Published online before print October 3, 2008
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* Division of Allergy and Immunology, Department of Pediatrics, and
Department of Anesthesiology and Critical Care Medicine, University of Pennsylvania School of Medicine and the Joseph Stokes Jr. Research Institute, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
1 Correspondence: Immunology Section, Room 1208A, Abramson Research Center, Children’s Hospital of Philadelphia, 3615 Civic Center Blvd., Philadelphia, PA 19104, USA. E-mail: kilpatrick{at}email.chop.edu
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Key Words: chemokines • MAPKs • calcium mobilization • receptor phosphorylation
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The biologic responses to SP are mediated by the SP-preferring neurokinin-1 receptor (NK1R), which is a G-protein-coupled receptor (GPCR) characterized by seven transmembrane domains and is a member of the pertussis toxin-insensitive Gq/11 family [19 ]. NK1R has been identified on immune cells [4 , 5 , 20 21 22 23 24 25 ], including monocytes/macrophages [5 , 26 ], neutrophils [27 ], T and B lymphocytes [22 , 28 , 29 ], and mast cells [30 ]. In vivo studies with NK1R antagonists and gene-targeted disruption of NK1R protected the lung from inflammatory injury, demonstrating that SP proinflammatory responses are mediated through binding to NK1R [6 , 31 ].
Two isoforms of NK1R have been identified. The human NK1R gene, through retention of introns in its genomic structure, produces splice variants: a full-length form (NK1R-F) composed of 407 aa and a naturally occurring splice variant with a truncated C-terminus {truncated NK1R (NK1R-T) [10 , 32 , 33 ]}, which has a 311-aa sequence with a short carboxyl-terminal sequence, extending only 7 aa residues after the end of the seventh transmembrane segment. The remaining amino acid sequence of the NK1R-T isoform is identical to that of NK1R-F [32 ]. The binding and signaling properties of NK1R-T or NK1R mutants have been compared with those of NK1R-F expressed in different cell systems, and differences have been observed [21 , 32 , 34 35 36 37 38 ]. When NK1R-T and NK1R-F were cloned and expressed in COS cells, NK1R-T binding affinity was tenfold less than NK1R-F [32 ]. Furthermore, NK1R-T elicited only weak electrophysiological responses in Xenopus oocytes (100-fold less) as compared with NK1R-F [32 ]. In the THP-1 cell line, we demonstrated differences in SP-induced calcium (Ca2+) mobilization between NK1R-F and NK1R-T [21 ]. The implications of differential expression of NK1R isoforms in different cell types are not known, and these different isoforms may regulate diverse cellular functions.
NK1R-F is the predominant form expressed at sites in the human brain, whereas NK1R-T is widespread throughout the CNS and is the predominant form in peripheral tissues [39 , 40 ]. Although the existence and expression of NK1R-F and NK1R-T have been documented in several cell types [32 , 38 , 39 , 41 ], the differences in their expression and physiological function have not been studied in human peripheral blood monocytes. Several studies have identified NK1R in human cells of monocyte/macrophage lineage; however, it is less certain which form of the receptor is expressed in human monocytes [5 , 9 , 18 , 21 , 24 , 42 , 43 ]. It has been variously reported that human monocytes express NK1R, do not express NK1R, or express an atypical form of the receptor [5 , 28 , 44 , 45 ].
We previously identified a critical interaction among SP, NK1R, and the HIV coreceptor CCR5 in human monocyte-derived macrophages [21
, 46
]. The NK1R antagonist CP-96,345 selectively inhibited M-tropic HIVs, which use CCR5 as a coreceptor for entry, but did not prevent infection by T-tropic HIVs, which use CXCR4 as a coreceptor [46
]. Thus, there is evidence that SP enhances M-tropic HIV infection [8
, 46
, 47
], which may result from crosstalk between the NK1R and CCR5 receptors. CCR5 is also a chemokine receptor, which regulates chemotaxis and cellular activation at inflammatory sites. The chemokine receptor CCR5 interacts with the ligands CCL5 (RANTES) as well as CCL3 (MIP-1
), CCL4 (MIP-1β), CCL8, and CCL7 [48
49
50
51
]. There is limited information available about regulation of chemokines and their receptors by SP and the role of this regulation in immune cell function. SP may mediate these effects through enhancement of CCR5 receptor function. In this study, we determined which form(s) of NK1R are expressed in human peripheral blood monocytes and determined the ability of NK1R to interact with CCR5 by examining functional effects of SP on CCL5-mediated cellular responses.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The U.S. Food and Drug Administration-approved NK1R antagonist, aprepitant (Emend®), manufactured by Merck and Co. (Whitehouse Station, NJ, USA), was purchased through the Children’s Hospital of Philadelphia Pharmacy (Philadelphia, PA, USA). The NK1R antagonist was extracted and purified by Dr. Ivan Alferiev (Children’s Hospital of Philadelphia). Briefly, aprepitant (Emend®) was extracted from a suspension in aqueous sodium carbonate with ethyl acetate, the organic phase was clarified on a cellulose CC-31 column, the solvent was evaporated, and the residue was crystallized from heptane/ethyl acetate and dried in vacuo. The product was uniform on TLC; HPLC analysis demonstrated a purity of 99%.
Preparation of human monocytes
Human peripheral blood monocytes were obtained from healthy adult donors through the Penn Center for AIDS Research Immunology Core (Philadelphia, PA, USA). Alternatively, monocytes were isolated from heparinized venous blood (10 U/ml) obtained from healthy adult donors following informed consent, in accordance with Institutional Review Board protocols at the Children’s Hospital of Philadelphia. In brief, heparinized blood was separated by centrifugation over lymphocyte separation medium (Organon Teknika Corp., Durham, NC, USA) [5
]. The mononuclear cell layer was collected and resuspended in DMEM (Gibco Laboratories, Grand Island, NY, USA), plated onto gelatin-coated flasks for monocyte adherence, and incubated for 45 min in 5% CO2 at 37°C. The flasks were washed with DMEM to remove lymphocytes and nonadherent cells. Adherent monocytes were then detached by incubation with 5 mM EDTA in DMEM containing 20% FCS for 15 min at 37°C. Monocytes were collected, resuspended in DMEM, and cultured overnight to up-regulate CCR5 expression. These cell populations were >97% monocytes, as determined by nonspecific esterase staining and FACS analysis using mAb against CD14 (Leu-M3) and low-density lipoprotein specific for monocytes and macrophages.
RT
Total RNA was extracted from human monocytes and human embryonic kidney (HEK)293 cells using the RNeasy mini RNA kit (Qiagen, Valencia, CA, USA). Total RNA (1 ug) was subjected to RT. The final reaction mixture (20 ul) contained the following elements: 5 mM MgCl2, 1x RT buffer, 500 µM each dNTPs, 1 unit/ul recombinant RNasin, 10–15 units avian myeloblastosis virus RT (Promega, Madison, WI, USA), and 50 ng random primers. RT was performed at 42°C for 1 h. The reaction was terminated by holding the reaction mixture at 99°C for 5 min. One-tenth (2 µl) of the resulting cDNA was used as a template for real-time PCR amplification.
Real-time PCR primers
The PCR primers used for quantitative measurement of NK1R-F and NK1R-T mRNA were modified from the method of Caberlotto et al. [39
]. The sequences of the primer pair used to amplify the NK1R-F were: (sense NK1R-F, forward) 5'-TCTTCTTCCTCCTGCCCTACATC-3'; (antisense NK1R-F, reverse-967) 5'-AGCACCGGAAGGCATGCTTGAAGCCCA-3', which is specific for the NK1R-F sequence. The sequences of the primer pair for the NK1R-T were: (sense NK1R-F, forward) 5'-TCTTCTTCCTCCTGCCCTACATC-3'; (antisense NK1R-T, reverse-1083) 5'-TGGAGAGCTCATGGGGTTGGGATCCT-3'. The forward primer is identical to the one used by Caberlotto et al. [39
]. However, the reverse primers for NK1R-F and NK1R-T are located further downstream from the forward primer to avoid potential nonspecific amplification as a result of the 50% sequence homology of reverse primers of NK1R-F and NK1R-T [40
]. The sequences of the primer pair for GAPDH were: 5'-GGTGGTCTCCTCTGACTTCAACA-3 ' (sense) and 5'-GTTGCTGTAGCCAAATTCGTTGT-3' (antisense) [52
]. The specificity of the modified primer pair for NK1R-T was confirmed by SYBR Green dye dissociation curve and agarose gel electrophoresis [40
]. The primers were resuspended in Tris-EDTA buffer and stored at –30°C.
Real-time PCR assay
The MyiQ iCycler system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used for real-time PCR analysis. Thermal cycling conditions were designed as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 10 s and 60°C for 1 min. Fluorescent measurements were recorded during each annealing step. At the end of each PCR run, data were analyzed automatically by the system, and amplification plots were obtained. For each PCR, 2 µl cDNA template was added to 23 µl iQTM SYBR Supermix (Bio-Rad Laboratories, Inc.) containing the primer pair for NK1R-F or -T or for GAPDH. A NK1R-F plasmid (a gift from Dr. Norma Gerard, Harvard University, Boston, MA, USA) was used to prepare standard curves and used as a specificity control for real-time PCR. The full-length plasmid was amplified by the primer pair for NK1R-F but not by the primer pair for NK1R-T. Using serial-diluted cDNA derived from pooled human brain tissue [40
], the amplification efficiency of the NK1R-F and -T primer pairs was similar (103% for NK1R-T and 102% for NK1R-F, respectively), thus enabling use of the NK1R-F standards to measure NK1R-T expression levels. All amplification reactions were performed in duplicate, and the average comparative threshold cycle numbers of the duplicates were used to calculate the expression levels (copy numbers) of NK1R-F and -T using the standard curve generated with the NK1R-F plasmid. To control the integrity of the RNA and normalize NK1R mRNA levels in these samples, a GAPDH mRNA fragment in the RNA was also amplified using our established real-time RT-PCR with iQTM SYBR Green Supermix (Bio-Rad Laboratories, Inc.), as reported previously [52
]. To generate a total RNA standard curve with GAPDH primers, a known amount of total RNA standard extracted from pooled human monocytes was reverse-transcribed and serial-diluted (ranging from 32 to 2000 ng), and the GAPDH fragment was amplified for 40 cycles. The human monocyte samples were amplified in the same plate with the standard under the identical conditions. The quantity (ng) of total RNA in the samples was calculated automatically by The MyiQ iCycler system based on the data obtained from the total RNA standard curve. All amplification reactions were performed in duplicate, and an average RNA quantity (ng) of the duplicates was used to normalize the NK1R-F and -T mRNA levels. The expression levels of NK1R-F and NK1R-T cDNA were normalized using endogenous cDNA, GAPDH. To normalize the NK1R-F and -T mRNA levels, the NK1R-F and -T copy numbers in cell samples were divided by the total RNA quantity (ng) determined by the GAPDH real-time RT-PCR in the same sample. The NK1R-F and NK1R-T mRNA levels are expressed as the mean copy number of NK1R mRNA/ng total RNA.
Immunofluorescence staining of NK1R-T and NK1R-F in human monocytes
Differential expression of human NK1R-T and NK1R-F was evaluated using antibodies directed against a common domain (aa 140–244) present in NK1R-T and NK1R-F (Novus Biologicals, #H00006869-A01) or the C-terminal of NK1R (aa 325–407; Santa Cruz Biotechnology, SC-15323). Monocytes and HEK293 cells transfected with NK1R-F were plated onto poly-L-lysine-coated glass slides, fixed in 2% paraformaldehyde, permeabilized with 0.2% Triton X-100, and blocked with 5% donkey serum in PBS with 5% BSA. The cells were incubated with normal rabbit IgG (1:500 dilution in blocking buffer) or rabbit anti-C-terminal NK1R (1:500) overnight at 4°C. The cells were washed and incubated with FITC-conjugated donkey anti-rabbit IgG (1:500 dilution) for 2 h at room temperature, rinsed, and blocked in 5% donkey serum in PBS with 5% BSA prior to incubation with normal mouse IgG (1:500 dilution) or the anti-mouse Novus NK1R antibody (1:500) overnight at 4°C. Fluorescence visualization was achieved using Alexa 568-conjugated donkey anti-mouse IgG (1:500) and nuclear counterstaining with 4',6-diamidino-2-phenylindole (DAPI; Vectashield, Vector Laboratories, Burlingame, CA, USA, #H-1200). Slides were examined on an Olympus BX61 microscope; images were acquired and analyzed using a Hammamatsu ORCA-ER digital camera and Volocity software (Improvision, Waltham, MA, USA).
Ca2+ measurements
Cytosolic-free Ca2+ concentrations were determined in Fura-2-loaded monocytes, which were incubated with 10 uM acetoxymethyl ester of Fura-2 in HEPES buffer at 37°C for 5 min, then diluted 10 times in HEPES buffer, and incubated a further 20 min at 37°C. Cells were washed and resuspended in buffer at a concentration of 5 x 106 cells/ml. Prior to use, cells were resuspended in fresh buffer at a concentration of 2 x 106 cells/200 ul. The kinetics of fluorescence changes were monitored in a 96-well plate at excitation wavelengths of 340 nm and 380 nm and an emission wavelength of 510 nm in a Flexstation fluorescence plate reader. Triton X-100 (1%) was added to measure Fmax (to calculate maximal Ca2+ concentration), and excess EGTA was added to measure Fo (to calculate minimal Ca2+). Cytosolic Ca2+ was calculated by the ratio method of Grynkiewicz et al. [53
]. Changes in peak cytosolic Ca2+ levels (calculated from resting to maximal intracellular Ca2+ concentrations) were used to compare Ca2+ levels under different experimental conditions.
For experiments in which monocytes were transfected with NK1R-F, Ca2+ measurements were carried out in single cells as described previously [21 ]. Monocytes were plated on poly-D-lysine-coated glass coverslips (Sigma Chemical Co.) in six-well plates. The coverslips were sized to fit a homeothermic perfusion chamber platform of an inverted Zeiss microscope. The cells were loaded with 2 µM Fura-2 acetoxymethylester as described previously [21 ]. The cells were then superfused with HBSS and supplemented with 1% FBS and 1.25 mM CaCl2 at 37°C at a flow rate of 1.5 ml/min. The excitation was performed at 334 and 380 nm with two narrow band-pass filters.
The emitted fluorescence was filtered (520 nm), captured with an Attofluor charged-coupled device video camera (512x480 pixel resolution), digitized (256 gray levels), and analyzed with Attofluor ratio vision (Version 6.00) software (Atto Instrument, Rockville, MD, USA). The intracellular Ca2+ concentration was calculated by comparing the ratio of fluorescence at each pixel with an in vitro two-point calibration curve. The Ca2+ concentration presented is that obtained by averaging the values of all pixels of a cell body. Data points were collected at 5-s intervals.
Transfection of NK1R by nucleofection
HEK293 cells were purchased from American Type Culture Collection (Rockville, MD, USA) and grown in DMEM, supplemented with 10% FBS, glutamine, and antibiotics at 37°C in 5% CO2. A NK1R-F plasmid (a gift from Dr. N. Gerard) was introduced into HEK293 cells as described previously [21
] and using Nucleofector II and cell line Nucleofection Kit V (Program Q-001), according to the manufacturer’s instructions (Amaxa, Gaithersburg, MD, USA). Transfected cells were selected with antibiotic (G418, 1 mg/ml) for 4–6 weeks. The antibiotic-resistant cells were further selected for by limited dilution in 96-well plates for another 3–5 weeks. The antibiotic-resistant clones were selected for further characterization by real-time RT-PCR quantitation of NK1R mRNA and by flow cytometry staining of the surface expression of NK1R. The expression levels of NK1R-F mRNA in HEK293 were 345 copies/ng RNA. Using flow cytometry, cell surface expression of NK1R in these cell lines was confirmed (data not shown).
For human peripheral blood monocytes, monocytes (5x106 cells) were suspended in 100 ul nucleofector solution. The NK1R-F gene containing plasmid (0.5 ug) was used for each nucleofection. Control monocytes were mock-transfected with an empty plasmid, pcDNA 3.0. The human monocyte nucleofection kit was used following the manufacturer’s protocol (Nucleofector Program #Y-01). Transfected cells then were transferred to six-well plates and incubated for 48 h before measuring Ca2+ mobilization.
Monocyte chemotaxis
Monocytes were suspended in DMEM + 0.2% BSA and incubated with calcein-AM for 30 min at 37°C. Cells were washed and resuspended in DMEM + 0.2% BSA at a concentration of 2 x 106 cells/ml and pretreated with 10–7M SP or buffer (DMEM+0.2% BSA) for 10 min at 37°C. For inhibitor experiments, cells were pretreated with buffer or the NK1R antagonist aprepitant (10–6M) for 15 min at 37°C. Following pretreatment with buffer, buffer + aprepitant, SP, or SP + aprepitant calcein-labeled monocytes (2x105) were placed in the upper compartment of the chemotaxis chamber (Neuroprobe, Cabin John, MD, USA). A filter (5 um pore-size) separated the upper and lower compartments. The lower compartment contained rhCCL5 at desired concentrations diluted in DMEM + 0.2% BSA. SP and/or aprepitant were not added to the lower compartment. The cells were incubated at 37°C for 90 min in humidified air with 5% CO2. The cells remaining in the upper chamber were removed, and 50 µl 5 mM EDTA was added to the top chamber to promote monocyte detachment in the filter. The cell plate was incubated for 60 min at 4°C and centrifuged, and the filter was removed. Monocyte migration was determined fluorometrically using a Flex Station fluorescence plate reader (Molecular Devices, Sunnyvale, CA, USA) at an excitation wavelength of 494 nm and an emission wavelength of 515 nm. The number of monocytes migrating was calculated from a standard curve prepared from calcein-loaded monocytes.
Measurement of ERK1/2 phosphorylation
Monocytes (8x106 cells/ml) were incubated with buffer, SP (10–7M), CCL5 (100 ng/ml), or SP (10–7M) + CCL5 (100 ng/ml) or pretreated with the NK1R antagonist aprepitant (10–7M) and then incubated with SP or SP + CCL5 for 10 min at 37°C. The cells were then harvested, and cell lysates were prepared as described above and run on 4–12% SDS-PAGE gels at a protein concentration of 30 ug/lane. ERK1/2 activation was determined by immunoblotting of cell lysates using a phospho-specific antibody for ERK1/2 (Thr202/Tyr204). Equal loading of ERK1/2 was confirmed by reprobing membranes using an antibody that recognizes phosphorylated and nonphosphorylated forms of ERK1/2, and ERK1/2 activation was quantitated by densitometry analysis of the immunoreactive bands using ImageJ software, Version 1.32j [National Institutes of Health (NIH), Bethesda, MD, USA]. Densitometry was measured in arbitrary densitometry units (ADU).
Immunoprecipitation (IP) of CCR5
Monocytes (10x106 cells/well) were incubated with buffer, SP (10–7M), CCL5 (100 ng/ml), or SP (10–7M) + CCL5 (100 ng/ml) or pretreated with aprepitant (10–7M) and then incubated with SP + CCL5 for 10 min at 37°C. Cell pellets were lysed in IP buffer and vortexed for 20 min at 4°C to solubilize the membrane fraction. The IP buffer consisted of 10 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM Na-orthovanadate, 20 µM 4-(2-aminoethyl)-benzenesulfonyl fluoride, 0.2% Nonidet P-40, 5 µg/ml leupeptin, Sigma phosphatase inhibitor cocktail, and Sigma protease inhibitor cocktail. Protein concentrations of the cell lysates were determined by the BCA protein assay kit according to the manufacturer’s instructions (Pierce). Cell lysates (250 µg protein) were incubated with anti-CCR5 (1 µg/200 ug cell lysates, Cell Sciences) overnight at 4°C and then with A/G PLUS agarose for 2 h at 4°C. The IgG agarose pellet was washed four times with lysis buffer, resuspended in 45 µl 2x SDS-PAGE sample buffer, and boiled for 5 min at 95°C to elute the immune complex. The samples were run on a 4–12% gradient SDS-PAGE at 100 V for 4 h. Serine phosphorylation of CCR5 was determined by Western blot analysis using a phosphoserine antibody. Total CCR5 levels were determined by Western blot analysis using a different CCR5 antibody (R&D Systems) directed to a different epitope of the receptor.
Statistical analysis
The results are expressed as means ± SE (n). Data were analyzed by Student’s t-test for two group comparisons or ANOVA for multiple comparisons. The Tukey-Kramer multiple comparisons post-test was used to evaluate the significance between experimental groups. Differences were considered significant when P < 0.05.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Differential protein expression of NK1R-F and NK1R-T in human peripheral blood monocytes was evaluated next using an antibody directed against a common domain of NK1R-T and NK1R-F (Novus Biologicals) and an antibody directed against the C-terminal of NK1R (Santa Cruz Biotechnology). The Novus NK1R antibody recognizes NK1R-F and NK1R-T, as it was produced against a common domain (aa 140–244), present in NK1R-T and NK1R-F. In contrast, the C-terminal NK1R antibody (Santa Cruz Biotechnology) was raised against a recombinant protein corresponding to amino acid residues 325–407 in the terminal sequence of the cytoplasmic domain of NK1R and detects only the NK1R-F. As shown in Figure 1 , monocytes stained positively with the antibody, which recognizes NK1R-F and NK1R-T (Fig. 1A) but did not bind the anti-C-terminal antibody (Fig. 1B) . In contrast, HEK293 cells transfected with NK1R-F stained positively when antibodies directed against the common domain of NK1R (Fig. 1E) or the C-terminal NK1R were used (Fig. 1F) . Thus, in contrast to transfected HEK cells (NK1R-F), monocytes express NK1R-T but not NK1R-F (see Fig. 1 D and H ). In summary, using mRNA and protein analysis, our studies demonstrate that human monocytes express NK1R and only express the NK1R-T.
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Figure 1. Immunofluorescence of NK1R in human monocytes and HEK293 cells transfected with NK1R-F. Monocytes (A–D) and HEK293 cells transfected with NK1R-F (E–H) were fixed, permeabilized, and immunostained with an antibody that recognizes NK1R-T and NK1R-F (red, A and E) and an antibody that recognizes the C-terminal of NK1R and only reacts with NK1R-F (green, B and F). The cells were mounted in Vectashield with DAPI (blue, C and G) and imaged using fluorescence microscopy. Merged images for monocytes and transfected HEK cells are shown in D and H, respectively (original magnification, x400) The data are representative of three independent experiments and donors. Rabbit and mouse IgG were used as negative controls (data not shown).
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Figure 2. (A) SP (10–7M) pretreatment enhances CCL5-mediated Ca2+ mobilization in human monocytes. An intracellular Ca2+ ([Ca2+]in) increase was measured in Fura-2-loaded monocytes following the addition of SP (10–7 M) or CCL5 (100 ng/ml). Changes in cytosolic Ca2+ were monitored at the dual excitation wavelengths at 340 and 380 nm and the emission wavelength of 510 nm (see Materials and Methods). The arrow indicates addition of CCL5 or SP. For experiments examining the effects of SP pretreatment on CCL5-elicited Ca2+ mobilization, SP (10–7 M) was added 5 min prior to the addition of CCL5 (representative experiment of n=9). (B) SP (10–6M) pretreatment enhances CCL5-mediated Ca2+ mobilization in human monocytes via NK1R. An intracellular Ca2+ increase was measured in Fura-2-loaded monocytes as described in A. Cells were activated by CCL5 (100 ng/ml; indicated by an arrow), stimulated by SP (10–6 M; arrow), pretreated with SP (10–6 M) and activated by CCL5 (100 ng/ml; arrow), and pretreated with SP (10–6 M) plus aprepitant (10–6 M) followed by stimulation with CCL5 (100 ng/ml; arrow; representative experiment of n=15). (C) SP (10–6M) priming of CCL5-mediated Ca2+ mobilization is the result of increased uptake of extracellular Ca2+. Intracellular Ca2+ increase was measured in Fura-2-loaded monocytes as described in A. Fura-2-loaded monocytes were pretreated with buffer or SP (10–6M) and incubated for 5 min at 37°C in the presence of 1.25 mM Ca2+ or 5 mM EGTA, followed by the addition of CCL5 (100 ng/ml), as indicated by the arrow (representative experiment of n=10).
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SP also enhanced Ca2+ mobilization elicited by another CCR5 ligand, CCL3. Pretreatment of human monocytes with SP, prior to the addition of CCL3, increased peak cytosolic Ca2+ levels 149 ± 10% as compared with peak cytosolic Ca2+ levels in response to CCL3 alone (control, n=4, P<0.01). Thus, SP priming of Ca2+ mobilization is not limited to CCL5, but SP also enhances Ca2+ mobilization in response to another CCR5 agonist, CCL3.
To determine whether the inability of SP to mobilize Ca2+ independently in human monocytes was a result of the absence of expression of NK1R-F, a NK1R-F gene-containing plasmid was introduced into isolated human monocytes. SP, at a concentration that did not elicit Ca2+ mobilization (10–6M) in monocytes lacking NK1R-F, stimulated a transient increase in intracellular Ca2+ in monocytes expressing NK1R-F (Fig. 3 ). These results indicate that the NK1R-F is required for SP-induced Ca2+ mobilization.
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Figure 3. Expression of NK1R-F is required for a SP-induced Ca2+ increase in human monocytes. Intracellular Ca2+ measurements in monocytes transfected with NK1R-F were carried out in single cells as described in Materials and Methods. SP (10–6 M) did not induce Ca2+ responses in Fura-2-loaded, mock-transfected monocytes ( , monocytes) but induced Ca2+ responses in monocytes transfected with a NK1R-F gene-containing plasmid ( , monocytes+NK1R-F). Each data point on the Ca2+ curves corresponds to mean values measured in six to eight individual cells. The data shown are representative of three independent transfection experiments.
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Figure 4. (A) CCL5-mediated chemotaxis in human monocytes is concentration-dependent. SP pretreatment potentiates CCL5-mediated chemotaxis. CCL5-directed migration was assessed in Transwell migration assays using calcein-labeled human monocytes, which were pretreated with buffer or SP (10–7 M) for 15 min prior to chemotaxis measurements. Different concentrations of CCL5 (0.01–100 ng/ml) were placed in the bottom wells, and the number of monocytes that migrated over the 90-min incubation period was determined fluorometrically as described in Materials and Methods. Values are mean ± SE, n = 5–8 separate monocyte preparations; *, CCL5 versus CCL5+SP: P < 0.01 at 0.01 ng/ml CCL5, P < 0.01 at 0.1 ng/ml CCL5, P < 0.05 at 1 ng/ml CCL5, P = NS at 10 ng/ml CCL5, P < 0.01 at 100 ng/ml CCL5; 0.01 ng/ml CCL5 (C0.01) versus C0.01 + SP, P < 0.01; 0.1 ng/ml CCL5 (C0.1) versus C0.1 + SP, P < 0.01; 1 ng/ml CCL5 (C1) versus C1 + SP, P < 0.05; 10 ng/ml CCL5 (C10) versus C10 + SP, P = NS; 100 ng/ml CCL5 (C100) versus C100 + SP (10–7 M), P < 0.01. (B) Potentiation of CCL5-mediated chemotaxis by SP is regulated via NK1R. Monocytes were pretreated in the absence or presence of the NK1R antagonist aprepitant (Aprep; 10–6M) for 30 min prior to the addition of CCL5 (100 ng/ml) or CCL5 (100 ng/ml) + SP (10–7 M). CCL5-directed chemotaxis was measured as described in A. Values are mean ± SE, n = 5, *, P < 0.02; CCL5 (100 ng/ml) versus CCL5 + SP (10–7 M), **, P < 0.01; CCL5 + SP versus CCL5 + SP + aprepitant (10–6M).
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SP pretreatment enhances CCL5-mediated activation of the MAPK ERK
SP activates the MAPK ERK in other cell types [55
, 56
], but it is not known which form(s) of NK1R activates ERK and whether ERK has a role in the potentiating effects of SP on CCL5-directed chemotaxis. As shown in Figure 5A
, at a concentration (10–7 M) that did not mobilize Ca2+, SP triggered phosphorylation of ERK2 but not ERK1. Activation of ERK2 by SP was ablated by pretreatment with aprepitant (10–6 M), indicating that SP-induced activation of ERK2 was mediated through NK1R-T. In contrast, treatment of monocytes with CCL5 (100 ng/ml) triggered phosphorylation of ERK1 and ERK2 (Fig. 5B)
. When monocytes were incubated with SP + CCL5 together, phosphorylation of ERK1 and ERK2 was increased significantly as compared with with CCL5 alone. The SP-mediated enhancement in ERK1 and ERK2 activation was inhibited by pretreatment with aprepitant (Fig. 5B)
. Pretreatment with aprepitant had no significant effect on CCL5-elicited ERK1 and ERK2 activation [CCL5+aprepitant=98.5±20% of control (CCL5 alone), mean±SE, n=5, P=NS]. These results indicate involvement of the SP:NK1R-T interaction with activation and phosphorylation of ERK1 as well as ERK2.
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Figure 5. Activation of the MAPK ERK in monocytes by SP and CCL5. (A) Activation of ERK2 by NK1R-T. Monocytes were incubated for 10 min with buffer alone, SP (10–7 M), or SP, following pretreatment with aprepitant (10–6 M) for 30 min. (Upper) ERK1/2 activation was determined in cell lysates by immunoblotting with antibodies specific to phosphorylated ERK1/2 (Phos-ERK1/2). Equal protein loading was determined by Western blot analysis for total ERK1/2. Representative Western blots. (Lower) Densitometry analysis of SP-mediated activation of ERK2. Values are expressed as mean ± SE (n=7 separate monocyte experiments) and are expressed in ADU. Statistical significance for phosphorylated ERK2: *, P <0.003, SP versus buffer; **, P < 0.001, SP + aprepitant versus SP. (B) Activation of ERK1 and ERK2 by CCL5: effect of SP. Monocytes were incubated for 10 min with SP (10–7 M), CCL5 (100 ng/ml), CCL5 + SP, or CCL5 + SP following pretreatment with aprepitant (10–7 M) for 30 min. (Upper) ERK1/2 activation was determined in cell lysates as described in A (representative Western blot from four different monocyte experiments). (Lower) Densitometry analysis of CCL5 and SP-mediated activation of ERK1 and ERK2. Values are expressed as mean ± SE (n=4) and are expressed in ADU. Statistical significance for phosphorylated ERK1 (pERK1): *, P < 0.05, CCL5 versus buffer; **, P < 0.001, CCL5 + SP versus CCL5 and CCL5 + SP versus SP; ***, P < 0.01, CCL5 + SP + aprepitant versus CCL5 + SP. Statistical significance for phosphorylated ERK2: *, P < 0.01, CCL5 versus buffer;  , P < 0.01, SP versus buffer; **, P < 0.01, CCL5 + SP versus CCL5 and CCL5 + SP versus SP; ***, P < 0.05, CCL5 + SP + aprepitant versus CCL5 + SP.
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Figure 6. Role of ERK in SP-mediated enhancement of CCL5-directed chemotaxis. CCL5-mediated chemotaxis was determined as described in Figure 4
. Monocytes were pretreated for 15 min with buffer or the MEK1/2 inhibitor PD 98059 (PD; 20 uM) prior to pretreatment with buffer or SP (10–7M). Monocyte chemotaxis was measured in response to CCL5 (100 ng/ml). Values are mean ± SE, n = 5, *, P < 0.01, CCL5 versus CCL5 + SP; **, P < 0.01, CCL5 + SP versus CCL5 + SP + PD.
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Figure 7. SP signaling through NK1R triggers phosphorylation of the CCR5 receptor. Monocytes were incubated for 10 min with CCL5 (100 ng/ml), SP (Aprep, 10–7 M), CCL5 + SP, or CCL5 + SP following pretreatment with aprepitant (Aprep, 10–7M). The CCR5 receptor was immunoprecipitated, and serine phosphorylation (PSer) of the receptor was determined by Western blotting with a phospho-specific antibody. Equal protein loading was determined by Western blot analysis for total CCR5. Representative Western blot from five separate monocyte preparations. Densitometry analysis of SP and CCL5-mediated phosphorylation of CCR5. Values are expressed as mean ± SE (n=5) in ADU. The addition of CCL5 (100 ng/ml) or SP (10–7M) to isolated monocytes triggered serine phosphorylation of CCR5 (*, buffer versus CCL5, P<0.005; *, buffer versus SP, P<0.05; buffer versus CCL5+SP, P<0.05). Treatment with CCL5 + SP did not produce additional phosphorylation (CCL5 versus CCL5+SP, P=NS). Serine phosphorylation of CCR5 was inhibited by pretreatment with (10–6 M; CCL5+SP versus CCL5+SP+aprepitant, **, P<0.02).
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The NK1R-T, however, was functional. Although SP:NK1R-T interactions did not mobilize Ca2+, SP did enhance CCL5-elicited Ca2+ mobilization in monocytes. In addition, aprepitant, a NK1R antagonist, inhibited the priming effect of SP on CCL5-elicited Ca2+ mobilization, indicating that the priming effect of SP was mediated via the NK1R-T receptor. The priming effect of SP on CCL5-elicited Ca2+ mobilization was rapid (within minutes) and also occurred when SP and CCL5 were added simultaneously (data not shown), indicating that the priming effect mediated through NK1R-T did not require de novo protein synthesis. SP enhanced Ca2+ mobilization elicited by CCL5 and CCL3, chemokines that are ligands for the CCR5 receptor. NK1R and CCR5 are members of the GPCR family, and there is extensive crosstalk between constituents of this receptor family. Heterologous interactions or crosstalk between GPCRs result in loss of function (desensitization) or alternatively, a gain or enhancement of function, which is often associated with increased intracellular Ca2+ mobilization [58 , 59 ]. Our studies establish that there is positive crosstalk, as demonstrated by SP-enhanced Ca2+ mobilization by two CCR5 ligands, CCL5 and CCL3.
CCL5 and CCL3 binding to the CCR5 receptor elicits a transient mobilization of Ca2+ from intracellular stores and the uptake of extracellular Ca2+ [60 ]. In the presence of EGTA, where cytosolic Ca2+ is derived solely from intracellular stores, SP did not prime CCR5-mediated Ca2+ mobilization, indicating that the enhancement in cytoplasmic Ca2+ concentrations was the result of increased uptake of extracellular Ca2+ and not release of intracellular Ca2+ stores. In neutrophils, SP also primed Ca2+ mobilization mediated by the chemokine IL-8, and this enhancement was dependent on extracellular Ca2+ uptake [61 ]. Thus, although SP does not mobilize Ca2+ in human monocytes, SP signaling through NK1R-T does enhance CCL5-mediated Ca2+ mobilization through increased extracellular Ca2+ uptake, indicating the NK1R-T is functional in human monocytes, and nanomolar concentrations of SP enhance CCL5-mediated cellular responses.
The finding that SP primes CCL5- and CCL3-elicited Ca2+ mobilization suggests that activation of NK1R-T modulates CCR5-mediated cellular responses. SP augments or potentiates the inflammatory response in macrophages and microglial cells [11 , 14 , 62 ], but little is known about whether signaling through NK1R-T also modulates inflammatory responses in monocytes. Chemotaxis or directed migration is an important component of the immune response, and our studies demonstrate that SP significantly enhanced CCL5-mediated chemotaxis (Fig. 4) . The priming effect of SP was mediated through the NK1R-T receptor, as pretreatment with the NK1R antagonist aprepitant significantly inhibited SP priming. These studies demonstrate that SP, through interactions with NK1R-T, amplifies the inflammatory response by enhancing multiple receptor-mediated responses.
SP might enhance CCL5-mediated cellular responses at several different levels. SP:NK1R-T interactions could increase cell surface CCR5 receptor expression, decrease CCL5-mediated receptor internalization, modify ligand-binding affinity, affect assembly of G-protein signaling complexes, and/or interact with selective signal transduction pathways [58 , 59 ]. The potentiating effects of SP on CCL5-directed migration may be the result of differential activation of a common signaling pathway. Indeed, unlike Ca2+ signaling, the NK1R-T activates signaling pathways required for activation of the MAPK ERK. Similar results were observed in a human colonic epithelial cell line, where SP (0.1 nM–10 uM) did not elicit increases in intracellular Ca2+, but SP activated ERK [63 ]. Our studies also show that CCL5 and SP elicited differential activation of the MAPK ERK1/2 in human monocytes (Fig. 5) . CCL5 triggered phosphorylation of ERK1 and ERK2, and SP, at a concentration that did not mobilize Ca2+, activated ERK2 only. However, the addition of CCL5 and SP together resulted in enhanced phosphorylation of ERK1 and ERK2. Although ERK1/2 was activated by CCL5, it was not required for CCL5-directed migration, as an inhibitor of ERK activation had no effect on CCL5-mediated chemotaxis (Fig. 6 and ref. [64 ]). In contrast, the potentiating effects of SP on CCL5-directed migration were ERK1/2-dependent, indicating a role for ERK1/2 only in SP-mediated potentiation of CCL5-directed migration. The activation of this common signaling pathway by SP and CCL5 may lead to altered cellular distribution of ERK1/2 and the phosphorylation of a distinct group of substrates. Our results suggest that crosstalk between CCL5 and SP signaling pathways is at the level of the ERK or upstream of the MAPKs, possibly at the level of the receptors [64 , 65 ].
CCL5 and CCL3 stimulate serine phosphorylation and activation of the CCR5 receptor [57 , 66 67 68 ]. A possible mechanism of crosstalk between the two GPCRs is at the level of the CCR5 receptor through regulation of phosphorylation. Our studies demonstrate that SP elicited serine phosphorylation of CCR5, which was NK1R-T-dependent. The current study did not identify specific serine residues but does demonstrate that SP-mediated phosphorylation was mediated by NK1R-T. SP:NK1R interaction is capable of transactivation of other GPCRs. SP:NK1R interactions trigger transactivation of the epidermal growth factor receptor, which mediates SP-induced mitogenic responses in human glioma cells, human colonocytes, and the U-373 MG cell line [56 , 69 , 70 ]. Whether NK1R-T is active in these other cell types is not known. Further studies are required to ascertain whether crosstalk is mediated through receptor:receptor heterodimerization in human monocytes and whether SP interaction with NK1R-T alters CCR5-binding affinity or cell surface expression.
In summary, the results of the present study demonstrate that human peripheral blood monocytes express only the NK1R-T, which is functional and is capable of crosstalk with other GPCRs. At physiological relevant concentrations of SP, SP interaction with NK1R-T enhanced CCL5-directed monocyte migration. The SP-mediated increase in CCL5-elicited cellular responses was associated with enhanced Ca2+ signaling and ERK1/2 activation, indicating that NK1R-T is capable of activating selective signaling pathways in human monocytes. Finally, NK1R-T interaction with SP elicited serine phosphorylation of CCR5, suggesting that crosstalk between CCL5 and SP-mediated signaling occurs at the level of the CCR5 receptor.
Received April 22, 2008; revised September 9, 2008; accepted September 13, 2008.
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