chemokine receptor biology at the immune system

Pharmacology & Therapeutics 119 (2008) 24–32

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Pharmacology & Therapeutics j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h a r m t h e r a

Associate editor: M. Teixeira

Imaging techniques: New insights into chemokine/chemokine receptor biology at the immune system Mario Mellado ⁎, Yolanda R. Carrasco ⁎ Department of Immunology and Oncology, National Centre of Biotechnology/CSIC, Darwin 3, UAM-Campus de Cantoblanco, Madrid E-28049, Spain

A R T I C L E

I N F O

A B S T R A C T Our current knowledge of molecular and cellular responses in vivo is based mainly on event reconstruction from time-freeze observations. Conventional biochemical and genetic methods consider the cell as an individual entity and ligand/receptor pairs as isolated systems. In addition, the data refer to the average behavior of a pool of cells and/or receptors removed from their real-life context. The use of new technologies, particularly real-time imaging approaches, is showing us that biological responses are highly dynamic and extremely dependent on the context in which they take place, and therefore much more diverse than initially envisaged. This review focuses on the mechanistic insights that new imaging techniques, such as those based on resonance energy transfer and two-photon microscopy, contribute to our understanding of how receptors work within a single cell, and how cells work within a tissue. Cell movement is a complex and regulated process; it has a key role in embryogenesis, organogenesis, wound-healing and tumor invasion. Nonetheless, it is in immune system homeostasis and response that cell movement becomes essential. For this reason, immunology is being radically transformed and enriched by these new approaches. We will discuss the use of these techniques for studying chemokine/chemokine receptors and their role in the immune system function, and comment on the potential contribution to the design of therapeutic strategies. © 2008 Elsevier Inc. All rights reserved.

Keywords: Chemokine receptors Immune cells Dynamics Imaging techniques

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The immune system: a highly dynamic machinery. . . . . . . . . . . . . . . . . . . . . 2.1. Two-photon microscopy in the immune system. . . . . . . . . . . . . . . . . . . 2.1.1. T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. B cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3. Other immune system cells . . . . . . . . . . . . . . . . . . . . . . . . 2.2. New challenges in the study of in vivo immune system dynamics in real time . . . . 3. Molecular dynamics of chemokine receptors at the plasma membrane . . . . . . . . . . . 3.1. Receptor dimerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Energy transfer-based techniques: a new approach to chemokine receptor physiology 3.2.1. The role of chemokines in receptor dimerization . . . . . . . . . . . . . . 3.2.2. The role of chemokine receptors and other GPCR in dimerization . . . . . . 3.3. New challenges: total internal reflection fluorescence microscopy (TIRFM) . . . . . . 4. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction ⁎ Corresponding authors. Tel.: +34 915854852; fax: +34 913720493. E-mail addresses: [email protected] (M. Mellado), [email protected] (Y.R. Carrasco). 0163-7258/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.pharmthera.2008.04.007

The immune system is formed by a complex network of different cell types such as lymphocytes, dendritic cells (DC), macrophages and neutrophils. Each has a specific function, but all of them share the

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ability to move continuously throughout the body, constantly surveying peripheral tissues and lymphoid organs for the presence of potential pathogens. These cells are able to change their homeostatic recirculation program when they detect an infection. Recruitment of immune cells to the infection site is critical for mounting an immune response to fight and clear the invaders. The chemokine/ chemokine receptor family leads the orchestration of the diverse patterns of immune cell migration (Cyster, 1999; Moser et al., 2004). Expression of distinct chemokines in a tissue- (skin versus intestine), location- (follicle versus paracortex) and condition- (inflammation versus homeostasis) restricted manner, together with differential and regulated expression of chemokine receptors within immune cells, constitutes the fine tuning of the immune system dynamics. The chemokines, a family of low molecular weight pro-inflammatory cytokines, have been the focus of exceptional interest over the last 25 years (Rossi & Zlotnik, 2000). Originally described as specific mediators of leukocyte directional movement, current perspectives implicate the chemokines in a much wider variety of cell types and functions (Mackay, 2001; Fernandez & Lolis, 2002; Raz, 2003; Lusso, 2006). Nearly 50 different chemokines have been described to date, all of which have remarkably similar three-dimensional structures despite considerable differences in amino acid sequence. The original classification system based on structural criteria (C, CC, CXC and CX3C chemokines) is being abandoned, and replaced by a functional classification that groups chemokines as constitutive or inducible (Proudfoot, 2002). Constitutive chemokines are generally implicated in homeostasis of the immune system, whereas inducible chemokine expression is regulated mainly during inflammatory processes. In addition, several viruses encode highly selective chemokine receptor ligands that act as agonists or antagonists, and may thus have a role in viral dissemination or evasion of host immune response (Murphy, 2000; Alcami, 2003). Chemokines exert their effects by interacting with G protein-coupled receptors (GPCR) at the target cell membrane (Rossi & Zlotnik, 2000). They can also be grouped into two major families, CCR and CXCR, which interact with the CC and CXC chemokines, respectively (Murphy et al., 2000). There is considerable promiscuity between chemokines and their receptors, with various ligands that can associate to the same receptor and distinct receptors that bind a single ligand. Based on their broad range of functions, chemokines and chemokine receptors must clearly be central factors in a variety of diseases characterized by inflammation and immune cell infiltration (Murdoch & Finn, 2000), such as asthma, atherosclerosis, rheumatoid arthritis, multiple sclerosis, colitis, Crohn's disease, and psoriasis. Finally, CXCR4 and CCR5 are the two main coreceptors for HIV-1 infection (Littman, 1998), and some chemokine receptors also participate in tumor metastasis (Muller et al., 2001; Strieter, 2001) and transplant rejection (el-Sawy et al., 2002). Thus, chemokines and chemokine receptors have become a major focal point as therapeutic targets. Here we will discuss latest data on the role of chemokines and chemokine receptors in the dynamics of immune cells within a tissue. In this area, the visualization of immune cell behavior in situ using two-photon microscopy is bringing important changes to our view of the workings of the immune system. We will also comment on recent data obtained regarding the molecular dynamics of chemokine receptors within a cell using new imaging technologies such as those based on resonance energy transfer. The possibility of studying these receptors in situ and in real time, not possible with conventional biochemical methods, is transforming our understanding of the field and adding new insights with potential therapeutic interest. 2. The immune system: a highly dynamic machinery The in vitro and in vivo approaches used to study the immune system to date have given us only limited information on its dynamic aspects. In vitro studies that attempt to address single cell function lack the

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complexity of the intact tissue environment; on the contrary, in vivo experiments maintain context while showing time-freeze observations, snapshots of how the immune system works. Our perception has changed markedly in recent years with the application of laser scanning microscopy, especially two-photon microscopy, to real time and in situ studies of the immune system. In this section, we will comment on progress in our understanding of immune system dynamics achieved by the application of two-photon microscopy, focusing on those aspects related to chemokine/chemokine receptor function. 2.1. Two-photon microscopy in the immune system Two-photon microscopy allows visualization of individual cell dynamics within intact organs in real time (Fig. 1A) (Cahalan et al., 2002; Sumen et al., 2004). The method is based on scanning with a pulsed infrared laser beam, which excites fluorescent dyes by the simultaneous absorption of two low-energy (long-wavelength) photons only at the plane of focus (Cahalan et al., 2002). Due to the high penetrance of infrared light into tissue, fluorescently labeled cells can be detected at depths of hundreds of microns (Fig. 1A). Thus far, most studies have been done in lymph nodes surgically exposed in anesthetized live mice (intravital imaging) (Miller et al., 2003; Mempel et al., 2004;) or as explanted preparations perfused with warmed, oxygenated medium (Miller et al., 2002; Okada et al., 2005); thymus, spleen and intestinal tissue explants have also been used (Bousso et al., 2002; Wei et al., 2002; Tutsch et al., 2004;). The manipulations involved with either approach may alter the physiology of the tissue. Intravital technology showed that lymphocyte movement stops after alterations of blood flow (Mempel et al., 2004); the lack of oxygenation has the same effect in the explants model (Y.R.C. unpublished observations; (Huang et al., 2007)). Also the temperature emerges as a critical factor for lymphocyte motility in both experimental set-ups (Miller et al., 2002; Mempel et al., 2004). Nevertheless, the dynamic measurements obtained so far via these two approaches have generally shown remarkable agreement (Miller et al., 2002, 2003; Mempel et al., 2004; Miller et al., 2004a; Okada et al., 2005), suggesting that both explants and intravital imaging models provide appropriate physiological conditions. The use of one approach or the other will mainly depend on the type of questions to be addressed. The Donnadieu group recently reported two-photon microscopy analysis of lymphoid tissue sections to analyze lymphocyte dynamics (Asperti-Boursin et al., 2007). This new approach provides an option for examining deep areas of the lymphoid organs that are inaccessible when working with the whole organ, due to technical limitations. Application of this method to other tissues in which infrared light penetrance is poor due to scattering (such as spleen, tumors and liver) will shed light on immune system dynamics in normal and pathological situations at these sites. 2.1.1. T cells Several aspects of T cell biology and responses have been analyzed by two-photon microscopy. Data obtained thus far (reviewed in (Cahalan & Parker, 2006; Henrickson & von Andrian, 2007)) have begun to illuminate key questions such as how T cells encounter antigen in vivo in real time. In homeostatic conditions, T cells are highly motile (average speed 10–12 μm/min) in the lymph node paracortex (Miller et al., 2002). This motility allows them to scan large numbers of dendritic cells (DC), facilitating detection of rare antigens within a reasonable period of time (Fig. 1B). Based on experimental data, a frequency is estimated of 500 contacts per hour of a single DC with CD8 T cells (Bousso & Robey, 2003), and of 5000 contacts per hour of a DC with CD4 T cells (Miller et al., 2004b). A recent in silico model indicates a similar DC scanning rate (2000 different T cells/h) and a scanning rate of 100 DC per T cell per hour (Beltman et al., 2007). Three distinct reports showed the relevance of CCR7 and its ligands CCL19 and CCL21 in regulating T cell basal motility within lymph nodes (Asperti-Boursin et al., 2007; Okada &

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Fig. 1. Lymphocyte dynamics in situ and in real time. (A) Scheme of two-photon imaging of lymphocyte dynamics in lymph nodes. The images show the projection of a z-series stack within a primary follicle at different time points; labeled B cells were adoptively transferred before imaging. The tracks of two different B cells are shown. (B) Illustration of distinct roles of chemokine and chemokine receptors in lymphocyte dynamics depicted by two-photon microscopy techniques.

Cyster, 2007; Worbs et al., 2007). Moreover, T cells deficient in key chemokine receptor signaling molecules rarely access the lymph node paracortex, lack the polarized morphology characteristic of migrating cells, and show markedly reduced motility (Hwang et al., 2007; Nombela-Arrieta et al., 2007). Although quite controversial until recently, the crucial role of chemokines in the orchestration of T cell basal motility is clear from these results (Fig. 1B). Chemokines are also reported to enhance T cell immunity by guiding non-random cell–cell interactions in the lymph node (Castellino et al., 2006; Hugues et al., 2007) (Fig. 1B). Specifically, CCR5 and its ligands CCL3 and CCL4 promote preferential interaction of CD8 T cells with mature, antigen presentation-competent DC. This mechanism increases the priming efficiency of the few antigenspecific CD8 T cells present in the naïve repertoire. Encounter with specific antigen results in a slow down of T cell motility (Mempel et al., 2004; Miller et al., 2004b). The magnitude of this decrease depends on the affinity of the T cell receptor (TCR) for the MHC (major histocompatibility complex)-antigenic peptide combination (Skokos et al., 2007). In vitro experiments showed that following antigen recognition, the T cell and the antigen-presenting cell (DC or B cell) form a stable, long-lasting interaction known as the immunological synapse (IS) (Monks et al., 1998; Grakoui et al., 1999). Although the in vivo relevance of the T cell IS is debated, recent work by Bousso and colleagues points out not only the existence of long-lived interactions after the first cognate T cell-DC encounter, but also their relevance for efficient T cell activation (Celli et al., 2007). The in vivo regulation of the different dynamic T cell stages, i.e., active migration, decreased motility, and stopped, is practically unknown. Large questions remain

as to how chemokine signaling regulates or is regulated to coordinate the complex in vivo dynamics of the T lymphocytes. 2.1.2. B cells Two-photon microscopy studies have also revealed interesting and sometimes unexpected data about B cell biology and responses to pathogens. In homeostasis, B cells survey the primary follicles of lymph nodes, searching for antigen at an average speed of 6 μm/min (Miller et al., 2002). This value is half that reported for T cells in basal conditions. Why this difference? One possibility is the distinct signals and/or substrate that support B and T cell migration. Follicular B cell basal motility depends on chemokine receptor signaling (Han et al., 2005), probably on the CXCL13/CXCR5 pair, although this has yet to be confirmed. In response to CCR7 ligands, however, B cells are reported to increase their speed in the paracortex (9 μm/min) (Okada et al., 2005). A second option is that intrinsic differences in biology or function determine differences in migration speed. In vivo, B cells recognize native antigen tethered to the surface of other cells (Carrasco & Batista, 2007; Junt et al., 2007; Phan et al., 2007); the antigen does not need to be processed. B cells may thus have more rapid access and greater likelihood of detecting a specific antigen compared to T cells. B cells would move more slowly, as they need to scan a smaller surface area in order to encounter specific antigen. Several studies show that antigen recognition by B cells leads to a decrease in their basal motility (Okada et al., 2005; Carrasco & Batista, 2007; Qi et al., 2006); it was also observed that stable, long-lived interactions are established with the antigen-presenting surface, resembling the formation of the B cell IS in vitro (Carrasco & Batista,

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2007). Moreover, the boundary area that limits the lymph node follicles with the subcapsular sinus appears to be a site for B cell antigen encounter (Carrasco & Batista, 2007; Junt et al., 2007; Phan et al., 2007). Basal B cell motility in this area is slower than in deeper follicular zones. Finally, while germinal center (GC) and follicular B cells show similar motility, GC B cells have highly dynamic shapes (Allen et al., 2007; Schwickert et al., 2007). Again we find distinct dynamic stages. Chemokine and chemokine receptor participation in regulating these complex dynamic changes in B cells is poorly understood. Seminal work on two-photon microscopy from the Cyster group shed light on the central role of chemokines on distinct events in B cell biology. They showed that following antigen exposure, B cells migrate preferentially toward the B cell-T cell boundary following a CCL21 gradient (Okada et al., 2005) (Fig. 1B); this is due to the increased CCR7 expression on antigen-activated B cells. The localization of antigenactivated B cells at the B-T cell bridge is critical for B cell encounter with T helper cells, and thus for T cell-dependent antibody responses. In a very recent study, although they do not use two-photon microscopy, they describe how the balance of ligands for CXCR5 and S1P1 regulates the follicle-marginal zone (MZ) shuttling of MZ B cells (Cinamon et al., 2008). This equilibrium is crucial for the continuous transport of blood-borne antigen from the marginal sinuses of the spleen to the white pulp, especially to the surface of the follicular dendritic cells where can be encountered by follicular B cells. This chemokine-regulated MZ-B cell shuttling thus provides an efficient mechanism for capture and follicular delivery of blood-borne antigens. 2.1.3. Other immune system cells The application of two-photon microscopy to the study of other cell lineages within the immune system has also been revealing. For example, regulatory T cells (Treg) were shown to exert their function as suppressors of the immune response in vivo by attenuating stable contact formation during DC priming of naïve T cells (Tadokoro et al., 2006; Tang et al., 2006). Natural killer (NK) cells also exhibit vigorous motility in a pattern reminiscent of other lymphocyte subsets (Bajenoff et al., 2006; Garrod et al., 2007). They patrol lymph nodes actively, searching for MHC-mismatched targets by interacting with other cells; formation of a stable conjugate (lasting N 5 min) leads to the subsequent elimination of the targeted cell. Moreover, in inflammatory conditions, NK cells recruited to the paracortex interact closely with DC and regulate T cell responses (Bajenoff et al., 2006). The chemokines and chemokine receptors involved in Treg and NK cell dynamics remain to be addressed. Two-photon studies have also helped explain the distinct barrier mechanisms to infectious agent entry. A report by Junt et al. shows that macrophages located in the floor of the draining lymph node subcapsular sinus capture lymph-borne pathogens and guide them to follicular areas for presentation to B cells (Junt et al., 2007). By this mechanism, macrophages prevent the systemic spread of lymphborne pathogens and facilitate the onset of B cell responses. The gastrointestinal tract is also a site of infectious agent entry; real time in situ experiments show that DC extend processes across the intestinal epithelium and capture bacteria in the lumen (Chieppa et al., 2006). This active DC sampling of the gut lumen is induced by inflammatory stimuli via Toll-like receptors on the epithelium. In this model, CCL20 expression is induced in the intestinal epithelium; however, the contribution of CCL20 to DC recruitment or to promoting DC extensions is not clear. The liver also provides an easy route of access to pathogens. A recent report in which intravital epifluorescence confocal microscopy is used instead of two-photon microscopy, shows that the natural killer T cells (NKT cells), which are abundant in the liver, provide intravascular immune surveillance by patrolling the liver sinusoids (Geissmann et al., 2005). Liver NKT cells express CXCR6, whose ligand CXCL16 is expressed as a membrane protein on sinusoidal epithelial cells. In this study, no role was observed for CXCR6 in NKT cell motility, although this receptor controls NKT cell abundance by enhancing cell survival.

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2.2. New challenges in the study of in vivo immune system dynamics in real time Two-photon microscopy studies of the immune response have centered mainly on well-known model antigens that do not induce tissue damage. The challenge now is to image infectious processes induced by bona fide pathogens. Pathogen infection promotes the recruitment of inflammatory immune cells such as neutrophils, granulocytes and monocytes to control pathogen spread. The inflammatory response results in damage and extensive changes in the tissue. Two-photon imaging of the immune response in this context should provide new insights into cell dynamics in a native inflammatory situation. To do this, we must not only develop adequate models of physiological infection to be imaged, but also tackle the imaging technology to overcome problems in visualizing the inflamed tissue. A second challenge in this type of study is “going smaller”, i.e., to visualize molecular dynamics and signaling events in vivo in real time. There are now some reports of success in imaging small antigenic particles and immune complexes (Carrasco & Batista, 2007; Phan et al., 2007); this has allowed detection of B cell antigen-particle gathering in a central cluster that resembles synapse formation and the deposition of immune complexes on the surface of follicular dendritic cells. The development of new tools such as quantum dots or the generation of new highly fluorescent green fluorescent proteins will open up the possibility of using two-photon microscopy to study receptor/ligand dynamics in a single cell within the tissue. It will also be both interesting and useful to image cell activation events in vivo; this will provide information about the outcome of certain cell interactions and about the lymphocyte activation process in the native in vivo environment. Lymphocyte calcium signaling was recently visualized in vivo under homeostatic and immunizing conditions using two-photon microscopy (Wei et al., 2007). The combination of creative mouse genetics and imaging technology could broaden our scope substantially in the study of gene expression dynamics in the in vivo immune response. Besides two-photon microscopy, the development and application of new imaging tools such us the multi-channel fluorescence spinning disk videomicroscopy give us the possibility of studying the immune response by new means. The spinning disk videomicroscopy seems very useful to analyze in vivo leukocyte behavior during recruitment and activation in the vasculature due to its very high-speed image acquisition in bright-field and multiple wide-field fluorescence channels (Chiang et al., 2007). The use of low doses of fluorophore-conjugated antibodies in vivo allows identification of leukocyte subpopulations and also visualization of the spatial distribution of plasma membrane receptors in distinct microdomains. Although the application of this technique is limited to accessible peripheral tissues and highly expressed cell-surface markers, it will aid greatly in the understanding of the inflammatory response at the vasculature in normal and pathological situations (Chiang et al., 2007; Norman et al., 2008). 3. Molecular dynamics of chemokine receptors at the plasma membrane 3.1. Receptor dimerization It was initially assumed that chemokine receptor function is governed entirely by Gi-mediated processes (Thelen, 2001), since most chemokine functions are blocked in cells pretreated with pertussis toxin (PTx) (L'Heureux et al., 1995; Arai et al., 1997). Gai protein subunits associate to chemokine receptors after ligand binding (Aragay et al., 1998). The chemokine-mediated signaling cascade is nonetheless more complex than originally thought; several studies, including those by our group, demonstrated chemokine receptor homo- and heterodimers and the functional relevance of these conformations (Mellado et al., 2001b). Receptor dimerization has been shown for CCR2, CCR5, CXCR4, CXCR1, CXCR2, among others

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(Rodriguez-Frade et al., 1999a,b; Vila-Coro et al., 1999; Trettel et al., 2003; Fernando et al., 2004). These structures are functionally relevant; for example, heterodimerization is the mechanism that underlies the delay in AIDS in HIV-1-infected patients bearing the CCR2V64I polymorphism (Mellado et al., 1999). There is further complexity, as heterodimers activate specific signaling cascades distinct from those triggered by homodimers (Mellado et al., 2001a; Molon et al., 2005). In contrast to the Gi activation triggered by homodimers, CCR2/CCR5 heterodimers promote activation of G11, which alters PI3K activation kinetics. As a consequence, the response threshold is reduced and cell adhesion is triggered, in contrast with homodimer-promoted cell migration. Chemokine receptors thus adopt distinct conformations at the cell surface, including homoand heterodimers as well as oligomers formed by groups of several receptors (Rodriguez-Frade et al., 2001). 3.2. Energy transfer-based techniques: a new approach to chemokine receptor physiology Biochemical approaches were used to determine both the expression of chemokine receptors at the cell membrane and the signaling molecules involved in chemokine function (Trettel et al., 2003; VilaCoro et al., 1999). Coimmunoprecipitation and western blot studies of chemokine receptors often detect individual receptors together with high molecular weight species that could correspond to receptor complexes. These techniques use disrupted cells, and the results were initially interpreted as artifacts due to incomplete solubilization or as a consequence of receptor clustering prior to internalization. In any case, the early experiments that demonstrated chemokine receptor dimerization combined immunoblot with coimmunoprecipitation, using wt chemokine receptors from primary cells or differentially N-terminustagged receptors (Rodriguez-Frade et al., 1999a). These experiments identified the presence of one tagged receptor in immunoprecipitates of the other; extracts of crosslinked cells analyzed in western blot showed individual receptor bands, as well as higher molecular weight species that indicated dimers and oligomers. To discard artifacts and to confirm specificity, these experiments used different solubilizing detergents, mixtures of cells expressing individual receptors, or dominant negative receptors that abrogate normal receptor signaling. For example, the non-functional CCR2Y139F mutant was shown to form inactive complexes with CCR2wt, indicating functional relevance for chemokine receptor dimerization (Rodriguez-Frade et al., 1999a). Despite their numerous drawbacks, these methods remain the most frequently used to determine receptor interactions. These types of strategies render a static view of the system that can lead to misinterpretation of the results. For example, by western blot and coimmunoprecipitation, we detected CCR2 dimers only after ligand activation, leading us to conclude that ligands had an active role in promoting receptor dimerization (Rodriguez-Frade et al., 1999a,b). Today's imaging-based techniques clearly show that dimers form in the absence of chemokines (Hernanz-Falcon et al., 2004; Percherancier et al., 2005; Wilson et al., 2005). Our initial conclusion was thus incorrect; the difficulty in detecting these complexes by immunoprecipitation might be due to instability of the dimer conformation in the absence of ligand. Newer methods to determine chemokine receptor oligomerization are based on resonance energy transfer (RET), in wide use for evaluating protein-protein interactions in live cells. These techniques are also useful for determining conformation dynamics, the role of ligand and receptor levels, and to define the dimerization site within the cell (Harrison & van der Graaf, 2006). There are two main types of RET, fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET); in the former, the donor fluorochrome transfers energy to an acceptor fluorochrome and in the latter, the donor molecule is luminescent (Pfleger & Eidne, 2006; Cardullo, 2007). Both techniques require the generation of fusion

proteins between the receptor and the fluorescent/luminescent donor and acceptor proteins, as well as the use of transfected cells (Boute et al., 2002). Although BRET has been used at the single cell level (Coulon et al., 2008), it is in fact an approach for cell suspensions (Pfleger et al., 2006). This technique allows measurement of energy transfer between receptors, independently of their expression pattern. Dimers at the plasma membrane cannot be distinguished from those that are being synthesized or that are trafficking through the endoplasmic reticulum. The use of BRET saturation curves nonetheless permits quantitation, as reported for CCR5/CCR2 heterodimers (ElAsmar et al., 2005) and CXCR4 homodimers (Percherancier et al., 2005). In contrast, FRET imaging techniques use confocal or wide field microscopy, allowing measurements in single cells and identification of cell locations (Fig. 2). FRET requires robust controls, including those to discard direct acceptor activation by the light used to excite the donor, to eliminate non-specific random collisions, and to monitor receptor overexpression (Marullo & Bouvier, 2007). The use of RET techniques made it clear that formation of homoand heterodimers between chemokine receptors is spontaneous (Issafras et al., 2002; Hernanz-Falcon et al., 2004; Percherancier et al., 2005). We now know that most CXC family receptors form both types of complexes; nonetheless, receptor behavior is not uniform, as some show higher FRET efficiency as homodimers, whereas others prefer to form heterodimeric entities (L.M. Martinez and M. Mellado, manuscript in preparation). 3.2.1. The role of chemokines in receptor dimerization Once it had been established that receptors preexist as dimeric complexes in live cells, the next step was to determine the role of the chemokines in stabilizing or disrupting these structures. Several reports indicate that the FRET ratio between CXCR4 homodimers tends to be higher when CXCL12 is added in a time-dependent manner (Babcock et al., 2003; Wilson et al., 2005), although the data are not statistically significant; however, other studies clearly show a significant increase in FRET efficiency in similar conditions (Angers et al., 2002; Toth et al., 2004). These discrepancies could be due to the cell area in which FRET was determined in each case (Fig. 2C); the first analyzed whole cells, whereas the latter restricted FRET determination to the cell membrane. A common concern when measuring RET of overexpressed fusion proteins is that high protein levels can increase the incidence of random collisions; these non-specific interactions could be attributed to specific RET. Furthermore, high receptor expression levels can overwhelm a cell's glycosylation capacity; it is thus not unusual to find a substantial fraction of BRET/FRETcompetent GPCR at intracellular membranes. This is especially relevant in the case of chemokine receptors, as only a small fraction of total receptors normally reach the extracellular membrane. Recent evidence on the extent of CXCR4 ligand-promoted BRET changes suggests that homodimers adopt different conformations that correlate with functional differences. Whereas the CXCR4 inverse agonist T14012 is predicted to induce minor rearrangements in the receptor complex, the weak agonist AMD3100 promotes important changes (Percherancier et al., 2005). These data correlate with the distinct effects promoted by these two ligands on the constitutively active CXCR4N119S mutant (Zhang et al., 2002) and indicate an active role for ligands in modulating chemokine receptor conformations. Internalization studies clearly indicate that, independently of the nature of the receptor complexes at the cell membrane, each chemokine triggers the specific internalization only of its own receptor, without affecting levels of other co-expressed receptors (Rose et al., 2004; Nasser et al., 2007). These data suggest that by binding to a specific receptor, chemokines disrupt heterodimeric complexes and stabilize the homodimer, thus modulating the receptor conformation at the cell surface. In the case of cells stably transfected with CCR5 and CCR2, simultaneous CCL5 + CCL2 addition reduces the response threshold and receptor internalization is not triggered,

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Fig. 2. FRET analysis of chemokine receptor dimerization. HEK293 cells were cotransfected with CXCR3-CFP and CXCR4-YFP, and FRET determined using an acceptor photobleaching method. The figure, displayed in quantitative pseudocolor, shows pre- (A) and post-bleaching (B) of CFP expression. (C) FRET efficiency determination at distinct cell locations (M1, membrane 1 region; M2, membrane 2 region; M3, membrane 3 region; I1, intracellular cytosolic region).

suggesting that the simultaneous presence of both ligands stabilizes the heterodimeric conformation (Mellado et al., 2001a). Several reports indicate that chemokines can also form homo- and heterodimeric complexes in solution, whose conformational stability depends on chemokine concentration and the pH of the medium (Nesmelova et al., 2005). Using activated T cells in the context of specific APC, CXCR4 and CCR5 were shown to form specific heterodimers that are recruited to the immunological synapse (Molon et al., 2005). This process is directed by the appropriate chemokines, CXCL12 and CCL5, and is blocked by PTx treatment or by neutralizing antibodies. Such data can obviously only be obtained using imaging-based techniques. These results showed that ligands have an active role in promoting heterodimeric complex formation, and that this conformation is functionally relevant, as it specifically associates Gq (Molon et al., 2005). All together, this evidence indicates that although chemokine receptor dimers exist in the absence of ligands, the chemokines stabilize the conformation that triggers function. 3.2.2. The role of chemokine receptors and other GPCR in dimerization The next important question was to evaluate whether receptor expression levels affect cell membrane homo- and heterodimer conformations. Some authors used biochemical approaches to show that CXCR1 neither homo- nor heterodimerizes with the analogous receptor, CXCR2 (Trettel et al., 2003). Saturation BRET experiments indicate, however, that the affinities at which CXCR1 forms homodimers and heterodimerizes with CXCR2 are very similar (Wilson et al., 2005); this process may take place during protein synthesis and maturation. We can thus hypothesize that unless these receptors are directed to degradation by the ER machinery, both CXCR1/CXCR2 hetero- and homodimers (CXCR1/CXCR1, CXCR2/CXCR2) exist and their ratio is determined by individual receptor levels. Although homodimers preexist at the cell surface, the coexpression of distinct receptors by a cell is sufficient to allow formation of heterodimeric complexes, as observed for CCR2/CCR5 (Mellado et al., 2001a; ElAsmar et al., 2005), CCR2/CXCR4 (Percherancier et al., 2005) and CXCR4/CCR5 (Molon et al., 2005), among others. The balance between homo- and heterodimers is thus modulated by receptor and/or ligand levels in the microenvironment. CXCR4/CCR2 heterodimers are ligand- modulated, although it is not clear whether BRET signals are due to changes in dimer numbers or to rearrangements of preformed dimers (Percherancier et al., 2005). Chemokine receptors also heterodimerize with opioid receptors, as demonstrated in coimmunoprecipitation and FRET experiments (Szabo et al., 2003; Chen et al., 2004; Pello et al., 2008). CXCR4 homodimers are disrupted as a consequence of δ-opioid receptor (DOR) expression, suggesting that homo- and heterodimers are in a dynamic equilibrium that is modulated by receptors levels. The effect

was reversed by treatment with the DOR ligand DPDPE, showing that both homo- and heterodimers are not only ligand- but also receptorregulated (Pello et al., 2008). In any case, inhibition of FRET signals does not necessarily reflect changes in protein association-dissociation, since a conformational change could be sufficient to modify the relative distance and/or orientation between donors and acceptors. In the case of the CXCR4/DOR complexes, recovery of FRET efficiency after DPDPE treatment correlated with DOR internalization data (Pello et al., 2008), indicating heterodimer disruption rather than conformational change. Dynamic evaluation of chemokine receptors will answer many other questions. For example, the site of GPCR biosynthesis and the mechanism by which these receptors are transported from the ER to the membrane are central aspects in the control of receptor expression. Some reports indicate that chemokine receptor mutants act as dominant negatives by promoting ER retention of the wild type receptor; this is the case of CCR5D32, whose dimerization with CCR5wt retains it in intracellular compartments, blocking its function (Liu et al., 1996; Samson et al., 1996). These observations suggest that chemokine receptor dimers are assembled during synthesis and transported to the cell membrane, and also validate FRET as a technique that can be used to determine the functional relevance of a receptor conformation. In addition, RET techniques will also shed light on the role of lipid rafts microdomains at the plasma membrane in chemokine receptor biology and function. It has been shown by FRET that ligand binding promotes association of chemokine receptors with lipid rafts; the disruption of this association alters chemokine receptor function (Jiao et al., 2005). 3.3. New challenges: total internal reflection fluorescence microscopy (TIRFM) The application of TIRFM to cell biology is breaking new ground, as it allows single-molecule visualization and analysis in living cells. It is used to observe the interface between two media with different diffractive indexes, such as water and glass; critical aspects of this technique are the illumination with an incident angle of the excitation laser beam at the interface, and the rise of an electromagnetic field (called “the evanescent field”) that excites the fluorophores near the interface (for a more complete explanation go to (Sako & Uyemura, 2002; Schneckenburger, 2005)). As a result, TIRFM reduces the excitation depth to approximately 100 nm, allowing the excitation of fluorophores located within or close to the cell-substrate interface. Using this approach, we can determine the dynamic and kinetic parameters of ligand/receptor units within the complexity of the structure and local environment in live cells. The first TIRFM studies of single-molecule imaging in live cells used epidermal growth factor (EGF) and its receptor (Sako et al., 2000); TIRFM has recently been

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applied to cells of the immune system to visualize the molecular dynamics of antigen-mediated lymphocyte activation in the nanoscale range (Yokosuka et al., 2005; Varma et al., 2006; Depoil et al., 2008). These studies show the formation of antigen/receptor microclusters, in which activation is initiated and sustained. Approximately 100 antigen receptors were estimated per microcluster. Highly dynamic exchange of signaling molecules is observed in these microclusters, depending on their location in the contact area between the lymphocyte and the antigen-presenting surface. Although TIRFM studies of chemokine receptors have yet to be reported, application of this technique will be very revealing in areas such as chemokine receptor conformation and dynamics, alone and with ligands in live cells. 4. Concluding remarks The possibility of visualizing immune response-related events in vivo and in real time has transformed our knowledge in the field and will continue to do so. One of the most revealing results is the great dynamism of immune cells in vivo and its relation to their function. Although several studies have already stressed the key role of chemokines and their receptors in this dynamism, many questions remain unanswered. This information about the workings of the immune system could then be used to design new approaches to fighting infection, autoimmune disorders and diseases such as cancer. It will be both interesting and useful to apply imaging technology to the in vivo visualization of the pharmacokinetics of substances and vaccines. The possible use of two-photon microscopy to study in situ immune system dynamics in autoimmune disorders and cancer will also be informative. The application of the RET imaging techniques discussed here has revealed an unexpected degree of complexity in chemokine receptor dynamics at the plasma membrane. In addition to the promiscuous interactions between ligands and receptors, the latter adopt several different conformations at the cell surface. These homo- and heterodimeric conformations can be modulated by the levels of chemokine receptors or of other GPCR, as well as by chemokine expression. There is also a correlation between the receptor conformation stabilized and the signaling cascade activated; it will be thus important to determine how distinct chemokine receptor conformations affect in vivo cell behavior. Chemokine receptor homoand heterodimers should thus be considered potential targets for drugs designed to modulate chemokine function. Acknowledgments We thank Dr. Jens Stein and members of the Chemokine Signaling Group for contributions and discussion, and especially Laura Martínez Muñoz for use of her FRET images. We thank Catherine Mark for editorial assistance. YRC is supported by a Ramon y Cajal contract from the Spanish Ministry of Education and Science. The work is supported by grants from the EU (INNOCHEM Project UE-518167; Molecular Imaging Project LSHG-CT-2003-503259), and from the Spanish Ministry of Education and Science (SAF-2005-03388). The Department of Immunology and Oncology was founded and is supported by the Spanish National Research Council (CSIC) and by Pfizer. References Alcami, A. (2003). Viral mimicry of cytokines, chemokines and their receptors. Nat Rev Immunol 3(1), 36−50. Allen, C. D., Okada, T., Tang, H. L., & Cyster, J. G. (2007). Imaging of germinal center selection events during affinity maturation. Science 315(5811), 528−531. Angers, S., Salahpour, A., & Bouvier, M. (2002). Dimerization: an emerging concept for G protein-coupled receptor ontogeny and function. Annu Rev Pharmacol Toxicol 42, 409−435. Aragay, A. M., Mellado, M., Frade, J. M., Martin, A. M., Jimenez-Sainz, M. C., Martinez, A. C., et al. (1998). Monocyte chemoattractant protein-1-induced CCR2B receptor desensitization mediated by the G protein-coupled receptor kinase 2. Proc Natl Acad Sci U S A 95(6), 2985−2990.

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