The Journal of Experimental Medicine o.24 o 1 Otbr2,20 20-78 www.jem.org/cgi/doi/ 2705-2718 October29, 2007 Vol. 204,No. 11, $30.00 JEM ©TheRockefeller University Press IP methionyl leucylphenylalanine; fl respectively; DNFB,dinitro- mokine ligandandreceptor, CXCL andCXCR,CXCche- tively; CI,chemotacticindex; ligand andreceptor,respec- CCL andCCR,CCchemokine bose; cADPR,cyclicADPR; ADPR, adenosinediphosphori- 2-aminoethoxydiphenylborate; Abbreviationsused:2-APB, fl Frances E.Lund: CORRESPONDENCE PTx, pertussistoxin. factor; PLC,phospholipase; tide; PAF,platelet-activating nicotinamide adeninedinucleo- formyl peptidereceptor;NAD Langerhans cell;mFPR,mouse [email protected] uorobenzene; fMLF, 3 , inositol trisphosphate;LC, N -formyl + , alternative chemokinereceptorpathwayisactivatedbyG Michael T.Borchers, and granulocytes transduction pathwayindendriticcells dependent chemokinereceptorsignal
make defectivecalciumandchemotacticresponsesuponstimulationwith and FrancesE.Lund critically importantfortheinitiationofinfl Regardless, thenovelalternativechemokinereceptorsignalingpathwayappearstobe alternative chemokinereceptorpathwaycontrolsthemigrationofonlyasubsetcells. chemokine receptorsignalingpathway.Similartothe ceptors containsthreesubunits: G The Gproteinsassociatedwith chemokinere- other keyintracellularsignalingmolecules( and transducessignalsfromthesereceptorsto directly associateswithchemokinereceptors model isthetrimericGproteincomplexthat ponents ofthischemokinereceptorsignaling has beenproposed( chemokine receptorsignaltransductionmodel migration havebeenidentifi proteins andsecondmessengersthatcontrolcell Overthelast15yr,manyofkeyintracellular receptors thatsignalinaCD38-dependentmannerandidentifi To determinethebasisforthissignalingheterogeneity,weexaminedchemokine that chemokinereceptorsignalinginleukocytesismorediversethanpreviouslyappreciated. CD38 controlsthechemotaxisofleukocytestosome,butnotall,chemokines,suggesting 4 3 2 1 Guixiu Shi, Identifi The activationofGproteins is inducedbythe lumbus Children S.Partida-S G.ShiandS.Partida-S Gq classofGproteins.Weshowthat However, unliketheclassicalpathway,alternativepathwayisalsodependenton after contactsensitization. sensitization andtheemigrationofmonocytesfrombonemarrowintoinfl migration ofDCsfromtheskintodraininglymphnodesafterfl In contrast,G stimulation withCCL2,CCL19,CCL21,andCXCchemokineligand(CXCL)12(DCs). methionyl leucylphenylalanineandCCchemokineligand(CCL)3(neutrophils),orupon Division ofBiology,CaliforniaInstituteTechnology,Pasadena,CA91125 Scottsdale, AZ85259 Division ofHematology/Oncology,DepartmentBiochemistryandMolecularBiology,MayoClinicArizona, Department ofInternalMedicine,PulmonaryDivision,UniversityCincinnatiCollegeCincinnati,OH45219 Trudeau Institute,SaranacLake,NY12983 á nchez ’ s Research Institute, Columbus, OH 43205. s ResearchInstitute, Columbus,OH43205. ’ cation ofanalternativeG s presentaddressisDept.ofPediatrics,Co- ␣ 1 SantiagoPartida-S q
-defi á nchez contributed equally to this work. nchez contributedequallytothiswork. 10.1084/jem.20071267 1 ). Oneofthecriticalcom- cient Tcell responsestoCXCL12andCCL19remainintact.Thus,the 1 ed, and a consensus ed, andaconsensus 2
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on November 30, 2007 2007 30, November on www.jem.org from Downloaded (for review see reference 17 ). Thus, G proteins are of central RESULTS importance in leukocyte traffi cking. Chemokine receptors can be divided into CD38-dependent Recently, we identifi ed the ectoenzyme CD38 as another and -independent subclasses important signaling protein in the chemokine receptor sig- We previously showed that the in vitro chemotaxis of mouse naling pathway ( 18 ). CD38, a nicotinamide adenine dinucle- formyl peptide receptor (mFPR) 1– expressing mouse bone otide (NAD) glycohydrolase and ADP-ribosyl cyclase ( 19 ), is marrow neutrophils to the chemoattractant N -formyl methi- expressed by many leukocyte populations, including neutro- onyl leucyl phenylalanine (fMLF) is dependent on CD38 and phils, monocytes, DCs, and lymphocytes (20, 21 ). The extra- at least one of its enzymatically generated products, cADPR cellular enzymatic domain of CD38 catalyzes the formation ( 28, 31). Indeed, neither WT neutrophils pretreated with the of cyclic adenosine diphosphoribose (cADPR), ADPR, and membrane-permeant cADPR antagonist 8Br-cADPR nor nicotinic acid adenine dinucleotide from its substrates NAD Cd38 / neutrophils migrate in transwell chambers in response and NADP (22 ). All three products of the CD38 enzyme to an optimal concentration of fMLF ( Fig. 1 A ). In contrast, reaction function as signaling molecules either by mobilizing chemotaxis of Cd38 / neutrophils and 8Br-cADPR– treated calcium from intracellular stores (23 ) or by activating cal- WT neutrophils to the CXC chemokine receptor (CXCR) cium entry from the extracellular space (24 – 27 ). We showed 1/CXCR2 ligand IL-8 ( Fig. 1 B ) is equivalent to chemotaxis that at least one of the metabolites made by CD38, cADPR, of normal WT neutrophils, indicating that there must be controls calcium infl ux in chemokine-stimulated cells, and at least two subclasses of chemokine receptors that can be that this CD38- and cADPR-dependent calcium response distinguished from one another based on their requirement is required for neutrophil and DC migration both in vitro for CD38. Downloaded from and in vivo (28, 29). Furthermore, loss of CD38-dependent In subsequent analyses of mouse and human leukocytes, chemokine receptor signals has important immunological we found additional examples of chemokine receptors that consequences, as mice defi cient in CD38 ( Cd38 − / − ) are more signal in either a CD38/cADPR-dependent or -independent susceptible to bacterial infections (28 ) and make attenuated manner ( 28, 29, 31 ). However, when we compared the
innate and adaptive immune responses to infl ammatory agents chemotactic response of peripheral (isolated from spleen and www.jem.org and immunogens ( 29, 30 ). LNs) mouse DCs and T cells with the same CC chemokine Based on these data, we concluded that CD38 is a criti- receptor (CCR) 7 ligand, CCL19, we found that the periph- / cal regulator of cell migration. However, unlike G i -con- eral Cd38 DCs were unable to migrate in response to the
taining G proteins, which are required for the chemotaxis of CCL19 gradient (Fig. 1 C), whereas CD4 T cells purifi ed on November 30, 2007 virtually all hematopoietic cells to all chemoattractants (17 ), from the same tissues of the same Cd38 / mice migrated CD38 is not universally required for cell migration. Instead, normally in response to CCL19 (Fig. 1 D ). Likewise, periph- we found that CD38 is required for leukocyte chemotaxis eral WT DCs pretreated with the cADPR antagonist 8Br- to some, but not all, chemokines (28, 31 ). These data sug- cADPR made a defective chemotactic response to CCL19 gested that the signals required to induce chemotaxis have ( Fig. 1 C), whereas the chemotactic response of WT T cells to be more diverse than previously appreciated, and we pretreated with 8Br-cADPR was equivalent to that observed hypothesized that assessing CD38-dependent pathways for the untreated WT T cells ( Fig. 1 D ). Collectively, these would allow us to identify new proteins involved in cell data showed that chemokine receptors can be divided into migration. In this study, we directly tested this hypothesis and diff erent subclasses and that the subclass of the chemokine discovered a novel “ alternative ” chemokine receptor signal- receptor is variable and dependent on the cell type expressing ing pathway that is required for cell chemotaxis. Similar to the chemokine receptor. the “ classical ” chemokine receptor signaling pathway, the ␣ alternative chemokine receptor signaling pathway is de- CD38-dependent chemokine receptors couple to G q pendent on G i . However, unlike the classical chemokine Our data suggested that there was considerably more diver- receptor signaling pathway, the alternative pathway is also sity or heterogeneity in the molecular signals that regulate dependent on G q -containing G proteins. Despite the abil- chemotaxis than previously appreciated. To better understand ity of G q proteins to directly activate several isoforms of the diversity between chemokine receptors, we examined / PLC (32, 33), G q is not required for chemokine-in- the response of WT and Cd38 neutrophils to platelet- duced IP 3 -mediated intracellular calcium release. Instead, activating factor (PAF), a ligand of the PAFR. We chose to G q , like CD38, regulates extracellular calcium entry in analyze signaling through this receptor, as it is one of the few chemokine-stimulated cells. Importantly, we found that known chemoattractant receptors that can induce calcium / G q -defi cient ( Gnaq ) DCs and monocytes are unable to release in a PTx-independent fashion, indicating that it must migrate to infl ammatory sites and LNs in vivo, demonstrat- functionally couple to other G proteins in addition to those ing that this alternative G q -coupled chemokine receptor containing G i ( 34– 36 ). Therefore, we loaded bone marrow signaling pathway is critically important for the initiation WT and Cd38 / neutrophils with calcium-sensing fl uo- of immune responses. The implications of these unexpected rescent dyes, stimulated the cells with PAF, and measured fi ndings for chemokine receptor biology and infl ammation accumulation of intracellular free calcium by FACS. As pre- are discussed. viously reported for human neutrophils (34 ), PAF-stimulated