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RECEPTORS AND SIGNAL TRANSDUCTION
Department of Pharmacology, National University of Singapore, Singapore
Submitted 12 February 2007 ; accepted in final form 3 May 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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/CCL3. The activating effect of SP on neutrophils was further evidenced by upregulation of the CD11b integrin, the activation marker of neutrophils. SP induced both the mRNA and protein expression of the chemokines MIP-1/CCL3 and MIP-2/CXCL2 in neutrophils and upregulated the chemokine receptors CC chemokine receptor (CCR)-1 and CXC chemokine receptor (CXCR)-2. This stimulatory effect on chemokine and chemokine receptor expression in neutrophils was further found to be neurokinin-1 receptor (NK-1R) specific. Pretreatment with selective NK-1R antagonists inhibited SP-triggered activation of neutrophils and chemokine and chemokine receptor upregulation. Moreover, SP-induced chemokine upregulation was NF-
B dependent. SP time dependently induced NF-B p65 binding activity, IB degradation, and NF-B p65 nuclear translocation in neutrophils. Inhibition of NF-B activation with its inhibitor Bay11-7082 (10 µM) abolished SP-induced NF-B binding activity and upregulation of MIP-1/CCL3 and MIP-2/CXCL2 in neutrophils. Together, these results suggest that SP exerts a direct stimulatory effect on the expression of chemokines and chemokine receptors in mouse neutrophils. The effect is NK-1R mediated, involving NF-B activation.
chemokines and receptors; neuro-immune interaction; neurokinin-1 receptor; primary leukocytes; NF-B activation
(TNF), interleukin (IL)-6 and IL-8, and other mediators from lymphocytes, macrophages, monocytes, and mast cells; and activation of nuclear factor-B (NF-B) (1, 6, 14, 20, 36).
Polymorphonuclear neutrophils are the first leukocyte subpopulation that appears at the site of inflammation and have a central role in the clearance of infectious pathogens and innate immunity. They have been implicated in mediating acute-phase inflammation in different disease models (3, 25, 33). Previous studies have shown that SP activates neutrophils during neurogenic inflammation. SP affects the migratory responses and cytotoxic functions of neutrophils and induces degranulation, respiratory burst, and production of reactive inorganic oxidants in the cells (22, 29, 30, 35). Additionally, SP is a priming agent affecting a variety of proinflammatory neutrophil functions, for example migration, IL-1
and TNF secretion, 2-integrin upregulation, leukotriene production, and calcium mobilization, (13, 18, 28, 32). Evidence from in vivo studies demonstrated that SP induces a rapid influx of neutrophils in human dermis. SP mediates neutrophil adherence to alveolar epithelial cells and induces IL-1 and TNF release (12, 30). Neutrophil accumulation to the lung is significantly inhibited in NK-1R knockout mice in inflammatory models (7), while other studies have reported that SP induces neutrophil accumulation in inflamed but not normal skin, using knockout animals (10, 24).
Blood neutrophil trafficking to the site of inflammation involves a complex cascade of events progressing from rolling and neutrophil activation to firm adhesion and transmigration. Chemokines, a special subtype of chemotactic cytokines, are critically involved in neutrophil recruitment under both inflammatory and homeostatic conditions (2). Chemokines fall into four subfamilies: CXC, CC, C, and CX3C, based on the spacing of the amino terminal cysteine residues (19). In general, the CXC chemokines are mainly characterized as neutrophil and T lymphocyte chemoattractants, while CC chemokines act on a wider spectrum of cell types including monocytes/macrophages, eosinophils, basophils, natural killer cells, and T lymphocytes (3, 19, 27). However, recent findings suggest that under certain inflammatory conditions or in response to specific inflammatory stimuli, neutrophils may also respond to some CC chemokines (4, 5, 11, 16, 31). Chemokine receptors are a family of G protein-coupled seven-transmembrane receptors. The expression pattern of chemokine receptors is a major determinant of the selectivity of chemokines for target cells.
Despite the plethora of studies on proinflammatory responses of neutrophils to SP, the role of SP in stimulating chemokine and chemokine receptor expression in these cells has not yet been addressed. In this study, we sought to investigate the modulatory effect of SP on chemokine production and chemokine receptor expression and the functional consequences of chemokine/chemokine receptor upregulation in mouse primary neutrophils. The SP-induced signaling pathway in the cells, particularly the involvement of NK-1R, and activation of the key transcription factor NF-B were also studied. Our results present novel evidence that SP stimulates increased expression of chemokines and their receptors in neutrophils via an NK-1R-specific, NF-B-dependent mechanism and functionally leads to enhanced migratory responses of the cells to both CXC and CC chemokines.
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MATERIALS AND METHODS |
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Reagents.
Ficoll-Paque Plus was obtained from Pharmacia Biotech (Uppsala, Sweden). Phenol red-free RPMI 1640 medium, the selective NK-1R antagonist L703,606, oxalate salt {cis-2-(diphenylmethyl)-N-[(2-iodophenyl)-methyl]-1-azabicyclo[2.2.2]octan-3 amine oxalate}, and the potent selective NK-2R antagonist GR159897 {(R)-1-[2-(5-fluoro-1H-indol-3-yl)ethyl]-4-methoxy-4-[(phenylsulfinyl)methyl]piperidine} were purchased from Sigma-Aldrich (St. Louis, MO). SP was purchased from Bachem (Torrance, CA). Recombinant macrophage inflammatory protein (rMIP)-2 and MIP-1/CCL3 (rMIP-1/CCL3) were purchased from R&D Systems. Molar concentrations of the chemokines were calculated based on molar masses of 8 and 7.8 kDa, respectively, as reported by the manufacturer. The selective NK-1R antagonist CP96,345 {2-(diphenylmethyl)-N-[(2-methoxyphenyl)methyl]-1-azabicyclo[2.2.2]octan-3 amine} was a gift from Pfizer Diagnostics. All other reagents and solvents were from Merck (Darmstadt, Germany).
Isolation of primary mouse neutrophils.
Mice were anesthetized, and 
1.4 ml of blood were drawn by cardiac puncture using heparinized needles and diluted with phosphate-buffered saline (PBS). The neutrophils were purified by density gradient centrifugation through Ficoll-Paque Plus (Amersham Pharmacia Biotech). Contaminating erythrocytes were removed by hypotonic lysis. Cells were resuspended in serum-free, phenol red-free RPMI 1640 medium (Sigma) and allowed to recover for 30 min at 37°C before treatment. Cytological examination of stained cells showed that 95% of the cells were neutrophils. Trypan blue staining confirmed that >98% of the cells were viable.
Treatment.
Cells (106 cells/ml) resuspended in serum-free RPMI 1640 were stimulated with 1 µM SP at 37°C for the times indicated. This dose of SP was adopted from previous work and preliminary experiments (13, 26). In some experiments, cells were pretreated with CP96,345 (1 µM), L703,606 (1 µM), or GR159897 (100 nM) for 15 min before SP stimulation. For NF-B inhibition experiments, cells were preincubated with 5, 10, or 30 µM Bay11-7082 (Calbiochem, San Diego, CA) for 1 h before stimulation with 1 µM SP for the periods indicated.
Cell migration assay.
Migratory responses of isolated neutrophils were examined using the Chemicon QCM Chemotaxis 3-µm 96-well Cell Migration Assay kit (Chemicon International), following the manufacturer's instructions. Briefly, the assay is based on the 3-µm pore size of Boyden chambers. Unstimulated or SP-stimulated cells (2 x 105 cells/ml) resuspended in serum-free, phenol red-free RPMI 1640 medium were placed in the upper chamber, and 0.1 nM to 10 µM rMIP-2 or 0.01 nM to 1 µM rMIP-1/CCL3 was placed in the lower chamber; cells were incubated at 37°C for 2 h. Cells that had migrated and fallen into the lower chamber were collected, and cells attached to the bottom of the insert membranes were dissociated from the membranes with the provided cell detachment buffer and collected. These cells were subsequently lysed and detected using the CyQuant GR dye (Molecular Probes) provided in the kit. The green fluorescent dye exhibits strong fluorescence enhancement when bound to cellular nucleic acids. Spontaneous migration was measured by placing the plain medium in the lower chamber. Data are expressed as "chemotactic index" which is the ratio between the migratory response toward the test chemokine and toward the medium control.
RT-PCR.
The effect of SP on the mRNA expression levels of CD11b, chemokines, and chemokine receptors in primary mouse neutrophils was examined by RT-PCR. Briefly, isolated cells (106 cells/ml) in serum-free RPMI 1640 medium were treated with SP (1 µM) for the indicated periods (see RESULTS) or left untreated at 37°C before total RNA was extracted using the RNeasy Mini kit (Qiagen, Hilden, Germany). RNA was quantitated spectrophotometrically by absorbance at 260 nm; 1 µg of total RNA was reverse transcribed using the iScript cDNA Synthesis kit (Bio-Rad). The cDNA synthesized was used as the template for PCR amplification, using iQ Supermix (Bio-Rad, Hercules, CA) in MyCycler (Bio-Rad). The PCR protocol consisted of 30–40 optimal cycles of denaturation at 95°C for 30 s, annealing for 30 s, and extension at 72°C for 30 s. The number of amplification cycles was optimized to assure that the reaction was terminated in the linear range of amplification for each gene. The following specific primer pairs (Proligo, Singapore) were used: CD11b, sense 5'-AGCCCAAGATCACATG-3' and antisense 5'-TGCAGAAGCATAACCC-3'; MIP-1/CCL3, sense 5'-ACTGCCCTTGCTGTTCTTCTCT-3' and antisense 5'-AGGCATTCAGTTCCAGGTCAGTGA-3'; MIP-2/CXCL2, sense 5'-TGCCTGAAGACCCTGCCAAGG-3' and antisense 5'-GTTAGCCTTGCCTTTGTTCAG-3'; CC chemokine receptor (CCR)-1, sense 5'-GACCAGCATCTACCTGTTCA-3' and antisense 5'-GCAGAAACAAATACACTCAG-3'; CXC chemokine receptor (CXCR)-2, sense 5'-TGTTCTTTGCCCTGACCTTGC-3' and antisense 5'-ACGCAGTACGACCCTCAAACG-3'; GAPDH, sense 5'-GCATCTGAGGGCCCACTGAAG-3' and antisense 5'-GTCCACCACCCTGTTGCTGTA-3'. Amplification of the GAPDH gene transcript was used as an internal control of RT-PCR reactions among samples. PCR products were analyzed on 1.5% (wt/vol) agarose gels containing 0.05 mg/100 ml ethidium bromide. Densitometry results from PCR products were normalized to the housekeeping gene GAPDH.
ELISA.
The effects of SP treatment on chemokine release from isolated neutrophils were examined using ELISA. Briefly, 2 x 106 isolated neutrophils in serum-free RPMI 1640 medium were treated with SP (1 µM) for the indicated periods (see RESULTS) or left untreated at 37°C. Cell suspension was centrifuged at 200 g for 5 min, and cell-free supernatant was collected for ELISA assays. MIP-1/CCL3 and MIP-2/CXCL2 concentrations were measured by specific Duoset ELISA kits (R&D Systems; detection levels 7.813 and 15.625 pg/ml, respectively) according to the manufacturer's instructions. The intra-assay variabilities for MIP-1 and MIP-2 ELISAs were 3.6 and 4.7%, respectively, whereas the interassay variabilities were 7.8 and 8.6%. Absorbance was measured at 450 nm by a microplate reader (Tecan, Maennedorf, Switzerland). Results were expressed as picograms per milliliter of each chemokine.
Flow cytometry.
The effect of SP on chemokine receptor expression in neutrophils was measured by immunofluorescent antibody staining and analyzed by flow cytometry. Briefly, 1 x 106 cells were fixed in 1% paraformaldehyde and permeabilized. The cell suspension was incubated with normal goat or rabbit serum (Biosource, Camarillo, CA) for 15 min on ice to block nonspecific binding. Cells were then incubated with the antibody CCR1 (8 µg/ml; Santa Cruz Biotechnology, Santa Cruz, CA) or CXCR2 (4 µg/ml, FabGennix) or isotype antibodies (Santa Cruz Biotechnology) for 30 min on ice and washed with PBS. Cells were then incubated with fluorochrome-conjugated secondary antibodies (Santa Cruz Biotechnology) for 30 min on ice, washed with PBS, and analyzed by a Cyan ADP flow cytometer (Dako, Carpinteria, CA). Neutrophils were identified and gated using forward- compared with side-scatter characteristics. Control sample-matched antibody isotypes were used to set negative staining criteria. Approximately 5,000 events were counted per sample. Data were analyzed using SUMMIT v4.2 software (Dako).
Immunofluorescence staining.
After SP treatment, suspended cells were fixed in 4% formaldehyde for 10 min at room temperature, washed, and resuspended in PBS. Approximately 1 x 103 cells were spun onto microscope slides at 8.5 x 103 rpm for 10 min using a CytoFuge 2 cytocentrifuge (StatSpin, Westwood, MA). Cytospin slides were then incubated with 1:100-diluted anti-mouse CD11b or CCR2 primary antibodies for 1 h at 37°C. After being washed in PBS, the slides were incubated with 1:200-diluted fluorescein isothiocyanate (FITC)-conjugated IgG secondary antibody (BD Pharmingen) for 30 min at 37°C. For NF-B p65 immunofluorescence staining, cytospin slides were incubated with 1:200-diluted rabbit anti-mouse NF-B p65 antibody (Serotec, Oxford, UK) for 2 h at 37°C, washed, and incubated with 1:200-diluted rhodamine-conjugated goat anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology). The slides were washed again and then mounted with mounting medium with DAPI (Vector Laboratories, Burlingame, CA). Staining was analyzed by fluorescence microscopy using a Leica DM IRB microscope.
NF-B transcription factor assay.
Nuclear extracts of neutrophils after SP stimulation were prepared using a nuclear extract kit (Active Motif, Carlsbad, CA) according to the manufacturer's protocol. Protein concentrations of the nuclear extracts were determined by Bradford assay using a commercial kit (Quick-Start, Bio-Rad). The effect of SP treatment on transcription factor NF-B activity in neutrophils was determined using the TransAM NF-B p65 Assay kit (Active Motif) according to the manufacturer's instructions. In brief, 5 ng of nuclear proteins were added to each well coated with an unlabeled oligonucleotide containing the consensus binding site for NF-B (5'-GGGACTTTCC-3') and incubated for 1 h. After a washing, a primary antibody directed against the NF-B p65 subunit was added and incubated for 1 h. Subsequently, a secondary antibody conjugated to horseradish peroxidase (HRP) was applied for 1 h. A colorimetric reaction was developed with addition of a developing solution and terminated by a stop solution. The readout was quantified using a Tecan Spectrofluor Plus fluorometer at an absorbance of 450 nm.
Western blot analysis.
After treatment, cells were lysed with RIPA buffer supplemented with 1 mM PMSF and protease inhibitor cocktail containing pepstatin, leupeptin, chymostatin, antipain, and aprotinin (5 µg/ml of each). Protein concentrations were determined by the Bio-Rad Protein Assay (Bio-Rad Laboratories); 50 µg of protein samples were separated on 12% SDS-polyacrylamide gel and electrophoretically transferred to nitrocellulose membranes. Nonspecific binding was blocked by 1-h incubation of the membranes in 5% nonfat dry milk in PBST (0.05% Tween 20 in PBS). The blots were then incubated overnight with the primary antibody IB (1:1,000 dilution; Cell Signaling Technology, Danvers, MA) in buffer containing 2.5% nonfat dry milk in PBST, after which they were washed four times with PBST and finally incubated for 1 h with goat anti-rabbit HRP-conjugated secondary antibody (Santa Cruz Biotechnology) at a 1:2,000 dilution in buffer containing 2.5% nonfat dry milk in PBST. The blots were developed for visualization using an enhanced chemiluminescence (ECL) detection kit (Pierce, Rockford, IL). Hypoxanthine-guanine phosphoribosyltransferase (HPRT; Santa Cruz Biotechnology) was used as the housekeeping protein.
Statistical analysis. Data are expressed as means + standard deviation (SD). Statistical analyses were performed by independent t-test or, when multiple comparisons were made, by one-way analysis of variance (ANOVA) with post hoc Tukey's test using SPSS program v13.0 (Chicago, IL). A P value <0.05 was considered a statistically significant difference.
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RESULTS |
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/CCL3.
The priming effect of SP on chemotaxis of primary mouse neutrophils to two recombinant chemokines, rMIP-2/CXCL2 and rMIP-1/CCL3, was examined. Isolated neutrophils were pretreated with either plain medium (resting cells) or 1 µM SP (primed cells) for 5 min before they were assayed for chemotactic responses to rMIP-2/CXCL2 or rMIP-1/CCL3. The rMIP-2/CXCL2 provoked chemotactic migration of resting neutrophils in a dose-dependent manner, with the maximal effect observed at 10–6 M rMIP-2/CXCL2. Compared with resting cells, SP-primed neutrophils exhibited significantly increased chemotactic responses to the chemokine. Both a left shift and an increase in the maximum of the chemokine dose-response curve were observed (Fig. 1A). In contrast, the CC chemokine rMIP-1/CCL3 did not induce chemotactic responses of resting neutrophils. However, when cells were pretreated with 1 µM SP for 5 min, they became responsive and displayed significant chemotactic responses to rMIP-1/CCL3. The response increased dose dependently in the chemokine dose range of 10–10 to 10–6 M (Fig. 1B). The results suggested that SP, at the micromolar concentration, enhanced chemotactic migration of primary mouse neutrophils not only to CXC but also to CC chemokines.
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B activation in primary neutrophils.
On the basis of the priming effect evoked by SP on neutrophil chemotactic responses, we studied whether SP induces activation of the transcription factor NF-B, which is a master regulator of chemokine and/or chemokine receptor gene transcription. Figure 3A shows the time course of NF-B p65 DNA binding activity in neutrophils stimulated with 1 µM SP. Isolated neutrophils were treated with 1 µM SP for 0, 15, 30, 60, and 90 min. Subsequently, nuclear extracts of the cells were prepared, and the binding activity of the NF-B p65 subunit to a consensus oligonucleotide sequence was assessed. The result showed that SP treatment increased NF-B p65 binding activity in neutrophils. The activity was maximal in cells treated with SP for 30 min and started decreasing in cells with longer treatment. The SP-induced increase in NF-B DNA binding activity was abolished by pretreatment of cells with the NK-1R antagonist CP96,345 or L703,606. However, preincubation with GR159897 (100 nM), a potent NK-2R antagonist, had no effect, suggesting that this effect was NK-1R specific (Fig. 3B). A specific NF-B inhibitor, Bay11-7082, shown to selectively and irreversibly inhibit the TNF-inducible phosphorylation of IB, dose dependently inhibited SP-induced NF-B p65 binding activity. Cells were pretreated with 5, 10, or 30 µM Bay11-7082 for 1 h before challenge with SP for 30 min. NF-B p65 binding activity was significantly inhibited in cells pretreated with 10 or 30 µM Bay11-7082 compared with untreated cells (Fig. 3C); 10 µM Bay11-7082, the lowest dose that gave a significant inhibitory effect on p65 binding activity, was used in the following experiments. SP-induced NF-B activation in neutrophils was further assessed by Western blot analysis of IB levels in the whole cell lysates. IB is a negative regulator of NF-B, and its degradation is a key mechanism of the classical NF-B activation pathway. Western blot results revealed that SP treatment caused a significant degradation of IB protein in the cells at 5 min of incubation. The protein levels were further decreased at 10 min of SP incubation but started recovering with longer incubation of SP (30 and 60 min) (Fig. 3D). Immunofluorescence staining of the NF-B p65 subunit revealed that a significant number of SP-treated neutrophils exhibited NF-B p65 translocation from the cytoplasm to the nucleus. Most of the untreated cells displayed diffuse cytoplasmic staining, and cells treated with SP showed intensified staining in the nucleus, indicative of NF-B p65 nuclear translocation. Figure 3E shows representative micrographs of control and SP-treated (30 min) cells for NF-B p65 staining.
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B-responsive proinflammatory chemokines by primary mouse neutrophils. Isolated neutrophils were incubated with SP (1 µM) for 30 min or 1, 2, or 4 h. Subsequently, total RNA was extracted from the cells, and mRNA expression levels of monocyte chemoattractant protein (MCP)-1, MIP-1/CCL3, and MIP-2/CXCL2 were examined by RT-PCR. The supernatants were harvested for determination of chemokine protein levels by ELISA. The results showed that both MIP-1/CCL3 and MIP-2/CXCL2 mRNA expression was significantly elevated in neutrophils treated with SP (1 h) compared with untreated cells (Fig. 4, A and B). The expression of another chemokine, MCP-1, was not changed by SP in neutrophils (data not shown). Pretreatment of cells with 1 µM CP96,345 for 15 min or with the NF-B inhibitor Bay11-7082 (10 µM) for 1 h reversed the stimulating effect of SP on the mRNA expression of both MIP-1/CCL3 and MIP-2/CXCL2 (Fig. 4, A and B).
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/CCL3 and MIP-2/CXCL2 production and release were significantly elevated from neutrophils with 4-h SP incubation. NK-1R antagonist CP96,345 or L703,606 (1 µM) pretreatment and NF-B inhibition attenuated the inducing effect of SP on chemokine protein production (Fig. 5, A and B). In contrast, NK-2R antagonist pretreatment did not significantly alter SP-triggered chemokine release. Together, these data suggested that SP induced mRNA and protein expression of some chemokines in primary mouse neutrophils, which was an NK-1R-specific effect and dependent on NF-B activation.
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/CCL3, and CCR2 is the sole MCP-1 receptor. CXCR2 is the only CXC receptor in mouse and is used by the potent neutrophil chemoattractant MIP-2/CXCL2. All three chemokine receptors were present on murine neutrophils (32). Stimulation of isolated neutrophils with SP (1 µM) for 1 h significantly upregulated the mRNA expression of CCR1 and CXCR2 (Figs. 6A and 7A). However, no significant effect of SP on CCR2 expression was observed, as confirmed by both RT-PCR analysis and immunofluorescence staining (data not shown). The stimulatory effect on CCR1 and CXCR2 mRNA expression was inhibited when the cells were preincubated with CP96,345 (1 µM) for 15 min.
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B inhibition by pretreatment of cells with Bay11-7082 had no effect on SP-induced upregulation of chemokine receptor expression. These results suggested that SP upregulated CCR1 and CXCR2 receptor expression in neutrophils via NK-1R, independent of NF-B. |
DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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B and induces chemokine production and release from these cells. This is coupled with upregulation of cell surface chemokine receptor expression and therefore enhanced chemotactic responses of SP-primed neutrophils to chemokines. Our study supports the interplay of inflammatory mediators and cells.
When neutrophils were exposed to a micromolar concentration of SP, the migratory responses of the cells to the CXC chemokine rMIP-2/CXCL2 were significantly enhanced compared with resting cells. Furthermore, SP-primed neutrophils became responsive, chemotactically, to the CC chemokine rMIP-1/CCL3. This suggested a priming effect of SP on neutrophils for their migratory responses not only to CXC but also to CC chemokines. Further investigation indicated that SP stimulation of neutrophils exerted differential effects on the expression of chemokines and chemokine receptors: some were upregulated in SP-stimulated neutrophils but not others. Chemokine MIP-1/CCL3 and MIP-2/CXCL2 but not MCP-1 expression was upregulated by SP in neutrophils. Receptor CCR1 and CXCR2 but not CCR2 expression was induced in cells by SP. The micromolar concentration of SP used in this study was adopted from preliminary experiments and others' reports (13, 26). This dose was found to effectively activate the cells and induce a rise in chemokine release, whereas lower doses were considerably less effective. At the same concentration, SP is reported to stimulate significant chemokine production by pancreatic acinar cells and prime neutrophils triggered by different stimuli to evoke various cellular responses including intracellular calcium changes, oxidative responses, and the formation of hydrogen peroxide and nitric oxide (13, 26, 32).
The biological functions of SP and other tachykinin family members are mediated by three types of G protein-coupled tachykinin receptors: NK-1R, NK-2R, and NK-3R. Different tachykinins bind to the receptors with differential preferences: SP shows the highest affinity for NK-1R, which is also shared by endokinins and hemokinins; NKA for NK-2R; and NKB for NK-3R (23). Moreover, the SP-induced responses in neutrophils may be mediated by both receptor-dependent and -independent mechanisms. The amphiphilic property of SP enables its cationic domain to directly interact with the carboxy terminus of G protein and activate G protein directly, bypassing the receptors. This has been proposed as an explanation for the histamine-releasing effects of SP on rat peritoneal mast cells and the promoting activities of SP on neutrophil locomotion (13). Conversely, SP-induced adherence of neutrophils to endothelial cells was suggested to reside in its carboxy terminus, mediated by NK-1R (13). We tested the possible involvement of different neurokinin receptors in SP-evoked responses in neutrophils by using specific antagonists of different receptors. NK-3R is not present on murine neutrophils, as demonstrated by RT-PCR detection of the gene transcript. Two selective NK-1R antagonists, CP96,345 and L703,606, and an NK-2R antagonist, GR159897, were evaluated for their inhibitory effects on SP-induced NF-B activation and chemokine synthesis in neutrophils. Both NK-1R antagonists blocked SP-induced responses with equal potency, whereas the NK-2R antagonist had no effect, suggesting that the effect was NK-1R specific.
Neutrophils are normally chemoattracted to CXC chemokines. However, SP-primed neutrophils exhibited chemotactic responses to the CC chemokine rMIP-1/CCL3. Consistently, expression of CCR1, the receptor for MIP-1/CCL3, was found to be upregulated on neutrophils stimulated by SP. Earlier studies have suggested that in response to certain inflammatory stimuli, neutrophils may respond to some CC chemokines (16, 31). CCR1 is also found to be induced on neutrophils stimulated by specific cytokines, suggesting the ability of neutrophils to respond directly to MIP-1/CCL3 (5, 11). In our study, SP stimulation did not affect CCR2 expression in neutrophils. To date, no single agonist has been found to induce CCR2 expression in neutrophils (31).
Chemokine and receptor expression is controlled by different signaling pathways and transcription factor activation. NF-B is a transcription factor with a crucial modulatory role in inflammation, immunity, cell proliferation, and apoptosis. We noticed a rapid degradation of IB and an activation of NF-B p65 in neutrophils induced by SP. The increase in NF-B DNA binding activity was dose dependently inhibited by the specific NF-B inhibitor Bay11-7082. Pretreatment of cells with Bay11-7082 substantially blocked SP-triggered chemokine production but did not change receptor expression. This indicates that chemokine receptor regulation was mediated by a different transduction pathway from the chemokine gene transcription.
In summary, our results demonstrate that SP plays a direct role in upregulating the expression of chemokines MIP-1/CCL3 and MIP-2/CXCL2 as well as chemokine receptors CCR1 and CXCR2 in neutrophils through an NK-1R-mediated mechanism. NF-B p65 activation was involved in the transcription activation of chemokine genes. These findings extend previous findings for SP regulation of the proinflammatory neutrophil functions and provide strong evidence for the role of SP in acute inflammation with enhancement of chemokine-directed neutrophil recruitment.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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P < 0.05 compared with SP for 1 h. Typical graphs from 3 separate experiments are shown. Original magnification: x100.
