CXCR2 Related Chemokine Receptors, CXCR1 and Regulation

Actin Filaments Are Involved in the Regulation of Trafficking of Two Closely Related Chemokine Receptors, CXCR1 and CXCR2 This information is current as of September 23, 2021. Alon Zaslaver, Rotem Feniger-Barish and Adit Ben-Baruch J Immunol 2001; 166:1272-1284; ; doi: 10.4049/jimmunol.166.2.1272 http://www.jimmunol.org/content/166/2/1272 Downloaded from

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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 © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Actin Filaments Are Involved in the Regulation of Trafﬁcking of Two Closely Related Chemokine Receptors, CXCR1 and CXCR2

Alon Zaslaver, Rotem Feniger-Barish, and Adit Ben-Baruch

The ligand-induced internalization and recycling of chemokine receptors play a significant role in their regulation. In this study, we analyzed the involvement of actin filaments and of microtubules in the control of ligand-induced internalization and recycling of CXC chemokine receptor (CXCR)1 and CXCR2, two closely related G protein-coupled receptors that mediate ELR-expressing CXC chemokine-induced cellular responses. Nocodazole, a microtubule-disrupting agent, did not affect the IL-8-induced reduction in cell surface expression of CXCR1 and CXCR2, nor did it affect the recycling of these receptors following ligand removal and cell recovery at 37°C. In contrast, cytochalasin D, an actin filament depolymerizing agent, promoted the IL-8-induced reduction Downloaded from in cell surface expression of both CXCR1 and CXCR2. Cytochalasin D significantly inhibited the recycling of both CXCR1 and CXCR2 following IL-8-induced internalization, the inhibition being more pronounced for CXCR2 than for CXCR1. Potent inhibition of recycling was observed also when internalization of CXCR2 was induced by another ELR-expressing CXC chemokine, granulocyte chemotactic protein-2. By the use of carboxyl terminus-truncated CXCR1 and CXCR2 it was observed that the carboxyl terminus domains of CXCR1 and CXCR2 were partially involved in the regulation of the actin-mediated process of

receptor recycling. The cytochalasin D-mediated inhibition of CXCR2 recycling had a functional relevance because it impaired the http://www.jimmunol.org/ ability of CXCR2-expressing cells to mediate cellular responses. These results suggest that actin filaments, but not microtubules, are involved in the regulation of the intracellular trafficking of CXCR1 and CXCR2, and that actin filaments may be required to enable cellular resensitization following a desensitized refractory period. The Journal of Immunology, 2001, 166: 1272–1284.

hemokines are small chemotactic cytokines that mediate which consist of the major cellular infiltrate in the course of acute inflammatory processes, hematopoiesis and angiogene- inflammation. Several investigations demonstrated that neutrophil C sis, and play a crucial role in AIDS pathogenesis (1–4). responses could be regulated and desensitized in processes involv- Chemokine-mediated responses are induced by the activation of ing chemokine receptor internalization (12–14). Chemokine recep- specific G protein-coupled receptors (GPCR),2 which are ex- tor internalization and recycling were also shown to be involved in by guest on September 23, 2021 pressed on target cells (5). In similarity to other members of the the regulation of HIV-1 infectivity, as illustrated by the ability of GPCR superfamily, the ability of chemokine receptors to transmit the RANTES antagonist aminooxypentane-RANTES to induce a intracellular signals may be rapidly attenuated, and is tightly reg- highly potent inhibition of HIV-1 entry to target cells. The ami- ulated by different mechanisms, including receptor phosphoryla- nooxypentane-RANTES-mediated inhibition was demonstrated to tion-dependent G protein-uncoupling and receptor internalization be a direct result of its ability to induce elevated levels of CCR5 (6–9). Agonist-induced internalization of GPCR depletes the internalization, and to prevent its recycling back to the plasma mem- plasma membrane of receptors for the agonist and, therefore, may brane, thereby removing a key element of the fusion complex (15). contribute to the desensitization of the functions mediated through Recent interest in the mechanisms regulating ligand-induced these receptors (6–9). Moreover, if the ligand that induced recep- GPCR internalization has led to observations suggesting that some tor internalization is removed, the receptors may be dephosphory- GPCR undergo endocytosis via clathrin-coated pits, in a process lated, recycled back to the plasma membrane and resensitized upon that involves the binding of arrestins to the receptors (8, 9, 16, 17). a subsequent exposure to the same ligand (9–11). Other factors that are potential regulators of the internalization and Chemokine-induced receptor internalization and recycling were recycling processes of GPCR are yet to be defined. In that regard, shown to play a significant role in the regulation of inflammatory elucidation of the involvement of actin filaments and/or microtu- processes, as indicated for example by studies on neutrophils, bules in ligand-induced GPCR intracellular trafficking is of major importance. The plasma membrane is functionally integrated with the cell “cortex” that consists of actin-based cytoskeleton (18). Department of Cell Research and Immunology, George S. Wise Faculty of Life Sci- Therefore, the trafficking of vesicles at the plasma membrane may ences, Tel-Aviv University, Tel-Aviv, Israel necessitate the active rearrangement of actin filaments, which may Received for publication April 10, 2000. Accepted for publication October 20, 2000. then be followed by actin-assisted vesicle budding and fusion at The costs of publication of this article were defrayed in part by the payment of page the plasma membrane. Moreover, microtubules may also play a charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. direct role in trafficking of receptors, and were shown to facilitate 1 Address correspondence and reprint requests to Dr. Adit Ben-Baruch, Department of transport along the trans-golgi network plasma membrane pathway Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel-Aviv (19–23). University, Tel-Aviv 69978, Israel. E-mail address: [email protected] Although of potentially major importance for the regulation of 2 Abbreviations used in this paper: GPCR, G protein-coupled receptors; CSB, cell ligand-induced receptor intracellular trafficking, the involvement sorter buffer; CXCR, CXC chemokine receptor; ELRϩ-CXC, ELR-expressing CXC; GCP-2, granulocyte chemotactic protein-2; HEK 293, human embryonal kidney 293; of cytoskeleton elements in GPCR trafficking was very minimally PTx, pertussis toxin; WT, wild-type. studied so far. Of high significance is the fact that the contribution

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 1273 of such elements to the regulation of trafficking of chemokine re- with high affinity (31, 38). Control transfections were performed with the ceptors was not addressed in any manner. In that respect, our study vector (pRc/CMV) alone, and the resulting cells did not specifically bind is focused on the elucidation of the role of cytoskeleton elements IL-8, GCP-2, or Abs specific for human CXCR1 or CXCR2 (29, 31, 38, 39). Parental and stable CXCR1-expressing 300-19 pre B cells (kindly do- in the ligand-induced internalization and recycling processes of nated by Dr. M. Wolf, Theodor Kocher Institute, University of Bern, Bern, CXC chemokine receptor (CXCR)1 and CXCR2, two closely re- Switzerland) were grown in RPMI 1640 medium, supplemented with 10% lated receptors that mediate the migration of neutrophils to inflam- FCS, 100 U/ml penicillin, 100 ␮g/ml streptomycin, 250 ng/ml amphoter- matory sites in response to ELR-expressing CXC (ELRϩ-CXC) icin, 2 mM L-glutamine, 1ϫ non essential amino acids (all purchased from Biological Industries, Beit Haemek, Israel), and 0.05% mM 2-ME (Sigma, chemokines (1, 5, 24). The use of CXCR1 and CXCR2 allowed us Rehovot, Israel). The DNA for CXCR1 transfection was expressed in a to compare between two chemokine receptors that have similar SR-␣-puro vector. Puromycin (1 ␮g/ml; Sigma) was used for selection. No general characteristics, but nevertheless are differentially regulated expression of human CXCR1 or CXCR2 was detected on the parental at multiple levels, including their internalization properties 300-19 cells, as determined by monoclonal mouse anti-CXCR1 or anti- (25–37). CXCR2 Abs (R&D Systems, Minneapolis, MN). The similarity between CXCR1 and CXCR2 is illustrated by the Preparation of human neutrophils fact that both receptors bind with high affinity the ELRϩ-CXC chemokine IL-8, and mediate potent cellular migration in response The preparation of human neutrophils was performed as previously described (40). Briefly, neutrophils (Ն94% purity) were separated by Ficoll- to this chemokine. However, the two receptors diverge in their Hypaque density gradient centrifugation followed by dextran sedimenta- ϩ ability to bind other members of the ELR -CXC subfamily of tion and hypotonic lysis of remaining erythrocytes. Because NH4Cl, a chemokines, in the intracellular signals they transduce and in the lysosomotropic agent, may inhibit receptor recycling on human neutrophils mechanisms that regulate their desensitization and internalization (33), the neutrophils used in these experiments were prepared without Downloaded from NH Cl. following the binding of high concentrations of ligands (25–37). 4 Based on their ability to undergo internalization and recycling, it Determination of receptor down-modulation by FACS analysis was suggested that CXCR1 is involved in mediating IL-8-induced Stable receptor-expressing HEK 293 cells were split, and a day later were chemotaxis at the site of inflammation, where the concentration of taken for the experiments. All the experimental steps were performed while IL-8 is high, whereas CXCR2 has an active role in the initiation the cells were in suspension. The analysis of receptor down-modulation

phase of neutrophil migration, distant from the site of inflamma- was performed as previously described (31). Briefly, aliquots of stable http://www.jimmunol.org/ tion, where the concentration of IL-8 is at the picomolar level (33). HEK 293 transfectants were removed and supplemented with 1000 ng/ml IL-8 (Dainippon, Japan) diluted in BSA medium (RPMI 1640 medium In search for further elucidation of the mechanisms regulating containing 1% BSA and 25 mM HEPES), while no IL-8 was added to CXCR1 and CXCR2 internalization, our previous studies demon- control tubes. The cells were incubated at 37°C for 60 or 90 min (see strated that potent CXCR1 internalization is induced by IL-8, but Results), washed in cell sorter buffer (CSB: PBS containing 1% FCS, ϩ not by other ELR -CXC chemokines, such as granulocyte che- 0.02% NaN3, and 25 mM HEPES) and incubated at 4°C with monoclonal motactic protein-2 (GCP-2) and neutrophil-activating protein-2. In mouse anti-CXCR1 or anti-CXCR2 Abs (R&D Systems). Baseline staining was obtained by adding CSB to the cells instead of anti-CXCR1 or anti- contrast, CXCR2 undergoes potent internalization in response to CXCR2 Abs. Following incubation and washings, the cells were incubated both IL-8 and GCP-2, but not to neutrophil-activating protein-2 with FITC-conjugated goat anti-mouse IgG Abs (Jackson ImmunoRe- (31, 32). search Laboratories, West Grove, PA), washed, and resuspended. The ef- by guest on September 23, 2021 In the present study we investigated by pharmacological means fects of cytoskeleton-disrupting drugs were determined by preincubating the cells with 10 ␮M nocodazole or 1 ␮M cytochalasin D (Sigma) for 30 the involvement of microtubules and of actin filaments in the reg- min at 37°C, as well as in the course of exposure to IL-8. Because both ulation of the cell surface expression of CXCR1 and CXCR2, fol- reagents were resuspended in DMSO and used in a 1:1000 final dilution, a lowing ELRϩ-CXC chemokine-induced internalization of these control of 1:1000 dilution of DMSO was also included. Incubations with receptors. Our study provides the first evidence for the involve- 100 ng/ml and 500 ng/ml pertussis toxin (PTx) were performed by incu- ment of actin filaments, but not microtubules, in the trafficking of bating the cells with the drug for 120 min at 37°C, followed by removal of the drug. In preliminary experiments, it was observed that nocodazole, chemokine receptors. Functionally intact actin filaments were cytochalasin D, and PTx did not have a notable effect on the basal expres- demonstrated to be required for potent recycling processes of both sion of CXCR1 and CXCR2. Analysis of the cells following nocodazole, CXCR1 and CXCR2. The critical role of actin filaments in the cytochalasin D, and PTx treatments indicated that cell viability was not regulation of receptor recycling was further substantiated by func- affected by these compounds. FACSort (Becton Dickinson, San Jose, CA), was used to analyze 5000 live cell events. Percent reduction in cell surface tional assays, suggesting that following a desensitized refractory expression was calculated from the mean channel fluorescence values of period, the resensitization of CXCR1 and CXCR2-expressing cells cells treated with ligand at 37°C vs the mean channel fluorescence of cells requires functionally intact actin filaments that will enable potent not treated with ligand, under similar conditions. Values of p were calcu- receptor re-expression, as well as migratory responses to occur. lated by the Student t test. Determination of receptor re-expression on the plasma Materials and Methods membrane DNAs for human CXCR1 and human CXCR2 The procedure used to determine receptor re-expression in HEK 293 cells Wild-type (WT) CXCR1, ⌬19 CXCR1, WT CXCR2 and ⌬20 CXCR2 was similar to that used to evaluate receptor down-modulation, only that DNAs were generated using PCR, shuttled into the expression vector pRc/ matched samples were allowed to undergo a receptor recovery process that CMV (Invitrogen, San Diego, CA), and subjected to full-length sequencing was performed as previously described (31). Briefly, internalization was as previously described (31, 38). The primary amino acid sequences of the induced by a 60- or 90-min exposure (see Results) to 1000 ng/ml IL-8, or predicated carboxyl terminus domains of the receptors are shown in Table II. by a 2-h exposure to 1000 ng/ml GCP-2. To allow receptor recovery, the stable receptor transfectants were washed once, resuspended in BSA me- Cell cultures, transfections, and characterization of receptor dium, and incubated for 90 min at 37°C in BSA medium. Following the expression by transfected cells incubation, the cells were washed and labeled at 4°C with anti-CXCR1 or anti-CXCR2 Abs as described above. The effects of cytoskeleton-disrupt- Human embryonal kidney 293 (HEK 293) cells (kindly donated by Dr. P. ing drugs were determined by incubating the cells at the time of cell re- Gray, ICOS, Bothell, WA) were grown and stably transfected as previously covery (but not of induction of internalization) with 10 ␮M nocodazole or described (31, 38). FACS analyses showed that a high percentage (over 1 ␮M cytochalasin D. Because both reagents were resuspended in DMSO 85%) of the transfected cells (WT CXCR1, ⌬19 CXCR1, WT CXCR2, and used in a 1:1000 final dilution, a control of 1:1000 dilution of DMSO ⌬20 CXCR2) expressed the receptor on the cell surface. All the transfected was also included. Analysis of the cells following nocodazole or cytocha- cells (WT CXCR1, ⌬19 CXCR1, WT CXCR2, ⌬20 CXCR2) bound IL-8 lasin D treatments indicated that cell viability was not affected by these 1274 ACTIN FILAMENTS IN TRAFFICKING OF CXCR1 AND CXCR2 compounds. Similar analysis of receptor re-expression in neutrophils was Results performed by a 10-min exposure to 1000 ng/ml IL-8 at 37°C, followed The effects of nocodazole and cytochalasin D on IL-8-induced by removal of the ligand and recovery of the cells for 40 min at 37°C. FACSort was used to analyze 5000 live cell events. Mean fluorescence down-modulation of CXCR1 and CXCR2 values of cells that were not exposed to IL-8, and of cells exposed to IL-8 To determine the effect of cytoskeleton-disrupting agents on the and allowed to undergo recovery, were used to determine the level of cell surface expression of CXCR1 and CXCR2 in the course of receptor re-expression. The percent of inhibition of receptor recovery was ligand-induced receptor internalization, we used CXCR1- or calculated for each experiment as follows: the percent inhibition ϭ 1 Ϫ (the percent of total expression in cells that underwent recovery in the CXCR2-expressing HEK 293 cells. The analysis of the regulation presence of cytochalasin D Ϫ the percent of total expression in cells that of CXCR1 and CXCR2 in neutrophils may be impeded by the fact underwent internalization)/(the percent of total expression in cells that un- that both receptors are expressed together on these cells (27, 28, derwent recovery in the presence of DMSO Ϫ the percent of total expres- 33, 34, 39), providing a possibility for the existence of intracellular ϫ sion in cells that underwent internalization) 100. Values of p were cal- interactions and cross-talk between the two receptors. Unlike neu- culated by the Student t test. trophils, the cells of our system allow us to dissociate the mechanisms regulating each of the receptors, and to elucidate by using Confocal analyses of receptor down-modulation receptor mutants the mechanisms that are involved in the regulation of receptor trafficking by cytoskeleton elements. This well- Stable CXCR2-expressing cells were split, and a day later, were taken for characterized system was shown to resemble neutrophils in terms the experiments. All the experimental steps were performed while the cells were in suspension. Aliquots of cells were either incubated in the presence, of ligand binding affinity, activation, homologous desensitization,

or in the absence of 1000 ng/ml IL-8 for 90 min at 37°C. The cells that certain characteristics of internalization of CXCR1 and CXCR2 Downloaded from were exposed to IL-8 were divided into two subgroups before exposure to and induction of migratory responses (29–32, 38, 41–44). In sim- IL-8: the cells of one group were exposed to 1 ␮M cytochalasin D for 30 ilarity to neutrophils, CXCR1- and CXCR2-expressing HEK 293 min at 37°C before, and in the course of incubation with IL-8, while the cells were shown to be activated in response to low concentrations cells of the other group were not exposed to cytochalasin D. All the pro- cedures from this stage forward were performed at room temperature. The of chemokines (10–100 ng/ml), and to undergo desensitization by cells were rinsed in PBS, fixed with 4% paraformaldehyde for 15 min and exposure to high concentrations of chemokines (500–1000 ng/ml) centrifuged onto 0.5% gelatin-coated slides. Permeabilized cells were ob- (29–32, 38, 42, 43). Furthermore, our previous studies have shown http://www.jimmunol.org/ tained by incubating the cells with 0.2% Triton X-100 (Sigma) for 30 min, by FACS analysis that the exposure of CXCR1- and CXCR2-ex- followed by blocking with 3% goat serum (Biological Industries), 0.25% pressing HEK 293 cells to high concentrations of chemokines gelatin (Sigma), and 0.15% saponin (Sigma) in PBS, for 60 min. After (CXCR1, 1000 ng/ml IL-8; CXCR2, 1000 ng/ml IL-8, 1000 ng/ml rinsing the cells in washing buffer (0.25% gelatin and 0.15% saponin in PBS), the cells were stained with polyclonal rabbit anti human CXCR2 Abs GCP-2), resulted in a prominent reduction in receptor cell surface (0.5 ␮g/60 ␮l; Santa Cruz Biotechnology, Santa Cruz, CA) for 90 min. expression (31, 32). Similar experiments that were performed at Following additional rinsing in washing buffer, the cells were stained with 4°C indicated that the preincubation of the cells with chemokines Rhodamine-conjugated goat anti-rabbit IgG (3.5 ␮g/60 ␮l, Jackson Immu- did not prevent the Abs from binding to cell surface-expressed noResearch Laboratories) and Alexa Fluor 488 phalloidin (Molecular CXCR1 and CXCR2. Confocal analyses performed under similar Probes, Eugene, OR) for 30 min. Following additional washings, stained conditions indicated that potent chemokine-induced receptor inter- by guest on September 23, 2021 cells were analyzed using a Zeiss confocal laser scanning microscope (Oberkochen, Germany). Zeiss LSM 410 invert was equipped with a 25 nalization gave rise to the observed reduction in cell surface ex- mW krypton-argon laser (488 and 568 maximum lines) and 10 mW HeNe pression of both receptors (Ref. 32 and data not shown). laser (633 maximum line). A 40ϫ NA/1.2 C-apochromat water-immersion The analysis of the effects of nocodazole or cytochalasin D was lens (Axiovert 135 M, Zeiss) was used for all imaging. performed by incubating WT CXCR1- or WT CXCR2-expressing cells with the drugs for 30 min at 37°C, followed by exposure to IL-8 for 90 min at 37°C. A highly notable IL-8-induced reduction Chemotaxis assays in the cell surface expression of CXCR1 and CXCR2 was ob- The migration of HEK 293 cells was assessed by a 48-well microchemo- served in the absence of nocodazole or of cytochalasin D (Fig. 1, taxis chamber technique as previously described (38). Briefly, the lower A and B). The extent of IL-8-induced CXCR1 or CXCR2 down- compartment of the chamber was loaded with aliquots of BSA medium, modulation following the treatment with the drugs was compared 100 ng/ml IL-8 (for CXCR1), or 50 ng/ml IL-8 (for CXCR2) diluted in BSA medium, while the upper compartment of the chamber was loaded with the extent of reduction in cell surface expression in their with cells (resuspended in BSA medium). The two compartments were absence, or in the presence of DMSO, which was the solubilizer of separated by a 10-␮m pore-sized polycarbonate polyvinylpyrrolidone the drugs and did not affect receptor down-modulation on its own coated with 50 ␮g/ml rat collagen type I (Collaborative Biomedical Prod- (Fig. 1, A and B). In preliminary experiments it was observed that ucts, Bedford, MA). Following 5–6 h of incubation at 37°C the filter was nocodazole and cytochalasin D did not have a notable effect on the removed, fixed and stained. The cells that were studied included: 1) cells basal expression of CXCR1 and CXCR2. that were not treated at all; 2) cells in which receptor down-modulation was induced by exposure to 1000 ng/ml IL-8; 3) cells in which receptor down- When receptor internalization was induced by exposure to 1000 modulation was induced by exposure to 1000 ng/ml IL-8, and recovery was ng/ml IL-8, nocodazole did not affect the ability of IL-8 to induce allowed (after removal of the ligand) for 90 min at 37°C in the absence of reduction in cell surface expression of either CXCR1 or CXCR2 cytochalasin D; 4) cells in which receptor down-modulation was induced (Fig. 1, A and B). However, it is important to note that the 30-min by exposure to 1000 ng/ml IL-8, and recovery was allowed (after removal pre-exposure to nocodazole was found to profoundly disrupt the of the ligand) for 90 min at 37°C in the presence of 1 ␮M cytochalasin D; and 5) cells that were incubated only with 1 ␮M cytochalasin D for 90 min microtubule cytoskeleton as was indicated by confocal analysis at 37°C. All the cells were washed before loading into the chemotaxis using Abs directed against ␤-tubulin (data not shown). chamber. In separate experiments, the effects of 100 ng/ml and 500 ng/ml Analysis of the effects of cytochalasin D on reduction in the cell PTx on migration were analyzed by the preincubation of the cells with the surface expression of CXCR1 or CXCR2, induced by incubation drug for 120 min at 37°C, followed by the removal of the drug, and loading with 1000 ng/ml IL-8 for 90 min, demonstrated that the drug in- of the cells in the chemotaxis chamber. Analysis of the cells following duced a moderate, but significant, effect on the cell surface ex- cytochalasin D and PTx treatments indicated that cell viability was not affected by the compounds. The baseline migration to BSA medium of the pression of both receptors. This was manifested by the fact that different types of cells was similar, and was subtracted from the response following the treatment with cytochalasin D, an increased reduc- to IL-8. Values of p were calculated by the Student t test. tion in cell surface expression of both CXCR1 and CXCR2 was The Journal of Immunology 1275

To visualize the process of receptor internalization in the presence of cytochalasin D, a confocal analysis was performed (the experiments were performed while the cells were in suspension). As shown in Fig. 2A, when CXCR2-expressing cells were not exposed to IL-8 and were not treated by the drug, a membranous expression of CXCR2 was noted, and an intact organization of actin ﬁlaments was observed. Following induction of internalization by 1000 ng/ml IL-8 (90 min, 37°C) in the absence of cytochalasin D, a clearly visible translocation of the receptors to intracellular cytoplasmatic regions of the cells was observed, while the actin network remained intact (Fig. 2B). A highly prominent intracellular localization of the receptors was observed also when IL-8-induced receptor internalization was performed in the presence of cytochalasin D, accompanied by a pronounced disruption of the actin cytoskeleton (Fig. 2C). These results provide evidence for notable receptor internalization occurring while actin depolymerization was induced by cytochalasin D.

The effects of nocodazole and cytochalasin D on the process of Downloaded from CXCR1 and CXCR2 re-expression on the cell membrane Studies of neutrophils, as well as our previous observations on our WT CXCR1- and WT CXCR2-transfected cells, indicated that if the internalization-inducing ligand was removed and the cells were allowed to recover at 37°C, the receptors were re-expressed on the

cell membrane (31–33, 41, 42). Complete receptor recovery, es- http://www.jimmunol.org/ pecially for CXCR2, required prolonged incubation at 37°C (for 6 h), whereas partial re-expression was noticed following 90 min of cell recovery (31, 32). Further analysis demonstrated that receptor re-expression following this shorter incubation period (90 min) was insensitive to cycloheximide, indicating that receptor recovery under these conditions did not result of de novo receptor synthesis (31, 32). Moreover, confocal analysis demonstrated that the receptors are not stored in pre-existing granules in these cells (Ref. 32 and data not shown). On the whole, these observations by guest on September 23, 2021 suggest that CXCR1 and CXCR2 re-expression on the cell surface following 90 min of recovery was the direct result of receptor recycling back to the plasma membrane. FIGURE 1. The effects of nocodazole and cytochalasin D on WT Determination of the effects of cytoskeleton-disrupting agents CXCR1 and WT CXCR2 down-modulation. HEK 293 cells were exposed on WT CXCR1 and WT CXCR2 recycling was performed by in- to nocodazole or cytochalasin D as described in Materials and Methods. Matched samples were incubated in the same conditions without exposure ducing receptor internalization by 1000 ng/ml IL-8 for 60 min at to the drugs, or in the presence of DMSO. Following these treatments, the 37°C (in the absence of nocodazole or cytochalasin D), and by cells were incubated with IL-8. Another group of cells was not exposed to allowing cell recovery for 90 min at 37°C, in the presence or in the any treatment. The cells were subjected to FACS analysis as described in absence of nocodazole or cytochalasin D. This time point of cell Materials and Methods. The data are presented as the percent of reduction recovery was chosen due to the fact that, at this time, receptor in cell surface expression as compared with cells that were not exposed to re-expression is the result of receptor recycling (as mentioned for any treatment, calculated as described in Materials and Methods. A, experiments with cycloheximide) and because in earlier time Ͻ ءء CXCR1-expressing cells. , p 0.002 for treatment with cytochalasin D points the level of receptor recovery is not high and stable enough ϭ ء vs DMSO treatment. B, CXCR2-expressing cells. , p 0.026 for treat- to allow for detection of relatively subtle effects of the cytoskel- ment with cytochalasin D vs DMSO treatment. Cyto-D, Cytochalasin D; eton-disrupting agents. The levels of receptor recycling in the pres- Nocod., nocodazole. Each value represents the mean Ϯ SD of three independent experiments. ence of the drugs were compared with the extent of receptor recycling in their absence or in the presence of DMSO, which was the solubilizer of the drugs and did not affect receptor recycling on observed (Fig. 1, A and B). Cytochalasin D enhanced the ability of its own (Figs. 3 and 4). IL-8 to induce reduction in cell surface expression of CXCR1 and As shown in Fig. 3A, disruption of the microtubule cytoskeleton of CXCR2 by 28.1% and by 29.3%, respectively, as compared by nocodazole did not affect the recycling process of WT CXCR1. with DMSO treatment (CXCR1, p Ͻ 0.002; CXCR2, p ϭ 0.026). In contrast, depolymerization of actin ﬁlaments by cytochalasin D The ability of cytochalasin D to promote IL-8-induced reduction in resulted in a marked, but partial, inhibition of the recycling of this cell surface expression of both receptors was time-dependent, be- receptor (Fig. 3, A and B). When compared with recycling levels cause the drug had no effect when internalization was induced by induced in the presence of DMSO alone, a notable inhibition of 60 min-exposure to IL-8 (data not shown). Altogether, these re- 51.7% was observed for WT CXCR1 recycling ( p ϭ 0.005). sults suggest that actin ﬁlaments, but not microtubules, are in- Analysis of the involvement of cytoskeleton elements in the volved in processes regulating the cell surface expression of both recovery of WT CXCR2 expression indicated that in similarity to CXCR1 and CXCR2 following IL-8-induced receptor WT CXCR1, microtubules were not involved in the recycling of internalization. CXCR2, as indicated by the inability of nocodazole to affect the 1276 ACTIN FILAMENTS IN TRAFFICKING OF CXCR1 AND CXCR2

FIGURE 2. Confocal analysis of WT CXCR2 internalization in the presence or in the absence of cytochalasin D. CXCR2-expressing HEK 293 cells were incubated in the absence or in the presence of IL-8. The incubation with IL-8 was performed either following exposure to cytochalasin D, or in its absence, as described in Materials and Methods. The cells were washed and stained with rabbit anti- CXCR2 Abs, followed by staining with rhodamine- conjugated goat anti-rabbit IgG, combined with Al- exa Fluor 488 phalloidin. The cells were then subjected to confocal analysis as described in Ma- terials and Methods. In all the pictures shown, the red color represents the expression of CXCR2 and the green color represents the distribution of actin ﬁlaments. A, Cells not exposed to cytochalasin D or to IL-8. B, Cells not exposed to cytochalasin D but incubated with IL-8. C, Cells exposed to cytochalasin D and incubated with IL-8. A representative experiment of three independent experiments performed is shown. The reference bar in the lower left Downloaded from corner represents 10 ␮m. recycling process (Fig. 4A). In contrast, a pronounced inhibition of ng/ml IL-8) ( p Ͻ 0.001). Similar effects of the drug were observed CXCR2 recycling was induced by cytochalasin D (Fig. 4, A and in 500 ng/ml (data not shown). In contrast to its effects on IL-8-

B), as indicated by the fact that the treatment with this drug abol- induced migratory responses, PTx did not interfere with the inter- http://www.jimmunol.org/ ished CXCR2 recycling (98.8% inhibition) as compared with cells nalization of CXCR1 and CXCR2, induced by 60-min exposure to treated with DMSO ( p ϭ 0.02; Fig. 4A). On the whole, the results 1000 ng/ml of the chemokine (Fig. 6B). The inability of the drug shown in Figs. 3 and 4 provide evidence for the signiﬁcant in- to modify the level of internalization was observed not only when volvement of actin ﬁlaments in processes that mediate CXCR1 and it was used in the concentration of 100 ng/ml (data not shown), but CXCR2 recycling to the cell surface. The extent of inhibition was also in the dose of 500 ng/ml (Fig. 6B). more prominent for CXCR2 recycling as compared with CXCR1. Our data on the effects of PTx activity suggest that the primary

The more rapid kinetics of CXCR1 recycling as compared with events of G␣i coupling do not regulate the process of CXCR1, or CXCR2 recycling (Fig. 3A vs Fig. 4A) suggest that the lower sen- of CXCR2 internalization, and hence probably do not contribute to sitivity of CXCR1 recycling to cytochalasin D results from the fact the differences observed between CXCR1 and CXCR2 with re- by guest on September 23, 2021 that recycling events of CXCR1 have taken place before the ac- spect to their sensitivity to cytochalasin D in the course of the more tivity of drug has come into a complete effect. delayed processes of receptor recycling. Because the fine control of neutrophil responses results from the coordinated activity of several ELRϩ-CXC chemokines (25, 26, The effects of cytochalasin D on CXCR1 and CXCR2 re- 28–32, 35, 36), we elucidated the role of actin filaments in the expression in cells of a hematopoietic origin regulation of CXCR2 trafficking in response to GCP-2, the only CXCR1 and CXCR2 are known to be expressed on cells of a other ELRϩ-CXC chemokine that was shown to induce potent hematopoietic origin, primarily on neutrophils (27, 28, 33, 34, 39). internalization of this receptor. As shown in Fig. 5, CXCR2 recy- Although the expression of both CXCR1 and CXCR2 on neutro- cling was inhibited not only when the internalization-inducing li- phils may complicate the analysis of the regulation of these recep- gand was IL-8, but also when internalization was induced by 1000 tors, similar experiments to those performed on HEK 293 cells ng/ml GCP-2 for2hat37°C, and cell recovery was allowed for 90 were performed on human neutrophils, to determine the effects of min at 37°C (82.6% inhibition; p ϭ 0.028), suggesting that actin cytochalasin D on CXCR1 and CXCR2 re-expression. The anal- filaments are involved in the trafficking of CXCR2 in a chemokine- ysis was performed on healthy independent donors. The basal ex- nonspecific manner, provided that the chemokine is able to induce pression levels of CXCR1 and CXCR2 in all donors were highly CXCR2 internalization and recycling. similar to the reported expression levels of these receptors on neu- The quantitative differences observed between CXCR1 and trophils (33), demonstrating higher expression levels of CXCR1 CXCR2 in their sensitivity to cytochalasin D effects in the course than CXCR2 (data not shown). Of note is the fact that the kinetics of the recycling process may be the result of differences in signal- of CXCR1 and CXCR2 internalization in neutrophils is more rapid ing events that are initiated by the receptors upon the first encoun- than in HEK 293 cells, and their recycling is quickly reaching a ter with the chemokine, at the stage of receptor internalization. To plateau level in neutrophils (33). Moreover, CXCR1 was shown to determine whether this may indeed be the case, the role of G pro- undergo a more efficient recycling in human neutrophils than tein coupling in the regulation of the internalization process was CXCR2, the later reaching only a maximal level of 40% recovery evaluated by the use of PTx, an effective inhibitor of signals trans- of receptor expression (33). mitted by members of the G␣i subclass of G proteins, which were To determine the effects of cytochalasin D on CXCR1 and demonstrated to be key mediators of IL-8-induced responses (45, 46). CXCR2 re-expression, the experimental conditions in neutrophils

First, the ability of PTx to uncouple the binding of G␣i was were set so that internalization and recycling levels would approx- determined by evaluating its ability to inhibit IL-8-induced migra- imately accommodate with those used in HEK 293 cells. To this tion of CXCR1- and CXCR2-expressing cells. As shown in Fig. end, neutrophils were exposed to 1000 ng/ml IL-8 for 10 min at 6A, 100 ng/ml PTx completely abolished IL-8-induced chemotac- 37°C, followed by removal of the ligand and cell recovery for 40 tic responses of both CXCR1 (100 ng/ml IL-8) and CXCR2 (50 min at 37°C. Cell recovery was performed in the presence of The Journal of Immunology 1277

DMSO, 1 ␮M cytochalasin D, or in their absence. As in HEK 293 cells, DMSO did not affect the re-expression of CXCR1 and CXCR2 following removal of IL-8 (data not shown). The exposure of neutrophils to IL-8 resulted in an average of 62.5% reduction in CXCR1 cell surface expression (Table I). The expression of CXCR1 was quickly restored following a 40-min recovery in the presence of DMSO, and reached an average of 51.8% of the basal expression level before induction of internalization (Table I). In each of the neutrophil samples analyzed, cytochalasin D did not affect the recycling process of CXCR1 (Table I). The inability of cytochalasin D to affect CXCR1 recycling may very well be affected by the rapid kinetics of the recycling process, and does not necessarily imply that the recycling of this receptor is not regulated by actin ﬁlaments (see Discussion). Similar experiments performed for CXCR2 internalization resulted in an average of 66% reduction in cell surface expression following exposure to IL-8 (Table I). In agreement with the previously observed low ability of CXCR2 to undergo recycling, no re-expression of

CXCR2 was observed following removal of the ligand in all donors. Downloaded from Being such, determination of the effects of cytochalasin D could not yield any conclusive results regarding the role of actin ﬁlaments in CXCR2 recycling, as was indeed the case (Table I). To nevertheless determine whether the effects of cytochalasin D on IL-8 receptors recycling that were observed in HEK 293 cells

are relevant to hematopoietic cells, similar analysis was performed http://www.jimmunol.org/ on 300-19 pre B cells that were transfected to stably express CXCR1. Furthermore, the use of this system allowed us to elucidate the regulation of CXCR1 recycling in a hematopoietic cell system in which the regulation of CXCR1 could be dissociated from that of CXCR2. The results of this analysis indicated that in similarity to HEK 293 cells, the recycling process of CXCR1 in 300-19 pre B cells was partially dependent on actin filaments, as observed by the fact that the recycling was inhibited by 45.6% by exposure to cytochalasin D, but was not dependent on microtu- by guest on September 23, 2021 bules. Following the exposure of 300-19 pre B cells to 1000 ng/ml IL-8 for 90 min at 37°C, a marked decrease in CXCR1 cell surface expression was noted, the expression levels reduced to 21.0 Ϯ 2.9% of the total expression before induction of internalization. The removal of the ligand and the recovery of the cells for 90 min at 37°C resulted in recovery of CXCR1 membrane expression to 53.9 Ϯ 0.35% and to 55.2 Ϯ 5% of the total receptor expression, observed when the recovery was performed in the absence or in the FIGURE 3. The effects of nocodazole and cytochalasin D on WT CXCR1 presence of DMSO, respectively. The cell surface expression level re-expression on the cell membrane following IL-8-induced internalization. of CXCR1 determined following the exposure to cytochalasin D in CXCR1-expressing HEK 293 cells were incubated in the absence or in the the course of the recovery phase were restored to only 39.6 Ϯ presence of IL-8. One group of IL-8-exposed cells was washed and stained 3.4% of the total expression level (inhibition of 45.6%), whereas immediately after exposure to IL-8 with anti-CXCR1-specific Abs. Another no significant effect of nocodazole was observed on CXCR1 ex- group of IL-8-exposed cells was washed and allowed to recover in the pres- pression (50.0 Ϯ 8.5% of total expression level before induction of ence of cytochalasin D, nocodazole, or DMSO, or in their absence. The cells internalization). The fact that CXCR1 recycling in 300-19 pre B were washed and subjected to FACS analysis as described in Materials and Methods. A, Recovery of CXCR1 expression following the different treat- cells is regulated by similar mechanisms to those of HEK 293 cells ments. The data are presented as the percent of total receptor expression in supports the similarities previously observed between hematopoi- cells that were not exposed to any treatment, calculated as described in Ma- etic cells and HEK 293 cells, and suggests that HEK 293 cells p ϭ 0.005 for treatment with cytochalasin D vs DMSO provide a legitimate and a reliable system for the analysis of the ,ء .terials and Methods treatment. Cyto-D, Cytochalasin D; Nocod., nocodazole. Each value repre- regulation of chemokine receptor trafficking by cytoskeleton sents the mean Ϯ SD of three independent experiments. B, FACS analysis elements. demonstrating the expression of CXCR1 after the different treatments as follows: 1) cells that were not exposed to any treatment; 2) cells that underwent IL-8-induced receptor down-modulation and no recovery at 37°C; 3) cells that The carboxyl terminus domains of CXCR1 and CXCR2 are underwent IL-8-induced receptor down-modulation and recovery at 37°C in partially involved in the regulation of the actin-mediated the absence of cytochalasin D, but in the presence of DMSO; and 4) cells that process of receptor recovery underwent IL-8-induced receptor down-modulation and recovery at 37°C in the presence of 1 ␮M cytochalasin D. Baseline represents cells stained with To determine the mechanisms that may be involved in the actin- CSB instead of Abs to CXCR1, counts represents relative cell number, and mediated process of receptor recycling, analysis of the role of car- FL1-H represents fluorescence. A representative experiment of three indepen- boxyl terminus domains of CXCR1 and CXCR2 was performed. In dent experiments performed is shown. preliminary experiments the optimal conditions for analyzing the 1278 ACTIN FILAMENTS IN TRAFFICKING OF CXCR1 AND CXCR2

response of WT and mutated CXCR1 and CXCR2 were determined. Based on these experiments, internalization was induced by exposure to IL-8 for 90 min at 37°C (unlike Figs. 3 and 4 in which internalization was induced by a 60-min exposure to IL-8), followed by cell recovery for 90 min at 37°C, in the presence or in the absence of cytochalasin D. A mutated CXCR1, termed ⌬19 CXCR1, was produced by carboxyl terminus truncation that resulted in the removal of eight of nine phosphorylation sites (Table II). High and similar levels of receptor internalization and recycling were observed for WT CXCR1 and ⌬19 CXCR1. However, whereas the recovery of WT CXCR1 was inhibited by 30.0% by cytochalasin D, ⌬19 CXCR1 recycling was not affected by the drug, demonstrating 0% inhibition (Table III). The levels of ⌬19 CXCR1 recovery in the presence of DMSO were 93.9 Ϯ 12.2%, and 95.1 Ϯ 15.4% when performed in the presence of cytochalasin D (Table III). Of note is the fact that in contrast to our previous study, ⌬19 CXCR1 demonstrated similar levels of internalization to WT CXCR1, probably resulting from the different experimental conditions used in the Downloaded from previous and present study, regarding primarily the densities of cells cultured for experiments that may affect actin polymerization and levels of internalization induced by exposure to IL-8 (data not shown). Similar analysis was performed for CXCR2, using the carboxyl terminus mutated ⌬20 CXCR2, in which 9 of 11 potential phos- http://www.jimmunol.org/ phorylation sites in the carboxyl terminus were removed (Table II). As compared with WT CXCR2, ⌬20 CXCR2 demonstrated relatively higher levels of internalization, possibly giving rise to the lower levels of receptor recycling under the experimental conditions used in this assay, indicating that the carboxyl terminus may affect the trafﬁcking of this receptor. Nevertheless, although being only partial, the recycling of ⌬20 CXCR2 was not affected by

cytochalasin D. In contrast to WT CXCR2, whose recycling was by guest on September 23, 2021 inhibited by cytochalasin D by 69.2%, the recycling of ⌬20 CXCR2 was not significantly affected by the drug, demonstrating only 6.9% inhibition (Table III). Following induction of IL-8-mediated internalization by exposure to 1000 ng/ml for 90 min at 37°C and recovery of the cells for 90 min at 37°C, 42.7 Ϯ 9.7% of ⌬20 CXCR2 re-expression were observed in the presence of DMSO. Similar levels of re-expression, at the level of 40.4 Ϯ 7.7%, were noticed in the presence of the drug (Table III). FIGURE 4. The effects of nocodazole and cytochalasin D on WT Of note is the fact that, in similarity to the experiments shown CXCR2 re-expression on the cell membrane following IL-8-induced internalization. CXCR2-expressing HEK 293 cells were incubated in the ab- in Table III (internalization, 90 min), reduced sensitivity of mu- sence or in the presence of IL-8. One group of IL-8-exposed cells was tated receptor recycling to cytochalasin D, as compared with WT washed and stained immediately after exposure to IL-8 with anti-CXCR2- receptors was observed also when the internalization was per- specific Abs. Another group of IL-8-exposed cells was washed and allowed formed by a 60-min exposure to the chemokine (data not shown). to recover in the presence of cytochalasin D, nocodazole or DMSO, or in As demonstrated above, the overall sensitivity to cytochalasin D of their absence. The cells were washed and subjected to FACS analysis as all the cells included in this study, was reduced following a 90-min described in Materials and Methods. A, Recovery of CXCR2 expression exposure to IL-8 (see Discussion), and enabled a clearer compre- following the different treatments. The data are presented as the percent of hension of the differences between the WT and the mutated recep- total receptor expression in cells that were not exposed to any treatment, -p ϭ 0.02 for treatment tors. Following a 60-min exposure to IL-8, the differences in sen ,ء .calculated as described in Materials and Methods ϳ with cytochalasin D vs DMSO treatment. Cyto-D, Cytochalasin D; Nocod., sitivity to cytochalasin D of WT and mutated receptors was 20% nocodazole. Each value represents the mean Ϯ SD of three independent for CXCR1, as compared with 30% difference in a 90-min expo- experiments. B, FACS analysis demonstrating the expression of CXCR2 sure to IL-8 (Table III; at the 60-min exposure to IL-8 there was a after the different treatments as follows: 1) cells that were not exposed to reduction from ϳ50% sensitivity for the WT receptors to 30% any treatment; 2) cells that underwent IL-8-induced receptor down-mod- sensitivity for the mutated receptor). For CXCR2, following a 60- ulation and no recovery at 37°C; 3) cells that underwent IL-8-induced min exposure to IL-8, about 60% difference in sensitivity to cy- receptor down-modulation and recovery at 37°C in the absence of cytocha- tochalasin D was detected between the WT and mutated receptors, lasin D, but in the presence of DMSO; and 4) cells that underwent IL-8- in similarity to ϳ60% difference between the WT and mutated induced receptor down-modulation and recovery at 37°C in the presence of 1 ␮M cytochalasin D. Baseline represents cells stained with CSB instead CXCR2 following a 90-min exposure to the ligand (Table III; at of Abs to CXCR2, counts represents relative cell number, and FL1-H rep- the 60-min exposure, reduction from ϳ100% sensitivity for WT resents fluorescence. A representative experiment of three independent ex- CXCR2 to 40% sensitivity for ⌬20 CXCR2). In all, the results of periments performed is shown. these experiments suggest that the dependence of the recycling The Journal of Immunology 1279

migration was not affected by incubation with DMSO alone (data not shown). Upon exposure to 1000 ng/ml IL-8, in a treatment that resulted in a pronounced receptor down-modulation (Fig. 1B), a marked inhibition in the migratory response of the cells was observed (Fig. 7, lane 2). When IL-8-induced CXCR2 down-modulation was followed by ligand removal and cell recovery for 90 min at 37°C, the expression of the receptors was partially restored (Fig. 4), and this process was accompanied by an almost-complete recovery of migratory responses to IL-8 (Fig. 7, lane 3). However, if cytochalasin D was present in the course of receptor recycling, its ability to inhibit receptor recycling (Fig. 4) was manifested by a pronounced impairment of migratory responses to the chemokine (Fig. 7, lane 4; p Ͻ 0.0001 for migration after recovery in the absence of cytochalasin D (lane 3) vs migration after recovery in the presence of cytochalasin D (lane 4)). It is important to note that because cytochalasin D depolymer- izes actin filaments that may be essential for migratory responses FIGURE 5. The effect of cytochalasin D on WT CXCR2 re-expression (48), its presence in the course of cell recovery may have affected on the cell membrane following GCP-2-induced internalization. CXCR2- migration through disruption of actin filaments that are required Downloaded from expressing HEK 293 cells were incubated in the absence or in the presence of GCP-2. One group of GCP-2-exposed cells was washed and stained immediately after exposure to GCP-2 with anti-CXCR2-specific Abs. An- other group of GCP-2-exposed cells was washed and allowed to recover at the presence of cytochalasin D, DMSO, or in their absence. The cells were washed and subjected to FACS analysis as described in Materials and

Methods. The data are presented as the percent of total receptor expression http://www.jimmunol.org/ in cells that were not exposed to any treatment, calculated as described in p ϭ 0.028 for treatment with cytochalasin D vs ,ء .Materials and Methods DMSO treatment. Cyto-D, Cytochalasin D. Each value represents the mean Ϯ SD of three independent experiments. process of both CXCR1 and CXCR2 on actin ﬁlaments is partially determined by the carboxyl terminus domain of these receptors. by guest on September 23, 2021 Cytochalasin D-induced inhibition of CXCR2 recycling results in impairment of CXCR2-mediated migratory responses As with many other receptors, it is expected that the level of receptor expression on the plasma membrane of the cells will affect the ability of the cells to respond to stimuli that are mediated by these receptors. Because both CXCR1 and CXCR2 mediate cellular migration in response to IL-8 (25, 26, 29, 47), the level of their expression on the cell surface may affect the migratory response of the cells that express these receptors. As was already demonstrated (Fig. 1, A and B), exposure of CXCR1- and CXCR2- expressing cells to high concentrations of IL-8 resulted in receptor internalization, as manifested by a decrease in receptor cell surface expression. Following ligand removal and cell recovery, the receptors were recycled in a cytochalasin D-sensitive process, and their expression was partially restored (Figs. 3 and 4). To test whether the effects of cytochalasin D on receptor recycling have a functional relevance, we determined the ability of cells that underwent these different treatments to migrate in response to IL-8 (the cells were washed before loading in the chemotaxis chamber). Due to the fact that the cytochalasin D-mediated effects were more FIGURE 6. The role of G protein coupling in the regulation of IL-8- prominent on CXCR2, we focused in this part of the study only on induced chemotaxis and internalization. A, The effect of PTx treatment on CXCR2-expressing cells. the migratory response of CXCR1- and CXCR2-expressing HEK 293 cells. To determine whether the effect of cytochalasin D on CXCR2 The cells were exposed to 100 ng/ml PTx for2hat37°C, washed, and recycling alters the migratory responses of these cells, CXCR2- subjected to chemotaxis assays in response to IL-8, as described in Mate- p Ͻ 0.001 for PTx treatment vs no treatment. A ,ءءء .rials and Methods expressing cells were exposed to internalization-inducing concen- representative experiment of three independent experiments performed is trations of IL-8, followed by recovery in the presence or in the shown. B, The effect of PTx treatment on the IL-8-induced internalization absence of cytochalasin D. All the cells were washed before load- of CXCR1 and CXCR2. The cells were exposed to 500 ng/ml PTx for 2 h ing into the chemotaxis chamber. at 37°C, washed and subjected to FACS analysis, as described in Materials The results shown in Fig. 7 demonstrate that untreated CXCR2- and Methods. Each value represents the mean Ϯ SD of two to three inde- expressing cells potently migrate to IL-8 (lane 1). The level of pendent experiments. 1280 ACTIN FILAMENTS IN TRAFFICKING OF CXCR1 AND CXCR2

Table I. The effect of cytochalasin D on CXCR1 and CXCR2 re- may enable the re-sensitization of cellular functions (29–33, 42, expression in human neutrophilsa 43). Such regulatory processes may play a significant role in the fine tuning of chemokine-induced inflammatory responses, and ne- Receptor cessitate a better understanding of the mechanisms that control the Re-expression (% of total)b intracellular trafficking of these receptors. Our present study is the % Decrease in first to provide evidence for novel findings regarding the regulation Cell Surface DMSO ϩ – of chemokine receptor trafficking, as indicated by the following ϩ Neutrophils Expression Cyto-D – three observations: 1) an increased reduction in cell surface ex- Donor 1 pression of CXCR1 and CXCR2 was observed when receptor in- CXCR1 61.0 58.9 57.7 ternalization was induced by IL-8 (without allowing receptor re- CXCR2 55.2 49.1 46.6 cycling) in the presence of an actin-disrupting agent but not of a Donor 2 CXCR1 62.5 52.7 57.8 microtubule-disrupting agent; 2) actin filaments are important con- CXCR2 79.2 23.6 24.2 tributors to the regulation of CXCR1 and CXCR2 recycling to the Donor 3 plasma membrane in HEK 293 cells and in 300-19 pre B cells. CXCR1 64.0 43.8 46.9 This observation has important physiological implications because CXCR2 63.8 33.7 27.7 the cytochalasin D-induced inhibition of CXCR2 recycling re- a Human neutrophils were incubated in the absence or in the presence of IL-8. One sulted in the inhibition of migratory responses. In contrast to actin group of IL-8-exposed cells was washed and stained immediately after exposure to filaments, microtubules apparently are not involved in the regula- IL-8 with anti-CXCR1- or anti-CXCR2-specific (non-cross-reactive) Ab. Another group of IL-8-exposed cells was washed and allowed to recover in the presence of tion of the recycling process of these receptors. 3) The carboxyl Downloaded from cytochalasin D or DMSO. The cells were washed and subjected to FACS analysis as terminus domains of CXCR1 and CXCR2 were demonstrated to be described in Materials and Methods. b The data are expressed as the percent of total receptor expression in cells that partially involved in the regulation of the actin-dependent process were not exposed to any treatment, calculated as described in Materials and Methods. of receptor recycling to the plasma membrane. The results of experiments performed on three healthy independent donors are The cytochalasin D-induced promotion of IL-8-induced reduc- demonstrated. tion in cell surface expression of CXCR1 and CXCR2 may be

interpreted in two different ways. The first is that actin filaments http://www.jimmunol.org/ for migration and not through its effects on receptor recovery. To should undergo a process of partial depolymerization to allow for determine this possibility, an additional control was included in the a highly potent receptor internalization to occur. The second pos- study, consisting of cells that were not exposed to IL-8-induced sibility, which is more likely in view of the direct involvement of internalization or recovery, but rather were only incubated with actin filaments in the regulation of receptor recycling, is that the cytochalasin D for 90 min at 37°C and washed before loading in cytochalasin D-mediated promotion of IL-8-induced reduction in the chemotaxis chamber. As shown in Fig. 7 (lane 5), this control receptor cell surface expression could actually be the direct result treatment resulted in a partial inhibition of migratory responses. of the ability of the drug to inhibit receptor recycling. With respect However, the extent of inhibition induced by exposure to cytocha- to this hypothesis, it is important to note that the level of receptor lasin D alone (treatment no. 5) was significantly lower than the one expression may be determined by rapid dynamics of receptor in- by guest on September 23, 2021 observed when receptor recovery, following IL-8-induced CXCR2 ternalization and recycling back to the plasma membrane. Obvi- internalization, was performed in the presence of cytochalasin D, ously, high levels of receptor recycling are observed following as shown in treatment no. 4 (Fig. 7, lane 4; p Ͻ 0.0001 for lane 4 induction of internalization, removal of the ligand and recovery of vs lane 5) (Note: If the cells of treatment no. 5 were not washed the cells at 37°C. However, it is possible that upon exposure to and loaded in the chemotaxis chamber in the presence of cytocha- internalization-inducing ligand, low levels of receptor recycling do lasin D, a complete inhibition of migration was observed). The occur concomitantly with high levels of receptor internalization. results shown in lane 5 demonstrate that the cytochalasin D treat- This may be manifested by the fact that in many systems of GPCR, ment may, to some extent, suppress the migratory responses by including the one used in this study, exposure of the cells to li- acting directly on actin filaments; however, its effect is much more gands in conditions inducing maximal internalization, does not pronounced when it is present during the recovery process, pro- result in a complete abolishment of receptor expression, and re- viding evidence to the fact that the ability of cytochalasin D to duction in cell surface expression often does not reach 100%. inhibit receptor recycling gives rise to prominent suppression of Therefore, the possibility exists that cytochalasin D treatment dur- migratory responses, and thus has a functional relevance. ing induction of internalization actually inhibited the low levels of receptor recycling that occurred concomitantly with the highly Discussion prominent events of receptor internalization. The net effect of such The two closely related chemokine receptors, CXCR1 and CXCR2 activity will be the observed promotion of reduction in cell surface are desensitized by exposure to high ligand concentrations, in a expression of CXCR1 and CXCR2. In support of this hypothesis is process which is accompanied by potent receptor internalization. the observation that the cytochalasin D-mediated effect was time- Following removal of the ligand, the recovery of the cells at 37°C dependent, and was observed only following longer incubation allows the recycling of the receptors to the plasma membrane, and periods with IL-8, in which more pronounced recycling processes

Table II. Primary amino acid sequences of the predicted carboxyl terminus domains of the receptorsa

Cells Amino Acid Sequences

WT CXCR1 RHGFLKILAMHGLVSKEFLARHRVTSYTSSSVNVSSNL ⌬19 CXCR1 RHGFLKILAMHGLVSKEFL WT CXCR2 FIGQKFRHGLLKILAIHGLISKDSLPKDSRPSFVGSSSGHTSTTL ⌬20 CXCR2 FIGQKFRHGLLKILAIHGLISKDSL

a Indicated in bold and italics are serine and threonine residues that serve as potential phosphorylation sites. The Journal of Immunology 1281

Table III. The effects of cytochalasin D on receptor re-expression following cell recovery

Receptor Re-expression (% of total)a Inhibition of % Decrease in Cell DMSO ϩ – Receptor Cells Surface Expression Cyto-D – ϩ Recovery (%)b

WT CXCR1 58.7 Ϯ 6.9 104.1 Ϯ 6.6 85.8 Ϯ 8.2 30.0 ⌬19 CXCR1 56.5 Ϯ 6.4 93.9 Ϯ 12.2 95.1 Ϯ 15.4 0.0 WT CXCR2 62.2 Ϯ 4.5 77.6 Ϯ 13.5 49.5 Ϯ 4.5 69.2 ⌬20 CXCR2 84.4 Ϯ 4.9 42.7 Ϯ 9.7 40.4 Ϯ 7.7 6.9

a The data are expressed as percent of total receptor expression in cells that were not exposed to any treatment, calculated as described in Materials and Methods. Each value represents the mean Ϯ SD of two to five independent experiments. b The percent of inhibition of receptor recovery was calculated as described in Materials and Methods. This value is the mean of the percent inhibition values derived for each of the experiments performed for each of the cell types, and was not calculated directly from the values of receptor expression (% of total) that are demonstrated in the table. may have started to take place. Moreover, when the effects of The understanding of the role of actin filaments in the regulation cytochalasin D on receptor recycling were determined, it was ob- of CXCR1 and CXCR2 recycling necessitates a thorough investi- served that the sensitivity to the drug was reduced when the ex- gation of the components that may be involved. In that respect, it Downloaded from posure to IL-8, in the course of internalization induction, was pro- is important to note that a recent report by Nakagawa and Miya- longed (Figs. 3 and 4 vs Tables II and III). This could be explained moto (49) has demonstrated that actin filaments localize on sorting by the fact that when the internalization-inducing exposure to the endosomes. Similarly, it is conceivable that a physical association chemokine was prolonged, recycling events were initiated during between polymerized actin filaments and recycling endosomes is the internalization process, before the addition of cytochalasin D required to allow for the adequate trafficking of CXCR1 and (added only in the course of recovery). Such processes may give CXCR2, and for their recycling back to the plasma membrane http://www.jimmunol.org/ rise to a receptor subpopulation that is not exposed to cytochalasin following their ligand-induced internalization. D, resulting in reduced sensitivity to the drug during receptor The significant involvement of actin filaments in processes of recycling. receptor recycling, observed upon ligand removal following receptor internalization, was noticed for both CXCR1 and CXCR2. However, it is interesting to note that quantitative differences were observed between CXCR1 and CXCR2, regarding the effect of cytochalasin D on their recycling. The effect was considerably much more evident for CXCR2 than for CXCR1, supporting our previous observations, as well as findings by others, suggesting by guest on September 23, 2021 that the two receptors may be differently regulated at multiple levels (25–37). The quantitatively different effect of the drug on CXCR1 recycling, as compared with CXCR2 suggests that CXCR1 recycling is differentially regulated than that of CXCR2. Our results suggest that the difference between the two receptors is

not the result of the divergent involvement of prime G␣i-mediated signaling events in induction of processes that were initiated by ligand-induced internalization. However, one cannot exclude the

possibility that non-G␣i-mediated signaling events are involved in the regulation of this process. An alternative possible explanation for the different extent of actin filament involvement in the recycling process of each of the receptors may be that their intracellular trafficking following internalization is somewhat different. Indeed, FIGURE 7. Cytochalasin D-induced inhibition of CXCR2 recycling re- it is possible that to some extent the two receptors undergo recy- sults in impairment of CXCR2-mediated migratory responses. CXCR2- cling through different endosomes, as indicated by the quicker re- expressing HEK 293 cells were either not incubated with IL-8 (lanes 1 and cycling kinetics of CXCR1 as compared with CXCR2 (see Fig. 3A 5) or incubated with 1000 ng/ml IL-8 (lanes 2-4). The cells that were not vs 4A; following 90 min of recovery, the expression of CXCR1 exposed to IL-8 were either not exposed to any treatment (lane 1)orex- was restored to 71.4 Ϯ 6.25% of total expression before induction posed to cytochalasin D for 90 min at 37°C (lane 5). The IL-8-exposed cells were subdivided into two groups. The cells of the first group were not of internalization, whereas the expression of CXCR2 was restored Ϯ allowed to undergo recovery at 37°C (lane 2). The cells of the other group only to 46.5 6.9% of total expression). Therefore, it is our in- were washed (to remove the ligand) and allowed to recover for 90 min at tention to study the nature of the intracellular organelles that direct 37°C in the absence (lane 3), or in the presence of cytochalasin D (lane 4). CXCR1 and CXCR2 in the course of IL-8-induced internalization The cells were washed and subjected to a chemotaxis assay in response to and recycling, in analysis based on the expression of the Rab fam- Ͻ ءءء 50 ng/ml IL-8, as described in Materials and Methods. , p 0.0001 for ily of small GTPases. The quicker recycling process of CXCR1, as migration after recovery in the absence of cytochalasin D (lane 3)vsmi- compared with CXCR2 further suggests that the lower ability of gration after recovery in the presence of the drug (lane 4), and for migration after recovery in the presence of cytochalasin D (lane 4) vs migration cytochalasin D to affect CXCR1 recycling results from the fact that after the treatment with cytochalasin D alone (lane 5). HPF, High power actin-dependent processes of CXCR1 recycling occurred before field. A representative experiment of four independent experiments per- the activity of the drug was complete (since the drug was added formed is shown. only at the time of recycling). In contrast, the slower kinetics of 1282 ACTIN FILAMENTS IN TRAFFICKING OF CXCR1 AND CXCR2

CXCR2 recycling may allow for cytochalasin D to fully depoly- endosomes is less dependent on functionally intact actin filaments. merize actin filaments, resulting in a more pronounced effect on Therefore, our results on the recycling of ⌬19 CXCR1 and ⌬20 the recycling process. CXCR2 suggest that the carboxyl terminus domains of CXCR1 The use of HEK 293 cells for the analysis of the role of actin and CXCR2 are significantly involved in the trafficking of these filaments in the internalization and recycling processes of CXCR1 receptors and on their dependence on cytoskeleton elements. This and CXCR2 allowed us to dissociate the mechanisms regulating hypothesis is supported by recent findings demonstrating that the the intracellular trafficking of each of the receptors, independently carboxyl terminus of several GPCR participate in the process of of the other, and to gain insight into these processes by the use of their intracellular sorting (54–56), and suggests that the removal mutated receptors. Similar analysis of the regulation of CXCR1 of carboxyl terminus motifs that normally mediate the trafficking and CXCR2 recycling in neutrophils demonstrated that cytocha- of the receptors into the recycling pathway, may result in their lasin D did not affect the process of CXCR1 recycling. The above- trafficking into the degradation pathway. mentioned suggestion that the sensitivity of the recycling process Because CXCR1 and CXCR2 both mediate migratory responses to cytochalasin D is affected by the kinetics of the process raises that necessitate polymerization of actin filaments (18, 48), our ob- the possibility that the inability of the drug to affect CXCR1 re- servations on the need for polymerized actin to participate in cycling in neutrophils results from the rapid kinetics of the process CXCR1 and CXCR2 recycling are of major importance. The treat- in these cells. It is possible that the crucial events of CXCR1 re- ment that was used to induce potent receptor internalization (1000 cycling take place more quickly in neutrophils than in HEK 293 ng/ml IL-8) was shown in our previous studies, as well as in stud- cells, before cytochalasin D activity has come into a complete ies by others, to induce receptor phosphorylation that resulted in effect, resulting in the apparent inability of the drug to affect the functional desensitization of the receptors (30, 38, 52, 53, 57). It Downloaded from recycling process in neutrophils. If this is indeed the case, the was observed that the desensitization of chemoattractant receptors inability of cytochalasin D to affect CXCR1 recycling does not is accompanied by actin depolymerization (58). Our present ob- necessarily mean that the process is actin-independent, but that the servations indicate that when the desensitizing/internalizing che- experimental approach taken cannot elucidate the role of actin fil- mokine was removed from the cell proximity, and the receptors aments in this process. However, the possibility cannot be ruled were allowed to recover, functionally intact actin filaments were out that the process is indeed independent of actin filaments in required to allow receptor recycling, providing a readily available http://www.jimmunol.org/ neutrophils, or that the cross-talk between CXCR1 and CXCR2, pool of potentially active receptors that may undergo resensitiza- shown to be differently regulated in many other aspects of the tion by subsequent stimuli. In the case of CXCR1 and CXCR2, trafficking process, hampers the ability to analyze this issue. Un- resensitization will be manifested by the ability of the receptors to fortunately, in contrast to other publications demonstrating low, transmit signals for migratory responses that also require actin po- but significant levels of CXCR2 recycling, no recycling of CXCR2 lymerization. Therefore, it is possible that following a desensitized was observed in neutrophils in our study, therefore, making it im- refractory period, functionally assembled actin filaments are re- possible to determine the regulation of CXCR2 recycling by actin quired for efficient receptor recycling, as well as for potent migra- filaments in neutrophils.

tory responses to occur. by guest on September 23, 2021 The inability to conclusively determine the role of actin fila- The involvement of functionally intact actin filaments in ments in CXCR1 and CXCR2 recycling in neutrophils emphasizes CXCR1 and CXCR2 recycling, as well as in migratory responses, the need for the use of other experimental systems. In that regard, suggests that chemokine receptors are subjected to a specialized it is important to note that the experiments performed on 300-19 pre B cells indicated that CXCR1 is subjected to similar regulation regulation, under which both recycling and migration depend on an in other hematopoietic cells; the recycling of CXCR1 in these cells interdependent mechanism. Further studies are required to eluci- was sensitive to cytochalasin D treatment (but not to nocodazole), date whether such regulation is a more generalized mechanism that the sensitivity being in the range observed in HEK 293 cells. This participates in the trafficking of other chemoattractant receptors. observation supports the possibility that indeed the recycling of As for the regulation of other members of the GPCR superfamily, CXCR1 and CXCR2 is regulated by actin filaments and provides the conflicting observations regarding the regulation of muscarinic us with experimental tools to further investigate this issue. More- cholinergic receptor and adrenergic receptor trafficking by cy- over, because CXCR1 and CXCR2 are expressed not only on he- toskeleton components, combined with our present study, suggest matopoietic cells but also on adherent cells, such as endothelial that different receptors that belong to the superfamily of GPCR are cells and several types of tumor cells (50, 51), the analysis of the divergently regulated in this respect (54, 59, 60). However, it is regulation of their trafficking in HEK 293 cells is of major interesting to note that in similarity to the role of actin filaments in relevance. the regulation of CXCR1 and CXCR2 recycling, the recycling pro- ␤ Further insight into the potential involvement of actin filaments cess of 2-adrenergic receptors was also dependent on actin fila- in the recycling process was provided by the use of the ⌬19 ments (54). Moreover, two additional reports, addressing the re- CXCR1 and the ⌬20 CXCR2 mutants, indicating that the carboxyl cycling of IgA receptor and of Tac (the IL-2 receptor ␣ subunit) terminus domains of CXCR1 and CXCR2 are partially involved in indicated that depolymerization of actin filaments by cytochalasin the regulation of the actin-mediated process of receptor recycling. D resulted in inhibition of receptor recycling (22, 61). When com- These findings are of major interest in view of observations on pared with CXCR1 and CXCR2, both IgA receptor and Tac are of other GPCR, suggesting that in the course of their recycling, these a totally different nature in terms of composition and signaling. receptors are dephosphorylated to enable subsequent re-sensitiza- Tac is of special interest because it may be internalized in a clath- tion (9–11). Such a process of dephosphorylation may not be re- rin-independent pathway (23, 61). In contrast to Tac, CXCR1, and quired for ⌬19 CXCR1 and ⌬20 CXCR2 recycling because they CXCR2, are seven transmembrane receptors that were demon- lack most of the carboxyl terminus phosphorylation sites that are strated to undergo clathrin-dependent internalization (17, 62). involved in their functional desensitization (30, 52, 53). In such a Therefore, it may be suggested that with regard to the regulation of case, it is possible that ⌬19 CXCR1 and ⌬20 CXCR2 undergo a their recycling, a line of similarity exists between receptors of ␤ divergent process of intracellular trafficking through different en- totally different nature (CXCR1, CXCR2, 2-adrenergic receptors, dosomes than the WT receptors, and that the trafficking of these IgA receptor and Tac), implying that receptor recycling is an event The Journal of Immunology 1283 that is tightly regulated by mechanisms that depend on the recep- 22. Maples, C. J., W. G. Ruiz, and G. Apodaca. 1997. Both microtubules and actin tors and ligands that are involved. filaments are required for efficient postendocytotic traffic of the polymeric im- munoglobulin receptor in polarized madin-darby canine kidney cells. J. Biol. Chem. 272:6741. 23. Subtil, A., and A. Dautry-Varsat. 1997. Microtubule depolymerization inhibits Acknowledgments clathrin coated-pit internalization in non-adherent cell lines while interleukin 2 We thank Dr. Levi and the staff of her laboratory (Laboratory of Infectious endocytosis is not affected. J. Cell Sci. 110:2441. Diseases, Department of Clinical Biochemistry, Faculty of Health Sci- 24. Ben-Baruch, A., D. F. Michiel, and J. J. Oppenheim. 1995. Signals and receptors ences, Ben-Gurion University of the Negev and Soroka Medical Center, involved in recruitment of inflammatory cells. J. Biol. Chem. 270:11703. 25. Wuyts, A., P. Proost, J.-P. Lenaerts, A. Ben-Baruch, J. Van Damme, and Beer Sheva, Israel) for their highly appreciated assistance in providing J. M. Wang. 1998. 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