Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.1205744 on May 2, 2006

Published online before print May 2, 2006
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(Journal of Leukocyte Biology. 2006;80:117-124.)
© 2006 by Society for Leukocyte Biology

Activation of kinin B1 receptors induces chemotaxis of human neutrophils

P. Ehrenfeld*, C. Millan{dagger}, C. E. Matus*, J. E. Figueroa{ddagger}, R. A. Burgos§, F. Nualart{dagger}, K. D. Bhoola and C. D. Figueroa*,1

Institutos de
* Histologia y Patologia,
{ddagger} Bioquimica, and
§ Farmacologia, Universidad Austral de Chile;
{dagger} Departmento de Biologia Celular, Universidad de Concepcion, Chile; and
Centre for Asthma and Allergy Research Institute, University of Western Australia, Perth

1 Correspondence: Instituto de Histología y Patología, Universidad Austral de Chile, Casilla 567, Valdivia, Chile. E-mail: cfiguero{at}uach.cl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Kinins are biologically active peptides that are powerful mediators of cellular inflammation. They mimic the cardinal signs of inflammation by inducing vasodilatation and by increasing vascular permeability and pain. Neutrophils are chemoattracted to sites of inflammation by several stimuli. However, the evidence concerning the chemotactic effect of kinin peptides has been contradictory. We analyzed the chemotactic effect of kinin B1 receptor agonists on neutrophils isolated from peripheral blood of human healthy subjects. Chemotaxis was performed using the migration under agarose technique. To test the effect of B1 receptor agonists, each assay was carried out overnight at 37°C in 5% CO2-95% air on neutrophils primed with 1 ng/ml interleukin-1ß. Simultaneous experiments were performed using unprimed cells or cells challenged with formyl-Met-Leu-Phe (fMLP). A clear chemotactic activity was observed when primed neutrophils were challenged with Lys-des[Arg9]-bradykinin (LDBK) or des[Arg9]-bradykinin at 10–10 M but not when unprimed cells were used. A reduction in the chemotactic response was observed after priming of cells in the presence of 0.5 mM cycloheximide and 10 µg/ml brefeldin A, suggesting that some protein biosynthesis is required. Techniques such as reverse transcriptase-polymerase chain reaction and in situ hybridization confirmed the expression of the B1 receptor mRNA, and immunocytochemistry and autoradiography demonstrated the expression of the B1 receptor protein. In contrast to other chemoattractants such as fMLP, cytosolic intracellular calcium did not increase in response to the B1 receptor agonist LDBK. A generation of kinin B1 receptor agonists during the early phase of acute inflammation may favor the recruitment of neutrophils to the inflammatory site.

Key Words: bradykinin • kallikrein • interleukin-1ß • inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Since the discovery of tissue kallikrein (hK1, true kallikrein, kininogenase) in human neutrophils during the late 1980s [1 ], several reports have confirmed its expression [2 3 4 ] and have indicated the occurrence of other protein components of the kinin system in these cells. In addition to this kallikrein, human neutrophils have been shown to contain kininogen [5 6 7 ]- and kinin-binding sites [8 ]. Using purified cell membranes, the kinin-binding sites identified were shown to be B1 and B2 kinin receptor subtypes [8 ]. Complementary experiments demonstrated that plasma prekallikrein localizes along the neutrophil cell membrane in close proximity to the H-kininogen molecule and that the complete array of contact factors is sited on the neutrophil surface [7 ].

Unlike the B2 receptor, the kinin B1 receptor is up-regulated during inflammation or by cytokines such as tumor necrosis factor (TNF) or interleukin (IL)-1ß [9 ]. Experiments in the mouse indicate that polymorphonuclear leukocyte accumulation into 6-day-old air pouches, in response to IL-1ß application, is attenuated by 50% in the presence of a kinin B1 receptor antagonist [9 ]. In support of the concept that kinin B1 receptors participate in the recruitment of neutrophils is the finding that the kinin B1 agonist des[Arg9]-bradykinin induces a chemotactic response only in air pouches pretreated with IL-1ß. These authors postulated that B1 receptors may be expressed on sensory C-fibers, as the IL-1ß-induced neutrophil accumulation into the murine air pouch was attenuated by chronic capsaicin treatment, neurokinin 1 receptor antagonists, and calcitonin gene-related peptide antagonism [9 ]. Further, it has recently been shown that kinin B1 receptor knockout mice have a decreased number of neutrophils migrating into the inflammatory milieu in a model of peritonitis [10 ].

Only one previous report has addressed the effect of kinin peptides on neutrophil chemotaxis under in vitro conditions using the Boyden chamber technique [11 ]. In their study, Paegelow et al. [11 ] concluded that bradykinin, a B2 receptor agonist, as well as two B1 receptor agonists, des[Arg9]-bradykinin and Lys-des[Arg9]-bradykinin LDBK, induce a concentration-dependent migration of the cells into the lower compartment of chemotaxis chambers. The fact that chemotaxis, provoked by the kinin B1 and B2 receptor agonists, was inhibited by a leukotriene B4 (LTB4) antagonist led Paegelow et al. [11 ] to suggest that kinins may induce the release of LTB4, which in turn, may be the cause of neutrophil migration.

Despite all this evidence, the expression of the kinin B1 receptor in human neutrophils has not been considered so far, as well as the use of techniques alternative to the Boyden chamber, to exclude the participation of other mediators in the chemotaxis mediated by kinin B1 receptor agonists. In this study, we demonstrate that the kinin B1 agonist LDBK specifically provokes chemotaxis of human neutrophils with the comcomitant expression of B1 receptor mRNA, the immunoreactive B1 receptor protein, and binding sites to a kinin B1-tritiated ligand.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Human neutrophils isolation
Cells were isolated from anticoagulated whole blood provided by young, healthy volunteers at the Pathology Laboratory, Universidad Austral de Chile (Valdivia). Sample collection followed the guidelines stipulated by the Medical Ethical Committee of Universidad Austral de Chile and the Declaration of Helsinki principles. All procedures were carried out under sterile conditions and using siliconized material. Blood (35 ml) collected in 50 ml sterile tubes containing 3.8% w/v trisodium citrate was mixed with 35 ml dextran (6% w/v, average molecular weight 300,000, Sigma-Aldrich, St. Louis, MO) and 105 ml phosphate-buffered saline (PBS; 10 mM sodium phosphate, 2.7 mM KCl, and 137 mM NaCl, pH 7.4) containing 0.4% w/v trisodium citrate. After 20 min at room temperature, the upper leukocyte-enriched plasma was centrifuged, and the cell pellet was resuspended in 3 ml of a 50% Percoll (Sigma-Aldrich) solution, which was placed onto a Percoll gradient to separate neutrophils as described previously [6 , 12 ]. Cell viability, assessed by trypan blue exclusion, was 99%.

HL-60 cell culture
The HL-60 cell line was subcultured to a density of 5 x 105 cells/ml in Iscove’s modified Dulbecco’s medium (Gibco-BRL, Grand Island, NY), supplemented with 10% fetal bovine serum, 10,000 U/ml penicillin, 10,000 U/ml streptomycin, fungizone, and 4 mM L-glutamine in a humidified atmosphere containing 5% CO2 at 37°C. For differentiation, HL-60 cells were centrifuged and resuspended at 5 x 105 cells/ml in the same medium containing 1.3% cell culture grade dimethyl sulfoxide (DMSO; Sigma-Aldrich) to initiate differentiation to the neutrophil lineage. After 5 days, the differentiated HL-60 cells were processed for mRNA extraction, reverse transcriptase-polymerase chain reaction (RT-PCR), and immunocytochemistry. Neutrophil differentiation was assessed by the nitroblue tetrazolium (NTB) reduction assay, expression of CD11b/CD18 (membrane-activated complex 1), and the disappearance of CD71 [13 ].

RT-PCR
The Oligotex direct mRNA kit (Qiagen Inc., Valencia, CA) or the RNAqueous-4PCR kit (Ambion, Inc., Austin, TX) for isolation of DNA-free RNA was used to extract RNA from HL-60 and human neutrophils following the manufacturers’ instructions. The cDNAs were synthesized using oligo(dT) primers and ThermoscriptTM RT (Invitrogen Inc., Carlsbad, CA) in a 20-µl reaction mixture containing reaction buffer (250 mM Tris acetate, pH 8.4, 375 mM potassium acetate, 40 mM magnesium acetate, 5 mM dithiothreitol, and stabilizers), 0.5 mM deoxy-unspecified nucleoside 5'-triphosphate (dNTP), 2.5 µM oligo(dT), 40 U RNaseOUTTM RNase inhibitor (Invitrogen Inc.), and 0.5 µg poly-A+ mRNA or 5 µg total RNA. The reaction was carried out for 1 h at 60°C. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was used as a housekeeping gene. Human kidney cDNA (Clontech Laboratories Inc., Mountain View, CA) was used as a positive control. Amplification of cDNA by PCR was performed using 25 µl reaction mixture containing PCR buffer (20 mM Tris HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl2), 0.2 mM dNTP, 1 U Taq polymerase (Invitrogen Inc.), 0.1 µM specific primers (Biosource International, Camarillo, CA), and 2 µl cDNA in a GeneAmp 2400 thermocycler (Perkin Elmer, Boston, MA). The primers used were for B1 receptor, 5'-TTCTTATTCCAGGTGCAAGCAG-3' (sense) and 5'-CTTTCCTATGGGATGAAGATAT-3' (antisense), generating a fragment of 214 base pairs (bp); for GAPDH, 5'-GCAGGGGGGAGCCAAAAGGG-3' (sense) and 5'-TGCCAGCCCCAGCGTCAAAG-3' (antisense), producing a fragment of 566 bp. PCR conditions were 95°C for 5 min, Start Hot at 80°C for 2 min, followed by denaturation at 95°C for 30 s, annealing at 55°C for 40 s, and extension at 72°C for 30 s during 35 cycles. PCR products were analyzed after separation by 1% agarose gels containing 20 µg/ml ethidium bromide for ultraviolet detection.

In situ hybridization
Human neutrophils were isolated as described above, attached onto slides precoated with 0.01% polylysine (Sigma-Aldrich), fixed with cold 4% paraformaldehyde-PBS, and then washed three times for 5 min each with cold PBS. The in situ hybridization protocol [14 ] was started by washing the cells with cold 2x saline sodium citrate (SSC; 1x SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) for 10 min. Cells were then incubated for 1 h at room temperature with a prehybridization mixture containing 50% formamide, 4x SSC, 0.5x Denhardt’s solution [0.02% Ficoll, 0.02% polyvinylpyrrolidone, and 0.02% bovine serum albumin (BSA)], 25 µg/ml yeast tRNA (Gibco-BRL), and 10% dextran sulfate. The hybridization step was carried out using digoxigenine-labeled oligonucleotides constructed according to the cloned human B1 receptor sequence [15 ]. Two antisense oligonucleotides (5'-TGGCTCTGGTTGGAGGATTGGA-3' and 5'-CTTTCCTATGGGATGAAGATAT-3') with their respective sense probes (5'-TCCAATCCTCCAACCAGAGCCA-3' and 5'-ATATCTTCATCCCATAGGAAAG-3') were diluted at a concentration of 0.03 pmoles/µl in the same prehybridization solution. Hybridization was performed in a moisture chamber overnight at 42°C, and when it was completed, the cells were washed rigorously with 2x SSC and 1x SSC for 1 h each at room temperature and with two changes of 0.5x SSC for 30 min each at 42°C and then at room temperature. Next, cells were incubated for 30 min in a blocking solution containing 100 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% BSA, and 0.3% Triton X-100. The hybridized probes were detected by incubating, for 4 h at room temperature, with an antidigoxigenine alkaline phosphatase-conjugated F(ab')2 antibody (Roche Diagnostics GmbH, Mannheim, Germany), diluted 1:100 in blocking solution. Cells were washed with 100 mM Tris-HCl/150 mM NaCl, pH 7.4, for 15 min and with 100 mM Tris-HCl/100 mM NaCl/50 mM MgCl2, pH 9.5, for 10 min. Finally, phosphatase activity was developed using a commercial 5-bromo-4-chloro-3-indolyl phosphate/NTB solution (Dako, Palo Alto CA), reaction was stopped with 10 mM Tris-HCl, pH 8.0, containing 1 mM EDTA, and slides were mounted with MowiolTM (Polysciences Inc., Warrington, PA).

Immunocytochemistry
After isolation, neutrophils were washed with PBS and fixed with periodate-lysine-paraformaldehyde for 20 min at room temperature. Excess of fixative was removed, and the cells were washed rigorously with PBS. Before immunostaining, the surface Fc receptors (FcRs) were blocked by incubation with 10% human serum diluted in PBS-0.1% immunoglobulin (Ig)-free BSA. Next, neutrophils were incubated overnight at 4°C with a rabbit antiserum (1:500) raised against a peptide corresponding to the C-terminal 16 amino acids ISSSHRKEIFQLFWRN of the kinin B1 receptor. Anti-B1 receptor antibody was diluted in PBS-0.1% BSA-0.3% saponin. This antibody is well-characterized and was kindly provided by Merck and Co. Inc. (West Point, PA) [16 ]. Bound rabbit Igs were detected using a gold (15 nm)-labeled probe diluted 1:50 and a silver enhancement kit (Amersham Pharmacia Biotech, Buckinghamshire, UK) as described previously [6 ]. Cells were then postfixed with 2.5% glutaraldehyde and smeared onto glass slides precoated with gelatin. Finally, slides were contrasted with hematoxylin and mounted with Canada balsam. Controls of the immunostaining procedure included omission of anti-receptor antibody, its replacement by nonimmune IgG of the same origin, at the same dilution, and preabsorption to the cognate peptide (50 µg/ml).

Autoradiography
Neutrophils were isolated and placed onto slides precoated with gelatin and stored at –80°C until used. Before incubation, slides were warmed to room temperature, freeze-dried, and washed with cold 25 mM N-tris[hydroxymethyl]methyl-2-amino-ethanesultonic acid, pH 6.8, containing 0.1% BSA, 10 µM captopril, and 1 µM 1,10 phenantroline (Sigma-Aldrich). Incubation with 6.6 nM [3H]-des[Arg10]-kallidin ([3H]LDBK Perkin Elmer) was performed overnight at 4°C in the same buffer supplemented with 140 µg/ml bacitracin (Sigma-Aldrich), 1 mM dithiotreitol (Sigma-Aldrich), and 1 µM phosphoramidon (Sigma-Aldrich) [17 ]. After incubation, the slides were washed with cold buffer (3x1 min) and distilled water (1x1 min) and dried with cold air. Next, the slides were exposed to paraformaldehyde fumes (2 h at 80°C), washed with increasing concentrations of ethanol and xylene, and then hydrated with decreasing concentrations of ethanol and distilled water. After drying overnight, the slides were dipped with light microscopy-1 photographic emulsion (Amersham Pharmacia Biotech) at 43°C. Exposure lasted for 2 weeks at 4°C in complete darkness. Labeling was achieved by using the D-19 Kodak developer for 5 min at 20°C and Kodak F-5 photographic fixative. Binding specificity was probed in the presence of 10 µM cold B1 receptor agonist LDBK, 10 µM HOE140 (kinin B2 antagonist, Aventis Pharma Deutschland GmbH, Frankfurt am Main, Germany), 10 µM kinin B1 antagonists des[Arg9]Leu8-bradykinin (Sigma-Aldrich), and Lys-Lys-Arg-Pro-Hyp-Gly-CpG-Ser-Dtic-CpG (B9958), kindly provided by Prof. John Stewart (University of Colorado, Boulder).

Chemotaxis assay
Isolated neutrophils were resuspended in macrophage culture serum-free medium (SFM; Gibco-BRL) and primed with 1 ng/ml IL-1ß (Calbiochem, San Diego, CA) for 30 min at 37°C in a humidified atmosphere containing 5% CO2. The macrophage SFM was supplemented with 10 mM glutamine, 10% heat-inactivated human serum, and a mixture of penicillin, streptomycin, and amphotericin. Chemotaxis was assayed using the technique described by Nelson et al. [18 ], which is based on migration of cells under agarose. Briefly, agarose was dissolved in sterile, distilled water by heating in boiling water for 10 min. After cooling to 48°C, the agarose was mixed with an equal volume of prewarmed 2x SFM supplemented with a final concentration of 10% heat-inactivated human serum. Approximately 8 ml of the mixture was delivered to each 100 x 15 mm tissue-culture dish and allowed to harden at 4°C. Six series of three wells, 3 mm in diameter and spaced 3 mm apart, were cut in each plate using a template and a stainless steel punch. The center well of each three-well series received 10 µl of the suspension containing 3 x 105 neutrophils (primed or unprimed); when cells primed with IL-1ß were used, the cytokine was maintained in contact with the neutrophils in the center well throughout the experiment. The outer well received 10 µl kinin B1 receptor agonist LDBK dissolved in 0.9% NaCl, whereas the inner well received 10 µl vehicle. Dishes were incubated overnight at 37°C in a humidified atmosphere containing 5% CO2 in air. Finally, dishes were fixed sequentially with 3 ml absolute methanol and 3 ml 4% formaldehyde, the gel removed, and the cells stained with toluidine blue.

To assess the kinin receptor involved in neutrophil migration, the cells were preincubated with the kinin B2 receptor antagonist HOE140 (Aventis Pharma Deutschland GmbH) or the kinin B1 receptor antagonist des[Arg9]Leu8-bradykinin (Sigma-Aldrich) before starting the chemotaxis assay. Both antagonists were used at a 10–8 M concentration. In another set of experiments, neutrophils were primed with IL-1ß in the presence of cycloheximide (0.5 mM) or Brefeldin A (10 µg/ml). Both inhibitors were maintained in contact with the neutrophils through the assay. Quantitation of migration was done by measurement of the linear distance the cells had moved from the margin of the well toward the chemotactic factor (distance A or chemotaxis in mm) and the linear distance the cells had moved from the margin of the well toward the vehicle (distance B or spontaneous migration). The real distance achieved by cells after chemotaxis was calculated by subtracting A – B.

Measurement of the intracellular calcium ([Ca2+]i)
To determine whether stimulation of neutrophils with the kinin B1 receptor agonist resulted in an increase of [Ca2+]i, cells were loaded with the ratiometric fluorescent indicator Fura-2/AM (Molecular Probes, Eugene, OR). Briefly, unprimed and primed neutrophils (1 ng/ml, 4 h at 37°C, and 5% CO2 in air) were incubated in a 2-µM solution of Fura-2/AM for 30 min at 37°C in the darkness and then washed with Hanks’ balanced saline solution-Ca2+ (0.4 mM KH2PO4, 0.3 mM Na2HPO4, 0.136 M NaCl, 6 mM glucose, 5 mM KCl, 0.9 mM CaCl2, pH 7.4), resuspended at 2 x 106 cells/ml in the same buffer, and placed on ice until used. Positive controls were performed on NIH 3T3 fibroblasts pretreated with IL-1ß and then loaded with Fura-2/AM in an identical manner. Measurements were carried out in a thermostatted cuvette compartment of a spectrofluorimeter LS55 (Perkin Elmer) operated in dual excitation mode (excitation 340 and 380 nm, emission 509 nm) and using 4 x 106 neutrophils or 106 NIH 3T3 fibroblast under continual stirring throughout each experiment. In addition, 100 nM formyl-Met-Leu-Phe (fMLP) or 20 nM epidermal growth factor was used as stimuli to increase [Ca2+]i in neutrophils and NIH 3T3 fibroblasts, respectively. As the kinin B1 receptor agonist elicited a biphasic chemotactic response in human neutrophils, concentrations ranging from 10–5 to 10–10 M were tested. Specificity was assessed by pretreatment of cells with an excess of two kinin B1 receptor antagonists, des[Arg9]Leu8-bradykinin (Sigma-Aldrich) and Lys-des[Arg9]Leu8-bradykinin (Bachem Inc., Torrance, CA).

Quantitative image analysis
The intensity of labeling after immunocytochemistry and in situ hybridization was quantified using an automated image digitizing system (Un-Scan-It, Silk Scientific Inc., Orem, UT) as described previously [12 ]. Labeling after autoradiography was evaluated by counting silver grains deposited on each cell.

Statistical analysis
Statistical evaluations were done using the nonparametric Mann-Whitney test to analyze differences between groups. A commercial statistical package (GraphPad InStatR, Version 3.01 for Windows 95/NT, San Diego, CA) was used for these analyses. Values are expressed as mean ± SE, and significance was considered acceptable at the 5% level (P <0.05).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Kinin B1 receptor mRNA expression in human neutrophils and in differentiated HL-60 cells
We probed for the expression of the kinin B1 receptor mRNA by RT-PCR using primers to amplify a 214-bp cDNA fragment derived from Exon 3 of the human B1 receptor. The assays carried out on human neutrophils and neutrophil-differentiated HL-60 cells revealed that kinin B1 receptor transcripts were amplified in both cell types. Controls in which RT was omitted (RT–) did not amplify the 214-bp cDNA fragment (Fig. 1 ). The transcripts amplified in neutrophils and HL-60 cells had identical size to the fragment detected in the human kidney (Fig. 1) .


Figure 1
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  		Figure 1. Human neutrophil leukocytes and HL-60 cells express kinin B1 receptors. PCRs were carried out for 35 cycles, and samples were analyzed by agarose gel electrophoresis. Lane 1, DNA molecular size ladder; lanes 2 and 3, human neutrophils (lane 2, RT+; lane 3, RT–); lane 4, human kidney (HK); lanes 5 and 6, HL-60 cells differentiated to neutrophil lineage (lane 5, RT+; lane 6, RT–).

 
The expression of kinin B1 receptor mRNA in human neutrophils was confirmed by in situ hybridization using two sets of antisense primers complementary to the B1 receptor sequence (Fig. 2a ). Absence of signal was observed when the hybridization procedure was carried out with the corresponding sense primers (Fig. 2a) .


Figure 2
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  		Figure 2. Visualization of kinin B1 receptor mRNA, receptor protein, and binding sites in human neutrophils. (a) In situ hybridization by means of probes labeled with digoxigenine at the 3' end and detected by an alkaline phosphatase-labeled F(ab')2antibody. (b) Immunocytochemistry using an anti-B1 receptor antibody raised against a 16-amino acid peptide of the C-terminal end. (c) Binding of tritiated des[Arg10]-kalllidin ([3H] LDBK) to human neutrophils and visualized by autoradiography. B1 Ant, Kinin B1 receptor antagonist (des[Arg9]Leu8-bradykinin); B2 Ant, B2 receptor antagonist (HOE140). *, P < 0.001.

 
Immunoreactive kinin B1 receptor protein and B1-binding sites
The method chosen to visualize the receptor protein was highly sensitive and allowed us to avoid the interference usually observed between endogenous peroxidase activity of human neutrophils and peroxidase-labeled probes. The antipeptide antibody raised to the C terminus of the human kinin B1 receptor showed intense immunoreactivity on saponin-permeabilized neutrophils, which appeared as dots of gold/silver-enhanced grains deposited over the cells (Fig. 2b) . Immunoreactivity was only observed when neutrophils were incubated with the antipeptide antibody but not when it was replaced by nonimmune rabbit serum or when the antiserum was used in the presence of an excess of the cognate peptide (Fig. 2b) .

Autoradiographic detection with the radioactive probe [3H]-des[Arg10]-kallidin revealed a receptor distribution that was largely congruent with the pattern of immunostaining obtained with the antipeptide antibody. The kinin B1-binding sites were significantly displaced by an excess of cold peptide or two kinin B1 receptor antagonists but not by an excess of HOE140, a kinin B2 receptor antagonist (Fig. 2c) .

Kinin B1 agonists induce chemotaxis of human neutrophils
The assay carried out to assess the chemotactic ability of kinin B1 receptor agonists revealed that human neutrophils have a clear response to these ligands and especially to LDBK (Fig. 3b ). The response was similar to that produced by fMLP, but it was only observed on neutrophils primed with 1 ng/ml IL-1ß before the experiment and during the assay (Figs. 3a and 3b , and 4a and 4b ). Unprimed, freshly isolated cells did not respond to any of the B1 receptor agonists tested under this assay (Figs. 3a and 4a ). The chemoattracted neutrophils displayed the characteristic morphological appearance of cells migrating along a concentration gradient, which is a wide, leading edge orientated toward the chemotactic kinin peptide and a long and thin tail or uropod (Fig. 3d and 3e) .


Figure 3
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  		Figure 3. The kinin B1 receptor agonist Lys-des[Arg10]-bradykinin (LDBK) induces chemotaxis of human neutrophils. The chemotaxis under agarose technique was used. (a) Unprimed neutrophils challenged with 10–10 M LDBK. (b) IL-1ß-primed cells challenged with 10–10 M LDBK. (c) Unprimed neutrophils challenged with 10–8 M of the chemotactic peptide fMLP. (d and e) Morphological appearance of neutrophils attracted by 10–10 M LDBK; the leading edge is indicated by triple arrows. The arrows on the right-hand side of each figure indicate the route of cell movement toward stimuli. *, Neutrophil nuclear lobes; U, uropod.

 

Figure 4
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  		Figure 4. Chemotactic response of unprimed and primed neutrophils to the kinin B1 receptor agonist LDBK. The chemotactic effect is observed only when IL-1ß-primed cells are used (b), whereas the kinin B1 agonist has no effect on unprimed neutrophils (a). (c) Chemotactic response of human neutrophils to the chemotactic peptide fMLP. *, P < 0.05.

 
The IL-1ß-primed neutrophils showed a typical dose-response curve, which reached a maximum as the B1 receptor agonist concentration increased to decline when the concentration of the peptide increased still more. The maximal chemoattraction was observed in primed neutrophils challenged with a 10–10 M concentration of LDBK (Fig. 4b) . Although this concentration was lower than that of fMLP, the distance reached by neutrophils challenged with LDBK was half of that achieved by cells challenged with fMLP (Fig. 4b and 4c) .

Neutrophil chemotaxis is inhibited by kinin B1 antagonists, cycloheximide and brefeldin A
Chemotaxis elicited by 10–10 M LDBK was clearly reduced when neutrophils were pretreated with 10–8 M of the B1 receptor antagonist des[Arg9]Leu8-bradykinin but not by the B2 receptor antagonist HOE140 (Fig. 5a ). Reduction in cell movement was also significantly affected by 0.5 mM cycloheximide and 10 µg/ml brefeldin A, although the reduction was less pronounced than that produced by the kinin B1 receptor antagonist (Fig. 5b) .


Figure 5
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  		Figure 5. The chemotactic response to the kinin B1 receptor agonist Lys-des[Arg10]-bradykinin (LDBK) is blocked by B1 receptor antagonists, cycloheximide and brefeldin A. (a) IL-1ß-primed neutrophils were pretreated with kinin B1 (des[Arg9]Leu8-bradykinin) or B2 (HOE140) receptor antagonists before challenge with 10–10 M des[Arg10]-kallidin (LDBK). (b) Priming and chemotaxis were carried out in the presence of cycloheximide (CX) and brefeldin A (BFA). *, P < 0.05; **, P < 0.01.

 
The kinin B1 agonist LDBK does not increase [Ca2+]i
Stimulation of nonprimed and primed neutrophils with LDBK did not increase [Ca2+]i mobilization (Fig. 6a 6b 6c ). It is important to emphasize that despite the wide range of peptide concentrations tested (10–5–10–10 M), none of them elicited [Ca2+]i mobilization. By contrast, the same cells stimulated with the chemotactic peptide fMLP produced a clear increase of [Ca2+]i (Fig. 6a and 6c) . Additional controls, carried out on 3T3 NIH fibroblasts pretreated with 1 ng/ml IL-1ß and then stimulated with the same doses of the peptide, resulted in an increase in [Ca2+]i mobilization (Fig. 6d) , which was abolished by two kinin B1 receptor antagonists (Fig. 6e and 6f) . The response of 3T3 NIH fibroblasts to 0.1 µM LDBK was smaller than that elicited by 0.02 µM EGF (Fig. 6d) .


Figure 6
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  		Figure 6. The kinin B1 receptor agonist LDBK does not provoke increase of [Ca2+]i. (a) Unprimed neutrophils. (b and c) IL-1ß-primed cells. (d–f) Response of IL-1ß-primed 3T3 fibroblasts without (a) or pretreated for 30 min with 1 µM of two different kinin B1 receptor antagonists (B1 Ant: e, des[Arg9]Leu8-bradykinin; f, Lys-des[Arg9]Leu8-bradykinin). The chemotactic peptide fMLP (0.1 µM) or epidermal growth factor (EGF; 0.02 µM) was used as a positive control for neutrophils and 3T3 fibroblasts, respectively. The Ca2+ ionophore ionomycin (2 µM) was also used as positive control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Pharmacological studies and experimental models of acute inflammation have suggested that neutrophil accumulation induced by the B1 agonist des[Arg9]-bradykinin is mediated by kinin B1 receptors expressed on sensory C-fibers [9 ]. In contrast, in vitro experiments performed on human neutrophils indicate that kinin B1 and B2 receptor agonists induce a concentration-dependent migration of the cells into the lower compartment of chemotaxis chambers [11 ]. However, a LTB4 antagonist inhibited migration, suggesting that the effect of B1 and B2 ligands on human neutrophils might be indirect [11 ]. Our results show that human neutrophils isolated from peripheral blood of healthy subjects express kinin B1 receptor mRNA, immunoreactive B1 receptor protein, and binding sites for the radiolabeled B1 agonist. The consistent results obtained by the three independent approaches demonstrate that human neutrophils express the kinin B1 receptor, a result that is in agreement with previous observations of Rajasekariah et al. [8 ], who determined ~720 B1 receptor sites/cell using a radioreceptor ligand-binding assay. Moreover, HL-60 cells, induced to differentiate to the neutrophil lineage with DMSO, also expressed the B1 receptor mRNA and the immunoreactive receptor protein (data not shown). In addition, when neutrophils were challenged with the kinin B1 receptor agonist LDBK in a chemotaxis assay, a typical dose-response curve was obtained. Nevertheless, migration under agarose was dependent of priming with IL-1ß, as no chemoattraction was observed using untreated neutrophils. The optimal dose of the kinin B1 agonist (10–10 M) was lower than that needed to achieve the maximal response with fMLP (10–8 M), but the distance covered in response to des[Arg10]-kallidin was half of that obtained after challenge with fMLP. Specificity of LDBK as a chemotactic peptide for human neutrophils was confirmed by the fact that migration was blocked by a kinin B1 receptor antagonist but not by the B2 antagonist HOE140. Analysis of the chemotactic potency of bradykinin and its fragments in the eukaryotic, ciliated model Tetrahymena pyriformis has revealed that truncation of the C-terminal end and exposure of the aromatic ring of Phe8 as kinin B1 receptor agonists result in a highly chemoattractant variant [19 ]. Thus, in addition to the reported neutrophil recruitment mediated by sensory C-fibers [9 ], the kinin B1 receptor agonist LDBK exerts a direct chemotactic effect on human neutrophils by acting on cell surface-sited B1 receptors.

An intriguing observation was the fact that migration in response to LDBK was dependent on priming of neutrophils with IL-1ß. Previous reports have shown that the kinin B1 receptor is up-regulated by cytokines such as TNF-{alpha} and IL-1ß [20 21 22 23 ]. When neutrophils were exposed to the protein translation inhibitor cycloheximide during priming and through the chemotaxis assay, the migration was decreased significantly. A similar result was obtained when the cells were primed and challenged in the presence of brefeldin A, an inhibitor of protein transport between the endoplasmic reticulum and the Golgi apparatus, and a blocking agent of secretion or transport to the membrane of proteins possessing a signal peptide, whithout blocking protein synthesis [24 ]. Based on the fact that human circulating neutrophils already express the kinin B1 receptor mRNA, one can assume that during contact with IL-1ß, translation and overexpression on the cell membrane may occur, steps that could be blocked by cycloheximide and brefeldin A, respectively. In other words, these observations strongly suggest that some active protein biosynthesis is necessary to achieve neutrophil chemotaxis when a kinin B1 receptor agonist is used. Another observation that speaks in favor of this idea is the fact that an overnight incubation was necessary to see neutrophil migration in response to LDBK. Expression of other plasma membrane receptors such as FcR for IgG (Fc{gamma}R)III, complement receptor 1 (CR1), CR3, and major histocompatibility complex class I can also be prevented by incubation of neutrophils with actinomycin D and cycloheximide, indicating that some receptors expressed on the neutrophil surface may be subjected to high turnover rates; that is, they may be removed continuously from the cell membrane and need to be replenished [25 ]. The results obtained by us using IL-1ß and cycloheximide are different than those achieved by Paegelow et al. [11 ], who reported no inhibition in response to treatment of neutrophils with these two substances. This discrepancy may be a result of the chemotaxis assays used, the Boyden chamber versus migration under agarose, which includes serum and locomotion of neutrophils over a plastic surface; further, we used a dose of IL-1ß, which is 10 times higher than that used by Paegelow et al. [11 ]. It has recently been proposed that B1 receptor homo-oligomerization is required for expression of functional receptors in the plasma membrane and that regulation at this level may be an additional, more rapid mechanism to regulate its expression [26 ]. It is therefore not unreasonable to propose that in addition to influence mRNA synthesis or stability, IL-1ß may prime the B1 receptor into a functionally active state by causing its homo-oligomerization on the cell surface.

Chemotactic substances such as IL-8 and fMLP also increase cytosolic Ca2+ in response to receptor activation. Similarly, stimulation of the kinin B1 receptor results in an increase of [Ca2+]i in several cell types kept in culture [23 ]. Nevertheless, most of these experiments have been performed on cells that synthesize extracellular matrix components such as IMR-90 fibroblasts [22 ], myofibroblasts of large arteries [27 ], tracheal smooth muscle cells [28 ], and mesangial cells [29 ]. It is surprising that our experiments carried out on unprimed and IL-1ß-primed neutrophils did not result in any changes in [Ca2+]i, although samples from several individuals were tested at low (10–10 M) and high (10–5 M) doses of LDBK. As expected, control experiments showed a clear increase in [Ca2+]i mobilization after stimulation of 3T3 NIH fibroblasts with the same peptide, a response that was blunted by two kinin B1 receptor antagonists. Some studies have considered that [Ca2+]i transients play a critical role during chemotaxis, but others have determined that it is not essential for chemotactic peptide-induced actin polymerization and the subsequent migration of neutrophils on albumin/serum-coated surfaces [30 31 32 ], such as the migration under agarose technique used in this study. Moreover, studies carried out on transgenic animals suggest that Ca2+ release is not required for in vitro and in vivo neutrophil chemotaxis [33 ]. The fact that the kinin B1 receptor agonist des[Arg10]-kallidin triggers neutrophil chemotaxis without changing [Ca2+]i suggests that other Ca2+-independent mechanisms such as the Rac and Rho-kinase pathways may be involved [34 ].

Finally, we have recently shown that human neutrophil enzymes, obtained by degranulation of cells isolated from peripheral blood of healthy subjects, have the capacity to generate bioactive kinin peptides that increase venular permeability and cause hypotension [12 ]. The results reported here suggest that the neutrophil kallikrein-kinin system may function in an autocrine/paracrine manner by which kinin generation may result in attraction of large numbers of neutrophils at the site of injury.


   ACKNOWLEDGEMENTS
 
This work was supported by Grants 1030258 from FONDECYT (Chile) to C. D. F. and 200510 from Direccion de Investigacion y Desarrollo, Universidad Austral de Chile, to P. E. The authors thank Aventis Pharma Deutschland GmbH (Frankfurt, Germany) for providing us the HOE140 kinin B2 receptor antagonist; Merck and Co. Inc. (West Point, PA) for the kind gift of anti-B1 receptor antibody; and Prof. John Stewart of University of Colorado (Boulder) for the B9958 kinin B1 antagonist. We record our deepest gratitude and appreciation for the conceptual support and advice given by Drs. L. Norambuena and I. Caorsi for this research project.

Received December 20, 2005; revised February 9, 2006; accepted February 17, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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