the functional competence of eosinophil-expressed Notch ligands, these data establish that Notch-mediatedautoregulatory signaling can occur in human blood eosinophils.Our data specifically identify downstream effects of autoregulatoryNotch signaling on eosinophil: (1) viability; (2) GM-CSF–inducedshape changes; and (3) GM-CSF–primed chemokinesis.

Inclusion of GSI in eosinophil cultures improved cell viability(Figure 6). Although the precise mechanism(s) is yet to bedetermined, cell death in the presence of intact Notch signalingoccurred by apoptosis, as determined by staining with annexin Vand PI. These findings may be particularly relevant in light of arecent clinical trial that revealed a mean increase of 48% in bloodeosinophils in patients receiving a "-secretase inhibitor comparedwith those patients receiving placebo treatment (P * .001 betweentreatment and placebo groups).18 Whether this increase in eosino-phil numbers reflects "-secretase inhibitor–mediated enhancementof eosinophil maturation,19 reduction in eosinophil apoptosis (asdemonstrated in this study; Figure 6), or a combination of bothmechanisms is yet to be determined. Moreover, more data areneeded to evaluate potential tissue eosinophilia and to determinethe clinical significance(s) of the rise in eosinophil counts.

Treatment with GSI also effectively inhibited eosinophil shapechanges, as determined by diminished eosinophil FSC and loss ofactin polarization (Figure 7A,Aii). Although the inhibitory effectsof GSI were observed in the absence of exogenous stimulation, it isunclear whether basal-level Notch-mediated shape change oc-curred independently of cytokine signals, as eosinophils are knownto actively secrete numerous cytokines with the potential forautostimulation, including IL-5, GM-CSF, eotaxin, and RANTES(regulated on activation normal T cell expressed and secreted).32

Effects of Notch signaling on eosinophil shape change were morepronounced upon inclusion of GM-CSF, indicative of synergybetween GM-CSF and Notch signaling pathways. Notably, ourfinding that GM-CSF–induced CD69 expression remained intact in

the presence of GSI suggests that inhibition of Notch signalingdoes not block eosinophil activation in general.

In our studies, synergy between Notch and GM-CSF signalingwas not observed in relatively short (1-hour) eosinophil cultures,regardless of whether or not cells were pretreated with GSI inadvance of GM-CSF stimulation (Figure 7C and data not shown).Several plausible explanations may account for the delayed actionof Notch-mediated effects, including the following: (1) inductionof specific Notch ligand(s) or critical expression thresholds may benecessary before induction of significant Notch signaling. (2) Thecontributions of Notch signaling to enhancement of GM-CSF–induced effects may be dependent upon downstream proteinsrequiring longer than 1 hour to be transcribed and translated. (3) Ashared signaling pathway component may be available for GM-CSF signaling at basal levels in circulating cells, whereas Notchsignaling contributes to its replenishment. Mitogen-activated pro-tein (MAP) kinase and nuclear factor (NF)–,B pathways areattractive candidates, as both are required for GM-CSF–inducedeosinophil shape change,33 and Notch signaling has been impli-cated in cross-talk with both of these pathways.34

Downstream of polarized cytoskeletal rearrangements, inhibi-tion of Notch signaling decreased GM-CSF–primed eosinophilchemokinesis (Figure 7E,F). Importantly, similar results wereobtained using 3 distinct "-secretase inhibitors to block the Notchsignaling pathway, or neutralizing Abs against Notch receptor 1,confirming the "-secretase dependence of the observed inhibition,and directly linking the Notch pathway to GM-CSF–primedeosinophil chemokinesis, respectively.

In this study, we demonstrate expression of Notch receptors andligands by mature human blood eosinophils and identify Notch signaling–dependent autoregulatory functions. Taken together, this work suggestsautocrine Notch signaling, enhanced in response to tissue- or inflamma-tory-derived signals, influences eosinophil migration and longevity, andas such may participate in the development of tissue eosinophilia andexacerbation or remediation of eosinophil effector functions. Thesignificance of these studies to eosinophil-associated pathologies lies inthe identification of Notch signaling pathway components as potentialtargets for therapeutic intervention to control eosinophil activation andviability. Moreover, as "-secretase inhibitors continue to be formulatedas potential therapies for Alzheimer disease and cancers, these findingsmay provide insights into potential unanticipated adverse effects oneosinophils.

Acknowledgments

We thank Dr Stephen Blacklow (Brigham and Women’s Hospital,Boston, MA) for helpful discussions throughout the course of this study.

This work was supported in part by National Institutes of Health(Bethesda, MD) grants AI020241 and AI051645 to P.F.W., and anInterest Section Award and the Women in Allergy Junior FacultyDevelopment Award to L.A.S. from the American Academy ofAllergy, Asthma & Immunology (AAAAI; Milwaukee, WI).

Authorship

Contribution: L.A.S., A.L.R., L.E.R., and R.C.N.M. performedresearch; L.A.S., L.E.R., A.L.R., and P.F.W. analyzed and inter-preted data; R.C.N.M. and A.M.D. interpreted EM data; and L.A.S.and P.F.W. designed research and wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: LisaA. Spencer, 330 BrooklineAve, E/CLS Room935, Boston, MA 02215; e-mail: lspencer@bidmc.harvard.edu.

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Mature human eosinophils express functional Notch ligands mediating eosinophil autocrine regulation

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