Hematopoietic Progenitor Cells + Adhesion and Proliferation of CD34

The CysLT1 Ligand Leukotriene D4 Supports α4β1- and α5β1-Mediated Adhesion and Proliferation of CD34 + Hematopoietic Progenitor Cells This information is current as of October 2, 2021. Andreas M. Boehmler, Adriana Drost, Lena Jaggy, Gabriele Seitz, Tina Wiesner, Claudio Denzlinger, Lothar Kanz and Robert Möhle J Immunol 2009; 182:6789-6798; ; doi: 10.4049/jimmunol.0801525 Downloaded from http://www.jimmunol.org/content/182/11/6789 References This article cites 68 articles, 33 of which you can access for free at: http://www.jimmunol.org/content/182/11/6789.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on October 2, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology ␣ ␤ The CysLT1 Ligand Leukotriene D4 Supports 4 1- and ؉ ␤ ␣ 5 1-Mediated Adhesion and Proliferation of CD34 Hematopoietic Progenitor Cells1 Andreas M. Boehmler,* Adriana Drost,* Lena Jaggy,* Gabriele Seitz,* Tina Wiesner,* Claudio Denzlinger,† Lothar Kanz,* and Robert Mo¨hle2* Cytokines and chemokines control hematopoietic stem and progenitor cell (HPC) proliferation and trafficking. However, the role of nonpeptide mediators in the bone marrow microenvironment has remained elusive. Particularly CysLT1, a G protein-coupled receptor recognizing inflammatory mediators of the cysteinyl leukotriene family, is highly expressed in HPCs. We therefore ؉ analyzed the effects of its ligands on human CD34 HPCs. The most potent CysLT1 ligand, LTD4, rapidly and significantly ␣ ␤ ␣ ␤ up-regulated 4 1 and 5 1 integrin-dependent adhesion of both primitive and committed HPC. LTD4-triggered adhesion was Downloaded from inhibited by specific CysLT1 antagonists. The effects of other CysLT1 ligands were weak (LTC4) or absent (LTE4). In serum-free liquid cultures supplemented with various hematopoietic cytokines including IL-3, only LTD4 significantly augmented the expan- sion of HPCs in a dose-dependent manner comparable to that of peptide growth factors. LTC4 and LTE4 were less effective. In ؉ CD34 cell lines and primary HPCs, LTD4 induced phosphorylation of p44/42 ERK/MAPK and focal adhesion kinase-related tyrosine kinase Pyk2, which is linked to integrin activation. Bone marrow stromal cells produced biologically significant amounts of cysteinyl leukotrienes only when hematopoietic cells were absent, suggesting a regulatory feedback mechanism in the hematopoietic microenvironment. In contrast to antagonists of the homing-related G protein-coupled receptor CXCR4, administration http://www.jimmunol.org/ ؉ of a CysLT1 antagonist failed to induce human CD34 HPC mobilization in vivo. Our results suggest that cysteinyl leukotriene may contribute to HPC retention and proliferation only when cysteinyl leukotriene levels are increased either systemically during inflammation or locally during marrow aplasia. The Journal of Immunology, 2009, 182: 6789–6798. oming of hematopoietic stem and progenitor cells diators like LTs, which regulate leukocyte migration and function (HPCs)3 to the bone marrow and their subsequent reten- at sites of inflammation, may also affect HPCs, particularly their H tion and survival in the hematopoietic microenviron- trafficking and proliferation in the bone marrow. ment are regulated by particular proteins and peptides (cytokines, Cysteinyl leukotrienes represent important lipid mediators in in- by guest on October 2, 2021 chemokines, and adhesion molecules) (1–3). However, the role of flamed tissues and have recognized roles in respiratory diseases, lipid mediators such as leukotrienes (LTs) in HPC homeostasis has cancer, and cardiovascular, gastrointestinal, skin, and immune dis- remained elusive, in contrast to their well-characterized involve- orders (10). They elicit many of their effects through seven-trans- ment in allergy and inflammation (4–6). In this context, it is worth membrane G protein-coupled receptors (GPCRs) that show struc- mentioning that the hematopoietic microenvironment shares sim- tural similarities to chemokine receptors such as CXCR4, which is ilarities with inflamed tissues and that the homing of HPCs resem- a key receptor involved in HPC homing to the bone marrow (11, bles extravasation of mature leukocytes during inflammation. For 12). To date, two GPCRs recognizing cysteinyl leukotrienes, instance, bone marrow endothelial cells (BMEC) constitutively ex- CysLT1 and CysLT2, have been cloned and characterized (10, 12– press the adhesion molecules E-selectin, P-selectin, and VCAM-1, 14). They show different binding affinities for their ligands, LTD4, which are found on the endothelium of other tissues only during LTC , and LTE (15). CysLT binds LTD with a 350-fold greater inflammation (7–9). It is therefore conceivable that nonpeptide me- 4 4 1 4 affinity than it does LTC4 (10). LTE4, which acts as a partial ag- onist, is the least active cysteinyl leukotriene (12). CysLT2,in † *Department of Medicine II, University of Tu¨bingen, Tu¨bingen, Germany; and De- contrast, binds LTD4 and LTC4 with equal affinity (13, 14). partment of Medicine III, Marienhospital, Stuttgart, Germany CysLT1 couples to pertussis toxin (PTX)-sensitive Gi/o and PTX- Received for publication May 9, 2008. Accepted for publication March 31, 2009. insensitive Gq proteins and activates signaling pathways that mod- The costs of publication of this article were defrayed in part by the payment of page ulate multiple cellular activities, including proliferation and mi- charges. This article must therefore be hereby marked advertisement in accordance gration. The role and function of CysLT is not completely with 18 U.S.C. Section 1734 solely to indicate this fact. 2 understood. This receptor may also activate G signaling path- 1 This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 510/A4 i/o and Deutsche JoseĆarreras Leuka¨miestiftung Grants DJCLS R04/13 and R08/24v. ways, although recent studies suggest that CysLT2 interacts with 2 Address correspondence and reprint requests to Dr. Robert Mo¨hle, Department of CysLT1 on the cell surface, resulting in the formation of het- Medicine II, University of Tu¨bingen, Otfried-Mu¨ller-Strasse 10, 72076 Tu¨bingen, erodimers and eventually in the attenuation of CysLT1-mediated Germany. E-mail address: [email protected] effects (16). 3 Abbreviations used in this paper: HPC, hematopoietic stem and progenitor cell; We have previously shown that CysLT1 is highly expressed in BMEC, bone marrow endothelial cell; CAFC, cobblestone area-forming cell; EIA, ϩ enzyme immunoassay; GPCR, G protein-coupled receptor; 5-LO, 5-lipoxygenase; CD34 progenitor cells (17). In contrast to CXCR4, the estab- LT, leukotriene; MPA, mycophenolic acid; PTX, pertussis toxin; rh, recombinant lished “homing receptor” for HPCs that is expressed much stron- human; SDF, stromal cell-derived factor. ger in mature mononuclear cells than in CD34ϩ HPCs (18), Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 CysLT1 expression appears to be more progenitor specific. Indeed, www.jimmunol.org/cgi/doi/10.4049/jimmunol.0801525 6790 LTD4 SUPPORTS PROGENITOR ADHESION AND PROLIFERATION even though mature leukocytes express some CysLT1 at the Flow cytometric analysis mRNA and protein level (17), it does not mediate typical func- Expression of glycophorin-A, CD14, CD15, CD34, CD38, CD41, and tions in these cells, e.g., calcium fluxes (19). In contrast, cir- CD45 was evaluated by incubating 2–5 ϫ 105 cells with the respective culating HPCs express high levels of CysLT1 and respond to FITC- or PE-labeled Abs (BD Biosciences) for 20 min at 4°C and analyzed on a FACSCalibur flow cytometer equipped with CellQuestPro software. LTD4 with much more pronounced calcium fluxes than those observed after stromal cell-derived factor (SDF)-1-induced Isotype-matched IgG Abs served as negative controls. A proportion of 1% or fewer false positive events were accepted in the negative control sam- CXCR4 activation (20). ples throughout. However, besides inducing considerable calcium fluxes and che- motaxis in vitro, the function of CysLT receptors and the potential Cell adhesion assays source of their ligands in the hematopoietic system in vivo remain to be characterized. Similar to CXCR4, which plays an important To prepare HUVEC monolayers, 1 ϫ 104 cells were seeded on gelatin- role in B cell trafficking and proliferation in addition to its function coated (0.1% in PBS), 96-well, flat-bottom tissue culture plates 48 h before the assay and grown to confluence in endothelial cell growth medium plus as a HPC homing receptor, CysLT1 as well may regulate the func- 10% FCS. Before adding HPCs, the monolayers were stimulated with 10 tion of different cell types, i.e., inflammatory effector cells ng/ml IL-1␤ (Boehringer Mannheim) for

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