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 differentiation, 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-1␣ and eotaxin (the selective ligands for CCR1 and CCR3, respectively); and exhibited specific calcium flux and chemotaxis responses to macrophage-inflammatory protein-1␣, 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-1␣ (MIP-1␣) 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-1␣ 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 Ј 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 ␮ 3 Abbreviations used in this paper: CCR, CC chemokine receptor; MIP-1␣, macro- to differentiate to eosinophil-like cells using 0.5 M butyric acid (Sigma) phage inflammatory protein-1␣; 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

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 ϫ 106/ml in PBS containing 2 ␮M 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 ϫ 106 cells/ml. Che- mokines were added at indicated times to 1 ϫ 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 fluorescence 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-derived eosinophils. The same Northern blot, containing total cellular RNA, Cells were harvested and total RNA prepared using a kit based on a gua- 15 ␮g/lane, from HL-60 clone 15 cells grown in the presence (ϩ)orab- Downloaded from nidine thiocyanate/phenol extraction method following the manufacturer’s sence (Ϫ)of0.5␮M butyric acid and 10 ng/ml IL-5 for the number of days instructions (Stratagene, La Jolla, CA). Isolated RNA (15 ␮g/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 ␮g/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 Ϫ70°C with an intensi- Schuell, Keene, NH) followed by UV cross-linking. Chemokine receptor fying screen for 16 h (CCR1 probe), 3 days (CCR3 probe), and 8 h (actin

probes were labeled using the Random Primer Labeling Kit (Boehringer probe). The blot was stripped between experiments, and removal of the http://www.jimmunol.org/ 32 Mannheim, Indianapolis, IN), and [ P]dCTP, 6000 Ci/mmol (Amersham preceding probe was confirmed. No signals were detected when the same Corp., Arlington Heights, IL). Northern blots were prehybridized in 50% RNA was tested with full-length CCR4 and CCR5 probes (not shown). formamide, 20% dextran sulfate, 5ϫ standard saline-phosphate-EDTA, 0.5% SDS, and 50 ␮g/ml denatured salmon sperm DNA for1hat37°C. 32 6 Denatured P-labeled probe at 2 ϫ 10 cpm/ml was hybridized to the blot into the plasmid vector pSC59 which contains a vaccinia strong early/ overnight at 37°C. Blots were then rinsed three times in 1ϫ SSC, 0.1% strong late promoter (H. Bazan, G. Alkhatib, C. Broder, and E. A. Berger, SDS at room temperature, washed at 60°C for 30 to 60 min in 1ϫ SSC, manuscript in preparation; S Chakrabarti and B. Moss, unpublished data). 0.1% SDS, and then exposed to x-ray film. The probes included the com- Effector HeLa cells were transfected by lipofection with this plasmid and plete open reading frame and, in most cases, some untranslated sequence. then infected with recombinant vaccinia virus vCB21R-LacZ containing They are: p4 cDNA, CCR1 (3); clone 3 cDNA, CCR3 (6); PCR-amplified the ␤-galactosidase gene linked to the T7 promoter (30). Cells were then by guest on September 26, 2021 CCR4 open reading frame (A. Sen and J.-L. Gao, unpublished probe); and mixed in an HL-60:HeLa ratio of 3:1, in a total volume of 200 ␮l, and 8c3 cDNA, CCR5 (13). A 50-mer oligonucleotide probe specific for human incubated at 37°C for 3 h. Relative cell fusion was recorded as ␤-galac- ␤-actin was used as a control for loading. ϫ tosidase activity (OD570 1000/min). Blocking experiments were conducted at the start of the coculture by adding either eotaxin or anti-receptor Chemokine binding assay Abs, including the anti-CCR3 mouse mAb 7B11 (16) and a rabbit poly- Recombinant human chemokines RANTES, MIP-1␣, MIP-1␤, MCP-3, clonal anti-CXCR4 Ab previously described (17). In separate experiments, MCP-1, IL-8, and eotaxin were purchased from Peprotech (Princeton, NJ). HeLa cells were coinfected with vCB21R-LacZ, and either vCB-43, en- 125I-labeled human MIP-1␣, MCP-3, and eotaxin, each having a sp. act. of coding Env from the prototypic macrophage-tropic HIV-1 strain Ba-L, or 2200 Ci/mmol, were purchased from New England Nuclear (Boston, MA). vCB-16, encoding a mutated nonfunctional Env, named Unc, derived from Cells were suspended in RPMI 1640 containing 1% BSA plus azide at 2 ϫ strain IIIB (31–33). Cells were then mixed with target cells and analyzed 107/ml. In a 1.5-ml microfuge tube, increasing concentrations of unlabeled as above. Both Ba-L and 89.6 Envs are able to induce cell fusion formation ligand and 0.4 nM 125I-ligand were added to 106 cells in a total volume of in this assay. 100 ␮l. Cells were incubated at room temperature for 1 h with occasional gentle shaking. One milliliter of 10% sucrose in PBS was then added, and Results the cells were pelleted in the microfuge tube for 2 min. The supernatants HL-60 clone 15-derived eosinophils express CCR1 and CCR3 were removed, and gamma emissions from the cell pellets were counted. Binding data were analyzed using the program LIGAND. We and others have previously shown that the clone 15 variant of Chemotaxis HL-60 cells can be induced by butyric acid treatment to differentiate within 2 days into cells having many of the characteristics of Cells were harvested and washed twice with PBS and then resuspended in peripheral blood eosinophils, including expression of eosinophil- serum-free RPMI 1640. Cells (175,000–190,000 per replicate) were loaded specific granule proteins (26, 27). Using Northern blot analysis, we in a total volume of 25 ␮l into the upper compartment of a microchemotaxis chamber (Neuroprobe, Cabin John, MD). Chemoattractants were were unable to detect mRNA for CCR1, -3, -4, or -5 in the unin- loaded in a final volume of 31 ␮l at indicated concentrations into the lower duced cells at time zero (Fig. 1 and data not shown). CCR4 and compartment. The two compartments were separated by a polyvinylpyrro- CCR5 mRNAs remained undetectable for at least 6 days after ␮ lidone-free polycarbonate filter with 5- m pores. The chemotaxis chamber treatment with butyric acid (not shown). In contrast, CCR1 mRNA was incubated at 37°C, 100% humidity, and 5% CO2 for 4 h. The filter was then removed, and the number of cells migrating into the bottom compart- was detectable 2 days after addition of butyric acid and increased ment was counted with a hemocytometer. All conditions were tested in progressively over the 5-day period of study (Fig. 1, top). CCR3 triplicate. mRNA was also induced by butyric acid but was not detected until ϳ Cell fusion assay 5 days after addition of butyric acid (Fig. 1, middle). Butyric acid induces expression of IL-5 receptors on HL-60 clone 15 cells, HIV Env-mediated cell fusion was quantitated using a vaccinia-based re- and the cells then proliferate in response to IL-5 (28). We observed porter gene activation assay (29). Induced and uninduced HL-60 clone 15 cells were coinfected overnight with recombinant vaccinia viruses vTF7–3 that the level of expression of CCR3 was markedly increased by encoding the T7 RNA polymerase (30) and vCB-3 encoding human CD4 the addition of IL-5 to the butyric acid-treated culture, the con- (31). The Env from the dual tropic primary HIV-1 isolate 89.6 was cloned centration previously shown to be optimal for proliferation of these The Journal of Immunology 1387 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. Binding of CCR1 and CCR3 ligands to HL-60 clone 15-derived eosinophils. Cells were incubated with 125I-MIP-1␣, 125I-MCP-3, or 125I-eotaxin (0.4 nM in each case), as indicated at the top of each panel, in the presence of 100 nM concentrations of the unlabeled chemokine indicated on the x-axis of each panel. A, Cells cultured for 3 days after addition of 0.5 ␮M butyric acid. B, Cells cultured for 6 days after addition of 0.5 ␮M butyric acid; 10 ng/ml IL-5 were added 2 days after addition of butyric acid. Results are expressed as the percentage of total binding, where total binding is the number of cell-associated counts per minute observed in the absence of unlabeled chemokine. Inhibition was statistically significant ( p Յ 0.005 by ANOVA) for all unlabeled chemokines tested except for IL-8 (all panels) and eotaxin inhibition of 125I-MIP-1␣ binding. Data are the mean Ϯ SEM of triplicate determinations from a single experiment representative of three separate experiments. cells. In contrast to its effects on steady state CCR3 mRNA, IL-5 since direct eotaxin binding and signaling was not observed (see had little effect on the amount of CCR1 mRNA detected (Fig. 1). below). The competition profile for the 125I-MCP-3-labeled site was similar, although not identical. The specificity of competition Characterization of CC chemokine-binding sites on HL-60 clone for the MIP-1␣ binding site is consistent with that established pre- 15-derived eosinophils viously for recombinant CCR1 (2). To test whether induction of CCR1 and CCR3 mRNA correlated For cells treated with both butyric acid and IL-5, the competition with induction of binding sites for known CCR1 and CCR3 li- binding profiles for 125I-MIP-1␣ and 125I-MCP-3 were similar to gands, we conducted radiolabeled chemokine binding assays. Spe- the profiles observed for cells treated with butyric acid alone (Fig. 125 ␣ 125 125 cific binding of I-MIP-1 , I-MCP-3, or I-eotaxin was not 2B). However, under these conditions, specific binding of 125I- detected on uninduced HL-60 cells when 0.4 nM radioligand was eotaxin was also detected (Fig. 2B). Eotaxin binding was strongly used as a probe (not shown). Three days after butyric acid was inhibited by 100 nM unlabeled eotaxin (75% inhibition) and added, specific binding of 125I-MIP-1␣ and 125I-MCP-3, but not MCP-3 (53%) but was affected only weakly by RANTES (30%), 125I-eotaxin, was detected (Fig. 2A). 125I-MIP-1␣ binding was MCP-1 (20%), and MIP-1␤ (15%) and not at all by IL-8 and MIP- strongly inhibited in the presence of 100 nM unlabeled MIP-1␣ 1␣. The specificity of competition for the eotaxin-binding site is (95% inhibition), RANTES (68%), MCP-3 (90%), MIP-1␤ (75%), consistent with that established previously for primary eosinophils and MCP-1 (65%), but not by the CXC chemokine IL-8 (0%). Eotaxin reduced binding by only 18%, indicating that the great and recombinant CCR3 (8, 15). Thus, at this time point eotaxin and ␣ majority of MIP-1␣- and eotaxin-binding sites are distinct. Al- MIP-1 appear to bind to separate sites on HL-60 clone 15-derived ␤ though statistically significant, the functional significance of this eosinophils, and MIP-1 , MCP-1, MCP-3, and RANTES appear to small amount of cross-competition is unclear, especially since we share binding determinants with both. The induced cells expressed did not observe it later in the course of cell differentiation, and 23,000 eotaxin-binding sites/cell with a Kd of 2.6 nM. Unlabeled 1388 EOSINOPHIL CHEMOKINE RECEPTORS Downloaded from http://www.jimmunol.org/

FIGURE 4. Calcium flux response of agonists for HL-60 clone 15-de- ␣ ϩ FIGURE 3. High affinity binding of eotaxin and MIP-1 to HL-60 rived eosinophils. [Ca2 ] was monitored in real time as the relative fluo- 125 i clone 15-derived eosinophils. A, I-eotaxin binding competition with un- rescence in fura-2-loaded HL-60 cells, cultured for 6 days in medium alone labeled eotaxin. Cells were cultured for 6 days after addition of 0.5 ␮M ␮ (uninduced) or after adding 0.5 M butryic acid for 6 days with 10 ng/ml by guest on September 26, 2021 butyric acid; IL-5, 10 ng/ml, was added 2 days after addition of butyric IL-5 added for the last 4 days (induced). Arrowheads mark the time when 125 ␣ acid. B, I-MIP-1 binding. Cells were cultured for 3 days after addition 50 nM concentrations of the chemokine indicated to the left of each cor- ␮ of 0.5 M butyric acid. Binding was measured in the presence of increas- responding row were added. Data are representative of at least three ex- ␣ f ‚ ⅜ ing concentrations of unlabeled MIP-1 ( ), RANTES ( ) or MCP-3 ( ). periments for each chemokine. Insets at the upper right of each panel show Scatchard analysis of the ␣ binding data indicating a Kd of 42 nM for MIP-1 (650,000 sites/cell) and a Kd of 2.6 nM for eotaxin (23,000 sites/cell). Data are the mean of triplicate determinations from a single experiment representative of three in- for CCR5, the only known MIP-1␤ receptor. The cells also did not dependent experiments. respond to MCP-1 or IL-8, which suggests that the IL-8 receptors CXCR1 and CXCR2, and the MCP-1 receptor CCR2 are also are not expressed in these cells, although we did not specifically probe MIP-1␣, RANTES, and MCP-3 competed for the 125I-MIP-1␣- for the corresponding mRNAs. ϳ labeled site on induced cells with IC50s 5, 20, and 50 nM, re- Consistent with the relative density of specific binding sites and spectively (Fig. 3B). The cells expressed 650,000 MIP-1␣ binding mRNA levels, the cell response to eotaxin was much weaker than

sites/cell with a Kd of 42 nM. the response to MIP-1␣. The responses to RANTES, MCP-3, and FMLP were similar in magnitude to MIP-1␣ and increased rapidly Calcium flux responses in HL-60 clone 15-derived eosinophils by day 2 and peaked on day 4 after addition of butyric acid (Figs. induced by CCR1 and CCR3 ligands 4 and 5, A and B, and data not shown). In contrast, the response to To test whether the chemokine-binding sites on HL-60 clone 15- eotaxin was never observed on day 2 after addition of butyric acid derived eosinophils were functional, we first monitored changes in but was consistently observed by day 4 (Fig. 5). Adding IL-5 to 2ϩ [Ca ]i in response to stimulation with chemokines. A rapid, tran- butyric acid-treated cells markedly enhanced responsiveness to sient calcium flux is typically observed when leukocytes are stim- eotaxin, beginning on day 4 and peaking on day 6, but had little ulated with chemokines, and it serves as a convenient measure of effect on responsiveness to MIP-1␣, RANTES, MCP-3, and FMLP chemokine receptor activation that can be followed in real time in (Fig. 5 and data not shown). The 50% effective concentration

fura-2-loaded cells. Uninduced cells and cells treated with IL-5 (EC50) of the calcium flux response to eotaxin for HL-60 clone 15 alone responded negligibly to MIP-1␣, RANTES, MCP-3, FMLP, cells induced with butyric acid and IL-5 was ϳ5 nM, a value ␤ eotaxin, MIP-1 , MCP-1, or IL-8. In contrast, the cells responded similar to the KD for eotaxin binding (Fig. 6). Eotaxin has similar well to ATP which activates purinergic receptors in myeloid cells potency for calcium flux at recombinant CCR3 expressed in HEK (Fig. 4 and data not shown). HL-60 clone 15 cells cultured in the 293 cells (7). The anti-CCR3 mAb 7B11 completely blocked the presence of butyric acid acquired responsiveness to MIP-1␣, calcium flux response to eotaxin in HL-60 clone 15 cells, but had RANTES, MCP-3, eotaxin, and FMLP (Fig. 4). The cells did not no effect on the response to MIP-1␣ (Fig. 7). It also specifically respond to MIP-1␤, consistent with absence of detectable mRNA blocked the eotaxin-induced calcium flux response in mouse pre-B The Journal of Immunology 1389

FIGURE 6. Eotaxin potency for inducing calcium ﬂux in HL-60 clone

15-derived eosinophils. HL-60 cells were cultured for 6 days after addition Downloaded from of 0.5 ␮M butyric acid; 10 ng/ml IL-5 were added 2 days after butyric acid. The peak response from individual ﬂuorescence tracings was plotted as a function of eotaxin concentration. The data are from a single experiment representative of 2 separate experiments.

ϳ ␣ http://www.jimmunol.org/ with EC50sof 5 and 20 nM for MIP-1 and eotaxin, respectively. Consistent with results from the calcium ﬂux assay, RANTES and MCP-3 (but not MIP-1␤, MCP-1, and IL-8) were able to chemoattract induced HL-60 clone 15 cells (data not shown). by guest on September 26, 2021

FIGURE 5. Time course of HL-60 clone 15 cell calcium flux responses ␣ 2ϩ to MIP-1 and eotaxin during differentiation into eosinophils. [Ca ]i was monitored as the relative fluorescence in fura-2-loaded cells stimulated with MIP-1␣ (A) or eotaxin (B) at 50 nM in each case. The peak response to each indicated agonist was plotted vs the time in culture for HL-60 clone 15 cells exposed to medium only (ᮀ) or after addition of butyric acid (BA) 0.5 ␮Mattime0(छ), IL-5 10 ng/ml alone at day 2 (⅜), or butyric acid 0.5 ␮M at time 0 with IL-5 10 ng/ml added at day 2 (‚). Each data point corresponds to the calcium flux peak height of a single tracing. The data are from a single experiment representative of at least three separate experiments.

cells transfected with recombinant CCR3 (J. E. Pease and P. M. Murphy, unpublished data), conﬁrming a previous report (16).

Chemotaxis in HL-60 clone 15-derived eosinophils induced by CCR1 and CCR3 ligands We next tested the ability of CCR1 and CCR3 ligands to induce chemotaxis in HL-60 clone 15-derived eosinophils (Fig. 8). Un- induced HL-60 clone 15 cells did not move in response to either MIP-1␣ or eotaxin (data not shown). Cells treated with butyric acid for 3 days moved weakly in response to MIP-1␣ with an optimal concentration of 5 nM, whereas the same cells did not FIGURE 7. HL-60 clone 15 cell calcium flux responses to eotaxin are ␮ move in response to eotaxin at any concentration tested (range, 1 mediated by CCR3. Cells were tested 6 days after addition of 0.5 M butyric acid and 4 days after addition of 10 ng/ml IL-5. Arrows mark the to 500 nM). In contrast, cells treated under optimal conditions for time of addition of the indicated substances. Chemokines were tested at 50 expression of CCR1 and CCR3 (butyric acid plus IL-5) exhibited nM; the anti-CCR3 mAb 7B11 was added at 6 ␮g/ml. 7B11 at the same ␣ robust chemotactic responses to both eotaxin and MIP-1 .As concentration also blocked eotaxin induction of calcium flux in CCR3- would be predicted from results of the calcium flux assay, MIP-1␣ transfected mouse pre-B cells, but not MIP-1␣ activation of CCR1-trans- was a much more effective agonist for chemotaxis. A typical bell- fected mouse pre-B cells, confirming a previous report (16) (J. E. Pease and shaped dose-response curve was observed for both chemokines, P. M. Murphy, data not shown). 1390 EOSINOPHIL CHEMOKINE RECEPTORS

the two Envs is not known but may reflect differences in levels of Env expression, differences in the CCR3 interaction sites for these Envs, and/or differential usage of other unknown coreceptors. RANTES and MCP-3, which bind to CCR3 with 30- and 100-fold lower affinity than eotaxin, respectively (8), were unable to block Ba-L Env-dependent fusion of induced clone 15 cells (Fig. 9D), consistent with previous results using National Institutes of Health 3T3 cells expressing recombinant CCR3 (25). Addition to the coculture of either anti-CXCR4 or anti-CCR3 Abs, shown previously to neutralize the corresponding HIV-1 coreceptor activities (17, 24), reduced 89.6 Env-dependent fusion activity in each case by ϳ50%, whereas control Abs had a negli- gible effect (Fig. 9, B and C). Consistent with previous reports of the ability of the 89.6 Env to interact with both recombinant CXCR4 and CCR3, we observed increased inhibition of fusion when both Abs were added together vs either added alone (Fig. 9C). Downloaded from Discussion Using an HL-60 cell line model of eosinophil differentiation, we have shown that CCR1 and CCR3 are differentially regulated by butyric acid and IL-5 late in maturation. Both receptors function in these cells as revealed by ligand-binding assays and three functional assays: chemokine-induced calcium flux and chemotaxis; http://www.jimmunol.org/ and HIV-1 Env-dependent cell fusion. The MIP-1␣-binding site on HL-60 clone 15-derived eosinophils is similar to that reported previously for HL-60 clone 7-derived eosinophils (34). The eotaxin-binding site on clone 15 cells is similar to that described on primary eosinophils (15). Taken together, the results are consistent with the model of chemokine binding established from studies of CCR1 and CCR3 in heterolo- ␣

gous transfected cells (2, 8). Specifically, MIP-1 and eotaxin bind by guest on September 26, 2021 to separate noninteracting sites, both of which overlap with binding determinants with other CC chemokines including RANTES ␣ FIGURE 8. HL-60 clone 15 cell chemotactic responses to MIP-1 and and MCP-3. eotaxin during differentiation. HL-60 clone 15 cells were cultured in the Although the relative levels of expression of CCR1 and CCR3 presence of 0.5 ␮M butyric acid for 3 days (छ)or0.5␮M butyric acid for in differentiated HL-60 clone 15 cells are reciprocal to those found 6 days plus 10 ng/ml IL-5 for days 2 to 6 (ᮀ). Cells (175,000 per replicate (छ; 190,000 per replicate (ᮀ)) were loaded into the upper compartment, in primary eosinophils (6, 8, 9, 15), the cell line is a useful sur- and the chemokines, eotaxin (A) and MIP-1␣ (B), were loaded at the rogate for eosinophils in studies requiring large numbers of cells or indicated concentrations into the lower compartment of a microchemotaxis observations over time in vitro. In this regard, our study provides chamber and incubated for4hat37°C. Closed symbols represent baseline new information about factors that regulate CCR1 and CCR3 ex- chemotaxis of cells in the absence of added chemokines. Data are from a pression, specifically butyric acid and IL-5. CCR1 and CCR3 single experiment representative of at least three experiments and are pre- mRNA levels accumulate in the HL-60 clone 15 cell line treated sented as the mean number of migrated cells Ϯ SEM of triplicate with butyric acid, which also induces the cells to terminally dif- determinations. ferentiate. Since the uninduced cells are arrested at the promyelocytic stage of myelopoiesis, CCR1 and CCR3 appear to be markers of mature eosinophils and not of precursor cells, although addi- HIV-1 Env-dependent cell fusion with HL-60 clone 15-derived tional studies with primary bone marrow-derived cells will be eosinophils needed to confirm this. CCR1 mRNA appeared much sooner than As a fourth test of CCR3 induction in butyric acid and IL-5-treated CCR3 mRNA and accumulated to much higher levels after butyric HL-60 clone 15 cells, we conducted HIV-1 Env-dependent cell acid induction. This correlated well with the ontogeny and mag- fusion assays (Fig. 9). We and others have previously shown that nitude of cell responses to MIP-1␣ and eotaxin, the selective ago- CCR3 facilitates membrane fusion and cell entry for certain HIV-1 nists for CCR1 and CCR3, respectively. CCR3 is expressed later in strains, including the prototypic dual tropic primary HIV-1 strain the HL-60 model of eosinophil maturation than other eosinophil 89.6 and the prototypic macrophage-tropic strain Ba-L (21, 22, 24, markers such as eosinophil-derived neurotoxin and eosinophil cat- 25) (H. Bazan, G. Alkhatib, C. Broder, and E. A. Berger, manu- ionic protein (26) and appears to represent one of the final steps in script in preparation). Uninduced HL-60 cells showed low levels the differentiation process. of fusion with cells expressing either Ba-L or 89.6 Env, that were Our study also provides the first evidence that IL-5 up-regulates markedly increased after treatment with butyric acid and IL-5 (Fig. CCR3 expression in differentiating eosinophils and is consistent 9). Addition of eotaxin to the coculture suppressed fusion in a with the extremely high levels of mouse CCR3 mRNA reported in ϳ dose-dependent manner, with an EC50 of 10 nM for the 89.6 Env IL-5 transgenic mice (35). Butyric acid is thought to activate genes and a threshold of ϳ100 nM for the Ba-L Env (Fig. 9, A and D). by blocking the action of deacetylases, thereby inhibiting histone The reason for the difference in blocking potency by eotaxin for binding to DNA due to increased histone acetylation (36). Thus, The Journal of Immunology 1391

FIGURE 9. Endogenous CCR3 facilitates HIV-1 Env-dependent cell fusion. Uninduced HL-60 clone 15 cells or cells cultured for 6 days after addition of 0.5 ␮M butyric acid and 4 days after addition of 10 ng/ml IL-5 (BA ϩ IL-5 induced), were infected with vaccinia virus encoding CD4 and then mixed with HeLa cells expressing the HIV-1 89.6 (A–C) or Ba-L (D) Envs. Total ␤-galactosidase activity in cell lysates is reported as a measure of cell fusion. Chemokines (A and D)orAbs Downloaded from (B and C) were added at the indicated concentrations during the3hofcell fusion; in C, anti-CCR3 and IgG2a were tested at 20 ␮g/ml, prebleed, and anti-CXCR4 was tested at 1 ␮g/ml. Unc designates effector cells express-

ing a nonfunctional HIV-1 Env de- http://www.jimmunol.org/ rived from strain IIIB; prebleed refers to preimmune serum for the rabbit anti- CXCR4 Ab; IgG2a refers to an isotype control Ab for the anti-CCR3 mAb 7B11. Data are mean Ϯ SEM of replicate samples. by guest on September 26, 2021

butyric acid may be acting at the chromosomal level to induce Although RANTES and MCP-3 are ligands for CCR3, neither CCR1 and CCR3 expression. Although CCR1 expression was not was an inhibitor of HIV-1 Env-dependent fusion. This is con- affected by IL-5 in HL-60 clone 15-derived eosinophils, it is up- sistent with their relatively lower afﬁnity and potency compared regulated by IL-2 treatment of T lymphocytes, as is CCR2 (37). with eotaxin established by studies of CCR3 in transfected IL-5 induction of CCR3 expression could potentially explain why pre-B cell lymphoma cells (8) and suggests that development of eosinophil recruitment is enhanced at sites injected with IL-5 (38). CCR3-directed antagonists of HIV-1 entry might be more suc- Previously, CCR3 was shown to function as an HIV-1 corecep- cessful starting from an eotaxin prototype than from RANTES tor (21, 22, 25, 39) (H. Bazan, G. Alkhatib, C. Broder, and E. A. or MCP-3. Berger, manuscript in preparation) that facilitates infection of mi- In summary, the HL-60 clone 15 cell line can be used as a model croglial cells in vitro (25), suggesting a potential role for CCR3 in of CCR1 and CCR3 regulation and CCR3 interactions with HIV-1 central nervous system infection by HIV-1. Our results suggest Envs. Not only can this cell line be used to follow expression of that CCR3 could also function as an HIV-1 coreceptor in maturing these receptors during development but it also can be used to study eosinophils. This is relevant since HIV-1 has been detected in bone receptor antagonists as they become available both for chemokines marrow eosinophils from certain HIV-1-infected individuals (40, and for HIV-1. The cell line should also be useful for detailed 41); however, the role of eosinophil infection in HIV-1 pathogen- studies of gene regulation and signal transduction for CCR1 esis is not known. and CCR3. In addition to increasing HIV-1 coreceptor expression on individual cells, IL-5 is an important eosinophilopoietin and, in References particular, regulates the hypereosinophilia associated with helminth infections (42). In underdeveloped world regions where 1. Murphy, P. M. 1996. Chemokine receptors: structure, function and role in mi- crobial pathogenesis. Cytokine Growth Factor Rev. 7:47. helminth infections are prevalent, these separate effects of IL-5 2. Neote, K., D. DiGregorio, J. Y. Mak, R. Horuk, and T. J. Schall. 1993. Molecular could conspire to facilitate HIV-1 transmission and to acceler- cloning, functional expression, and signaling characteristics of a C-C chemokine ate immune system deterioration and progression to AIDS in receptor. Cell 72:415. 3. Gao, J.-L., D. B. Kuhns, H. L. Tiffany, D. McDermott, X. Li, U. Francke, and infected individuals by providing the virus with a greatly ex- P. M. Murphy. 1993. Structure and functional expression of the human macro- panded target area. phage inﬂammatory protein-1␣/RANTES receptor. J. Exp. Med. 177:1421. 1392 EOSINOPHIL CHEMOKINE RECEPTORS

4. Charo, I. F., S. J. Myers, A. Herman, C. Franci, A. J. Connolly, and HIV-1 isolate that uses fusin and the ␤-chemokine receptors CKR-5, CKR-3, and S. R. Coughlin. 1994. Molecular cloning and functional expression of two mono- CKR-2b as fusion co-factors. Cell 85:1149. cyte chemoattractant protein 1 receptors reveals alternative splicing of the car- 23. Berger EA. 1997. HIV-1 fusion and tropism: the chemokine receptor connection. boxyl-terminal tails. Proc. Natl. Acad. Sci. USA 91:2752. AIDS 11(Suppl. A):S3. 5. Combadiere, C., S. K. Ahuja, J. Van Damme, H. L. Tiffany, J.-L. Gao, and 24. He, J., Y. Chen, M. Farzan, H. Choe, A. Ohagen, S. Gartner, J. Susciglio, P. M. Murphy. 1995. Monocyte chemoattractant protein-3 is a functional ligand X. Yang, W. Hofmann, W. Newman, C. R. Mackay, J. Sodroski, and D. Gabuzda. for CC chemokine receptors 1 and 2B. J. Biol. Chem. 270:29671. 1997. CCR3 and CCR5 are co-receptors for HIV-1 infection of microglia. Nature 6. Combadiere, C., S. K. Ahuja, and P. M. Murphy. 1995. Cloning and functional 385:645. expression of a human eosinophil CC chemokine receptor. J. Biol. Chem. 270: 25. Alkhatib, G., E. A. Berger, P. M. Murphy, and J. E. Pease. 1997. Determinants 16491. [Published erratum appears in 1995 J. Biol. Chem. 270:30235]. of HIV-1 coreceptor function on CC chemokine receptor 3: importance of both 7. Kitaura, M., T. Nakajima, T. Imai, S. Harada, C. Combadiere, H. L. Tiffany, extracellular and transmembrane/cytoplasmic regions. J. Biol. Chem. 272:20420. P. M. Murphy, and O. Yoshie. 1996. Molecular cloning of human eotaxin, an 26. Tiffany, H. L., F. Li, and H. F. Rosenberg. 1995. Hyperglycosylation of eosin- eosinophil-selective CC chemokine, and identification of a specific eosinophil ophil ribonucleases in a promyelocytic leukemia cell line and in differentiated eotaxin receptor, CC Chemokine Receptor 3. J. Biol. Chem. 271:7725. peripheral blood progenitor cells. J. Leukocyte Biol. 58:49. 8. Ponath, P., S. Qin, T. W. Post, J. Wang, L. Wu, N. P. Gerard, W. Newman, 27. Fischkoff, S. A. 1988. Graded increase in probability of eosinophilic differenti- C. Gerard, and C. R. Mackay. 1996. Molecular cloning and characterization of a ation of HL-60 promyelocytic leukemia cells induced by culture under alkaline human eotaxin receptor expressed selectively on eosinophils. J. Exp. Med. 183: conditions. Leuk. Res. 12:679. 2437. 9. Combadiere, C., S. K. Ahuja, and P. M. Murphy. 1995. Cloning, chromosomal 28. Plaetinck, G., J. Van der Heyden, J. Tavernier, I. Fache, T. Tuypens, localization and RNA expression of a novel human ␤ chemokine receptor-like S. A. Fischkoff, W. Fiers, and R. Devos. 1990. Characterization of interleukin 5 gene. DNA Cell Biol. 14:673. receptors on eosinophilic sublines from human promyelocytic leukemia (HL-60) 10. Power, C. A., A. Meyer, K. Nemeth, K. B. Bacon, A. J. Hoogewerf, cells. J. Exp. Med. 172:683. A. E. I. Proudfoot, and T. N. C. Wells. 1995. Molecular cloning and functional 29. Nussbaum, O., C. C. Broder, and E. A. Berger. 1994. Fusogenic mechanisms of expression of a novel CC chemokine receptor cDNA from a human basophilic enveloped-virus glycoproteins analyzed by a novel recombinant vaccinia virus- based assay quantitating cell fusion-dependent reporter gene activation. J. Virol. cell line. J. Biol. Chem. 270:19495. Downloaded from 11. Hoogewerf, A. J., D. Black, A. E. I. Proudfoot, T. N. C. Wells, and 68:5411. C. A. Power CA. 1996. Molecular cloning of murine CC CKR-4 and high affinity 30. Fuerst, T. R., E. G. Niles, F. W. Studier, and B. Moss. 1986. Eukaryotic transient- binding of chemokines to murine and human CC CKR-4. Biochem. Biophys. Res. expression system based on recombinant vaccinia virus that synthesizes bacte- Commun. 218:337. riophage T7 RNA polymerase. Proc. Natl. Acad. Sci. USA 83:8122. 12. Samson, M., O. Labbe, C. Mollereau, G. Vassart, and M. Parmentier. 1996. 31. Broder, C. C., D. S. Dimitrov, R. Blumenthal, and E. A. Berger. 1993. The block Molecular cloning and functional expression of a new human CC chemokine to HIV-1 envelope glycoprotein-mediated membrane fusion in animal cells ex- receptor gene. Biochemistry 35:3363. pressing human CD4 can be overcome by a human cell component(s). Virology 13. Combadiere, C., S. K. Ahuja, H. L. Tiffany, and P. M. Murphy. 1996. Cloning 193:483. and functional expression of CC CKR5, a human monocyte CC chemokine re- 32. Alkhatib, G., C. C. Broder, and E. A. Berger. 1996. Cell type-specific fusion http://www.jimmunol.org/ ceptor selective for MIP-1␣, MIP-1␤, and RANTES. J. Leukocyte Biol. 60:147. cofactors determine human immunodeficiency virus type 1 tropism for T-cell 14. Raport, C. J., J. Gosling, V. L. Schweickart, P. W. Gray, and I. F. Charo. 1996. lines versus primary macrophages. J. Virol. 70:5487. Molecular cloning and functional characterization of a novel human CC chemo- 33. Broder, C. C., and E. A. Berger. 1995. Fusogenic selectivity of the envelope kine receptor (CCR5) for RANTES, MIP-1␤, and MIP-1␣. J. Biol. Chem. 271: glycoprotein is a major determinant of human immunodeficiency virus type 1 17161. tropism for CD4ϩ T-cell lines vs. primary macrophages. Proc. Natl. Acad. Sci. 15. Ponath, P. D., S. Qin, I. Ringler, I. Clark-Lewis, J. Wang, N. Kassam, H. Smith, USA 92:9004. G.-Q. Jia, W. Newman, J.-C. Gutierrez-Ramos, and C. R. Mackay. 1996. Cloning 34. Van Riper, G., D. W. Nicholson, M. P. Scheid, P. A. Fischer, M. S. Springer, and of the human eosinophil chemoattractant, eotaxin: expression, receptor binding H. Rosen. 1994. Induction, characterization, and functional coupling of the high and functional properties provide a mechanism for the selective recruitment of affinity chemokine receptor for RANTES and macrophage inflammatory pro- eosinophils. J. Clin. Invest. 97:604. tein-1 ␣ upon differentiation of an eosinophilic HL-60 cell line. J. Immunol. 16. Heath, H., S. Zin, P. Rao, L. Wu, G. LaRosa, N. Kassam, P. D. Ponath, and

152:4055. by guest on September 26, 2021 C. R. Mackay. 1997. Chemokine receptor usage by human eosinophils: the im- 35. Gao, J.-L., A. I. Sen, M. Kitaura, O. Yoshie, M. E. Rothenberg, P. M. Murphy, portance of CCR3 demonstrated using an antagonistic monoclonal antibody. and A. D. Luster. 1996. Identification of a mouse eosinophil receptor for the CC J. Clin. Invest. 99:178. chemokine eotaxin. Biochem. Biophys. Res. Commun. 223:679. 17. Feng, Y., C. C. Broder, P. E. Kennedy, and E. A. Berger. 1996. HIV-1 entry 36. Klehr, D., T. Schlake, K. Maass, and J. Bode. 1992. Scaffold-attached regions co-factor: functional cDNA cloning of a seven-transmembrane G-protein coupled (SAR elements) mediate transcriptional effects due to butyrate. Biochemistry 31: receptor. Science 272:872. 3222. 18. Alkhatib, G., C. Combadiere, C. C. Broder, Y. Feng, P. E. Kennedy, 37. Loetscher, P., M. Seitz, M. Baggiolini, and B. Moser. 1996. Interleukin-2 regu- P. M. Murphy, and E. A. Berger. 1996. CC CKR5: a RANTES, MIP-1␣, MIP-1␤ lates CC chemokine receptor expression and chemotactic responsiveness in T receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 272:1955. lymphocytes. J. Exp. Med. 184:569. 19. Deng, H., R. Liu, W. Ellmeier, S. Choe, D. Unutmaz, M. Burkhart, P. DiMarzio, S. Marmon, R. E. Sutton, C. M. Hill, D. Littman, and N. R. Landau. 1996. 38. Collins, P. D., S. Marleau, D. A. Griffiths-Johnson, P. J. Jose, and T. J. Williams. Identification of a major co-receptor for primary isolates of HIV-1. Nature 381: 1995. Cooperation between interleukin-5 and the chemokine eotaxin to induce 661. eosinophil accumulation in vivo. J. Exp. Med. 182:1169. 20. Dragic, T., V. Litwin, G. P. Allaway, S. R. Martin, Y. Huang, K. A. Nagashima, 39. Dittmar M. T., A. McKnight, G. Simmons, P. R. Clapham, and R. A. Weiss. C. Cayanan, P. J. Maddon, R. A. Koup, J. P. Moore, and W. A. Paxton. 1996. 1997. HIV-1 tropism and co-receptor use. Nature 385:495. HIV-1 entry into CD4ϩ cells is mediated by the chemokine receptor CC CKR-5. 40. Freedman, A., F. Gibson, S. Fleming, C. Spry, and G. Griffin. 1991. Human Nature 381:667. immunodeficiency virus infection of eosinophils in human bone marrow cultures. 21. Choe, H., M. Farzan, Y. Sun, N. Sullivan, B. J. Rollins, P. D. Ponath, L. Wu, J. Exp. Med. 174:1661. C. R. Mackay, G. LaRosa, W. Newman, N. P. Gerard, C. Gerard, and J. Sodroski. 41. Weller. P., W. Marshall, D. Lucey, T. Rand, A. Dvorak, and R. Finberg. 1995. 1996. The ␤-chemokine receptors CCR3 and CCR5 facilitate infection by pri- Infection, apoptosis and killing of mature human eosinophils by human immu- mary HIV isolates. Cell 85:1135. nodeficiency virus-1. Am. J. Respir. Cell Mol. Biol. 10:610. 22. Doranz, B. J., J. Rucker, Y. Yi, R. J. Smyth, M. Samson, S. C. Peiper, 42. Weller, P. F. 1991. The immunobiology of eosinophils. N. Engl. J. Med. 324: M. Parmentier, R. G. Collman, and R. W. Doms. 1996. A dual tropic primary 1110.