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ADP RiboseLigands Is Differentially Regulated by CyclicPhagocytes to Formyl Peptide Receptor Chemotaxis and Calcium Responses of

Oppenheimer, Ji Ming Wang and Frances E. LundMoreno-García, Ji-Liang Gao, Philip M. Murphy, Norman Santiago Partida-Sánchez, Pablo Iribarren, Miguel E.

http://www.jimmunol.org/content/172/3/1896doi: 10.4049/jimmunol.172.3.1896

2004; 172:1896-1906; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2004 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Chemotaxis and Calcium Responses of Phagocytes to FormylPeptide Receptor Ligands Is Differentially Regulated by CyclicADP Ribose1

Santiago Partida-Sanchez,* Pablo Iribarren,† Miguel E. Moreno-Garcıa,* Ji-Liang Gao,‡

Philip M. Murphy, ‡ Norman Oppenheimer,§ Ji Ming Wang,† and Frances E. Lund2*

Cyclic ADP ribose (cADPR) is a calcium-mobilizing metabolite that regulates intracellular calcium release and extracellularcalcium influx. Although the role of cADPR in modulating calcium mobilization has been extensively examined, its potential rolein regulating immunologic responses is less well understood. We previously reported that cADPR, produced by the ADP-ribosylcyclase, CD38, controls calcium influx and chemotaxis of murine neutrophils responding to fMLF, a peptide agonist for twochemoattractant receptor subtypes, formyl peptide receptor and formyl peptide receptor-like 1. In this study, we examine whethercADPR is required for chemotaxis of human monocytes and neutrophils to a diverse array of chemoattractants. We found thata cADPR antagonist and a CD38 substrate analogue inhibited the chemotaxis of human phagocytic cells to a number of formylpeptide receptor-like 1-specific ligands but had no effect on the chemotactic response of these cells to ligands selective for formylpeptide receptor. In addition, we show that the cADPR antagonist blocks the chemotaxis of human monocytes to CXCR4, CCR1,and CCR5 ligands. In all cases, we found that cADPR modulates intracellular free calcium levels in cells activated by chemokinesthat induce extracellular calcium influx in the apparent absence of significant intracellular calcium release. Thus, cADPR regulatescalcium signaling of a discrete subset of chemoattractant receptors expressed by human leukocytes. Since many of the chemoat-tractant receptors regulated by cADPR bind to ligands that are associated with clinical pathology, cADPR and CD38 representnovel drug targets with potential application in chronic inflammatory and neurodegenerative disease.The Journal of Immunol-ogy, 2004, 172: 1896–1906.

A denosine 5�-diphosphate ribosyl cyclases (cyclases),such as the mammalian ecto-enzyme CD38, transformNAD� into several products including the calcium-mo-

bilizing metabolite cyclic ADP-ribose (cADPR)3 (1, 2). SinceCD38 is a member of a highly conserved family of cyclases iso-lated from plants, invertebrates, and vertebrates (2), it has beenhypothesized that CD38, via its production of cADPR, is likely tobe an important regulator of calcium-based signal transduction.cADPR modulates the level of intracellular free calcium in cells inat least two ways. In combination with free cytosolic calcium,cADPR induces intracellular calcium release from ryanodine re-ceptor-gated stores by a process referred to as calcium-inducedcalcium release (3). In addition, cADPR has been shown to regu-

late the influx of extracellular calcium (4, 5), possibly by activatingthe store-operated calcium release-activated calcium current channels(Icrac) (6). Although it is very clear that cADPR modulates intracel-lular free calcium levels in cells, less is known about which receptorsrely on cADPR for signaling. In addition, very little is understoodabout the role(s) for cADPR in regulating important cellular processessuch as development, growth, and differentiation.

To address which receptors utilize cADPR for signaling, severallaboratories have now synthesized a number of different cADPRinhibitors including 8-Br-cADPR, a potent cADPR antagonist (7)and N(8-Br-A)D�, a NAD� analogue that can be cyclized byCD38 into the cADPR antagonist 8-Br-cADPR (5). These antag-onists have recently been successfully used to identify receptorssuch as the muscarinic receptor that mobilizes calcium in acADPR-dependent fashion (8, 9). To assess the in vivo signalingrole of cADPR, we produced mice that lack CD38 (10), one of thetwo known mammalian ADP-ribosyl cyclases (2). Using bonemarrow neutrophils isolated from the CD38 knockout (KO) mice,we demonstrated that calcium signaling induced upon ligation ofthe classical chemoattractant formyl peptide receptor (FPR) is de-pendent on CD38 and cADPR (5). Importantly, we also found thatchemotaxis of mouse neutrophils to the FPR ligand fMLF is reg-ulated by cADPR and CD38 (5). Furthermore, we showed thatpretreatment of normal mouse neutrophils with either 8-Br-cADPR or N(8-Br-A)D� inhibited the chemotactic response ofthese normal neutrophils to fMLF (5, 11). Together, these datashowed that cADPR and the ADP-ribosyl cyclase CD38 modulateFPR-induced signal transduction and control the chemotactic re-sponses of mouse neutrophils to fMLF.

The G protein-coupled FPR is one of the founding members ofthe chemoattractant receptor superfamily (12–15). Like many of

*Trudeau Institute, Saranac Lake, NY 12983; †Laboratory of Molecular Immuno-regulation, National Cancer Institute-Frederick Cancer Research and DevelopmentCenter, National Institutes of Health, Frederick, MD 21702; ‡Laboratory of HostDefenses, National Institute of Allergy and Infectious Diseases, National Institutes ofHealth, Bethesda, MD 20892; and §Department of Pharmaceutical Chemistry, Uni-versity of California, San Francisco, CA 94143

Received for publication June 11, 2003. Accepted for publication November19, 2003.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by Trudeau Institute (to F.E.L., S.P.-S, and M.E.M.-G).2 Address correspondence and reprint requests to Dr. Frances E. Lund, TrudeauInstitute, 154 Algonquin Avenue, Saranac Lake, NY 12983. E-mail address:flund@trudeauinstitute.org3 Abbreviations used in this paper: cADPR, cyclic ADP-ribose; cGDPR, cyclic GDP-ribose; FPR, formyl peptide receptor; FPRL1, FPR-like 1; Icrac, calcium release-activated calcium current channel; IP3, inositol trisphosphate; mFPR, mouse FPR;NGD, nicotinamide guanine dinucleotide; SAA, serum amyloid A; KO, knockout;MIP, macrophage-inflammatory protein; CI, chemotaxis index; PMN, polymorpho-nuclear cell; SDF-1, stromal cell-derived factor 1.

The Journal of Immunology

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00

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the chemoattractant receptors, FPRs are constitutively expressedby myeloid lineage cells such as neutrophils, monocytes/macro-phages, and dendritic cells (15). The prototype FPR ligand fMLFinduces a number of biologic activities in myeloid cells, includingchemokinesis, chemotaxis, cytokine production, and superoxidegeneration (13, 14, 16–18). Since N-formylated peptides are de-rived from bacterial (19–21) or mitochondrial proteins (22), in-vestigators initially proposed that the primary function of FPRs isto promote the traffic of phagocytes to sites of infection and tissuedamage where they can exert their antibacterial effector functionsand also clear cell debris. Indeed, mice deficient in one of theknown FPRs, mouse FPR (mFPR)-like 1, have been shown to bemore susceptible to bacterial infections (23).

Recently, it has become clear that regulation of inflammatoryprocesses by FPRs is more complex than originally assumed. Inaddition to the “classical” high-affinity FPR, it is now known thathuman myeloid cells also express a second related receptor, re-ferred to as FPR-like 1 (FPRL1) (24). FPRL1, unlike FPR, is alow-affinity receptor for fMLF and is only activated by high (mi-cromolar) concentrations of fMLF (25). A wide variety of newligands for the different FPRs have recently been identified. Theseinclude peptide library-derived agonists that activate FPR and/orFPRL1 such as MMK1 (26), W (27), and A5 (28) peptides. FPRand FPRL1 can also be activated by peptides derived from hostand pathogen proteins. In particular, FPR is activated by the T20peptide derived from gp41 of HIV-1 (29), while FPRL1 can beactivated by F peptide and V3 peptide derived from HIV-1 gp120(30, 31). Likewise, FPR binds a peptide derived from the endog-enous protein annexin 1 (lipocortin) (32), while FPRL1 binds se-rum amyloid A (SAA) (33), � amyloid peptide (34), prion proteinpeptide (35), and the lipid metabolite lipoxin A4 (36).

Given that several of the naturally occurring FPRL1 ligands areassociated with the pathology that accompanies diseases such asamyloidosis, Alzheimer’s disease, and prion disease, it has beenspeculated that cells responding to FPRL1 ligands may contributeto the inflammatory pathology observed in the diseased tissues (18,37, 38). Since cADPR regulates fMLF-induced calcium signalingin mouse neutrophils (5), we hypothesized that this metabolitemight also regulate signaling through FPRs in human cells. Todetermine whether cADPR regulates signaling through FPR,FPRL1, or both receptors, we have now examined the effect ofcADPR antagonists or CD38 substrate analogues on the calciumand chemotactic responses of human neutrophils and monocytes toFPR and FPRL1-specific ligands as well as to a number of addi-tional inflammatory and homeostatic chemokines. Our resultsdemonstrate that cADPR regulates calcium mobilization and che-motaxis of human neutrophils and monocytes that have been stim-ulated with a discreet, biochemically distinct subset of chemoat-tractant receptors.

Materials and MethodsMice, cell lines, and NAD�/cADPR analogues

mFPRI-deficient mice (backcrossed to C57BL/6J for six generations) wereproduced as previously described (23) and wild-type C57BL/6J controlanimals were purchased from The Jackson Laboratory (Bar Harbor, ME).Animals were housed in the Trudeau Institute Animal Breeding Facility inaccordance with all Trudeau Institute Institutional Animal Care and UseCommittee guidelines. The N9 murine microglial cell line (39) was a kindgift from Dr. P. Ricciardi-Castagnoli (Universita Degli Studi di Milano-Bicocca, Milan, Italy). The cells were grown in IMDM supplemented with5% heat-inactivated FBS, 2 mM glutamine, 100 U/ml penicillin, 100 �g/mlstreptomycin, and 50 mM 2-ME. The cADPR antagonist 8-Br-cADPR wasobtained from Sigma-Aldrich (St. Louis, MO) and N(8-Br-A)D� was syn-thesized according to the protocol of Abdallah et al. (40).

Isolation of neutrophils and monocytes

Mouse bone marrow neutrophils were prepared by flushing bone marrowfrom tibias and femurs of mFPRI-deficient and C57BL/6J mice and thenpositively selecting the neutrophils using biotinylated GR-1 (BD PharM-ingen, San Diego, CA) and MACS Streptavidin Microbeads (Miltenyi Bio-tec, Auburn, CA). Purity was �95% as assessed by FACS. Human leuko-cytes were isolated from fresh peripheral blood donated by healthyvolunteers in accordance with the Trudeau Institute Institutional ReviewBoard regulations (samples kindly provided by the Blood Donor Center,Champlain Valley Plattsburgh Hospital, Plattsburgh, NY). Neutrophilswere purified (�95% purity) using a one-step Ficoll gradient (RobbinsScientific, Sunnyvale, CA). Monocytes were isolated by enriching formononuclear cells using the one-step Ficoll density gradient centrifugationmethod and then purified (�95%) by MACS using a CD14 monocyteisolation kit (Miltenyi Biotec). All purified cells were washed and resus-pended in HBSS supplemented with 1% FBS.

Analysis of CD38 expression and cyclase activity in humanperipheral blood leukocytes

Human peripheral blood leukocytes were isolated from whole fresh pe-ripheral blood by Ficoll density gradient centrifugation and then assessedfor CD38 expression by FACS. Cell suspensions were stained with mouseanti-human CD38-biotin (Caltag Laboratories, Burlingame, CA) or biotin-ylated mouse isotype control Ab (Zymed Laboratories, San Francisco, CA)and anti-human CD15-FITC (BD Biosciences, San Jose, CA) and thenstreptavidin-allophycocyanin (BD Biosciences), and were then analyzed byflow cytometry using a FACSCalibur (BD Biosciences).

To measure CD38-dependent ADP ribosyl cyclase activity in neutrophillysates, purified human peripheral blood neutrophils (1.5 � 107 cells) weredisrupted with lysis buffer containing 2 mM EDTA, 1 mM DTT, 2 �g/mlleupeptin, 1 �g/ml pepstatin, 50 �g/ml PMSF, and 1% Triton X-100 (v/v;Sigma-Aldrich). Solubilized proteins were recovered from the lysate bycentrifugation and were incubated with biotinylated mouse anti-humanCD38 (Caltag Laboratories) or a biotinylated mouse IgG1 Ab (ZymedLaboratories) along with streptavidin agarose beads (Sigma-Aldrich). Theprecipitated protein-bead complexes were extensively washed and then re-suspended in 40 �l of HBSS. CD38-dependent GDP-ribosyl cyclase ac-tivity was determined by measuring accumulation of the fluorescent prod-uct, cyclic GDP-ribose (cGDPR), as previously described (41). Aliquots ofthe protein-bead complex (10 �l) were placed in individual wells of opaque96-well plates (Corning, Rochester, NY) containing 80 �l of HBSS/welland were allowed to settle for 30 min. NGD� (10 �l, 40 �M final con-centration) was added to each well and the plates were incubated for 20min at 37°C. Cyclase activity was then determined by monitoring the ac-cumulation of cGDPR in the reaction using a SpectraMax GeminiXS mi-croplate fluorometer (Molecular Devices, Sunnyvale, CA) that was cali-brated for an excitation wavelength of 300 nm and an emission wavelengthof 415 nm. Relative fluorescence units are reported.

Chemoattractants

The chemokines used in this study were obtained from either Sigma-Al-drich (IL-8 and C5a) or R&D Systems (macrophage-inflammatory protein(MIP) 1� and RANTES; Minneapolis, MN). Chemotactic peptides used inthis study included the following FPRL1 agonists: the synthetic A5, W, andMMK-1 peptides (26–28), amyloid �1–42 peptide (42), HIV-derived F pep-tide (HIV-1, gp120 C4-V4 region) (30), HIV-derived V3 peptide (HIV-1,gp120 V3 region) (31), and SAA polypeptide (33). In addition, the studyused the following FPR agonists: HIV-derived T20 peptide (HIV-1 gp41)(29) and fMLF. N-formylated fMLF peptide was purchased from Sigma-Aldrich. A5 peptide and F peptide were purchased from New EnglandPeptide (Fitchburg, MA). Amyloid �1–42 peptide was purchased from Pen-insula Laboratories (San Carlos, CA) and V3 peptide was obtained fromGlobal Peptide Services (Fort Collins, CO). T20 peptide, MMK-1 peptide,and W peptide were synthesized and purified by the Department of Bio-chemistry, Colorado State University (Fort Collins, CO). The amino acidcomposition of all peptides was verified by mass spectrometry and thepurity was shown to be �90%. No endotoxin was detected in the solubi-lized peptides.

Chemotaxis assays

Chemotaxis assays with mouse neutrophils and human neutrophils andmonocytes were performed using 24-well Transwell plates (Costar, Cam-bridge, MA) with a 3-�m (for neutrophil chemotaxis assays) or a 5-�m (formonocyte chemotaxis assays) pore size polycarbonate filter. Chemotaxisassays for N9 cells were performed with 48-well chemotaxis chambers(NeuroProbe, Cabin John, MD) using polycarbonate filters with an 8-�m

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pore size. Chemoattractants were diluted in HBSS and placed in the lowerchamber, upper chamber, or both upper and lower chambers of the Trans-well. In most experiments, cells were first pretreated for 15–20 min witheither 8-Br-cADPR (0–100 �M; Sigma-Aldrich) or N(8-Br-A)D� (500�M) and then added to the upper chamber of the Transwell in the contin-ued presence of the drug. For neutrophil assays, 1 � 106 cells/Transwellwere incubated at 37°C for 45 min, whereas for monocyte and microglialcell assays 1 � 105 cells/Transwell were incubated at 37°C for 90 min. Thetransmigrated cells were collected from the lower chamber, fixed, andcounted on a flow cytometer. To determine the absolute number of cells ineach sample, a standard number of 20 �m size fluorescent microspheres(Polysciences, Warrington, PA) was added to each tube and counted alongwith the cells. The total number of transmigrated cells � the number ofcounted neutrophils � the total number of beads/the number of beadscounted. In some cases the results are expressed as the mean � SD of thechemotaxis index (CI). The CI represents the fold increase in the numberof untreated or inhibitor-pretreated cells that migrated in response to thechemoattractant divided by the basal migration of untreated or antagonistpretreated cells migrating in response to control medium.

Intracellular calcium mobilization

Purified human peripheral blood neutrophils or monocytes were resus-pended in cell-loading medium (HBSS plus 1 mM Ca2� plus 1 mM Mg2�

plus 1% FBS plus 4 mM probenecid) at 1 � 107 cells/ml and loaded witha mixture of the calcium-sensitive dyes Fluo-3 AM (4 �g/ml) and Fura-Red AM (10 �g/ml; Molecular Probes, Eugene, OR). The cells were in-cubated at 37°C for 30 min, washed twice, and resuspended in cell-loadingmedium at 1 � 106 cells/ml. Cells were preincubated in the presence orabsence of 8-Br-cADPR (100 �M; Sigma-Aldrich) for 20 min and thenstimulated with various chemokines and chemoattractants in calcium-con-taining or calcium-depleted (plus 2 mM EGTA) medium. The accumula-tion of intracellular free calcium was assessed by FACS over the next 7min by measuring the fluorescence emission of Fluo-3 in the FL-1 channeland Fura-Red in the FL-3 channel. Data were analyzed by FACS analysissoftware FlowJo 4.0 (Tree Star, San Carlos, CA) using the kinetic platform.The relative intracellular free calcium levels were expressed as the ratiobetween Fluo-3 and Fura-Red mean fluorescence intensity over time.

Statistical analysis

Data sets were analyzed using GraphPad Prism version 3.0a for Macintosh(GraphPad Software, San Diego, CA). Student’s t test analyses wereapplied to the data sets to determine statistically significant differencesbetween groups. Differences were considered significant when p valueswere �0.05.

ResultsChemotaxis of mouse bone marrow neutrophils to mFPR1 andmFPR2 agonists is regulated by cADPR

Murine neutrophils express at least two functional fMLF receptors(mFPR1 and mFPR2) that can be activated by a number of differ-ent agonists, including fMLF, HIV-derived peptides, SAApolypeptide, and amyloid � peptide (14, 15). We previouslyshowed that 8-Br-cADPR, a competitive antagonist of cADPR thatacts by blocking cADPR-induced calcium mobilization (7), alsoinhibits chemotaxis of mouse bone marrow neutrophils to fMLF(5, 43). To test whether 8-Br-cADPR inhibits the migration ofmouse neutrophils to other mFPR1 or mFPR2 ligands, we mea-sured the chemotactic response of 8-Br-cADPR-treated neutro-phils isolated from the bone marrow of C57BL/6 and mFPR1-deficient mice (Ref. 23; mFPR1 KO) to peptides that activatemFPR1 and/or mFPR2. In agreement with previous studies (44),we found that HIV-derived T20 peptide induced a strong migra-tory response in C57BL/6J neutrophils and essentially no responsein mFPR1 KO neutrophils (Fig. 1), indicating that T20 peptidepreferentially activates mFPR1. In contrast, fMLF, A5 peptide,and the HIV-derived F peptide induced the chemotaxis of mFPR1KO neutrophils (Fig. 1B), indicating that these ligands can activatemFPR2 in mouse bone marrow neutrophils. However, all peptidesincluding fMLF, A5 peptide, and F peptide induced even greatermigration of C57BL/6J neutrophils compared with mFPR1 KOneutrophils (cf Fig. 1, A vs B), suggesting that these ligands, atleast at the concentrations used in this experiment, may activateboth mFPR1 and mFPR2 in mouse bone marrow neutrophils. In-terestingly, 8-Br-cADPR treatment blocked the migration of bothC57BL/6J and mFPR1 KO neutrophils to all of the mFPR1 andmFPR2 ligands tested (Fig. 1). This 8-Br-cADPR-mediated inhi-bition of chemotaxis to mFPR1 and mFPR2 ligands was specificfor FPR ligands as 8-Br-cADPR treatment had no effect on thechemotaxis of mouse neutrophils to the CXCR2 agonists IL-8 andMIP-2 (data not shown and Ref. 5). Together, these data show thata cADPR antagonist can be used to inhibit the migration of mousebone marrow neutrophils to a variety of FPR ligands and suggestthat cADPR regulates the signaling of both mFPR1 and mFPR2 inmouse neutrophils.

FIGURE 1. Chemotaxis of mouse bone marrow neutrophils to mFPR1 and mFPR2 ligands is dependent on cADPR. Bone marrow neutrophils isolatedfrom either C57BL/6J (A) or mFPR1-deficient mice (B) were preincubated for 15 min in medium (�) or medium containing 8-Br-cADPR (100 �M, f).Cells were then placed in the upper wells of chemotaxis chambers that contained medium or peptide chemoattractants that activate mFPR1 (HIV-derivedT20 peptide) or mFPR1 and mFPR2 (fMLF, HIV-derived F peptide, and A5 peptide) in the lower wells of the chamber. Transmigrated cells were collectedafter 45 min and enumerated by flow cytometry. The results are expressed as the mean � SD of triplicate wells for each experimental condition and arerepresentative of at least four similar experiments. Values of p were determined by Student’s t test. �, p � 0.02; ��, p � 0.001; ���, p � 0.0003.

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Human neutrophils express a functional ADP-ribosyl cyclase

The cADPR antagonist 8-Br-cADPR inhibited the chemotaxis ofmouse neutrophils to a number of different FPR ligands (Fig. 1).These data suggested the intriguing possibility that compoundsthat block cADPR-dependent signaling could be used therapeuti-cally to modulate inflammatory responses mediated by the poten-tially pathogenic FPRL1 ligands. Since CD38 is the primary andbest-characterized mammalian ADP-ribosyl cyclase (cyclase) (11,45, 46), we first performed FACS analysis to determine whetherCD38 is expressed by different subpopulations of human periph-eral blood leukocytes. As has been previously reported (47), onlya subset of the CD15neg lymphocytes expressed CD38 while es-sentially all of the CD15low monocytes expressed CD38 (Fig. 2A).Although it has been previously suggested that CD38 is not ex-pressed by human neutrophils (47), we found that the majority ofthe highly granular CD15high-expressing neutrophils expressedCD38, albeit at lower levels compared with the CD38-expressingmonocytes and lymphocytes (Fig. 2A).

Primary human lymphocytes and monocytes have been previ-ously shown to exhibit CD38-dependent ADP-ribosyl cyclase ac-tivity (48, 49); however, it has not been tested whether primaryhuman neutrophils express a functional cyclase. To test whetherthe cyclase reaction could be catalyzed by neutrophil-derivedCD38, we immunoprecipitated CD38 from lysates of purified pe-ripheral blood neutrophils and then incubated the purified CD38protein with a synthetic substrate, nicotinamide guanine dinucle-otide (NGD�). Cyclases, such as CD38, utilize NGD� as a sub-strate and catalyze production of the highly fluorescent cyclicproduct cGDPR, which can be detected using a fluorometer (41).As shown in Fig. 2B, CD38 isolated from human neutrophils rap-idly produced cGDPR when incubated with NGD�. Similar resultswere obtained upon analysis of polymorphonuclear cell (PMN)samples from multiple donors (data not shown) indicating that hu-man neutrophils, like human lymphocytes and monocytes, expressCD38 and can produce cyclic metabolites such as cADPR.

The cADPR antagonist 8-Br-cADPR blocks the chemotaxis ofhuman neutrophils to FPRL1-specific ligands

Human neutrophils express at least two FPRs, the high-affinityfMLF-binding receptor FPR and the low-affinity fMLF-bindingreceptor FPRL1 (15). The human FPRL1 is considered to be anorthologue of the mFPR2 and both have a similar low affinity forfMLF (25, 50–52). Likewise, the human FPR is thought to be mostclosely related to the mFPR1, although the affinity of the mFPR1for fMLF is 100- to 500-fold lower than the affinity of the humanFPR for fMLF (25, 50–52). Using cells transfected with eitherFPR or FPRL1, it has been demonstrated that some agonists, suchas fMLF and T20 peptide, specifically activate FPR while others,like A5 and amyloid � peptide, preferentially activate FPRL1 (re-viewed in Refs. 14 and 15). Since the cADPR antagonist 8-Br-cADPR blocked the chemotaxis of mouse neutrophils to all of theFPR ligands that we tested, including agonists that are known tospecifically activate human FPR or FPRL1, we predicted that 8-Br-cADPR would also inhibit the chemotaxis of human neutrophils toall of the different FPR- and FPRL1-binding ligands. To test thishypothesis, we incubated human peripheral blood neutrophils inthe presence or absence of 8-Br-cADPR and then measured themigration of these cells to fMLF and to the synthetic FPRL1-specific agonist A5 peptide (28). Interestingly, treatment of humanneutrophils with 8-Br-cADPR had no effect on the migration ofthese cells to nanomolar concentrations of fMLF (Fig. 3A). How-ever, the cADPR antagonist did inhibit the migration of humanneutrophils to the A5 peptide (Fig. 3B) in a dose-dependent man-

ner (Fig. 3C). Thus, the cADPR antagonist blocked chemotaxis ofhuman PMNs to a FPRL1-specific ligand but not to a FPR-specificligand.

Since the cADPR antagonist inhibited neutrophil migration tothe A5 peptide, a FPRL1-specific ligand, but had no effect onchemotaxis to very low concentrations of fMLF, we postulated thatthe cADPR antagonist inhibited FPRL1 dependent, but not FPR

FIGURE 2. Human neutrophils and monocytes express CD38, an en-zyme with ADP-ribosyl cyclase activity. A, CD38 expression levels weredetermined on human peripheral blood leukocytes by FACS using fluoro-chrome-conjugated anti-human CD38 (or isotype control Ab) and anti-human CD15. Cell size (forward scatter, FSC) and granularity (side scatter,SSC) of the peripheral blood leukocytes was analyzed to identify lympho-cytes (Lym), monocytes (MN) and PMNs. The expression of CD38 wasthen assessed in PMN (CD15high), monocyte (CD15low), and lymphocyte(CD15neg) cells. The percentage of cells in each quadrant is indicated. B,ADP-ribosyl cyclase activity in lysates from human peripheral blood neu-trophils was measured using a fluorometric assay. CD38 was immunopre-cipitated from protein lysates prepared from human peripheral blood neu-trophils and was then incubated in medium (f) or in the presence of theCD38 substrate, NGD� (F). Accumulation of the fluorescent cyclic prod-uct, cGDPR, was measured over time and is expressed as relative fluores-cence units (RFU). The data are representative of at least 10 similar ex-periments using neutrophils from different donors.

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dependent, signaling in human neutrophils. To test this hypothesis,we incubated human neutrophils in the presence or absence of8-Br-cADPR and then performed migration assays using IL-8 anda variety of different chemoattractants with demonstrated specific-ity for either FPR or FPRL1. As shown in Fig. 4A and Table I, wefound that 8-Br-cADPR treatment of human neutrophils had noeffect on migration of the cells to IL-8. However, unlike what wehad observed with murine neutrophils (Fig. 1), 8-Br-cADPR treat-

ment of human neutrophils had minimal effect on the chemotacticresponse of the cells to fMLF or T20 peptide (Fig. 4A and TableI). Strikingly, however, the migration of human neutrophils to allof the FPRL1 ligands tested, including HIV-derived F peptide,HIV-derived V3 peptide, amyloid � peptide, MMK-1 peptide, andA5 peptide, was significantly inhibited by 8-Br-cADPR treatment(Fig. 4A and Table I). Taken together, these data indicate that thecADPR antagonist 8-Br-cADPR specifically inhibits the migration

FIGURE 3. A cADPR antagonist inhibits chemotaxis of human neutrophils to a FPRL1-specific ligand but has no effect on chemotaxis to fMLF, ahigh-affinity FPR ligand. Human peripheral blood neutrophils were preincubated in medium (�) or medium containing 8-Br-cADPR (100 �M, f) for 15min and then placed in the upper wells of the chemotaxis chamber. The lower wells of the chambers contained increasing concentrations of fMLF (A) orthe FPRL1-specific ligand A5 peptide (B). Transmigrated cells were collected after 45 min and enumerated by flow cytometry. The results are expressedas the mean � SD of triplicate wells for each experimental condition and are representative of at least four similar experiments using neutrophils fromdifferent donors. Values of p were determined by Student’s t test. �, p � 0.001. C, Human peripheral blood neutrophils were preincubated in mediumcontaining increasing concentrations of 8-Br-cADPR (1–100 �M) for 15 min and were then tested in a chemotaxis Transwell assay using 1 �M A5 peptideas the chemoattractant. One micromolar 8-Br-cADPR inhibited chemotaxis of neutrophils to A5 peptide by 50%.

FIGURE 4. Calcium mobilization and migration of human neutrophils to multiple FPRL1 ligands is dependent on cADPR. A, Migration of humanneutrophils to IL-8, FPR-specific ligands (fMLF and T20 peptide), and FPRL1-specific ligands (MMK-1 peptide, HIV V3 and F peptides, amyloid �1–42

peptide, and A5 peptide) was measured using Transwell chambers. Human peripheral blood neutrophils were preincubated in medium (�) or mediumcontaining 8-Br-cADPR (100 �M, f) for 15 min and then placed in the upper well of the chemotaxis chamber. The cells that migrated to the bottomchamber in response to the chemotactic stimulus were collected after 45 min and then quantitated by flow cytometry. The results are expressed as themean � SD of the CI, which represents the fold increase in the number of untreated or 8-Br-cADPR-pretreated cells that migrated in response to thechemoattractant divided by the basal migration of untreated or 8-Br-cADPR-treated cells to control medium. The data are averaged from a minimum ofthree independent experiments using neutrophils from different donors and were performed in triplicate for each experimental condition. Values of p weredetermined by Student’s t test. �, p � 0.001; ��, p � 0.0004; ���, p � 0.0001. B, Peripheral blood human neutrophils were loaded with fluorescentcalcium-detecting dyes Fluo-3 and Fura-Red and preincubated in the presence (red line) or absence of 100 �M 8-Br-cADPR (blue and green lines) for 15min. Cells were then stimulated with IL-8 or peptides that preferentially activate either FPR (fMLF and HIV T20 peptide) or FPRL1 (A5 and MMK-1synthetic peptides) in medium containing extracellular calcium (blue line and red line) or calcium-free (plus 2 mM EGTA) medium (green line). Intra-cellular free calcium levels were measured by FACS for 7 min. Data are from a representative experiment of a total of at least three similar experimentsusing neutrophils isolated from different donors.

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of human neutrophils to FPRL1-specific, but not FPR-specific,ligands.

cADPR regulates calcium mobilization in FPRL1-stimulatedhuman neutrophils

Since cADPR is a known calcium-mobilizing metabolite (2), wepredicted that cADPR might regulate intracellular free calciumlevels in FPRL1-stimulated human neutrophils. To test this hy-pothesis, we measured the accumulation of intracellular free cal-cium in primary human neutrophils stimulated with IL-8, FPR-specific agonists, or FPRL1-specific agonists (Fig. 4B). Asexpected, calcium mobilization in response to IL-8 was essentiallyidentical between 8-Br-cADPR-treated and control neutrophils(Fig. 4B). Likewise, 8-Br-cADPR treatment had minimal effect onthe calcium response of human neutrophils stimulated with theFPR-specific ligands fMLF and T20 peptide (Fig. 4B). In contrast,the calcium response of 8-Br-cADPR-treated neutrophils to theFPRL1-specific ligands, A5 and MMK-1 peptides, was reducedcompared with the neutrophils that were not pretreated with 8-Br-cADPR (Fig. 4B). Interestingly, calcium mobilization in FPRL1-stimulated neutrophils was completely dependent on the presenceof external calcium because the calcium signal was abolishedwhen external calcium was depleted from the medium usingEGTA (Fig. 4B). This is in contrast to what we observed when thecells were stimulated with IL-8, T20 peptide, or fMLF, as a sub-stantial calcium response was seen even when extracellular cal-cium was depleted from the medium (Fig. 4B). Thus, these dataindicate that FPR and FPRL1 induce distinct calcium responses,with FPR inducing a calcium response that occurs independentlyof cADPR and is largely, although not exclusively, due to intra-cellular calcium release, while FPRL1 induces a calcium responsethat is regulated by cADPR and is primarily due to extracellularcalcium influx.

cADPR antagonists block FPRL1-mediated chemotaxis, but notFPRL1-induced chemokinesis

Treatment of human neutrophils with the cADPR antagonist didnot completely block migration of the neutrophils to the FPRL1ligands (e.g., Fig. 4A). However, we have previously shown that8-Br-cADPR specifically blocked the chemotaxis (chemoattrac-tant-induced directional migration) of murine bone marrow neu-trophils to fMLF, but had minimal effect on fMLF-induced che-mokinesis (chemoattractant-induced nondirectional migration) (5).

Therefore, we concluded that cADPR-dependent signaling is mostcritical for mediating directional movement toward FPR ligands.To determine whether cADPR is required for the directional move-ment of FPRL1-stimulated human neutrophils, we incubated pe-ripheral blood neutrophils in the presence or absence of 8-Br-cADPR and then performed a Transwell checkerboard assay tomeasure basal migration, chemokinesis, and chemotaxis. Asshown in Fig. 5, A and B, pretreatment of human neutrophils with8-Br-cADPR had no effect on basal migration, chemokinesis, orchemotaxis of neutrophils stimulated with the FPR ligands, T20peptide, and fMLF. Likewise, treatment of neutrophils with 8-Br-cADPR had no effect on the basal migration or migration due tochemokinesis of neutrophils activated with the FPRL1 ligands, V3peptide, F peptide, amyloid � peptide, and A5 peptide (Fig. 5,C–F). In striking contrast, 8-Br-cADPR treatment inhibited thedirectional migration of the neutrophils responding to the FPRL1ligands and reduced the migration that can be specifically attrib-uted to chemotaxis by �90% (Fig. 5, C–F). Therefore, these datademonstrate that the cADPR antagonist specifically blocks the di-rectional movement of human neutrophils that are responding toFPRL1-specific signals.

The NAD� analogue N(8-Br-A)D� inhibits chemotaxis ofFPRL1-activated neutrophils

Our experiments demonstrate that treatment of human neutrophilswith a cADPR antagonist blocks the directional migration of theseneutrophils to a number of FPRL1-specific ligands including in-flammatory mediators such as amyloid � peptide. These datastrongly suggest that compounds that either inhibit the activity ofthe cyclase(s) that produce cADPR or that alter the products thatare produced by these cyclase(s) could also be used to block themigration of neutrophils responding to FPRL1-specific agonists.One easily synthesized compound that can be used to alter productformation by ADP-ribosyl cyclases is the NAD� analogue N(8-Br-A)D� (40). ADP-ribosyl cyclases, like CD38, utilize thisNAD� analogue as a substrate, but instead of producing cADPR,cells incubated with this substrate produce 8-Br-cADPR, thecADPR antagonist (7). In previous experiments using mouse neu-trophils, we showed that treatment of neutrophils with N(8-Br-A)D� inhibited the chemotactic response of the neutrophils tofMLF in a CD38- and cADPR-dependent manner (5, 11).

Since human neutrophils express CD38 and are dependent oncADPR for FPRL1-induced chemotactic responses, we predicted

Table I. Migration of human neutrophils to FPRL1 ligands is inhibited by a cADPR antagonist

Preincubationa

Chemoattractantb

Neutrophil Migrationc

MediumNo Attractant

8-Br-cADPR� Attractant

MediumNo Attractant

8-Br-cADPR� Attractant

A5 peptide 6,101 � 737 6,877 � 3,380 161,289 � 12,353 76,272 � 11,893�Amyloid � 6,092 � 1,956 10,731 � 997 69,532 � 4,727 26,491 � 2,107��HIV V3 1,775 � 278 1,515 � 1,540 12,393 � 242 4,718 � 1,179��HIV F 3,493 � 20 4,545 � 1,836 13,761 � 1,352 6,574 � 719�MMK-1 3,625 � 130 3,404 � 130 109,122 � 4,808 67,323 � 1,168��HIV T20 2,084 � 176 2,899 � 422 118,109 � 4,011 127,470 � 4,204fMLF 1,732 � 307 2,250 � 840 186,265 � 11,714 185,900 � 37,785IL-8 4,442 � 189 5,000 � 1,108 190,126 � 29,459 175,434 � 8,906

a Human peripheral blood neutrophils were preincubated for 15 min in medium alone or 100 �M 8-Br-cADPR and thenassessed in Transwell migration assays.

b The chemoattractants (or media control) were placed in the bottom well of the Transwell chambers at the concentrationslisted in Fig. 4 legend.

c The number of neutrophils that migrated to the bottom chamber in 45 min was determined by FACS and the mean and SDof triplicate wells for each condition are shown. The results are representative of at least four separate experiments for eachchemoattractant analyzed.

�, p � 0.001; ��, p � 0.0004, Student’s t test.

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that treatment of human neutrophils with N(8-Br-A)D� would in-hibit the capacity of these cells to respond to chemotactic gradientsof FPRL1-specific agonists. To test this prediction, we preincu-bated human peripheral blood neutrophils with N(8-Br-A)D� and

then assessed the chemotactic potential of these cells in checker-board assays using FPR and FPRL1 agonists as the chemoattrac-tants. As shown in Fig. 6A, treatment of neutrophils with N(8-Br-A)D� had absolutely no effect on the chemokinetic or chemotactic

FIGURE 5. FPRL1-induced chemotaxis, butnot chemokinesis, is controlled by cADPR. The ef-fect of 8-Br-cADPR on chemokinesis and chemo-taxis of human neutrophils responding to FPR andFPRL1 agonists was evaluated using a checker-board assay. Cells were pretreated with medium(�) or 8-Br-cADPR (100 �M, f) and were thenplaced into the top wells of chemotaxis chambers.The chemoattractants fMLF (A), HIV-derived T20peptide (B), HIV-derived V3 peptide (C), HIV-de-rived F peptide (D), amyloid �1–42 peptide (E), orA5 peptide (F) were placed in either the upper,lower or both upper and lower wells of the chemo-taxis chambers and checkerboard migration assayswere performed. Migration due to chemokinesis isdetermined when an equivalent concentration ofthe chemoattractant is present in the top and bottomchamber of the Transwell. Total migration due tochemokinesis plus chemotaxis is determined whenthe chemoattractant is present in only the bottomchamber of the Transwell. Migration due to che-motaxis alone is determined by subtracting the mi-gration due to chemokinesis from the migrationdue to both chemokinesis and chemotaxis. Trans-migrated cells were collected after 45 min and enu-merated by flow cytometry. The results are ex-pressed as the mean � SD of triplicate wells foreach experimental condition and are representativeof at least three similar experiments using neutro-phils from different donors. Values of p were de-termined by Student’s t test. �, p � 0.003; ��, p �0.001.

FIGURE 6. A substrate analogue of CD38 inhibits FPRL1- but not FPR-induced chemotaxis of human neutrophils. Human peripheral blood neutrophilswere preincubated in medium (�) or in medium containing N(8-Br-A)D� (500 �M, f) for 15 min and then placed in the upper wells of chemotaxischambers. Checkerboard assays (as described in Fig. 5 legend) were performed using FPR-specific (fMLF, A) or FPRL1-specific (amyloid �1–42 orHIV-derived V3 peptide, B and C, respectively) ligands. The cells that migrated to the bottom chamber in 45 min were collected and enumerated by flowcytometry. The mean � SD of the triplicate wells is shown. The data are representative of three independent experiments using neutrophils from differentdonors. Values of p were determined by Student’s t test. �, p � 0.001.

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response of the cells to the FPR ligand fMLF. In addition, theNAD� analogue did not affect the chemokinetic response of theneutrophils to the FPRL1 ligands amyloid � peptide and HIV-derived V3 peptide (Fig. 6, B and C). However, the chemotacticresponse of neutrophils responding to FPRL1 ligands was dramat-ically reduced when the cells were preincubated with N(8-Br-A)D� (Fig. 6, B and C). Together, these results show that bothNAD� analogues and cADPR antagonists can be used to block thedirectional migration of human neutrophils to inflammatory che-moattractants that activate FPRL1 but not FPR.

8-Br-cADPR inhibits FPRL1/mFPR2-mediated chemotaxis ofmultiple cell types

Neutrophils are not the only cells that express FPRL1 and migratedirectionally in response to FPRL1 ligands. Indeed, FPRL1 is con-stitutively expressed by monocytes (15) and is inducibly expressedby microglial cells (39), which reside in the CNS and are of my-eloid origin. Since FPRL1-dependent migration is regulated bycADPR in neutrophils, we postulated that the cADPR antagonist8-Br-cADPR could also be used to block the migration of othercell types to FPRL1 ligands. To examine this possibility, we testedthe effect of 8-Br-cADPR treatment on migration of human mono-cytes in response to FPRL1-specific ligands. First, we assessed thechemotactic response of 8-Br-cADPR-treated human peripheralblood monocytes to FPR and FPRL1-specific ligands. As previ-ously reported, peripheral blood monocytes migrated in responseto a variety of FPR and FPRL1 ligands including HIV-derivedpeptides (Fig. 7A), amyloid peptides (Fig. 7B), and synthetic pep-tides (Fig. 7, A and B). Treatment of monocytes with 8-Br-cADPRhad no effect on the chemotactic responses of these cells to theFPR ligands, fMLF and HIV-derived T20 peptide (Fig. 7, A andB). In contrast, the migration of the 8-Br-cADPR-treated mono-cytes was significantly reduced in response to the synthetic FPRL1ligands MMK-1 and A5 peptides, the HIV-derived F peptide, andthe amyloid peptides SAA and amyloid � peptide (Fig. 7, A and B).Therefore, in complete accordance with our data using peripheralblood neutrophils, we found that the cADPR antagonist inhibitedthe chemotaxis of primary human peripheral blood monocytes to anumber of different FPRL1-specific ligands.

Next, we examined whether cADPR controls the migration ofmouse myeloid-derived microglial cells to mFPR1/mFPR2 li-gands. To do so, we used a murine microglial cell line, N9, thatexpresses typical markers of resting mouse microglia and are fre-quently used for functional analyses of microglial cells (18, 39,53). Importantly, when N9 cells or normal mouse microglial cellsare stimulated with LPS the ADP-ribosyl cyclase CD38 is ex-pressed at low levels on the plasma membrane (data not shown),and mFPR2, the mouse orthologue of human FPRL1, is also up-regulated (18, 39). As expected, after 24 h of LPS treatment, theN9 cells expressed mFPR2 and could migrate in response to sev-eral mFPR2 ligands including the synthetic W and A5 peptides andhigh micromolar concentrations of fMLF (Fig. 7C). However,when the N9 cells were first pretreated with the cADPR antagonist8-Br-cADPR, the cells were significantly impaired in their abilityto migrate in response to any of the agonists (Fig. 7C). Interest-ingly, the cADPR antagonist had no effect on the chemotaxis of themicroglial cells to another chemoattractant, C5a (Fig. 7C). Takentogether, these data indicate that the cADPR antagonist specificallyblocks FPRL1/mFPR2-dependent chemotaxis in myeloid-derivedmonocytes and microglial cells.

Treatment of monocytes with a cADPR antagonist blockscalcium influx and chemotaxis to homeostatic and inflammatorychemokines

The data from the experiments with human leukocytes indicatedthat cADPR is required for FPRL1-dependent chemotaxis but is

FIGURE 7. cADPR controls the migration of monocytes and myeloid-de-rived microglial cells to FPRL1-specific ligands. A and B, Human peripheralblood monocytes were preincubated with medium (�) or medium containing8-Br-cADPR (100 �M, f) for 15 min and then placed in the upper wells ofchemotaxis chambers. The lower wells of the chambers contained peptides thatpreferentially activate FPR (fMLF and HIV T20 peptide) or FPRL1 (A5 pep-tide, amyloid �1–42 peptide, HIV-derived F and V3 peptides, SAA polypep-tide, and MMK-1 peptide). Transmigrated cells were collected after 90 minand were enumerated by flow cytometry. The mean � SD of the triplicatewells is shown. The data are representative of three independent experimentsusing neutrophils from different donors. C, N9 microglial cells were stimulatedwith 300 ng/ml LPS for 48 h and then preincubated with medium (�) or with100 �M 8-Br-cADPR (f). Cell migration in response to the synthetic Wpeptide (106 M), fMLF (105 M), the synthetic A5 peptide (50 �g/ml), orC5a (100 ng/ml) was then determined by counting the number of cells presenton the Transwell filter (number of cells per four high-power fields). Themean � SD of the triplicate wells is shown. The data are representative of twoindependent experiments. Values of p were determined by Student’s t test. �,p � 0.02; ��, p � 0.001; ���, p � 0.0004.

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not needed for CXCR1/2 (IL-8 receptors), FPR, or C5a receptor-dependent chemotaxis. These data suggested that cADPR mustregulate signaling through only a subset of the chemoattractantreceptors expressed by monocytes. Human peripheral blood mono-cytes are reported to express a number of additional chemokinereceptors including CCR1, CCR2, CCR5, and CXCR4 (31, 54). Totest whether cADPR regulates signaling through some of theseother chemokine receptors, we measured calcium and chemotacticresponses in 8-Br-cADPR-treated human monocytes that werestimulated with CCR1/CCR5 and CXCR4 agonists (Fig. 8). Asexpected, the chemotactic response of 8-Br-cADPR-treated mono-cytes stimulated with the FPR ligand fMLF was completely nor-mal (Fig. 8A). Likewise, the fMLF-induced calcium response wasunaffected by the presence of 8-Br-cADPR (Fig. 8B). In contrast,8-Br-cADPR treatment did block the migration of monocytes inresponse to the CCR1/CCR5 ligands RANTES and MIP-1� and tothe CXCR4 ligand stromal cell-derived factor 1 (SDF-1) (Fig. 8A).Interestingly, migration of CCR1-expressing murine neutrophils toMIP-1� was also found to be cADPR and CD38 dependent (datanot shown). Although 8-Br-cADPR treatment did not completelyinhibit calcium mobilization in SDF-1- or MIP-1�-stimulatedmonocytes, the response was significantly reduced (Fig. 8B).When the cells were stimulated in calcium-free medium, no cal-cium mobilization was observed after SDF-1 and Mip-1� stimu-lation (Fig. 8B). Thus, as we observed after stimulation withFPRL1-specific ligands (see Fig. 4B), calcium mobilization in-duced by ligation of CCR1/CCR5 and CXCR4 is almost exclu-sively due to extracellular calcium influx. Taken together, cADPRappears to modulate calcium influx in cells that have been acti-vated by chemoattractant receptors that induce calcium mobiliza-tion via a calcium influx-dependent mechanism. This cADPR-reg-ulated calcium influx signal is required for FPRL1-, CCR1/CCR5-,and CXCR4-induced chemotaxis but is not obligate for FPR-,CXCR1/2-, or C5aR-mediated chemotaxis.

DiscussionThe data presented in this manuscript demonstrate that cADPRregulates calcium signaling in a discreet subset of chemokine

and chemoattractant receptors. The chemokine receptors thatutilized cADPR for signaling could be distinguished from theother non-cADPR-dependent receptors because the receptorsthat utilized cADPR mobilized calcium in a unique fashion.Typically, it is believed that engagement of chemokine recep-tors leads to the generation of the calcium-mobilizing secondmessenger inositol trisphosphate (IP3) which in turn leads torelease of intracellular calcium from IP3 receptor-gated storesin the endoplasmic reticulum (55). The initial, very rapid, short-lived release of calcium is often, although not always, accom-panied by a delayed and sustained influx of extracellular cal-cium (55). This classical calcium response to chemoattractantreceptor engagement was observed after engagement of recep-tors such as C5aR (data not shown), CXCR1/2 (Fig. 4), and thehuman high-affinity FPR (Fig. 4); receptors that all signal in acADPR-independent fashion. In striking contrast and in agree-ment with what we previously observed with mFPR receptors(5), engagement of the chemoattractant receptors that do utilizecADPR, such as FPRL1, CXCR4, CCR1, and CCR5, resulted inminimal intracellular calcium release that was accompanied bya very strong influx of extracellular calcium (Figs. 4 and 8).Although it is not known how cADPR regulates extracellularcalcium influx in any cell type, it has been suggested thatcADPR regulates the activation of L-type calcium channels andstore-operated Icrac channels (6, 56). Interestingly, human neu-trophils have been reported to express L-type channels as wellas Icrac channels (57, 58). Regardless of the exact mechanism bywhich cADPR regulates calcium influx in leukocytes, the dataare clear that a specific cADPR antagonist partially inhibits thecalcium response and blocks chemotaxis of human leukocytesresponding to FPRL1, CXCR4, and CCR1/CCR5 ligands (i.e.,Fig. 4).

These cADPR-dependent chemoattractant receptors are not thefirst chemoattractant receptors to be identified that mobilize cal-cium primarily by a calcium influx-dependent mechanism. Indeed,ligation of the platelet-activating factor receptor, CCR2, andCX3CR1 induces minimal intracellular calcium release and strongcalcium influx (59–62). Interestingly, no one has yet identified the

FIGURE 8. Calcium influx and chemotaxis of human monocytes to CCR5 and CXCR4 ligands is regulated by cADPR. A, Migration of humanmonocytes to fMLF (FPR-specific ligand), SDF-1 (CXCR4 ligand), and to RANTES and MIP-1� (CCR1/CCR5 ligands) was measured using Transwellchambers. Human peripheral blood monocytes were preincubated in medium (�) or medium containing 8-Br-cADPR (100 �M, f) for 15 min and thenplaced in the upper well of the chemotaxis chamber. The cells that migrated to the bottom chamber in response to the chemotactic stimulus were collectedafter 90 min and then quantitated by flow cytometry. The mean � SD of the triplicate wells is shown. The data are representative of at least threeindependent experiments using neutrophils from different donors. Values of p were determined by Student’s t test. �, p � 0.04; ��, p � 0.001; ���, p �0.0003. B, Peripheral blood human monocytes were loaded with fluorescent calcium-detecting dyes Fluo-3 and Fura-Red and preincubated in the presence(red line) or absence of 100 �M 8-Br-cADPR (blue and green lines) for 15 min. Cells were then stimulated with fMLF, SDF-1, or MIP-1� in mediumcontaining extracellular calcium (blue line and red line) or calcium-free (plus 2 mM EGTA) medium (green line). Intracellular free calcium levels weremeasured by FACS for 7 min. Data are from a representative experiment of a total of at least three similar experiments using neutrophils isolated fromdifferent donors.

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calcium-mobilizing second messenger that potentiates calcium in-flux upon ligation of these receptors although it has been shownthat IP3 is apparently not involved in signaling through CCR2 (60).Therefore, based on these data we predict that other chemoattrac-tant receptors such as platelet-activating factor receptor, CCR2,and CX3CR1 that primarily mobilize calcium via a calcium influx-dependent mechanism may also utilize cADPR for signaling.

Chelating extracellular calcium with EGTA (Figs. 4 and 8) orusing calcium-free medium (data not shown) appeared to com-pletely abolish the calcium response of all the chemoattractant re-ceptors that require cADPR for optimal calcium mobilization.However, the cADPR antagonist only partially inhibited calciuminflux in response to FPRL1, CCR1/CCR5, and CXCR4 agonists(Figs. 4 and 8). These data indicate that other calcium-mobilizingsecond messengers, such as sphingosine-1-phosphate or perhapsIP3, must also play a role in modulating calcium influx in thesecells (63, 64). In addition, the data indicate that at least somechemoattractant-induced signals are likely to induce functionalchanges in the neutrophils even in the absence of cADPR. In fact,the neutrophils treated with the cADPR antagonist or the NAD�

analogue were activated by FPRL1 ligands and were perfectlycompetent to migrate nondirectionally (Figs. 5 and 6), indicatingthat activation-induced nondirectional movement is not controlledby cADPR but instead may be regulated by other calcium-mobi-lizing metabolites. Taken together, the data indicate that cADPR/CD38 antagonists are not simply global inhibitors of calcium mobi-lization in chemoattractant-activated leukocytes and that theseantagonists modulate the extracellular calcium influx induced by aselective group of chemoattractants and regulate only a subset of thebiologic functions induced upon binding of these chemoattractants.

Our observation that two highly related human chemoattractantreceptors, FPR and FPRL1, can be distinguished by their relianceon cADPR for calcium signaling is of potential therapeutic impor-tance. FPRL1-dependent calcium mobilization is primarily due toextracellular calcium influx and utilizes cADPR, whereas FPR-dependent calcium mobilization is due in large part to intracellularcalcium release and is cADPR independent. This result was ini-tially somewhat surprising to us since we had previously shownthat cADPR and CD38 are necessary for chemotaxis of mouseneutrophils to the prototypic FPR receptor ligand fMLF (5). In-deed, the mFPR1 and human FPR are believed to be orthologuesof one another and were presumed to signal through similar mech-anisms (14, 15). However, the data presented here, as well as pre-vious published data, indicate that there are significant differencesbetween these two receptors. First, the affinity of fMLF for humanFPR is quite high (nanomolar range) whereas the affinity of fMLFfor the mFPR1 is �400 times lower (51). Second, mFPR1 ex-pressed by mouse bone marrow neutrophils appears to be a morepromiscuous receptor as it can be activated by high concentrationsof ligands such as HIV-derived F peptide and the synthetic A5peptide (Fig. 1), which do not efficiently activate the high-affinityhuman FPR (15, 28). Third, chemotaxis induced upon mFPR1 en-gagement is inhibited by the cADPR antagonist (Fig. 1), whereaschemotaxis induced upon ligation of the human FPR is not affectedby the cADPR antagonist (Fig. 3). Finally, fMLF only weaklyinduces extracellular calcium influx in human peripheral bloodneutrophils (Fig. 4), while it strongly induces calcium entry infMLF-stimulated mouse bone marrow neutrophils (5). Thus, whilemFPR1 is most homologous at a structural level to human FPR(52), its affinity for fMLF, its reliance on cADPR, and the patternof calcium mobilization induced by receptor engagement appearsmore similar to that seen with the human FPRL1 and mFPR2receptors.

Despite the biochemical and functional differences betweenmFPR1 and human FPR, it is clear from the data presented herethat a cADPR antagonist as well as a CD38 substrate analogue canbe used to inhibit the chemotactic response of human and mouseneutrophils to a diverse array of mFPR2 and human FPRL1-spe-cific ligands including several synthetic peptides, multiple HIVgp120-derived peptides, and at least two different amyloidogenicpeptides (Figs. 4 and 6). This result was not limited to neutrophilsas we also found that chemotaxis of human monocytes and mousemyeloid-derived microglial cells to these same ligands was inhib-ited by the cADPR antagonist (Fig. 7). Therefore, based on thesedata, we suggest that the signal transduction pathways engagedupon mFPR2/FPRL1 ligation in all three cell types are similarlydependent on cADPR. Furthermore, we predict that cADPR an-tagonists should be effective inhibitors of FPRL1-dependent che-motaxis of all myeloid-derived human cells. Finally, given that anumber of the FPRL1-specific ligands are present in diseased tis-sue and are postulated to exacerbate the inflammatory responsewithin these tissues (38), we predict that compounds like 8-Br-cADPR and N(8Br-A)D� are likely to be useful for assessing theimpact of FPRL1-induced inflammatory cell recruitment on theprogression of pathology within the diseased tissues.

AcknowledgmentsWe thank Troy D. Randall for critical reading of this manuscript and help-ful advice.

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