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Maturation of Eosinophilic HL-60 Cells Differentially Regulated by IL-5

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Maturation of Eosinophilic HL-60 Cells Differentially Regulated by IL-5 CC Chemokine Receptors 1 and 3 Are Differentially Regulated by IL-5 During Maturation of Eosinophilic HL-60 Cells This information is current as H. Lee Tiffany, Ghalib Alkhatib, Christophe Combadiere, Edward of September 26, 2021. A. Berger and Philip M. Murphy J Immunol 1998; 160:1385-1392; ; http://www.jimmunol.org/content/160/3/1385 Downloaded from References This article cites 40 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/160/3/1385.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 26, 2021 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 © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. CC Chemokine Receptors 1 and 3 Are Differentially Regulated by IL-5 During Maturation of Eosinophilic HL-60 Cells1 H. Lee Tiffany,* Ghalib Alkhatib,† Christophe Combadiere,* Edward A. Berger,† and Philip M. Murphy2* CC chemokine receptors 1 and 3 (CCR1 and CCR3) are expressed by eosinophils; however, factors regulating their expression and function have not previously been defined. Here we analyze chemokine receptor expression and function during eosinophil dif- ferentiation, using the eosinophilic cell line HL-60 clone 15 as a model system. RNA for CCR1, -3, -4, and -5 was not detectable in the parental cells, and the cells did not specifically bind CC chemokines. Cells treated with butyric acid acquired eosinophil characteristics; expressed mRNA for CCR1 and CCR3, but not for CCR4 or CCR5; acquired specific binding sites for macro- Downloaded from phage-inflammatory protein-1a and eotaxin (the selective ligands for CCR1 and CCR3, respectively); and exhibited specific calcium flux and chemotaxis responses to macrophage-inflammatory protein-1a, eotaxin, and other known CCR1 and CCR3 agonists. CCR3 was expressed later and at lower levels than CCR1 and could be further induced by IL-5, whereas IL-5 had little or no effect on CCR1 expression. Consistent with the HIV-1 coreceptor activity of CCR3, HL-60 clone 15 cells induced with butyric acid and IL-5 fused with HeLa cells expressing CCR3-tropic HIV-1 envelope glycoproteins, and fusion was blocked specifically by eotaxin or an anti-CCR3 mAb. These data suggest that CCR1 and CCR3 are markers of late eosinophil differentiation that are http://www.jimmunol.org/ differentially regulated by IL-5 in this model. The Journal of Immunology, 1998, 160: 1385–1392. hemokine receptors form a large family of seven-trans- In addition to their role in leukocyte migration, specific chemo- membrane-domain G protein-coupled receptors on leu- kine receptors also act in concert with CD4 as HIV-1 coreceptors, C kocytes that are important for leukocyte migration to mediating the first step in the viral life cycle, fusion of the viral sites of inflammation (reviewed in Ref. 1). The family includes envelope with the target cell membrane (17–22). The viral deter- two major subdivisions containing receptors specific for either CC minant of fusion is the envelope glycoprotein (Env). Different viral or CXC chemokines. Each receptor has a unique specificity for strains interact with specific chemokine receptors as determined by leukocyte subtypes and chemokines, but there can be extensive sequences in the gp120 component of Env (reviewed in Ref. 23). by guest on September 26, 2021 overlap in specificity among different receptors (1–14). The CC When CCR3 is expressed in foreign cells, it can support cell fusion chemokine receptors CCR13 and CCR3, which are the focus of this reactions mediated by Envs from diverse strains of HIV-1, includ- report, illustrate this particularly well. The recombinant receptors ing those used separately by the major HIV-1 coreceptors CCR5 both bind the CC chemokines RANTES and monocyte chemoat- and CXCR4 (17–22, 24, 25) (H. Bazan, G. Alkhatib, C. Broder, tractant protein (MCP)-3, yet eotaxin is selective for CCR3 and and E. A. Berger, manuscript in preparation). Moreover, endoge- macrophage inflammatory protein-1a (MIP-1a) is selective for nous CCR3 has recently been shown to support HIV-1 infection of CCR1 (2, 3, 5, 7, 8); both receptors are expressed in eosinophils microglial cells (24). However, its importance for HIV-1 infection (6–9, 15). The importance of CCR3 for eosinophil responses to of eosinophils and for HIV-1 pathogenesis has not been eotaxin, RANTES, and MCP-3 has been demonstrated by the demonstrated. Here we use a cultured HL-60 cell model of eosinophil differ- blocking effects of an anti-CCR3 mAb (16); the role of CCR1 in entiation to address the ontogeny and regulation of eosinophil che- eosinophil responses to MIP-1a is less well-defined. mokine receptor expression. Our results indicate that CCR1 and CCR3 are expressed late during eosinophil differentiation in this model and are differentially regulated by IL-5. Moreover, the data suggest that endogenous eosinophil CCR3 can functionally inter- Laboratories of *Host Defenses and †Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 act with HIV-1 Envs to facilitate membrane fusion reactions. Received for publication March 20, 1997. Accepted for publication October 9, 1997. 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 Materials and Methods with 18 U.S.C. Section 1734 solely to indicate this fact. Cell culture 1 This study was funded in part by the National Institutes of Health Intramural AIDS Targeted Antiviral Program. G.A. was supported by the Dr. Nathan Davis Award The promyelocytic cell line HL-60 clone 15 (CRL 1964, American Type from the American Medical Association Education and Research Foundation to Culture Collection, Rockville, MD) was maintained in RPMI 1640 with E.A.B. L-glutamine (Biofluids, Rockville, MD) containing 10% heat-inactivated 9 2 Address correspondence and reprint requests to Dr. Philip M. Murphy, Building 10, FCS (HyClone, Logan, UT) and 25 mM N-[2-hydroxyethyl]piperazine-N - Room 11N113, National Institutes of Health, Bethesda, MD 20892. E-mail address: [2-hydroxypropanesulfonic acid] (Sigma Chemical Co., St. Louis, MO), [email protected] pH 7.6, at 37°C in an atmosphere containing 5% CO2. Cells were induced m 3 Abbreviations used in this paper: CCR, CC chemokine receptor; MIP-1a, macro- to differentiate to eosinophil-like cells using 0.5 M butyric acid (Sigma) phage inflammatory protein-1a; MCP, monocyte chemoattractant protein; Env, en- as previously described (26, 27). Previously, it has been demonstrated that velope glycoprotein of HIV-1; EC50, 50% effective concentration. stimulation with butyric acid for 2 days renders these cells responsive to Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 1386 EOSINOPHIL CHEMOKINE RECEPTORS IL-5 owing to induction of surface IL-5 receptor expression (28). There- fore, in some experiments, 10 ng/ml IL-5 (R&D Systems, Minneapolis, MN) were added to the culture 2 days after addition of butyric acid. After addition of butyric acid, the medium was not replenished, including when IL-5 was added. Calcium flux assay Cells were suspended at 1 to 3 3 106/ml in PBS containing 2 mM fura- 2/AM (Molecular Probes, Eugene, OR) and incubated for 30 to 60 min at 37°C in the dark. They were then washed twice in HBSS (BioWhittaker, Walkersville, MD) and resuspended in HBSS at 1 3 106 cells/ml. Che- mokines were added at indicated times to 1 3 106 cells in a 2-ml volume in a continuously stirred cuvette at 37°C in a Model MS-III fluorimeter (Photon Technology, Inc., South Brunswick, NJ). The relative ratio of flu- orescence emitted at 510 nm following sequential excitation at 340 and 380 nm was recorded every 200 ms. Blocking experiments were conducted with the anti-CCR3 mAb 7B11 (generously provided by Charles MacKay), using methods previously described (16). RNA analysis FIGURE 1. CCR1 and CCR3 gene expression in HL-60 clone 15-de- rived eosinophils. The same Northern blot, containing total cellular RNA, Cells were harvested and total RNA prepared using a kit based on a gua- 15 mg/lane, from HL-60 clone 15 cells grown in the presence (1)orab- Downloaded from nidine thiocyanate/phenol extraction method following the manufacturer’s sence (2)of0.5mM butyric acid and 10 ng/ml IL-5 for the number of days instructions (Stratagene, La Jolla, CA). Isolated RNA (15 mg/sample) was electrophoresed in a 1% agarose gel in 10 mM 3-morpholinopropanesul- indicated above each lane, was hybridized sequentially with full-length fonic acid buffer, 5 mM sodium acetate, and 1 mM EDTA at pH 7.0 con- cDNA probes for CCR1 (top) and CCR3 (middle), followed by hybrid- taining 2% formaldehyde and 10 mg/ml ethidium bromide. The RNA was ization with an actin oligonucleotide probe (bottom). After a washing at blotted overnight to Nytran using a Turboblotter apparatus (Schleicher & high stringency, the blot was exposed to x-ray at 270°C with an intensi- Schuell, Keene, NH) followed by UV cross-linking.
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