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Chemokine receptor binding and signal transduction in native cells of the central nervous system
Methods 29 (2003) 326–334 www.elsevier.com/locate/ymeth
Chemokine receptor binding and signal transduction in native cells of the central nervous system Christopher N. Davis,a Shuzhen Chen,a Stefen A. Boehme,b Kevin B. Bacon,c and Jeffrey K. Harrisona,* a
c
Department of Pharmacology & Therapeutics, University of Florida College of Medicine, Gainesville, FL 32610-0267, USA b Genicon Sciences, Inc., 11535 Sorrento Valley Road, San Diego, CA 92121, USA Department of Biology, Bayer Yakuhin, Ltd., Kyoto Research Center, 6-5-1-3, Kunimidai, Kizu-cho, Soraku-gun, Kyoto 619-0216, Japan Accepted 10 December 2002
Abstract Chemokine receptors belong to the superfamily of seven-transmembrane-spanning, G-protein-coupled receptors, and their expression by central nervous system cells is clearly documented. As this gene family has become the target of novel therapeutic development, the analysis of these receptors requires radioligand binding techniques as well as methods that entail assessing receptor stimulation of signal transduction pathways. Herein, we describe specific protocols for measuring radiolabeled chemokine binding to their cognate receptors on cultured glial cells as well as to receptors expressed in heterologous cell systems. Multiple downstream signaling pathways, including intracellular calcium influx and receptor-dependent kinase activation, are associated with chemokine receptor stimulation. Protocols for measuring these signaling events in chemokine-receptor-expressing cells are also presented. 2003 Elsevier Science (USA). All rights reserved. Keywords: Radioligand; Iodination; Receptor; Microglia; Calcium; MAPK; Akt/PKB; Immunoprecipitation; Western blot
1. Introduction G-protein-coupled receptors (GPCRs) embody a large and diverse gene family. Historically they are well represented as targets of the pharmaceutical industry in the development of novel pharmacologic agents. The characterization of new therapeutics requires a multifaceted approach, including a range of pharmacologically based approaches that include radioligand binding techniques as well as analysis of the signal transduction processes activated by specific receptors. In recent years, the chemokine receptor family has become the object of intense study, as this receptor subfamily poses unique opportunities for drug development. The use of radiolabeled chemokine binding assays has proved necessary in characterizing the biological functions of chemokine receptors, and these studies often complement information gained from functional assays. Radiolabeled *
Corresponding author. Fax: 1-352-392-9696. E-mail address: [email protected]fl.edu (J.K. Harrison).
chemokine binding techniques have proven absolutely critical for identifying receptors expressed in heterologous cell expression systems and native cells, including cells resident in the central nervous system (CNS). Methods related to measuring chemokine binding to cultured cells of the CNS parallel techniques used for study of chemokine receptor binding to peripheral leukocyte populations as well as binding methods utilized for study of other GPCRs, most notably receptors for peptide ligands. Like many peptide ligand GPCRs, the endogenous chemokines are agonists for their respective receptors, thus binding assays can be sensitive to functional states of the receptor. Furthermore, the often promiscuous nature of chemokine receptor binding profiles with overlapping selectivities lends difficulties to the interpretation of data. Here we discuss methodological protocols for measuring chemokine receptors on cultured cells of the CNS as well as those expressed in heterologous expression systems. Specific protocols for assessing downstream signaling events associated with chemokine receptor activation are also presented.
1046-2023/03/$ - see front matter 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S1046-2023(02)00356-0
C.N. Davis et al. / Methods 29 (2003) 326–334
2. Chemokine receptor binding Little information is available on chemokine binding properties of native brain cells or tissues. Table 1 outlines studies in which methods of radiolabeled or biotinylated chemokine binding to whole cells, membranes, or brain sections were utilized. Specific biotinylated MCP-1 and MIP-1a binding sites have been identified on cultured human astrocytes [1]. Using unlabeled chemokines to compete for these binding sites, it was determined that these MCP-1 and MIP-1a binding sites present on these glial cells displayed pharmacological properties similar to those of CCR1, CCR2, and CCR5. Immunocytochemistry using anti-chemokine receptor antibodies was also used to demonstrate the presence of these receptors in the astrocyte cell cultures, thus validating the binding data. The properties and distribution of 125 I-SDF-1a binding to CXCR4 in rat brain sections have been characterized [2]. Competition and kinetic binding studies revealed high-affinity SDF-1a binding sites in specific brain structures. Duffy antigen receptor for chemokines (DARC) has been demonstrated to be present on Purkinje cells of human cerebellum [3]. Immunoreactive DARC, detected by monoclonal antibodies (mAb) (Fy6), and radiolabeled MGSA binding established the presence of this chemokine binding protein in membrane fractions from human cerebellum. The binding of 125 I-MGSA was blocked by Fy6, and not by a mAb to CXCR2, and was displaced by an excess of unlabeled ligands. CXCR2 was found on projection neurons in many regions of the brain using mAbs specific for this receptor. CXCR3 expression in brain tissue in a model of rat focal stroke has been characterized by IP-10 binding analysis [4]. Cortical membranes of sham rats and those exposed to focal brain ischemia displayed
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specific binding of 125 I-IP-10 that was displaced by unlabeled chemokine. Radiolabeled chemokine binding has also been demonstrated in differentiated human neurons derived from the cell line NTera 2 (hNT cells). Displacement of 125 I-interleukin-8, 125 I-SDF-1, and 125 IMIP-1a by varying concentrations of unlabeled chemokine in the hNT neurons has been shown [5]. The presence of CX3CR1 on rat astrocytes and microglia has been determined by the specific and saturable binding kinetics of 125 I-labeled fractalkine [6,7]. Regarding CX3CR1 on astrocytes, no significant differences were seen in competition binding using unlabeled full-length fractalkine or fractalkine chemokine domain. In addition, steady state binding was blocked by preincubation of the cells with anti-CX3CR1 Ab. When developing protocols for measuring specific binding of chemokines to their receptors and, most importantly, for proper interpretation of the resultant data, it is necessary to consider the following. Receptor binding should be specific, time-dependent, and reversible. Specificity of chemokine binding is determined by the following experiments: (1) binding of the chemokine of interest and not an irrelevant protein should be observed, (2) saturation of binding should be achieved with high affinity, and (3) competitive inhibition should be observed by unlabeled chemokine homologues. Measuring the rate and extent of chemokine binding to native tissue or recombinant cells can provide information on the number of binding sites, as well as their affinity and accessibility. The use of chemokine- and/or chemokine receptor-specific antibodies to inhibit binding is also appropriate in determining the specificity of the ligand/receptor interaction. Given that some chemokines bind to multiple receptors, the use of the latter type of antibody in the binding analysis can aid in
Table 1 Methods used for chemokine binding CNS tissue
Ligand
Competitors
Whole cell binding on astrocytes Slide-mounted coronal sections of brain
Biotinylated MCP-1 and MIP-1a 125 I-SDF-1a
MCP-3, MIP-1b; and RANTES SDF-1a
Membranes from cerebellum and cerebral cortex Cortical membranes
125
I-MGSA
IL-8, RANTES, MCP-1
125
I-IP-10
IP-10
NTera 2 neurons
125
Whole cell binding on astrocytes Whole cell binding on microglia
I-SDF-1, 125 I-IL-8, and 125 I-MIP-1a 125 I-Fkn
SDF-1, MGSA, and MIP-1a Fkn or Fkn-CD
125
Fkn-CD
I-Fkn
Binding buffer
Ref. [1]
MEM, 1% BSA, 250 KIU/ml Trasylol, and 0.25 mM Tris–HCl, pH 7.4 PBS 50 mM Hepes, pH 7.4, 1 mM CaCl2; 4 mM MgCl2; and 0.1% BSA PBS 25 mM Hepes, pH 7.4, 1 mM CaCl2; 4 mM MgCl2 , and 0.1% BSA HBSS, 0.1% BSA
[2]
[3] [4]
[5] [6]
[7]
IL-8, interleukin 8; MEM, minimal essential medium; BSA, bovine serum albumin; PBS, phosphate-buffered saline; HBSS, HanksÕ balanced salt solution.
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the identification of the specific chemokine receptor. Of course, immunohistochemical analysis using specific receptor antibodies can provide alternative and complementary information in determining which chemokine receptor is present. Three general approaches are normally used to characterize binding of a ligand to its cognate receptor. These include kinetic, saturation, and competition binding analyses. In kinetic analysis, rates of association and dissociation of the ligand to and from the receptor are measured. From this, affinity constants can be calculated. In saturation binding experiments, the concentration of the labeled ligand is varied and binding to the receptor at steady state is determined. The relationship between binding and ligand concentration allows direct determination of the ligand affinity (Kd ), as well as the total number of binding sites (Bmax ) in the receptor preparation, i.e., cells or membranes. Competitive binding experiments measure the steady-state binding of a single concentration of radioligand in the presence of various concentrations of an unlabeled competitor. Competition binding experiments have significant advantages compared to theoretically simpler saturation binding experiments. In saturation experiments, the signal-to-noise ratio decreases with increased ligand concentration, and the binding information gained is that of the labeled analog, not the natural ligand [8]. Competition binding experiments should be designed so that concentrations of both the receptor and the ligand are kept as low as possible and always well below the Kd values. Experimentally, the need to keep both the level of nonspecific binding and the signal-to-noise ratio low often dictates the concentration of receptor and ligand used in the assay. These three general experimental approaches for studying the binding of a ligand to a receptor are complementary. Preliminary kinetic binding experiments should be performed in order to determine the time at which the receptor/ligand interaction has reached steady state. This is valuable information for proper design and interpretation of results from both saturation and competition binding analysis. When binding ligand to whole cells, one must also take receptor internalization into consideration. Binding at lower temperatures will slow the energy-dependent process of receptor internalization. However, it will also take longer for the ligand binding reaction to reach steady state. This shift in time to reach steady state should be taken into consideration when performing binding analysis at lower temperatures. An alternative approach is to measure the binding in the presence of an inhibitor of mechanisms of internalization. Given that this process is active and requires energy, mitochondrial poisons such as azide can be included. Concentrations of sodium azide that are sufficient to inhibit receptor internalization are in the range of 0.01 to 0.1%.
Binding of certain chemokines to their respective receptors can be sensitive to the specific incubation buffer components. We have determined that fractalkine will bind to CX3CR1 and MCP-1 to CCR2 in a buffer system containing HanksÕ balanced salt solution (HBSS) and bovine serum albumin (BSA). In addition, these chemokines bind their respective receptors equally well in a Hepes-based binding buffer (described below). However, we could not detect any specific 125 I-MIP-1a binding to CCR5 in the HBSS/BSA-based buffer system despite readily detecting this interaction in the Hepes binding buffer. The dependence of binding buffer components on the binding of other chemokines to their respective receptors is also documented. For instance, the level of 125 I-SDF-1a nonspecific binding was determined to be dependent on the concentration of salt in the binding buffer; high concentrations of salt in the buffer lead to high nonspecific binding [2]. Eotaxin binding to eosinophils, as well as functional responses of the cells to this chemokine, is also influenced by salt concentration and pH [9]. Thus, if embarking on characterizing a novel chemokine receptor in a given cell or tissue, it is important to establish optimal conditions for readily detecting specific chemokine binding. Our group has successfully characterized radiolabeled chemokine binding to freshly isolated microglia cells from newborn rats. Specific methods for radiolabeling chemokines, culturing microglia, and binding to whole cells are described in detail below. A section on chemokine binding to recombinant cells is included as well.
3. Analysis of chemokine-mediated signal transduction pathways Chemokines can activate multiple signaling pathways upon binding their appropriate receptors. For instance, fractalkine (CX3CL1) binding to the CX3CR1 receptor in microglial cells initiates multiple signaling cascades resulting in cell migration [6,10] and increased cell viability by modulating the functional status of Bcl-2 family proteins [11]. These downstream effects are triggered by the activation of receptor-mediated signaling proteins, such as Ga, Gbc, and receptor-associated kinases [12]. The fractalkine receptor, CX3CR1, is a G-proteincoupled receptor expressed by microglia and astrocytes in the CNS that induces microglial cell migration upon ligand binding. Fractalkine binding to CX3CR1 induces a rapid, transient mobilization of intracellular calcium, and this response can be blocked by pretreating cells with pertussis toxin, suggestive of a Gai -mediated signaling process [6]. This response can be measured experimentally by fluorimetry. Downstream signaling events involved in microglial cell migration include
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activation of the Rho family member Rac1. This, in turn, leads to activation of the PAKs (p21-activated serine/threonine kinases) in a process involving binding to the vav and nck adapter proteins. These protein/ protein interactions can be demonstrated by immunoprecipitation and Western blot analysis. Further downstream the signaling pathway leading to microglial cell migration is the activation of p42/p44 mitogen-activated protein (MAP) kinases, which occurs within 1 min of fractalkine stimulation and is dependent on PAK activation (K.B.B., unpublished observations). Activation of myosin light chain kinase is dependent on p42/p44 MAP kinase, and this ultimately results in the phosphorylation of the myosin light chain and actin rearrangement in microglial cells (S.A.B. and K.B.B., unpublished observations). Activation of the PAKs, p42/p44 MAP kinase, and myosin light chain kinase can be experimentally demonstrated by immunoprecipitation followed by in vitro kinase assays or, in the case of the MAP kinases, by Western blotting analysis probing with phosphorylation state-specific antibodies.
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Fractalkine also has the ability to act as a survival factor for microglial cells cultured in the absence of MCSF/GM-CSF and block Fas-mediated microglial cell death [6,10]. This occurs in part due to the activation of the phosphatidylinositol 3 (PI-3)-kinase/protein kinase Ba signal transduction pathway. Activation of protein kinase Ba leads to the phosphorylation and blockade of the proapoptotic functions of the Bcl-2 family member Bad. Furthermore, fractalkine upregulates the expression of antiapoptotic protein Bcl-XL, and inhibits the cleavage of the antiapoptotic protein Bid [11]. Taken together, these observations demonstrate that fractalkine binding to CX3CR1 in microglial cells results in the activation of a number of distinct signaling pathways leading to a range of disparate functional outcomes. Fig. 1 depicts a schematic of the pathways and outlines the general experimental approaches used to monitor them. In the sections to follow, we describe in detail methods to assess the activation of these signal transduction pathways.
Fig. 1. Schematic depicting signal transduction pathways regulated by chemokine receptors in microglia. The general methodological approaches used to measure them are highlighted.
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4. Specific methods 4.1. Primary microglial cell culture 4.1.1. Materials The following materials are used. • Poly-l-lysine (Sigma, St. Louis, MO, USA): 0.01 g/ liter in H2 O • Solution D: 137 mM NaCl, 5.4 mM KCl, 0.2 mM NaH2 PO4 , 0.2 mM KH2 PO4 , 1 g/liter glucose, 20 g/ liter sucrose, 0.25 lg/liter Fungizone, 106 U/liter penicillin/streptomycin, pH 7.4 • Complete medium: DulbeccoÕs modified EagleÕs medium (Life Technologies, Rockville, MD, USA) containing 10% fetal bovine serum (Life Technologies) 4.1.2. Methods Just prior to establishment of the cultures, flasks are coated with poly-l-lysine and incubated at 37 C for at least 1 h (20 ml in a 175-cm2 cell culture flask). Before use, poly-l-lysine is aspirated and the flasks are washed with solution D once. Cerebra are dissected from newborn Sprague–Dawley rats, stripped of meninges, and mechanically minced with a scalpel blade in solution D. The tissues are then digested with 0.25% trypsin solution D (10 ml total for 10–15 brains) in a conical tube at 37 C on a bidirectional rotator for 30 min. An equal amount of complete medium is added to stop the trypsin and the mixture is passed through 130-lm Nitex filter (Tetko, Inc., Brairdiff Manor, NY, USA). The mixture is then centrifuged at 400g for 10 min. The pellet is resuspended in complete medium, filtered through a 40lm Nitex filter, and plated into T175 flasks (Sarstedt, Newton, NC, USA) at a density of 1.5 brains per flask. The cultures are incubated with complete medium at 37 C, 5–8% (v/v) CO2 . After 3 days, the medium is changed and the cells are left for another 4–7 days to favor the proliferation of microglial cells. To harvest microglial cells, the flasks are shaken on an orbital shaker at 100 rpm for 60 min. The supernatant is collected and centrifuged at 400g for 10 min. The cells are counted using a hemacytometer and replated into appropriate cell culture plates in complete medium and at the indicated cell density for each experiment. Plates are incubated at 37 C, 5–8% CO2 for 60 min. The medium is aspirated to remove any nonadherent cells and replaced with fresh complete medium. The adherent cells contain more than 95% microglia. 4.2. Preparation of
125
I-labeled chemokines
Although many 125 I-labeled chemokines are available from commercial sources, we generally radiolabel our own chemokines. The advantage of this lies primarily in a cost savings. However, our methods typically yield chemokine peptides with specific activities approxi-
mately 1/10 (200 Ci/mmol) of the specific activity of the commercially prepared reagent. For binding to recombinant receptors in heterologous cell expression systems or even primary cultures of rat microglia, this is not a problem. However, if the investigator wishes to characterize a chemokine receptor that is expressed at a relatively low level, the use of high specific activity radiolabeled chemokine will enhance receptor detectability. This is especially important if the assay suffers from high nonspecific binding. The general method of chemokine iodination involves incubating the chemokine with sodium iodine in the presence of an oxidizing agent. We use Iodo-Beads (Pierce, Rockford, IL, USA) as the source of this oxidizing agent. Iodo-Beads utilize a nonporous polystyrene bead which has N-chlorobenzenesulfonamide covalently linked. The chief advantage of the IodoBead, as opposed to soluble chloramine-T, for instance, is that the reaction is more easily terminated by simply pipetting the reaction away from the bead. The extent of iodination is generally reproducible and easily controlled by varying the incubation time. Our specific protocol for iodinating chemokines, which we have used successfully to label fractalkine, SDF-1, MIP-1a, and MCP-1 is as follows. 4.2.1. Solutions and reagents The following materials are used. • Iodo-Beads (Pierce) • 100 mM phosphate buffer, pH 7.6 (PB) • HBSS with 0.1% BSA • 0.5 M potassium iodide (KI) • 5 lg chemokine peptide, carrier free [dissolved in phosphate-buffered saline (PBS) at 1 lg=ll] • Na 125 I (Perkin–Elmer, Palo Alto, CA, USA) • Desalting column (D-Salt Dextran, plastic desalting column, 5 ml, Pierce), preequilibrated with 30 ml of HBSS/0.1% BSA 4.2.2. Procedure All procedures are conducted at room temperature, in a well-ventilated biosafety hood, behind sufficient lead shielding in order to minimize exposure to the radioactive iodine. Place one Iodo-Bead into a 1.5-ml Eppendorf tube. Add 1 ml of 0.1 M PB, pH 7.6, briefly vortex, and then remove bead by shaking onto a dry Kimwipe. Transfer washed bead to a fresh 1.5-ml tube. Add 100 ll of PB followed by 1 mCi of Na125 I (approximately 3 ll). Allow reaction to proceed for 6 min. Add 5 lg of chemokine peptide (5 ll volume) and allow reaction to continue for an additional 9 min. At the end of this incubation, carefully pipet the reaction out of the tube (leaving the bead behind) and place into a tube containing 11 ll of 0.5 M KI. A 1-ll sample is pulled from this mixture and added to 500 ll of HBSS/0.1% BSA. Fifty microliters of 100% trichloroacetic acid (TCA) is
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then added and the reaction is allowed to sit on ice for 5 min. The TCA solution containing the aliquot of the iodination reaction is filtered through a GF/C filter and rinsed with ice-cold 10% TCA, and the radioactivity associated with the filter is determined using a gamma counter. The specific activity of the radiolabeled peptide is determined by dividing the counts associated with the filter by the amount of chemokine subjected to the TCA precipitation. In this example, 50 ng of chemokine was analyzed. The molecular mass of the particular chemokine and the specific activity of the 125 I (2200 Ci/mmol) allow one to convert the specific activity of the labeled chemokine into units of Ci/mmol. The volume of the remainder of the iodination reaction is doubled by the addition of 125 ll of HBSS/0.1% BSA. This solution is mixed and carefully applied to a preequilibrated desalting column. Ten individual fractions are collected by sequentially adding 500 ll of HBSS/0.1% BSA and moving a rack of tubes prior to each 500-ll addition. From each fraction, a 5-ll aliquot is analyzed by gamma spectroscopy. Knowing the specific activity of the labeled peptide allows one to determine the amount of labeled peptide in the 5-ll aliquot and hence its concentration. 4.3. Binding to native cells Whole cell binding is performed on 500,000 cells/ well. Microglial cells are seeded into 12-well cell culture plates (Corning). The cells are grown at 37 C, 5–8% CO2 for 1–2 days before the binding assay. Appropriate incubation buffer containing labeled and/or unlabeled chemokine is prepared at room temperature just prior to cell washing. Cells are washed once with 1 ml room temperature sPBS and incubated in 500 ll incubation buffer (HBSS, 0.1% BSA, pH 7.4) at room temperature for 60 min (or whatever has been experimentally determined to be steady state). Cells are washed three times with 1 ml ice-cold incubation buffer (this step is performed as quickly as possible to prevent disassociation of ligand). Cells are lysed with 500 ll 0.2 M NaOH. The cell lysates are collected into tubes and are counted on an appropriate gamma counter. 4.4. Binding to recombinant receptors in heterologous cell systems 4.4.1. Materials Typically, receptor is expressed in recombinant cells and the cells are passed with antibiotic to select for those containing receptor. Binding is usually performed on whole cells seeded directly from flasks of cells. Unlabeled chemokines are available from many sources and are generally recombinantly expressed or generated through peptide synthesis. The following materials are used:
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• Incubation buffer: 50 mM Hepes, pH 7.2, containing 0.5% BSA, 5 mM MgCl2 , 1 mM CaCl2 , 10 lg/ml each of chymostatin, leupeptin, and aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride (PMSF). • All inhibitors are maintained as 1000 stocks and added to buffer just prior to use. Chymostatin stock is in dimethyl sulfoxide (DMSO). Leupeptin and aprotinin stocks are in water. PMSF stock is in methanol. • Wash buffer is 25 mM Hepes, pH 7.2, containing 0.5 M NaCl. 4.4.2. Methods Whole cell binding is performed on 800,000 cells/ well. Cells are seeded into 12-well cell culture plates (Corning) containing appropriate medium at a density of 500,000 cells/well. Cells are grown overnight in a 37 C, 5% CO2 cell incubator. Appropriate incubation buffer containing labeled and/or unlabeled chemokine is prepared at room temperature just prior to cell washing. Total volume for each well is 500 ll. We typically dilute labeled chemokine into appropriate polypropylene or polystyrene tubes and split into 1.5-ml aliquots (for triplicate wells). Unlabeled competitor would be added directly to 1.5-ml aliquots to produce appropriate dilutions. Wells are washed once with 1 ml room temperature sPBS approximately 24 h after seeding. Five hundred microliters of binding solution (containing both labeled and unlabeled chemokine) is pipetted onto the cells. The plates are incubated at room temperature for 60 min (in our hands 60 min has been experimentally determined to be steady state). After 60 min, cells are washed three times with 1 ml ice-cold wash buffer. This step is performed as quickly as possible. Cells are then lysed with 500 ll 0.2 M NaOH and the contents of the well is collected into scintillation tubes and counted on an appropriate gamma counter. 4.5. Signaling: preparing cell lysates for biochemical analysis 4.5.1. Solutions and reagents Cell lysis solution consists of 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40 (Calbiochem), 0.25% sodium deoxycholate, 5 mM EDTA, and protease and phosphatase inhibitors (1 mM PMSF, 10 lg/ml aprotinin, 10 lg/ml leupeptin, 1 mM sodium orthovana_ M tetrasodium pyrophosphate, 1 mM EGTA, date, 1m 100 lg/ml b-glycerophosphate). The protease and phosphatase inhibitors should be made in 100 or 1000 stock solutions, aliquoted, and kept frozen until ready for use. Complete lysis solution, containing the protease and phosphatase inhibitors, should be at 4 C prior to use. It should be noted that this cell lysis cocktail does not lyse the nuclear membrane, thus, nuclei remain intact.
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4.5.2. Procedure Cells to be examined should be pelleted at 4 C, transferred to a 1.5-ml Eppendorf tube in 1 ml of icecold PBS, and centrifuged for 5 min at 4 C to pellet the cells. After all of the supernatant is decanted, the cells should be vigorously resuspended in complete cell lysis solution and incubated on ice for 15–60 min with periodic vortexing. The volume of lysis solution used should be minimal (e.g., 20 ll) to ensure a high protein concentration. After the cells are incubated on ice, the lysate should be centrifuged at 12,000g for 15 min at 4 C to pellet the nuclei and other insoluble material. The lysates, as well as any unused cell lysis solution, can be frozen at this time at )20 C. Alternatively, the protein concentration in each sample can be quantitated, using the excess lysis solution as a background standard for each experimental sample. When doing time-course experiments or studies having a large number of experimental conditions, the cells should be placed into a tube on ice containing a large excess of ice-cold PBS to effectively stop all cellular processes at the desired point. From this point on, all of the samples can be processed together. 4.6. Western blotting procedure On isolation and quantification of the protein levels in the cell lysates, the samples should be diluted with an equal volume of 2 Laemmli sample buffer and heated to 100 C for 5 min and either frozen or loaded onto a polyacrylamide gel (Novex/Invitrogen, San Diego, CA, USA). If frozen samples are to be run, they should be heated to 37 C for 5 min to ensure that all the SDS is dissolved before the gel is loaded. The samples should contain sufficient protein to allow for detection by Western analysis (25–50 lg). Attention must be given at this point to whether denaturing or nondenaturing conditions should be used and the percentage polyacrylamide gel to be used. As for the latter point, it should be taken into account which proteins will be blotted for, as the membranes can undergo multiple antibody probings. The percentage of polyacrylamide gel should be chosen to give sufficient separation of the proteins probed for. It is also advisable to use molecular weight markers with standards in the molecular weight range of the proteins to be examined. Following protein separation by electrophoresis, protein is transferred to a nitrocellulose membrane using the XCELL II system (Novex/Invitrogen). Following transfer, the nitrocellulose is washed briefly in water, and the nonspecific protein binding sites are blocked by incubating the membrane in blocking solution [2% BSA, 0.5% ovalbumin, 2.5% nonfat dry milk (Bio-Rad, Hercules, CA, USA), 10 mM Tris, pH 8, 150 mM NaCl, 0.2% thimerosal] for 1 h at 25 C. This step onward should be carried out on a shaker set at 70 rpm. The
membrane can then be probed by incubating the blot in 10 ml of fresh blocking solution containing the primary antibody for 1–2 h at 25 C or overnight at 4 C in a covered container. Following incubation with the primary antibody, the blot is washed in TBS-T (20 mM Tris, pH 7.5, 0.5 M NaCl, 0.05% Tween 20), three times for 5 min, and incubated with the appropriate peroxidase-conjugated secondary antibody diluted in blocking buffer for 1 h at 25 C. The filter should again be washed three times for 5 min in TBS-T, three times for 5 min in TBS (20 mM Tris, pH 7.5, 0.5 M NaCl), and once for 5 min in H2 O. The protein of interest can then be visualized using the chemiluminescent peroxidase substrate Super Signal Ultra (Pierce). For subsequent probing, the membrane can be stripped using 7 M guanidine, 50 mM glycine, 50 lM EDTA, 100 mM KCl, 20 mM 2mercaptoethanol, pH 10.8, for 10 min at 25 C, followed by three washes in H2 O. The blot can then be stored in TBS-T at 4 C indefinitely or placed in blocking solution to start another probing. 4.7. Immunoprecipitation protocol Proteins can be specifically isolated by immunoprecipitation (IP) to examine protein–protein interactions or by in vitro kinase assays. Immunoprecipitation assays are carried out by taking cell lysates of all the samples to be examined, with equal protein content, and incubating them with 1–5 lg of the antibody specific for the protein of interest conjugated to protein A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The incubations should be carried out in low volumes rotating in 1.5-ml Eppendorf tubes at 4 C. The immunoprecipitation step should proceed for a minimum of 1 h, but may go overnight. As controls, one immunoprecipitation should be done with fresh, complete lysis buffer only, and another IP should be done with a cell lysate but only protein A/G beads, with no conjugated antibody. These conditions should control for nonspecific binding of protein from either the cells or the lysis buffer to the beads. After the cell lysates are incubated with antibody-coated beads, the beads should be gently pelleted (400g, 1–2 min) and the supernatant transferred to a fresh tube and saved for possible further analysis. The pelleted beads should be washed once in a 100–500ll volume of fresh, complete lysis buffer. After the beads are spun down and the supernatant is removed, the IP should be washed again in fresh, complete lysis solution containing 0.5 M NaCl. After the beads are gently centrifuged and the wash buffer is removed, the immunoprecipitation is ready for further analysis. For subsequent Western analysis, the beads should be resuspended in 20 ll of 2 Laemmli sample buffer and boiled for 5 min. This heating step will dissociate both the immunoprecipitated protein from the capture antibody and the antibody from the agarose bead. Post-
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heating, the samples should be briefly centrifuged to concentrate the sample and either loaded on an SDS– PAGE gel, with a small bore pipette tip to exclude the beads, for immediate analysis or frozen at )20 C indefinitely. 4.8. In vitro kinase assay In vitro kinase assays can be used to examine the activity of various protein kinases involved in numerous intracellular signaling pathways. The first protocol below (the following two sections) is used for assaying the activity of the p42/p44 MAP kinases, and a protocol for assaying the activity of protein kinase Ba (also known as Akt) follows. 4.8.1. Solutions and reagents The following materials are used. • p42/p44 MAP kinase washing buffer (used to wash the immunoprecipitated ERK kinases): 25 mM Tris–HCl, pH 7.4, 40 mM MgCl2 , 137 mM NaCl, and 10% (v/v) glycerol • p42/p44 MAP kinase assay buffer: 42.5 mM Hepes, 42.5 mM MgCl2 , 0.21 mM ATP, 50 mM myelin basic protein (Upstate Biotechnology, Inc., Lake Placid, NY, USA), and 50 lCi [c-32 P]ATP (>3000 Ci/mmol sp act) (Amersham, Arlington Heights, IL, USA). 4.8.2. Procedure Following immunoprecipitation of the ERK1 and ERK2 kinases and subsequent washes in cell lysis solution 0:5 M NaCl (see Calcium mobilization analyses), the immunoprecipitated material is washed once in MAP kinase washing buffer. At this point, ERK enzyme activity is assessed in an in vitro kinase assay. The immunoprecipitation beads are suspended in 20 ll of kinase assay buffer for 30 min at 30 C. Following this incubation, an equal volume of 2 Laemmli buffer is added, and the samples are heated to 100 C for 5 min, briefly centrifuged, and cooled on ice. The samples may be frozen for future analysis or directly loaded onto a 16% Tris–glycine gel (Novex/Invitrogen) using the XCELL II apparatus (Novex/Invitrogen). Proteins are then transferred to nitrocellulose membranes using the Western blotting protocol (see Western blotting procedure). Precautions should be taken, however, as the samples are radioactive ð32 PÞ. Phosphorylation of the myelin basic protein substrate can be visualized using Biomax MR autoradiography film (Eastman Kodak, Rochester, NY, USA). Multiple exposures of the autoradiograph should be taken, to allow for accurate quantitation of the amount of phosphorylated myelin basic protein in each sample by densitometry. Analysis of the amount of phospho-ERKs in each sample can also be determined by probing blots with anti-phospho-MAP kinase antibodies (Upstate Bio-
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technology, Inc.) using the Western analysis protocol outlined above. Equal loading of the immunoprecipitates or cell lysates can be assessed by stripping the Western blot of the phospho-MAP kinase antibodies and reprobing the blot with anti-MAP kinase antibodies (Upstate Biotechnology, Inc.), as outlined under Western blotting procedure. 4.8.3. Solutions and reagents for measuring protein kinase Ba activity The following materials are used. • Protein kinase Ba wash buffer: 20 mM Hepes, pH 7.4, 10 mM MgCl2 , and 10 mM MnCl2 • Protein kinase Ba reaction buffer: 20 mM Hepes, pH 7.4, 10 mM MgCl2 , 10 mM MnCl2 , 0.05 mg/ml histone 2B (Boehringer Mannheim, Mannheim, Germany), 5 lM ATP, 1 mM dithiothreitol, and 10 lCi ½c-32 PATP (Amersham). 4.8.4. Procedure for protein kinase Ba in vitro kinase assay The procedure for assaying protein kinase Ba is similar to the MAP kinase assay, with just a few changes outlined below. Immunoprecipitations of protein kinase Ba are washed twice in complete cell lysis buffer, once in H2 O, and once in protein kinase Ba wash buffer. After all of the supernatant from the last wash is decanted, the immunoprecipitation beads are resuspended in 20 ll of protein kinase Ba reaction buffer and incubated at 30 C for 30 min. At this point, the samples can be handled in the same manner as in the MAP kinase assay, again using precaution as the sample contains 32 P. The phosphorylated histone 2B can be visualized by autoradiography and quantitated by densitometry. The amount of active [phosphorylated protein kinase Ba at positions 308 (threonine) and 473 (serine)] can also be assayed by probing Western blots with anti-phosphoprotein kinase Ba antibodies (Upstate Biotechnology), and the total amount of protein kinase Ba can be determined by probing the stripped blot with anti-protein kinase Ba antibodies (Upstate Biotechnology). The molecular mass of protein kinase Ba is 60 kDa. 4.9. Calcium mobilization analyses Chemokine binding to cells expressing cognate chemokine receptors results in a rapid, transient mobilization of Ca2þ . This event can be measured and quantitated using intracellular, UV-excitable, ratiometric Ca2þ indicators, such as indo-1 AM and fura-2 AM (Molecular Probes, Eugene, OR, USA), and a fluorimeter. First, the cells are loaded with esters of indo-1 or fura-2, which passively diffuse inside the cell. Once inside, the Ca2þ indicators (indo-1 AM and fura-2 AM, in which the AM represents an acetoxymethyl group) are cleaved by intracellular esterases to yield
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cell-impermeant fluorescent indicators. Once intracellular stores of Ca2þ are released or extracellular calcium enters the cells, the emission wavelength of the calcium indicator changes. It is important to note that both indo-1 AM and fura-2 AM need to be resuspended in anhydrous DMSO at a concentration of 1 mg/ml. Microglial cells are isolated, resuspended in 1 ml of complete medium, and incubated with the calcium-sensitive fluorescent dye indo-1 AM (3 lmol/liter final concentration). The labeling procedure is carried out for 45 min at room temperature, with the cells in a 15-ml conical tube wrapped in aluminum foil (to shield from light), on a rotating platform. The cells are subsequently pelleted and washed once in prewarmed HBSS containing 1% BSA. The labeled cells are then resuspended in 1 ml of prewarmed HBSS/1% BSA and kept in a dark environment until used. The cells are then placed in 1-ml cuvettes containing a small stir bar, at a concentration of 1 106 cells/ml of HBSS/1% BSA in the cuvette and analyzed using a PTI fluorimeter (South Brunswick, NJ, USA). The results obtained are from the ratios of dual emission (405/490 nm), and the results represent nanomolar shifts in calcium mobilization.
5. Concluding remarks As we unravel the mysteries of the role of chemokines in CNS physiology it will continue to be necessary to use radioligand binding techniques and signal transduction assays to characterize the presence and function of these receptors on cells of the CNS. These methods are useful in identifying the properties of chemokine receptors, including types and numbers of chemokine receptors as well as the battery of signal transduction cascades they activate. As new chemokine receptors are discovered it will be appropriate to use these methods to establish their
presence and function in the CNS. In the long term, the development of methodological approaches that allow measurement of chemokine-dependent cell signaling in vivo or in situ will enhance our understanding of the mechanisms of these molecules in the intact CNS. As important, it will be necessary to decipher the physiological end points associated with chemokine receptor stimulation of specific signaling pathways.
References [1] A.V. Andjelkovic, D. Kerkovich, J. Shanley, L. Pulliam, J.S. Pachter, Glia 28 (1999) 225–235. [2] G. Banisadr, E. Dicou, T. Berbar, W. Rostene, A. Lombet, F. Haour, J. Neuroimmunol. 110 (2000) 151–160. [3] R. Horuk, A.W. Martin, Z. Wang, L. Schweitzer, A. Gerassimides, H. Guo, Z. Lu, J. Hesselgesser, H.D. Perez, J. Kim, J. Parker, T.J. Hadley, S.C. Peiper, J. Immunol. 158 (1997) 2882– 2890. [4] X. Wang, X. Li, D.B. Schmidt, J.J. Foley, F.C. Barone, R.S. Ames, H.M. Sarau, Mol. Pharmacol. 57 (2000) 1190–1198. [5] J. Hesselgesser, M. Halks-Miller, V. DelVecchio, S.C. Peiper, J. Hoxie, D.L. Kolson, D. Taub, R. Horuk, Curr. Biol. 7 (1997) 112–121. [6] D. Maciejewski-Lenoir, S. Chen, L. Feng, R. Maki, K.B. Bacon, J. Immunol. 163 (1999) 1628–1635. [7] S. Chen, D. Luo, W.J. Streit, J.K. Harrison, J. Neuroimmunol. 133 (2002) 46–55. [8] B.L. Daugherty, S.J. Siciliano, M.S. Springer, Methods Mol. Biol. 138 (2000) 129–134. [9] D.J. Dairaghi, E.R. Oldham, K.B. Bacon, T.J. Schall, J. Biol. Chem. 272 (1997) 28206–28209. [10] J.K. Harrison, Y. Jiang, S. Chen, Y. Xia, D. Maciejewski, R.K. McNamara, W.J. Streit, M.N. Salafranca, S. Adhikari, D.A. Thompson, P. Botti, K.B. Bacon, L. Feng, Proc. Natl. Acad. Sci. USA 95 (1998) 10896–10901. [11] S.A. Boehme, F.M. Lio, D. Maciejewski-Lenoir, K.B. Bacon, P.J. Conlon, J. Immunol. 165 (2000) 397–403. [12] M. Mellado, C. Martinez-A., J.M. Rodriguez-Frade, Methods 27 (2002) 349–357.
Chemokine receptor binding and signal transduction in native cells of the central nervous system Christopher N. Davis,a Shuzhen Chen,a Stefen A. Boehme,b Kevin B. Bacon,c and Jeffrey K. Harrisona,* a
c
Department of Pharmacology & Therapeutics, University of Florida College of Medicine, Gainesville, FL 32610-0267, USA b Genicon Sciences, Inc., 11535 Sorrento Valley Road, San Diego, CA 92121, USA Department of Biology, Bayer Yakuhin, Ltd., Kyoto Research Center, 6-5-1-3, Kunimidai, Kizu-cho, Soraku-gun, Kyoto 619-0216, Japan Accepted 10 December 2002
Abstract Chemokine receptors belong to the superfamily of seven-transmembrane-spanning, G-protein-coupled receptors, and their expression by central nervous system cells is clearly documented. As this gene family has become the target of novel therapeutic development, the analysis of these receptors requires radioligand binding techniques as well as methods that entail assessing receptor stimulation of signal transduction pathways. Herein, we describe specific protocols for measuring radiolabeled chemokine binding to their cognate receptors on cultured glial cells as well as to receptors expressed in heterologous cell systems. Multiple downstream signaling pathways, including intracellular calcium influx and receptor-dependent kinase activation, are associated with chemokine receptor stimulation. Protocols for measuring these signaling events in chemokine-receptor-expressing cells are also presented. 2003 Elsevier Science (USA). All rights reserved. Keywords: Radioligand; Iodination; Receptor; Microglia; Calcium; MAPK; Akt/PKB; Immunoprecipitation; Western blot
1. Introduction G-protein-coupled receptors (GPCRs) embody a large and diverse gene family. Historically they are well represented as targets of the pharmaceutical industry in the development of novel pharmacologic agents. The characterization of new therapeutics requires a multifaceted approach, including a range of pharmacologically based approaches that include radioligand binding techniques as well as analysis of the signal transduction processes activated by specific receptors. In recent years, the chemokine receptor family has become the object of intense study, as this receptor subfamily poses unique opportunities for drug development. The use of radiolabeled chemokine binding assays has proved necessary in characterizing the biological functions of chemokine receptors, and these studies often complement information gained from functional assays. Radiolabeled *
Corresponding author. Fax: 1-352-392-9696. E-mail address: [email protected]fl.edu (J.K. Harrison).
chemokine binding techniques have proven absolutely critical for identifying receptors expressed in heterologous cell expression systems and native cells, including cells resident in the central nervous system (CNS). Methods related to measuring chemokine binding to cultured cells of the CNS parallel techniques used for study of chemokine receptor binding to peripheral leukocyte populations as well as binding methods utilized for study of other GPCRs, most notably receptors for peptide ligands. Like many peptide ligand GPCRs, the endogenous chemokines are agonists for their respective receptors, thus binding assays can be sensitive to functional states of the receptor. Furthermore, the often promiscuous nature of chemokine receptor binding profiles with overlapping selectivities lends difficulties to the interpretation of data. Here we discuss methodological protocols for measuring chemokine receptors on cultured cells of the CNS as well as those expressed in heterologous expression systems. Specific protocols for assessing downstream signaling events associated with chemokine receptor activation are also presented.
1046-2023/03/$ - see front matter 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S1046-2023(02)00356-0
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2. Chemokine receptor binding Little information is available on chemokine binding properties of native brain cells or tissues. Table 1 outlines studies in which methods of radiolabeled or biotinylated chemokine binding to whole cells, membranes, or brain sections were utilized. Specific biotinylated MCP-1 and MIP-1a binding sites have been identified on cultured human astrocytes [1]. Using unlabeled chemokines to compete for these binding sites, it was determined that these MCP-1 and MIP-1a binding sites present on these glial cells displayed pharmacological properties similar to those of CCR1, CCR2, and CCR5. Immunocytochemistry using anti-chemokine receptor antibodies was also used to demonstrate the presence of these receptors in the astrocyte cell cultures, thus validating the binding data. The properties and distribution of 125 I-SDF-1a binding to CXCR4 in rat brain sections have been characterized [2]. Competition and kinetic binding studies revealed high-affinity SDF-1a binding sites in specific brain structures. Duffy antigen receptor for chemokines (DARC) has been demonstrated to be present on Purkinje cells of human cerebellum [3]. Immunoreactive DARC, detected by monoclonal antibodies (mAb) (Fy6), and radiolabeled MGSA binding established the presence of this chemokine binding protein in membrane fractions from human cerebellum. The binding of 125 I-MGSA was blocked by Fy6, and not by a mAb to CXCR2, and was displaced by an excess of unlabeled ligands. CXCR2 was found on projection neurons in many regions of the brain using mAbs specific for this receptor. CXCR3 expression in brain tissue in a model of rat focal stroke has been characterized by IP-10 binding analysis [4]. Cortical membranes of sham rats and those exposed to focal brain ischemia displayed
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specific binding of 125 I-IP-10 that was displaced by unlabeled chemokine. Radiolabeled chemokine binding has also been demonstrated in differentiated human neurons derived from the cell line NTera 2 (hNT cells). Displacement of 125 I-interleukin-8, 125 I-SDF-1, and 125 IMIP-1a by varying concentrations of unlabeled chemokine in the hNT neurons has been shown [5]. The presence of CX3CR1 on rat astrocytes and microglia has been determined by the specific and saturable binding kinetics of 125 I-labeled fractalkine [6,7]. Regarding CX3CR1 on astrocytes, no significant differences were seen in competition binding using unlabeled full-length fractalkine or fractalkine chemokine domain. In addition, steady state binding was blocked by preincubation of the cells with anti-CX3CR1 Ab. When developing protocols for measuring specific binding of chemokines to their receptors and, most importantly, for proper interpretation of the resultant data, it is necessary to consider the following. Receptor binding should be specific, time-dependent, and reversible. Specificity of chemokine binding is determined by the following experiments: (1) binding of the chemokine of interest and not an irrelevant protein should be observed, (2) saturation of binding should be achieved with high affinity, and (3) competitive inhibition should be observed by unlabeled chemokine homologues. Measuring the rate and extent of chemokine binding to native tissue or recombinant cells can provide information on the number of binding sites, as well as their affinity and accessibility. The use of chemokine- and/or chemokine receptor-specific antibodies to inhibit binding is also appropriate in determining the specificity of the ligand/receptor interaction. Given that some chemokines bind to multiple receptors, the use of the latter type of antibody in the binding analysis can aid in
Table 1 Methods used for chemokine binding CNS tissue
Ligand
Competitors
Whole cell binding on astrocytes Slide-mounted coronal sections of brain
Biotinylated MCP-1 and MIP-1a 125 I-SDF-1a
MCP-3, MIP-1b; and RANTES SDF-1a
Membranes from cerebellum and cerebral cortex Cortical membranes
125
I-MGSA
IL-8, RANTES, MCP-1
125
I-IP-10
IP-10
NTera 2 neurons
125
Whole cell binding on astrocytes Whole cell binding on microglia
I-SDF-1, 125 I-IL-8, and 125 I-MIP-1a 125 I-Fkn
SDF-1, MGSA, and MIP-1a Fkn or Fkn-CD
125
Fkn-CD
I-Fkn
Binding buffer
Ref. [1]
MEM, 1% BSA, 250 KIU/ml Trasylol, and 0.25 mM Tris–HCl, pH 7.4 PBS 50 mM Hepes, pH 7.4, 1 mM CaCl2; 4 mM MgCl2; and 0.1% BSA PBS 25 mM Hepes, pH 7.4, 1 mM CaCl2; 4 mM MgCl2 , and 0.1% BSA HBSS, 0.1% BSA
[2]
[3] [4]
[5] [6]
[7]
IL-8, interleukin 8; MEM, minimal essential medium; BSA, bovine serum albumin; PBS, phosphate-buffered saline; HBSS, HanksÕ balanced salt solution.
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the identification of the specific chemokine receptor. Of course, immunohistochemical analysis using specific receptor antibodies can provide alternative and complementary information in determining which chemokine receptor is present. Three general approaches are normally used to characterize binding of a ligand to its cognate receptor. These include kinetic, saturation, and competition binding analyses. In kinetic analysis, rates of association and dissociation of the ligand to and from the receptor are measured. From this, affinity constants can be calculated. In saturation binding experiments, the concentration of the labeled ligand is varied and binding to the receptor at steady state is determined. The relationship between binding and ligand concentration allows direct determination of the ligand affinity (Kd ), as well as the total number of binding sites (Bmax ) in the receptor preparation, i.e., cells or membranes. Competitive binding experiments measure the steady-state binding of a single concentration of radioligand in the presence of various concentrations of an unlabeled competitor. Competition binding experiments have significant advantages compared to theoretically simpler saturation binding experiments. In saturation experiments, the signal-to-noise ratio decreases with increased ligand concentration, and the binding information gained is that of the labeled analog, not the natural ligand [8]. Competition binding experiments should be designed so that concentrations of both the receptor and the ligand are kept as low as possible and always well below the Kd values. Experimentally, the need to keep both the level of nonspecific binding and the signal-to-noise ratio low often dictates the concentration of receptor and ligand used in the assay. These three general experimental approaches for studying the binding of a ligand to a receptor are complementary. Preliminary kinetic binding experiments should be performed in order to determine the time at which the receptor/ligand interaction has reached steady state. This is valuable information for proper design and interpretation of results from both saturation and competition binding analysis. When binding ligand to whole cells, one must also take receptor internalization into consideration. Binding at lower temperatures will slow the energy-dependent process of receptor internalization. However, it will also take longer for the ligand binding reaction to reach steady state. This shift in time to reach steady state should be taken into consideration when performing binding analysis at lower temperatures. An alternative approach is to measure the binding in the presence of an inhibitor of mechanisms of internalization. Given that this process is active and requires energy, mitochondrial poisons such as azide can be included. Concentrations of sodium azide that are sufficient to inhibit receptor internalization are in the range of 0.01 to 0.1%.
Binding of certain chemokines to their respective receptors can be sensitive to the specific incubation buffer components. We have determined that fractalkine will bind to CX3CR1 and MCP-1 to CCR2 in a buffer system containing HanksÕ balanced salt solution (HBSS) and bovine serum albumin (BSA). In addition, these chemokines bind their respective receptors equally well in a Hepes-based binding buffer (described below). However, we could not detect any specific 125 I-MIP-1a binding to CCR5 in the HBSS/BSA-based buffer system despite readily detecting this interaction in the Hepes binding buffer. The dependence of binding buffer components on the binding of other chemokines to their respective receptors is also documented. For instance, the level of 125 I-SDF-1a nonspecific binding was determined to be dependent on the concentration of salt in the binding buffer; high concentrations of salt in the buffer lead to high nonspecific binding [2]. Eotaxin binding to eosinophils, as well as functional responses of the cells to this chemokine, is also influenced by salt concentration and pH [9]. Thus, if embarking on characterizing a novel chemokine receptor in a given cell or tissue, it is important to establish optimal conditions for readily detecting specific chemokine binding. Our group has successfully characterized radiolabeled chemokine binding to freshly isolated microglia cells from newborn rats. Specific methods for radiolabeling chemokines, culturing microglia, and binding to whole cells are described in detail below. A section on chemokine binding to recombinant cells is included as well.
3. Analysis of chemokine-mediated signal transduction pathways Chemokines can activate multiple signaling pathways upon binding their appropriate receptors. For instance, fractalkine (CX3CL1) binding to the CX3CR1 receptor in microglial cells initiates multiple signaling cascades resulting in cell migration [6,10] and increased cell viability by modulating the functional status of Bcl-2 family proteins [11]. These downstream effects are triggered by the activation of receptor-mediated signaling proteins, such as Ga, Gbc, and receptor-associated kinases [12]. The fractalkine receptor, CX3CR1, is a G-proteincoupled receptor expressed by microglia and astrocytes in the CNS that induces microglial cell migration upon ligand binding. Fractalkine binding to CX3CR1 induces a rapid, transient mobilization of intracellular calcium, and this response can be blocked by pretreating cells with pertussis toxin, suggestive of a Gai -mediated signaling process [6]. This response can be measured experimentally by fluorimetry. Downstream signaling events involved in microglial cell migration include
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activation of the Rho family member Rac1. This, in turn, leads to activation of the PAKs (p21-activated serine/threonine kinases) in a process involving binding to the vav and nck adapter proteins. These protein/ protein interactions can be demonstrated by immunoprecipitation and Western blot analysis. Further downstream the signaling pathway leading to microglial cell migration is the activation of p42/p44 mitogen-activated protein (MAP) kinases, which occurs within 1 min of fractalkine stimulation and is dependent on PAK activation (K.B.B., unpublished observations). Activation of myosin light chain kinase is dependent on p42/p44 MAP kinase, and this ultimately results in the phosphorylation of the myosin light chain and actin rearrangement in microglial cells (S.A.B. and K.B.B., unpublished observations). Activation of the PAKs, p42/p44 MAP kinase, and myosin light chain kinase can be experimentally demonstrated by immunoprecipitation followed by in vitro kinase assays or, in the case of the MAP kinases, by Western blotting analysis probing with phosphorylation state-specific antibodies.
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Fractalkine also has the ability to act as a survival factor for microglial cells cultured in the absence of MCSF/GM-CSF and block Fas-mediated microglial cell death [6,10]. This occurs in part due to the activation of the phosphatidylinositol 3 (PI-3)-kinase/protein kinase Ba signal transduction pathway. Activation of protein kinase Ba leads to the phosphorylation and blockade of the proapoptotic functions of the Bcl-2 family member Bad. Furthermore, fractalkine upregulates the expression of antiapoptotic protein Bcl-XL, and inhibits the cleavage of the antiapoptotic protein Bid [11]. Taken together, these observations demonstrate that fractalkine binding to CX3CR1 in microglial cells results in the activation of a number of distinct signaling pathways leading to a range of disparate functional outcomes. Fig. 1 depicts a schematic of the pathways and outlines the general experimental approaches used to monitor them. In the sections to follow, we describe in detail methods to assess the activation of these signal transduction pathways.
Fig. 1. Schematic depicting signal transduction pathways regulated by chemokine receptors in microglia. The general methodological approaches used to measure them are highlighted.
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4. Specific methods 4.1. Primary microglial cell culture 4.1.1. Materials The following materials are used. • Poly-l-lysine (Sigma, St. Louis, MO, USA): 0.01 g/ liter in H2 O • Solution D: 137 mM NaCl, 5.4 mM KCl, 0.2 mM NaH2 PO4 , 0.2 mM KH2 PO4 , 1 g/liter glucose, 20 g/ liter sucrose, 0.25 lg/liter Fungizone, 106 U/liter penicillin/streptomycin, pH 7.4 • Complete medium: DulbeccoÕs modified EagleÕs medium (Life Technologies, Rockville, MD, USA) containing 10% fetal bovine serum (Life Technologies) 4.1.2. Methods Just prior to establishment of the cultures, flasks are coated with poly-l-lysine and incubated at 37 C for at least 1 h (20 ml in a 175-cm2 cell culture flask). Before use, poly-l-lysine is aspirated and the flasks are washed with solution D once. Cerebra are dissected from newborn Sprague–Dawley rats, stripped of meninges, and mechanically minced with a scalpel blade in solution D. The tissues are then digested with 0.25% trypsin solution D (10 ml total for 10–15 brains) in a conical tube at 37 C on a bidirectional rotator for 30 min. An equal amount of complete medium is added to stop the trypsin and the mixture is passed through 130-lm Nitex filter (Tetko, Inc., Brairdiff Manor, NY, USA). The mixture is then centrifuged at 400g for 10 min. The pellet is resuspended in complete medium, filtered through a 40lm Nitex filter, and plated into T175 flasks (Sarstedt, Newton, NC, USA) at a density of 1.5 brains per flask. The cultures are incubated with complete medium at 37 C, 5–8% (v/v) CO2 . After 3 days, the medium is changed and the cells are left for another 4–7 days to favor the proliferation of microglial cells. To harvest microglial cells, the flasks are shaken on an orbital shaker at 100 rpm for 60 min. The supernatant is collected and centrifuged at 400g for 10 min. The cells are counted using a hemacytometer and replated into appropriate cell culture plates in complete medium and at the indicated cell density for each experiment. Plates are incubated at 37 C, 5–8% CO2 for 60 min. The medium is aspirated to remove any nonadherent cells and replaced with fresh complete medium. The adherent cells contain more than 95% microglia. 4.2. Preparation of
125
I-labeled chemokines
Although many 125 I-labeled chemokines are available from commercial sources, we generally radiolabel our own chemokines. The advantage of this lies primarily in a cost savings. However, our methods typically yield chemokine peptides with specific activities approxi-
mately 1/10 (200 Ci/mmol) of the specific activity of the commercially prepared reagent. For binding to recombinant receptors in heterologous cell expression systems or even primary cultures of rat microglia, this is not a problem. However, if the investigator wishes to characterize a chemokine receptor that is expressed at a relatively low level, the use of high specific activity radiolabeled chemokine will enhance receptor detectability. This is especially important if the assay suffers from high nonspecific binding. The general method of chemokine iodination involves incubating the chemokine with sodium iodine in the presence of an oxidizing agent. We use Iodo-Beads (Pierce, Rockford, IL, USA) as the source of this oxidizing agent. Iodo-Beads utilize a nonporous polystyrene bead which has N-chlorobenzenesulfonamide covalently linked. The chief advantage of the IodoBead, as opposed to soluble chloramine-T, for instance, is that the reaction is more easily terminated by simply pipetting the reaction away from the bead. The extent of iodination is generally reproducible and easily controlled by varying the incubation time. Our specific protocol for iodinating chemokines, which we have used successfully to label fractalkine, SDF-1, MIP-1a, and MCP-1 is as follows. 4.2.1. Solutions and reagents The following materials are used. • Iodo-Beads (Pierce) • 100 mM phosphate buffer, pH 7.6 (PB) • HBSS with 0.1% BSA • 0.5 M potassium iodide (KI) • 5 lg chemokine peptide, carrier free [dissolved in phosphate-buffered saline (PBS) at 1 lg=ll] • Na 125 I (Perkin–Elmer, Palo Alto, CA, USA) • Desalting column (D-Salt Dextran, plastic desalting column, 5 ml, Pierce), preequilibrated with 30 ml of HBSS/0.1% BSA 4.2.2. Procedure All procedures are conducted at room temperature, in a well-ventilated biosafety hood, behind sufficient lead shielding in order to minimize exposure to the radioactive iodine. Place one Iodo-Bead into a 1.5-ml Eppendorf tube. Add 1 ml of 0.1 M PB, pH 7.6, briefly vortex, and then remove bead by shaking onto a dry Kimwipe. Transfer washed bead to a fresh 1.5-ml tube. Add 100 ll of PB followed by 1 mCi of Na125 I (approximately 3 ll). Allow reaction to proceed for 6 min. Add 5 lg of chemokine peptide (5 ll volume) and allow reaction to continue for an additional 9 min. At the end of this incubation, carefully pipet the reaction out of the tube (leaving the bead behind) and place into a tube containing 11 ll of 0.5 M KI. A 1-ll sample is pulled from this mixture and added to 500 ll of HBSS/0.1% BSA. Fifty microliters of 100% trichloroacetic acid (TCA) is
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then added and the reaction is allowed to sit on ice for 5 min. The TCA solution containing the aliquot of the iodination reaction is filtered through a GF/C filter and rinsed with ice-cold 10% TCA, and the radioactivity associated with the filter is determined using a gamma counter. The specific activity of the radiolabeled peptide is determined by dividing the counts associated with the filter by the amount of chemokine subjected to the TCA precipitation. In this example, 50 ng of chemokine was analyzed. The molecular mass of the particular chemokine and the specific activity of the 125 I (2200 Ci/mmol) allow one to convert the specific activity of the labeled chemokine into units of Ci/mmol. The volume of the remainder of the iodination reaction is doubled by the addition of 125 ll of HBSS/0.1% BSA. This solution is mixed and carefully applied to a preequilibrated desalting column. Ten individual fractions are collected by sequentially adding 500 ll of HBSS/0.1% BSA and moving a rack of tubes prior to each 500-ll addition. From each fraction, a 5-ll aliquot is analyzed by gamma spectroscopy. Knowing the specific activity of the labeled peptide allows one to determine the amount of labeled peptide in the 5-ll aliquot and hence its concentration. 4.3. Binding to native cells Whole cell binding is performed on 500,000 cells/ well. Microglial cells are seeded into 12-well cell culture plates (Corning). The cells are grown at 37 C, 5–8% CO2 for 1–2 days before the binding assay. Appropriate incubation buffer containing labeled and/or unlabeled chemokine is prepared at room temperature just prior to cell washing. Cells are washed once with 1 ml room temperature sPBS and incubated in 500 ll incubation buffer (HBSS, 0.1% BSA, pH 7.4) at room temperature for 60 min (or whatever has been experimentally determined to be steady state). Cells are washed three times with 1 ml ice-cold incubation buffer (this step is performed as quickly as possible to prevent disassociation of ligand). Cells are lysed with 500 ll 0.2 M NaOH. The cell lysates are collected into tubes and are counted on an appropriate gamma counter. 4.4. Binding to recombinant receptors in heterologous cell systems 4.4.1. Materials Typically, receptor is expressed in recombinant cells and the cells are passed with antibiotic to select for those containing receptor. Binding is usually performed on whole cells seeded directly from flasks of cells. Unlabeled chemokines are available from many sources and are generally recombinantly expressed or generated through peptide synthesis. The following materials are used:
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• Incubation buffer: 50 mM Hepes, pH 7.2, containing 0.5% BSA, 5 mM MgCl2 , 1 mM CaCl2 , 10 lg/ml each of chymostatin, leupeptin, and aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride (PMSF). • All inhibitors are maintained as 1000 stocks and added to buffer just prior to use. Chymostatin stock is in dimethyl sulfoxide (DMSO). Leupeptin and aprotinin stocks are in water. PMSF stock is in methanol. • Wash buffer is 25 mM Hepes, pH 7.2, containing 0.5 M NaCl. 4.4.2. Methods Whole cell binding is performed on 800,000 cells/ well. Cells are seeded into 12-well cell culture plates (Corning) containing appropriate medium at a density of 500,000 cells/well. Cells are grown overnight in a 37 C, 5% CO2 cell incubator. Appropriate incubation buffer containing labeled and/or unlabeled chemokine is prepared at room temperature just prior to cell washing. Total volume for each well is 500 ll. We typically dilute labeled chemokine into appropriate polypropylene or polystyrene tubes and split into 1.5-ml aliquots (for triplicate wells). Unlabeled competitor would be added directly to 1.5-ml aliquots to produce appropriate dilutions. Wells are washed once with 1 ml room temperature sPBS approximately 24 h after seeding. Five hundred microliters of binding solution (containing both labeled and unlabeled chemokine) is pipetted onto the cells. The plates are incubated at room temperature for 60 min (in our hands 60 min has been experimentally determined to be steady state). After 60 min, cells are washed three times with 1 ml ice-cold wash buffer. This step is performed as quickly as possible. Cells are then lysed with 500 ll 0.2 M NaOH and the contents of the well is collected into scintillation tubes and counted on an appropriate gamma counter. 4.5. Signaling: preparing cell lysates for biochemical analysis 4.5.1. Solutions and reagents Cell lysis solution consists of 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40 (Calbiochem), 0.25% sodium deoxycholate, 5 mM EDTA, and protease and phosphatase inhibitors (1 mM PMSF, 10 lg/ml aprotinin, 10 lg/ml leupeptin, 1 mM sodium orthovana_ M tetrasodium pyrophosphate, 1 mM EGTA, date, 1m 100 lg/ml b-glycerophosphate). The protease and phosphatase inhibitors should be made in 100 or 1000 stock solutions, aliquoted, and kept frozen until ready for use. Complete lysis solution, containing the protease and phosphatase inhibitors, should be at 4 C prior to use. It should be noted that this cell lysis cocktail does not lyse the nuclear membrane, thus, nuclei remain intact.
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4.5.2. Procedure Cells to be examined should be pelleted at 4 C, transferred to a 1.5-ml Eppendorf tube in 1 ml of icecold PBS, and centrifuged for 5 min at 4 C to pellet the cells. After all of the supernatant is decanted, the cells should be vigorously resuspended in complete cell lysis solution and incubated on ice for 15–60 min with periodic vortexing. The volume of lysis solution used should be minimal (e.g., 20 ll) to ensure a high protein concentration. After the cells are incubated on ice, the lysate should be centrifuged at 12,000g for 15 min at 4 C to pellet the nuclei and other insoluble material. The lysates, as well as any unused cell lysis solution, can be frozen at this time at )20 C. Alternatively, the protein concentration in each sample can be quantitated, using the excess lysis solution as a background standard for each experimental sample. When doing time-course experiments or studies having a large number of experimental conditions, the cells should be placed into a tube on ice containing a large excess of ice-cold PBS to effectively stop all cellular processes at the desired point. From this point on, all of the samples can be processed together. 4.6. Western blotting procedure On isolation and quantification of the protein levels in the cell lysates, the samples should be diluted with an equal volume of 2 Laemmli sample buffer and heated to 100 C for 5 min and either frozen or loaded onto a polyacrylamide gel (Novex/Invitrogen, San Diego, CA, USA). If frozen samples are to be run, they should be heated to 37 C for 5 min to ensure that all the SDS is dissolved before the gel is loaded. The samples should contain sufficient protein to allow for detection by Western analysis (25–50 lg). Attention must be given at this point to whether denaturing or nondenaturing conditions should be used and the percentage polyacrylamide gel to be used. As for the latter point, it should be taken into account which proteins will be blotted for, as the membranes can undergo multiple antibody probings. The percentage of polyacrylamide gel should be chosen to give sufficient separation of the proteins probed for. It is also advisable to use molecular weight markers with standards in the molecular weight range of the proteins to be examined. Following protein separation by electrophoresis, protein is transferred to a nitrocellulose membrane using the XCELL II system (Novex/Invitrogen). Following transfer, the nitrocellulose is washed briefly in water, and the nonspecific protein binding sites are blocked by incubating the membrane in blocking solution [2% BSA, 0.5% ovalbumin, 2.5% nonfat dry milk (Bio-Rad, Hercules, CA, USA), 10 mM Tris, pH 8, 150 mM NaCl, 0.2% thimerosal] for 1 h at 25 C. This step onward should be carried out on a shaker set at 70 rpm. The
membrane can then be probed by incubating the blot in 10 ml of fresh blocking solution containing the primary antibody for 1–2 h at 25 C or overnight at 4 C in a covered container. Following incubation with the primary antibody, the blot is washed in TBS-T (20 mM Tris, pH 7.5, 0.5 M NaCl, 0.05% Tween 20), three times for 5 min, and incubated with the appropriate peroxidase-conjugated secondary antibody diluted in blocking buffer for 1 h at 25 C. The filter should again be washed three times for 5 min in TBS-T, three times for 5 min in TBS (20 mM Tris, pH 7.5, 0.5 M NaCl), and once for 5 min in H2 O. The protein of interest can then be visualized using the chemiluminescent peroxidase substrate Super Signal Ultra (Pierce). For subsequent probing, the membrane can be stripped using 7 M guanidine, 50 mM glycine, 50 lM EDTA, 100 mM KCl, 20 mM 2mercaptoethanol, pH 10.8, for 10 min at 25 C, followed by three washes in H2 O. The blot can then be stored in TBS-T at 4 C indefinitely or placed in blocking solution to start another probing. 4.7. Immunoprecipitation protocol Proteins can be specifically isolated by immunoprecipitation (IP) to examine protein–protein interactions or by in vitro kinase assays. Immunoprecipitation assays are carried out by taking cell lysates of all the samples to be examined, with equal protein content, and incubating them with 1–5 lg of the antibody specific for the protein of interest conjugated to protein A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The incubations should be carried out in low volumes rotating in 1.5-ml Eppendorf tubes at 4 C. The immunoprecipitation step should proceed for a minimum of 1 h, but may go overnight. As controls, one immunoprecipitation should be done with fresh, complete lysis buffer only, and another IP should be done with a cell lysate but only protein A/G beads, with no conjugated antibody. These conditions should control for nonspecific binding of protein from either the cells or the lysis buffer to the beads. After the cell lysates are incubated with antibody-coated beads, the beads should be gently pelleted (400g, 1–2 min) and the supernatant transferred to a fresh tube and saved for possible further analysis. The pelleted beads should be washed once in a 100–500ll volume of fresh, complete lysis buffer. After the beads are spun down and the supernatant is removed, the IP should be washed again in fresh, complete lysis solution containing 0.5 M NaCl. After the beads are gently centrifuged and the wash buffer is removed, the immunoprecipitation is ready for further analysis. For subsequent Western analysis, the beads should be resuspended in 20 ll of 2 Laemmli sample buffer and boiled for 5 min. This heating step will dissociate both the immunoprecipitated protein from the capture antibody and the antibody from the agarose bead. Post-
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heating, the samples should be briefly centrifuged to concentrate the sample and either loaded on an SDS– PAGE gel, with a small bore pipette tip to exclude the beads, for immediate analysis or frozen at )20 C indefinitely. 4.8. In vitro kinase assay In vitro kinase assays can be used to examine the activity of various protein kinases involved in numerous intracellular signaling pathways. The first protocol below (the following two sections) is used for assaying the activity of the p42/p44 MAP kinases, and a protocol for assaying the activity of protein kinase Ba (also known as Akt) follows. 4.8.1. Solutions and reagents The following materials are used. • p42/p44 MAP kinase washing buffer (used to wash the immunoprecipitated ERK kinases): 25 mM Tris–HCl, pH 7.4, 40 mM MgCl2 , 137 mM NaCl, and 10% (v/v) glycerol • p42/p44 MAP kinase assay buffer: 42.5 mM Hepes, 42.5 mM MgCl2 , 0.21 mM ATP, 50 mM myelin basic protein (Upstate Biotechnology, Inc., Lake Placid, NY, USA), and 50 lCi [c-32 P]ATP (>3000 Ci/mmol sp act) (Amersham, Arlington Heights, IL, USA). 4.8.2. Procedure Following immunoprecipitation of the ERK1 and ERK2 kinases and subsequent washes in cell lysis solution 0:5 M NaCl (see Calcium mobilization analyses), the immunoprecipitated material is washed once in MAP kinase washing buffer. At this point, ERK enzyme activity is assessed in an in vitro kinase assay. The immunoprecipitation beads are suspended in 20 ll of kinase assay buffer for 30 min at 30 C. Following this incubation, an equal volume of 2 Laemmli buffer is added, and the samples are heated to 100 C for 5 min, briefly centrifuged, and cooled on ice. The samples may be frozen for future analysis or directly loaded onto a 16% Tris–glycine gel (Novex/Invitrogen) using the XCELL II apparatus (Novex/Invitrogen). Proteins are then transferred to nitrocellulose membranes using the Western blotting protocol (see Western blotting procedure). Precautions should be taken, however, as the samples are radioactive ð32 PÞ. Phosphorylation of the myelin basic protein substrate can be visualized using Biomax MR autoradiography film (Eastman Kodak, Rochester, NY, USA). Multiple exposures of the autoradiograph should be taken, to allow for accurate quantitation of the amount of phosphorylated myelin basic protein in each sample by densitometry. Analysis of the amount of phospho-ERKs in each sample can also be determined by probing blots with anti-phospho-MAP kinase antibodies (Upstate Bio-
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technology, Inc.) using the Western analysis protocol outlined above. Equal loading of the immunoprecipitates or cell lysates can be assessed by stripping the Western blot of the phospho-MAP kinase antibodies and reprobing the blot with anti-MAP kinase antibodies (Upstate Biotechnology, Inc.), as outlined under Western blotting procedure. 4.8.3. Solutions and reagents for measuring protein kinase Ba activity The following materials are used. • Protein kinase Ba wash buffer: 20 mM Hepes, pH 7.4, 10 mM MgCl2 , and 10 mM MnCl2 • Protein kinase Ba reaction buffer: 20 mM Hepes, pH 7.4, 10 mM MgCl2 , 10 mM MnCl2 , 0.05 mg/ml histone 2B (Boehringer Mannheim, Mannheim, Germany), 5 lM ATP, 1 mM dithiothreitol, and 10 lCi ½c-32 PATP (Amersham). 4.8.4. Procedure for protein kinase Ba in vitro kinase assay The procedure for assaying protein kinase Ba is similar to the MAP kinase assay, with just a few changes outlined below. Immunoprecipitations of protein kinase Ba are washed twice in complete cell lysis buffer, once in H2 O, and once in protein kinase Ba wash buffer. After all of the supernatant from the last wash is decanted, the immunoprecipitation beads are resuspended in 20 ll of protein kinase Ba reaction buffer and incubated at 30 C for 30 min. At this point, the samples can be handled in the same manner as in the MAP kinase assay, again using precaution as the sample contains 32 P. The phosphorylated histone 2B can be visualized by autoradiography and quantitated by densitometry. The amount of active [phosphorylated protein kinase Ba at positions 308 (threonine) and 473 (serine)] can also be assayed by probing Western blots with anti-phosphoprotein kinase Ba antibodies (Upstate Biotechnology), and the total amount of protein kinase Ba can be determined by probing the stripped blot with anti-protein kinase Ba antibodies (Upstate Biotechnology). The molecular mass of protein kinase Ba is 60 kDa. 4.9. Calcium mobilization analyses Chemokine binding to cells expressing cognate chemokine receptors results in a rapid, transient mobilization of Ca2þ . This event can be measured and quantitated using intracellular, UV-excitable, ratiometric Ca2þ indicators, such as indo-1 AM and fura-2 AM (Molecular Probes, Eugene, OR, USA), and a fluorimeter. First, the cells are loaded with esters of indo-1 or fura-2, which passively diffuse inside the cell. Once inside, the Ca2þ indicators (indo-1 AM and fura-2 AM, in which the AM represents an acetoxymethyl group) are cleaved by intracellular esterases to yield
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cell-impermeant fluorescent indicators. Once intracellular stores of Ca2þ are released or extracellular calcium enters the cells, the emission wavelength of the calcium indicator changes. It is important to note that both indo-1 AM and fura-2 AM need to be resuspended in anhydrous DMSO at a concentration of 1 mg/ml. Microglial cells are isolated, resuspended in 1 ml of complete medium, and incubated with the calcium-sensitive fluorescent dye indo-1 AM (3 lmol/liter final concentration). The labeling procedure is carried out for 45 min at room temperature, with the cells in a 15-ml conical tube wrapped in aluminum foil (to shield from light), on a rotating platform. The cells are subsequently pelleted and washed once in prewarmed HBSS containing 1% BSA. The labeled cells are then resuspended in 1 ml of prewarmed HBSS/1% BSA and kept in a dark environment until used. The cells are then placed in 1-ml cuvettes containing a small stir bar, at a concentration of 1 106 cells/ml of HBSS/1% BSA in the cuvette and analyzed using a PTI fluorimeter (South Brunswick, NJ, USA). The results obtained are from the ratios of dual emission (405/490 nm), and the results represent nanomolar shifts in calcium mobilization.
5. Concluding remarks As we unravel the mysteries of the role of chemokines in CNS physiology it will continue to be necessary to use radioligand binding techniques and signal transduction assays to characterize the presence and function of these receptors on cells of the CNS. These methods are useful in identifying the properties of chemokine receptors, including types and numbers of chemokine receptors as well as the battery of signal transduction cascades they activate. As new chemokine receptors are discovered it will be appropriate to use these methods to establish their
presence and function in the CNS. In the long term, the development of methodological approaches that allow measurement of chemokine-dependent cell signaling in vivo or in situ will enhance our understanding of the mechanisms of these molecules in the intact CNS. As important, it will be necessary to decipher the physiological end points associated with chemokine receptor stimulation of specific signaling pathways.
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