Supplemental Material can be found at: http://www.jlr.org/content/suppl/2011/04/26/jlr.M008045.DC1 .html ␣ TNF- promotes LPA1 - and LPA3 -mediated recruitment of leukocytes in vivo through CXCR2 ligand chemokines Chenqi Zhao , 1, * Anne Sardella , 1, * Jerold Chun , † Patrice E. Poubelle , * Maria J. Fernandes , * and Sylvain G. Bourgoin 2, * Rheumatology and Immunology Research Center,* CHUQ-CHUL Research Center and Faculty of † Medicine, Laval University , Québec City, Québec, Canada ; and Department of Molecular Biology, The Scripps Research Institute , La Jolla, CA Downloaded from Abstract Lysophosphatidic acid (LPA) is a bioactive lyso- Lysophosphatidic acid (LPA) is a simple lipid with a sin- phospholipid present in low concentrations in serum and gle fatty acyl chain, a glycerol backbone, and a free phos- biological fl uids but in high concentrations at sites of infl am- phate group ( 1 ). Despite the simplicity of its structure, www.jlr.org mation. LPA evokes a variety of cellular responses via binding LPA has been implicated in various physiological (cell to and activation of its specifi c G protein-coupled receptors growth, differentiation, migration, and survival) and path- (GPCR), namely LPA1-6 . Even though LPA is a chemoattrac- tant for infl ammatory cells in vitro, such a role for LPA in ological (angiogenesis, cancer, and autoimmunity) situa- at Kresge Library, The Scripps Research Institute, on November 14, 2012 vivo remains largely unexplored. In the present study, we tions. LPA is present in micromolar concentrations in used the murine air pouch model to study LPA-mediated serum and biological fl uids and in higher concentrations leukocyte recruitment in vivo using selective LPA receptor at sites of infl ammation and tumor ( 2 ). LPA evokes a vari- agonist/antagonist and LPA3 -defi cient mice. We report that ety of cellular responses via binding to and activation of its 1 ) LPA injection into the air pouch induced leukocyte re- specifi c G protein-coupled receptors (GPCR) ( 3 ). So far, cruitment and that both LPA1 and LPA3 were involved in this fi ve and possibly six GPCRs have been identifi ed as specifi c process; 2 ) LPA stimulated the release of the pro-infl amma- LPA receptors, namely, LPA ( 4 ). Two additional GPCRs, tory chemokines keratinocyte-derived chemokine (KC) and 1-6 interferon-inducible protein-10 (IP-10) in the air pouch; 3 ) GPR87 ( 5 ) and P2Y10 ( 6 ), have been suggested to be acti- tumor necrosis factor- ␣ (TNF- ␣ ) injected into the air pouch vated by LPA. prior to LPA strongly potentiated LPA-mediated secretion LPA has been shown to be a chemoattractant for human of cytokines/chemokines, including KC, IL-6, and IP-10, infl ammatory cells in in vitro studies. Chettibi et al. showed which preceded the enhanced leukocyte infl ux; and 4 ) block- that LPA was a powerful stimulator of human neutro- ing CXCR2 signifi cantly reduced leukocyte infi ltration. phil polarization and motility ( 7 ). Similarly, Fischer et al. We suggest that LPA, via LPA1 and LPA3 receptors, may play described chemoattractive effects of LPA toward human a signifi cant role in inducing and/or sustaining the massive PMNs under agarose ( 8 ). LPA also causes an increase in infi ltration of leukocytes during infl ammation. — Zhao, C., binding of monocytes and neutrophils to human aortic A. Sardella, J. Chun, P. E. Poubelle, M. J. Fernandes, and S. ␣ endothelial cells ( 9 ). Emerging evidence suggests a role G. Bourgoin. TNF- promotes LPA1 - and LPA3 -mediated re- cruitment of leukocytes in vivo through CXCR2 ligand for autotaxin (ATX)-LPA in facilitating lymphocyte migra- chemokines. J. Lipid Res. 2011. 52: 1307–1318. tion into lymphoid and chronically infl amed nonlymphoid tissues ( 10, 11 ). The study of Kanda et al. demonstrated Supplementary key words infl ammation • murine air pouch model • that LPA, locally generated by ATX in high endothelial LPA receptor agonist/antagonist • LPA receptor defi cient mice • CXC venules, facilitates lymphocyte transendothelial migration chemokines Abbreviations: ATX, autotaxin; COX-2, cyclooxygenase-2; GPCR, G protein-coupled receptor; IL, interleukin; IP-10, interferon-␥ -inducible This work was supported by grants from the Arthritis Society of Canada protein-10; KC, keratinocyte-derived chemokine; KO, knock out; LPA, (RG07/092) and the Canadian Arthritis Network (S.G.B). C.Z. is the recipient lysophosphatidic acid; MCP-1, monocyte chemotactic protein-1; MIP-2, of the Canadian Arthritis Network Post-Doctoral Fellowship Award. A.S. is the macrophage infl ammatory protein-2; PGE2 , prostaglandin E2 ; RA, recipient of the Canadian Arthritis Network Master Student Trainee Award. rheumatoid arthritis; TNF- ␣ , tumor necrosis factor- ␣ ; WT, wild type. J.C. was supported by National Institutes of Health Grants MH-51699 and 1 C. Zhao and A. Sardella contributed equally to this work. A. Sardella HD-050685. The contents are solely the responsibility of the authors and do not necessarily represent the offi cial views of the National Institutes of Health or performed this work in partial fulfi llment of her MSc degree at the other granting agencies. Faculty of Medicine, Laval University, Québec, Canada. 2 To whom correspondence should be addressed. Manuscript received 27 April 2010 and in revised form 21 May 2011. e-mail: [email protected] Published, JLR Papers in Press, April 25, 2011 The online version of this article (available at http://www.jlr.org) DOI 10.1194/jlr.M008045 contains supplementary data in the form of three fi gures. Copyright © 2011 by the American Society for Biochemistry and Molecular Biology, Inc. This article is available online at http://www.jlr.org Journal of Lipid Research Volume 52, 2011 1307 Supplemental Material can be found at: http://www.jlr.org/content/suppl/2011/04/26/jlr.M008045.DC1 .html via stimulating chemokinesis rather than chemotaxis (10 ). control (clone 54447) were purchased from R&D Systems The role of LPA in the recruitment of leukocytes to in- Inc. (Minneapolis, MN). KC/MIP-2 ELISA kit and Proteome TM fl ammatory sites in vivo, however, remains elusive. Profi ler Mouse Cytokine Array Panel A were purchased from R&D Systems. The mouse Cytokine/Chemokine Luminex Multi- Accumulating evidence suggests that LPA-induced cell plex Immunoassay kit was from Millipore Corporation (St. migration may be mediated, in part, by chemokines. We Charles, MO). recently reported that LPA induces the expression of a potent chemoattractant, interleukin (IL)-8 ( 12 ). Chemo- Mice kines are small chemoattractant peptides that play key Ϫ / Ϫ LPA3 receptor knock out (KO) mice (LPA3 ) were previ- roles in the accumulation of infl ammatory cells at the ously generated ( 22 ). Mice were subjected to genotyping for site of infl ammation. Some chemokines, particularly CXC LPA3 alleles as described previously (22, 25 ). Female Balb/ chemokines containing the ELR motif (including CXCL1, c (wild-type) mice 6-8 weeks old (from Charles River, St.- 2, 3, 5, 6, 7, and 8) are crucial for neutrophil recruitment Colomban, Canada) were used as background controls. All ani- via binding to and activation of their receptors CXCR1 or mal exper imental procedures were approved by the Animal Care CXCR2 ( 13, 14 ). In the mouse, IL-8/CXCL8 and a func- Committee at CHUL Research Center, Laval University and con- formed to National Institutes of Health (NIH) guidelines and tional homolog of CXCR1 are absent, and keratinocyte- public law. Downloaded from derived chemokine (KC, Gro ␣ , CXCL1) and macrophage infl ammatory protein-2 (MIP-2) appear to be the major The air pouch model ELR CXC chemokines expressed at sites of tissue infl am- Air pouches were raised on the dorsum of mice by subcutane- mation following injury and/or infection ( 15–19 ). It is as- ous injection of 3 ml sterile air on days 0 and 3 as previously sumed that neutrophil migration in this species is mediated described ( 26 ). Before the injection of air, mice were briefl y mainly by KC and MIP-2 via binding to their sole receptor anesthetized with isofl urane. On day 7, LPA or LPA receptor www.jlr.org CXCR2 ( 20, 21 ). LPA was shown to regulate the expres- agonist/antagonist in 1 ml of the diluent (PBS + 0.1% BSA) ␣ sion of cyclooxygenase-2 (COX-2) and the production of were injected into air pouches. In some experiments, TNF- was injected into air pouches 16 h prior to stimulation with LPA prostaglandin E (PGE ) in studies using LPA -defi cient at Kresge Library, The Scripps Research Institute, on November 14, 2012 2 2 3 or LPA receptor agonist. To test a systemic effect of LPA recep- mice ( 22 ). Additionally, LPA has been shown to induce tor antagonism on LPA-induced leukocyte recruitment, LPA1/3 the synthesis of PGE2 , cytokines and chemokines in syn- antagonist VPC32183 was intravenously injected 15 min prior to oviocytes isolated from patients with rheumatoid arthritis LPA injection into the air pouch. To defi ne the role of CXCR2 (RA) ( 12, 23 ), a chronic infl ammatory disease character- and CXCL1 chemokines in LPA-induced leukocyte recruit- ized by extensive leukocyte infi ltration into the affected ment, the CXCR2 antagonist SB225002 and blocking antibod- joint space ( 24 ). ies against KC, MIP-2, and CXCR2 were intravenously injected In the present study, we employed an in vivo experimen- 15 min prior to LPA injection into the air pouch. At specifi c tal model of infl ammation, the murine air pouch model, times, mice were euthanized by asphyxiation using CO2 . The air pouch exudates were collected by washing once with 1 ml of to objectively evaluate the effect of LPA on leukocyte re- PBS-5 mM EDTA, and then twice with 2 ml of PBS-5 mM EDTA. cruitment in vivo. The LPA receptor dependency and the After 5 min centrifugation at 1,200 rpm, cell pellets were resus- mechanisms of LPA-induced leukocyte recruitment in vivo pended, stained with acetic blue, and the leukocytes were were investigated using both a pharmacological (LPA re- counted.