Chemotaxis and Calcium Responses of Phagocytes to Formyl Peptide Receptor Ligands Is Differentially Regulated by Cyclic ADP Ribose 1 Santiago Partida-Sa ´nchez,* Pablo Iribarren, † Miguel E. Moreno-Garcı ´a,* Ji-Liang Gao, ‡ Philip M. Murphy, ‡ Norman Oppenheimer, § Ji Ming Wang, † and Frances E. Lund 2 * Cyclic ADP ribose (cADPR) is a calcium-mobilizing metabolite that regulates intracellular calcium release and extracellular calcium influx. Although the role of cADPR in modulating calcium mobilization has been extensively examined, its potential role in regulating immunologic responses is less well understood. We previously reported that cADPR, produced by the ADP-ribosyl cyclase, CD38, controls calcium influx and chemotaxis of murine neutrophils responding to fMLF, a peptide agonist for two chemoattractant receptor subtypes, formyl peptide receptor and formyl peptide receptor-like 1. In this study, we examine whether cADPR is required for chemotaxis of human monocytes and neutrophils to a diverse array of chemoattractants. We found that a cADPR antagonist and a CD38 substrate analogue inhibited the chemotaxis of human phagocytic cells to a number of formyl peptide receptor-like 1-specific ligands but had no effect on the chemotactic response of these cells to ligands selective for formyl peptide 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 chemokines that induce extracellular calcium influx in the apparent absence of significant intracellular calcium release. Thus, cADPR regulates calcium 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 represent novel 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, transform NAD + into several products including the calcium-mo- bilizing metabolite cyclic ADP-ribose (cADPR) 3 (1, 2). Since CD38 is a member of a highly conserved family of cyclases iso- lated from plants, invertebrates, and vertebrates (2), it has been hypothesized that CD38, via its production of cADPR, is likely to be an important regulator of calcium-based signal transduction. cADPR modulates the level of intracellular free calcium in cells in at 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-induced calcium release (3). In addition, cADPR has been shown to regu- late the influx of extracellular calcium (4, 5), possibly by activating the store-operated calcium release-activated calcium current channels (I crac ) (6). Although it is very clear that cADPR modulates intracel- lular free calcium levels in cells, less is known about which receptors rely on cADPR for signaling. In addition, very little is understood about the role(s) for cADPR in regulating important cellular processes such as development, growth, and differentiation. To address which receptors utilize cADPR for signaling, several laboratories have now synthesized a number of different cADPR inhibitors including 8-Br-cADPR, a potent cADPR antagonist (7) and N(8-Br-A)D + , a NAD + analogue that can be cyclized by CD38 into the cADPR antagonist 8-Br-cADPR (5). These antag- onists have recently been successfully used to identify receptors such as the muscarinic receptor that mobilizes calcium in a cADPR-dependent fashion (8, 9). To assess the in vivo signaling role of cADPR, we produced mice that lack CD38 (10), one of the two known mammalian ADP-ribosyl cyclases (2). Using bone marrow neutrophils isolated from the CD38 knockout (KO) mice, we demonstrated that calcium signaling induced upon ligation of the classical chemoattractant formyl peptide receptor (FPR) is de- pendent on CD38 and cADPR (5). Importantly, we also found that chemotaxis of mouse neutrophils to the FPR ligand fMLF is reg- ulated by cADPR and CD38 (5). Furthermore, we showed that pretreatment of normal mouse neutrophils with either 8-Br- cADPR or N(8-Br-A)D + inhibited the chemotactic response of these normal neutrophils to fMLF (5, 11). Together, these data showed that cADPR and the ADP-ribosyl cyclase CD38 modulate FPR-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 of the 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 Development Center, National Institutes of Health, Frederick, MD 21702; ‡ Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 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 November 19, 2003. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 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, Trudeau Institute, 154 Algonquin Avenue, Saranac Lake, NY 12983. E-mail address: flund@trudeauinstitute.org 3 Abbreviations used in this paper: cADPR, cyclic ADP-ribose; cGDPR, cyclic GDP- ribose; FPR, formyl peptide receptor; FPRL1, FPR-like 1; I crac , calcium release- activated calcium current channel; IP 3 , 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