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Originally published online as doi:10.1189/jlb.1103546 on January 23, 2004

Published online before print January 23, 2004
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(Journal of Leukocyte Biology. 2004;75:631-640.)
© 2004 by Society for Leukocyte Biology

Stimulated human neutrophils form biologically active kinin peptides from high and low molecular weight kininogens

Miguel Stuardo*, Carlos B. Gonzalez{dagger}, Francisco Nualart{ddagger}, Mauricio Boric§, Jenny Corthorn, Kanti D. Bhoola|| and Carlos D. Figueroa*,1

* Institutos de Histología/Patología and
{dagger} Fisiología, Universidad Austral de Chile, Valdivia;
{ddagger} Departamento de Biología Celular, Facultad de Ciencias Biológicas, Universidad de Concepcion, Chile;
§ Departamento de Ciencias Fisiológicas and
Centro de Investigaciones Médicas, P. Universidad Católica, Santiago, Chile; and
|| Asthma and Allergy Research Institute, School of Medicine and Pharmacology, University of Western Australia, Perth

1Correspondence: 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
 
Human neutrophils play a pivotal role in acute inflammation. However, their capacity to generate bioactive kinin peptides has not been established as yet. We have examined the ability of neutrophil enzymes to release biologically active kinins in vitro from purified human H- and L-kininogens. Neutrophils isolated from human blood were stimulated with f-Met-Leu-Phe, thrombin, or human immunoglobulin G adsorbed to silica particles. Supernatants were incubated with iodinated kininogens, and polyacrylamide gel electrophoresis analyzed aliquots taken after a range of incubation times. A time-course analysis demonstrated that supernatants from stimulated neutrophils caused a rapid hydrolysis of both substrates, resulting in an accumulation of fragments ranging from 20 to less than 10 kDa. Radioimmunoassay (RIA) revealed that all supernatants were able to generate kinins in vitro. High-performance liquid chromatography of the generated peptides indicated that they had a retention time similar to that of bradykinin and Met-Lys-bradykinin, clearly recognized as kinin peptides when the corresponding fractions were tested by RIA. The kinin-immunoreactive fractions produced lowering of blood pressure and a dramatic increase in venular permeability. Biological activity of the neutrophil-generated kinins was completely abolished by the B2 receptor antagonist HOE140, indicating that over the time-course of the experiments, only kinin B2 agonists appeared to have been generated and that cellular actions of these were mediated by kinin B2 receptors. Together, our results demonstrate that human neutrophil proteases can release kinins from both plasma kininogens, suggesting that these peptides may participate actively during acute inflammation.

Key Words: neutrophil kallikrein • degranulation • kinin B2 receptor • inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inflammation is a cellular response to injury, in which chemotactic factors and cytokines initiate the recruitment of circulating neutrophils to the site of injury and their subsequent diapedesis. Concomitantly, an increase in vascular permeability takes place, induced by various chemical mediators including kinins, which have been suggested to originate after activation of the plasma kallikrein cascade. It has also been reported that neutrophils actively participate in the enhancement of vascular permeability that takes place during acute inflammation [1 ]. However, the mechanism(s) involved in the diapedesis of neutrophils is as-yet not fully understood. A novel hypothesis based on the local release of kinins on the neutrophil surface, to open a corridor between endothelial cells and facilitate its migration into the interstitial tissue space, has already been proposed [2 3 4 ].

Evidence supports the concept of a major role of kinins in the acute, inflammatory response. First, bradykinin injections mimic the cardinal signs of inflammation, namely erythema and the increase in vascular permeability (edema) and pain. Second, various components of the kallikrein-kinin pathway have been detected at the sites of injury, and the local conditions that prevail during tissue injury are favorable for kinin accumulation. One such condition is an acid environment, which is known to reduce the activity of kinin-hydrolyzing enzymes [2 , 5 ]. Finally, increased kinin levels have been found during several inflammatory disorders including those characterized by an accumulation of a large number of neutrophils at the site of injury [6 ]. Human neutrophils have so far been shown to contain, in addition to kallikrein, a pool of neutral kininogenases capable of liberating a kinin-like substance from plasma at neutral pH [7 , 8 ]. The kinin-like peptide, generated by these kininogenases, contracted the rat uterus, and intradermal injections of the purified protease in the guinea pig induced hyperemia and an increase of vascular permeability when it was mixed with kininogen before injection [9 ]. Tissue kallikrein (KLK1, true kallikrein, kininogenase) was first described in human neutrophils based on the use of inhibitors, bioactive peptide formation, and immunoreactivity recognized by five antikallikrein (urinary origin) antibodies in radioimmunoassays (RIAs) and also on tissue sections or cell smears prepared from whole peripheral blood [10 , 11 ]. Moreover, antikallikrein antibodies inhibited the enzymatic activity present in the neutrophil homogenates [11 ]. Later, it was reported that when neutrophils are stimulated by thrombin, they release an immunoreactive kallikrein, suggesting a possible linkage between blood coagulation and inflammation [12 ]. More recently, the expression of KLK1 (tissue kallikrein or true kallikrein) in human neutrophils was confirmed using reverse transcriptase-polymerase chain reaction [13 ] in situ hybridization and antibodies raised against the recombinant protein and against an 11-amino acid peptide present in the proenzyme but not in the active protease [14 ]. Other components of the kallikrein-kinin system, such as high (H) and low (L) molecular weight kininogens, are also expressed by human neutrophils. In fact, H-kininogen was first identified in lysates of washed neutrophils by a competitive immunoassay [15 ]. Further, kininogen-binding sites can be determined using the iodinated proteins and specific monoclonal antibodies [3 , 16 ]. These observations suggested the presence of surface receptors that capture circulating kininogens or H-kininogen plasma-prekallikrein complexes. 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 [4 ]. Similarly, Kemme et al. [17 ] have shown that tissue prokallikrein localizes bound to the neutrophil surface in addition to the intracellular compartment. Ligand blot assays suggest that tissue prokallikrein may attach to the cell membrane also by interacting with H-kininogen [18 ]. In addition, binding experiments using purified cell membranes have shown that neutrophils display kinin-binding sites that have been characterized as B1 and B2 kinin receptor subtypes [19 ].

On the other side, a recent report shows that human neutrophil elastase can produce a new 17-amino acid peptide from H-kininogen [20 ]. This peptide enhances vascular permeability and induces hypotension in a magnitude comparable with that produced by bradykinin [20 ]. Nevertheless, the capacity of neutrophil enzymes, released after cell activation, to generate bioactive kinin peptides from purified human H- and L-kininogens has, so far, not been analyzed comprehensively. We describe here 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 can increase venular permeability and reduce blood pressure by acting on kinin B2 receptors. Moreover, the typical 62-kDa band, which is obtained from low molecular weight kininogen after kinin release, was observed in the presence of antielastase antibodies, indicating that kallikrein or other neutrophil enzymes, different from elastase, can release kinin peptides from low molecular weight kininogen. Kinin peptides generated by such a mechanism may aid neutrophils to modulate vascular permeability and to mobilize across the venular wall.


   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). All procedures were performed under sterile conditions and using siliconized material. Blood (35 ml) collected in 7 ml tubes containing 10.5 mg EDTA/tube 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 at 700 g for 15 min, and the cell pellet was resuspended in 3 ml of a 50% Percoll (Sigma Aldrich) solution as described previously [3 ]. Neutrophils were then separated on a Percoll gradient by centrifugation at 270 g for 26 min. Completing this procedure, the neutrophils were collected from the interface, washed with PBS–0.4% sodium citrate, and stimulated as described below.

Preparation of radiolabeled human kininogens
Purified H- and L-kininogens were kindly provided by Prof. Alvin Schmaier and Dr. Yong Ping Jiang of the Department of Internal Medicine and Pathology, University of Michigan (Ann Arbor). Both proteins were radiolabeled with 125I-Na using the method of Fraker and Speck Jr. [21 ] under conditions described previously by Schmaier et al. [22 ]. Briefly, purified kininogens (10 µg) were mixed with 5 µl 0.5 M phosphate buffer/1 M NaCl, pH 7.5, and 0.5 mCi 125I-Na (Comision Chilena de Energia Nuclear, La Reina, Santiago, Chile) in a propylene tube precoated with 4 µg iodogen and incubated for 12 min on ice. The reaction was stopped by dilution with 100 µl of the same phosphate buffer, and free iodine was separated from the 125I-labeled protein by gel filtration on a 30 x 1 cm column of Sephadex G-50 equilibrated in 0.02 M Tris-HCl/1 M NaCl, pH 8.0, containing 0.25% gelatin. Fractions of 1 ml were collected, and 10 µl aliquots were taken from each tube to count radioactivity in a Packard {gamma} counter. Labeled fractions were stored at −20°C until used.

Neutrophil degranulation
Polymorphonuclear leukocytes (95–98% neutrophils), isolated by dextran sedimentation and percoll gradients, were resuspended in Hanks/0.1% gelatin (tissue-culture grade; Sigma Aldrich) and pretreated with cytochalasin B (Sigma Aldrich) for 10 min at room temperature. Next, the cells (15x106 cells in a volume of 500 µl) were incubated for 45 min at 37°C in an atmosphere of 5% CO2–95% air with the chemotactic peptide f-Met-Leu-Phe (fMLP; 10−4 M); human thrombin (10−6 M) or human immunoglobulin G (IgG) adsorbed to silica particles (ODS/C-18 silica). For coupling, silica beads were washed with PBS–0.4% sodium citrate, and then 100 µl wet gel was incubated with 4 mg human IgG for 15 min at room temperature. Finally, an excess (10 mg) of human serum albumin was added to block the free sites that could be still available. Control experiments were performed by incubating the neutrophils under the same conditions but in the absence of stimulus. Supernatants from stimulated and control neutrophils were stored at −20°C, and the corresponding cells were processed for conventional electron microscopy or immunocytochemistry. To assess effective cell degranulation, supernatants were routinely analyzed for myeloperoxidase content at the end of each experiment using 3,3',5,5'-tetramethyl-benzidine and a colorimetric detection. Cell viability, assessed by trypan blue exclusion before and after degranulation, was 99% and 90% before and after degranulation, respectively.

Immunocytochemistry
In addition to myeloperoxidase assay, immunocytochemistry was used to evaluate effectiveness of neutrophil degranulation after stimulation. Smears prepared from control and stimulated cells were air-dried and fixed for 15 min with 2% (w/v) paraformaldehyde in PBS, pH 7.4. After fixation, cell smears were washed with PBS and treated with 0.1 M glycine to block aldehyde groups. After two washes with PBS, cells were incubated with antibodies directed to neutrophil cathepsin G (Dako Corporation, Carpinteria CA, 1:500), elastase (ICN Pharmaceuticals Ltd., Hampshire, UK, 1:1000), and human urinary kallikrein (1:500–1:1000) as described previously [3 , 4 , 10 ]. Antibodies were diluted in 0.05 M Tris-HCl, pH 7.8, containing 1% Ig-free bovine serum albumin (BSA; Sigma Aldrich). After overnight incubation at 22°C in a moisture chamber, smears were incubated with anti-rabbit IgG (Dako, 1:80) and peroxidase/antiperoxidase complexes (Dako, 1:100) for 30 min each. When the first antibody was produced in species different from the rabbit, an additional step, using a rabbit-produced IgG (1:1000 for 30 min) raised against the other IgG species, was introduced. Peroxidase activity was developed using a solution of 0.1% 3,3'diaminobenzidine–0.03% hydrogen peroxide for 15 min in 0.05 M Tris buffer. In addition, myeloperoxidase activity was cytochemically evaluated by incubating cell smears directly in the diaminobenzidine-hydrogen peroxide solution as described above. Controls of the technique included omission and replacement of the first antibody by nonimmune IgG of the same origin at the same dilution. As a control for neutrophil peroxidase, this enzymatic activity was inhibited by addition of sodium azide to the diaminobenzidine solution.

Effect of human neutrophil-released proteases on human radiolabeled kininogens
Supernatants (5 µl for silica–IgG and fMLP and 80 µl for thrombin-stimulated cells) containing the neutrophil-released enzymes were incubated at 37°C with iodinated H- and L-kininogens for 2, 5, 10, 20, and 45 min in a final volume of 150 µl 0.1 M Tris-HCl, pH 7.4. An exact determination of the amount of protein present in each supernatant was not feasible, as neutrophils had been suspended in Hank’s solution containing 0.1% gelatin to avoid cell aggregation. Nevertheless, it was possible to find out that supernatants obtained after stimulation with fMLP (fMLP supernatant) and silica–IgG (silica–IgG supernatant) contained between 0.1 and 0.17 µg/µl released proteins, whereas supernatants from thrombin-stimulated neutrophils (thrombin supernatant) contained approximately 0.08 µg/µl released proteins. Supernatants from control experiments contained an undetectable amount of proteins.

For comparison, both iodinated kininogens were also incubated with purified human urinary kallikrein (Calbiochem, San Diego, CA). In other experiments, to assess the relevance of tissue kallikrein and elastase, kininogen hydrolysis by neutrophil supernatants was performed in the presence of an excess (25 µg purified IgG in an incubation volume of 150 µl) of antibodies directed to human urinary kallikrein and human neutrophil elastase, previously passed through agarose-soya bean trypsin inhibitor and agarose-aprotinin columns to remove remnant protease activity [23 ]. After incubation, proteins were precipitated with 10% trichloroacetic acid and centrifuged at 10,000 g for 20 min, and the pellet was washed in ether and finally resuspended in sodium dodecyl sulfate (SDS)–4% ß-mercaptoethanol polyacrylamide gel electrophoresis (PAGE) buffer and boiled for 2 min before separation by SDS-PAGE.

SDS-PAGE and Western blotting
Hydrolysis products of radiolabeled kininogens, incubated with neutrophil supernatants, were run on a 12.5% polyacrylamide gel for ~3 h. The gel was then removed from glass plates and stained with Coomassie blue, dried, and exposed to X-Omat or Biomax MS Kodak films for 2–3 days at −20°C. In addition, supernatants were tested for kallikrein presence using already well-characterized antibodies to human urinary kallikrein [10 ]. For this purpose, 35 µl was taken from each supernatant, mixed with an equal amount of electrophoresis buffer, and boiled for 2 min. Each gel included one lane containing 1 µg purified human urinary kallikrein (Calbiochem). Proteins were separated by SDS-PAGE and electrotransferred to nitrocellulose membranes. After blocking of the nonspecific binding sites with 5% skimmed milk, blots were incubated overnight with anti-human urinary kallikrein (1:2000) in 0.05 M Tris-HCl containing 0.1% Tween 20 (Sigma Aldrich) and 5% BSA. Bound antibodies were detected using a peroxidase-labeled anti-rabbit IgG and a chemiluminesce kit (Pierce, Rockford, IL). Controls included omission or replacement of the anti-kallikrein antibody by nonimmune serum from the same origin.

Kinin release by neutrophil proteases
To assess kinin release by neutrophil enzymes acting on purified human kininogens, the protocol described by Shimamoto et al. [24 ] was followed. Reaction was performed in 1.5 ml polypropylene tubes containing 25 µg H- or L-kininogens and 10 µl (obtained after silica–IgG or fMLP stimulation) or 80 µl supernatant (obtained after thrombin stimulation). The final incubation volume was adjusted to 150 µl using 0.1 M Tris-HCl buffer, pH 7.4, supplemented with 30 mM EDTA, 3 mM phenanthroline, 10 µM phosphoramidon, and 20 µM captopril. These inhibitors were used only in these experiments to assess the total amount of kinin peptides generated by neutrophil enzymes, but they were excluded from other experiments in which the product was further analyzed by high-performance liquid chromatography (HPLC), as they produced enormous interference. The reaction mixture was incubated for 15 min at 37°C. When the incubation was completed, 150 µl of a 0.1% HCl in absolute ethanol solution was added, and the tubes were kept for 60 min at −20°C. Next, samples were centrifuged at 5000 g for 10 min at 4°C. Supernatants were kept, pellets were washed with 150 µl absolute ethanol and centrifuged, and supernatants were mixed with those obtained after the first centrifugation step. All supernatants were evaporated in an oven at 50°C. The dried tubes were kept at −20°C until they were resuspended and tested by RIA.

Kinin RIA
The amount of kinin peptides present after acid precipitation was measured by using a previously standardized RIA [25 , 26 ]. A standard curve was prepared using a 1-µg/ml stock solution of commercial bradykinin diluted in 1 mM oxalic acid, and the labeled peptide used was [125I]-Tyr8-bradykinin. The standard curve prepared ranged from 10 to 100 pg/tube. All points were performed in duplicates, and samples were measured in triplicates. Incubation of samples with the radioactive bradykinin was performed at 4°C for 16 h in polypropylene tubes.

HPLC
Experiments were performed in a HPLC (Pharmacia LKB Biotechnology, Bromma, Sweden) using a Lichrospher C-18 column (particle size: 5 µm). The following buffer system was used: buffer A, consisting of 0.1% trifluoroacetic acid in water; buffer B, consisting of 0.080–0.095% trifluoroacetic acid in acetonitrile/water, 60:40 (v/v). All experiments were performed at room temperature at a flow rate of 400 µl/min. Gradients were run as indicated in the figures. Monitoring at 216 nm assessed the elution profile of standard peptides and supernatants [27 ]. Aliquots of 5 µg each standard (bradykinin, lys-bradykinin, and met-lys-bradykinin) obtained commercially (Sigma Aldrich) were used. Peaks with a retention time similar to that of commercial kinin standards, and additional fractions eluted before and after those peaks were collected and further analyzed by RIA.

Effect of neutrophil kinins on vascular permeability and arterial blood pressure
The effect of kinin peptides, released by neutrophil proteases from purified kininogens, on vascular permeability was analyzed using the method described by Gawlowski et al. [28 ], modified by Boric et al. [29 ]. Briefly, adult male golden Syrian hamsters (Mesocricetus aureatus), 90–120 g, were anesthetized with 60 mg/Kg sodium pentobarbital intraperitoneally. The trachea, left carotid artery, and the jugular vein were cannulated, and the right cheek pouch was prepared for intravital microscopy as described previously [30 ]. An observation chamber was placed on top of the pouch and superfused with bicarbonate buffer, pH 7.4, 35°C, equilibrated with 95% N2/5% CO2 using a peristaltic pump at a flow of 1 ml/min. A glass cover-slide was used to isolate the chamber from room air and to prevent optical disturbances. Once surgery was completed, the animal was placed on the stage of a Nikon Optiphot microscope. Arterial carotid pressure was registered with a Statham transducer and monitored continually on a polygraph. Following a 45-min period of stabilization, commercial bradykinin (250 µl 10–7 and 10–6 M) and the various fractions collected after HPLC and RIA were added for periods of 30–50 min. The amount of immunoreactive kinin peptides, separated by HPLC, added to the preparation, ranged from 150 ng to 300 ng. The half-life of the kinin peptides was enhanced by systemic blockade of kininase II with enalapril, a drug that was injected as an intravenous (i.v.) bolus of 200 µg, followed by a continuous i.v. infusion at a rate of 10 µg/min per animal. Fluorescein isothiocyanate–dextran of 150,000 molecular weight (FITC–Dx150) was used as a macromolecular tracer. A 100-mg/Kg i.v. bolus of FITC–Dx150 was administered, and the fluorescence of the tracer was detected by epi-illumination with a halogen lamp and a Nikon B filter. Two to three selected fields were observed before and at several time-intervals following peptide administration. When the effect of the peptides on blood pressure was assessed, the standards and the fractions collected from HPLC were injected through the cannulated jugular vein. Commercial bradykinin, used as a standard, was injected as a 60- or a 90-ng bolus. To verify that the observed effects produced by HPLC fractions on vascular permeability and blood pressure were a result of activation of kinin receptors, the fractions were also tested under the influence of an excess of the kinin B2 receptor antagonist HOE140. For microvascular permeability studies, HOE140 (6.5 µg in 250 µl) was added to the superfusion buffer 5 min before applying the commercial bradykinin or HPLC fractions. For systemic pressure experiments, HOE140 was given as an i.v. bolus of 200 µg/Kg. The kinin B2 receptor antagonist, HOE140, was kindly provided by Aventis (Frankfurt am Main, Germany).

Quantitative image analysis
The intensity of labeling after immunocytochemistry in control and stimulated neutrophils and the intensity of protein bands detected after SDS-PAGE were quantified using an automated image digitizing system (UN-SCAN-IT, Silk Scientific, Orem, UT) as described previously [31 ].

Statistical analysis
Statistical evaluation was performed using the Student’s t-test. Values are expressed as means ± SE, and significance was considered acceptable at the 5% level (P<0.05).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Morphological appearance and immunocytochemistry of degranulated human neutrophils
Electron microscopy revealed a dramatic reduction in the number of cytoplasmic granules in stimulated neutrophils in comparison with nonstimulated cells. Stimulated cells also displayed numerous empty vesicles in their cytoplasm. The appearance of stimulated neutrophils was similar with the three stimuli used. Further, when silica–IgG was used as a stimulus, neutrophils were seen in close contact with the silica particles (Fig. 1C ). The three stimuli used to induce neutrophil degranulation resulted in the release of various proteins, which when analyzed by SDS-PAGE, covered a range of 95–20 kDa in molecular mass (not shown). This observation was in agreement with the release of variable amounts of neutrophil myeloperoxidase into the supernatants as assessed by a colorimetric enzymatic method (not shown). Furthermore, the stimulated neutrophils showed a significant decrease in the content of myeloperoxidase, elastase, cathepsin G, and tissue kallikrein when compared with nonstimulated controls by cytochemistry and immunocytochemistry (Fig. 2 ).



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  		Figure 1. Ultrastructural appearance of control and stimulated neutrophils. (A) Control, nonstimulated neutrophil. g, Granule. (B) fMLP-stimulated neutrophil. (C) Neutrophil stimulated with IgG-coated silica (s) particles. (D) Thrombin-stimulated neutrophil. Asterisks point out empty cytoplasmic vesicles present in the stimulated cells. Original bar size represents 0.5 µm.

 


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  		Figure 2. Visualization of myeloperoxidase (MPO), neutrophil elastase (NE), cathepsin G (CG), and kallikrein (KALL) in control and stimulated neutrophils. Original bar size represents 7 µm. AU, arbitrary units. *, P < 0.05; **, P < 0.01.

 
Effect of the released neutrophil proteases on human kininogens
Incubation of iodinated H- and L-kininogens with various volumes of supernatants obtained from stimulated neutrophils resulted in the hydrolysis of both kinin-containing substrates (Figs. 3 and 4 ). Preliminary experiments showed that after 45 min of incubation with 5 µl (0.5–0.85 µg protein) silica–IgG and fMLP supernatants, both kininogens were almost completely transformed into polypeptides ranging from 20 to less than 10 kDa. By comparison, a similar effect was obtained with thrombin supernatants but only after using 80 µl (6 µg protein) supernatant (not shown). In accordance with this observation, all experiments involving silica–IgG and fMLP supernatants were performed with 5 µl supernatant, whereas 80 µl was used when the effect of thrombin supernatants was analyzed.



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  		Figure 3. Time-course effect of fMLP, silica–IgG (5 µl), and thrombin (80 µl) supernatants on iodinated H-kininogen. (A) After incubation of iodinated kininogen with the various neutrophil supernatants, proteins were separated by SDS-PAGE, and the gel was dried and exposed to photographic film. (B) Densitometry of most representative bands was estimated at 0, 5, and 45 min of hydrolysis with a supernatant obtained from silica–IgG-stimulated neutrophils. Gels are representative of three independent experiments (n=3). Mr, Molecular weight.

 


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  		Figure 4. Time-course effect of fMLP, silica–IgG (5 µl), and thrombin (80 µl) supernatants on iodinated L-kininogen. (A) After incubation of iodinated kininogen with the various neutrophil supernatants, proteins were separated by SDS-PAGE, and the gel was dried and exposed to photographic film. (B) Densitometry of most representative bands was estimated at 0, 5, and 45 min of hydrolysis with a supernatant obtained from silica–IgG-stimulated neutrophils. Gels are representative of four independent experiments (n=4).

 
Time-course experiments showed that silica–IgG supernatants produced, after 5 min of being in contact with the H-kininogen molecule, its complete hydrolysis into several radiolabeled proteins with a molecular mass of less than 66 kDa (Fig. 3) . After 20 min of incubation, most of the molecule was transformed to a major band of 40 kDa and to small polypeptides of less than 20 kDa (Fig. 3) . The relative amount of these low molecular mass polypeptides increased progressively with the time of incubation (Fig. 3) . A similar but less-pronounced effect was observed when the same supernatant was used on L-kininogen. The 68-kDa intact molecule was partially converted after 5 min incubation with silica–IgG supernatants into a 62-kDa protein (heavy chain) that rapidly disappeared to give radiolabeled proteins of 45 kDa or less (Fig. 4) . In contrast, the formation of small radiolabeled polypeptides was less pronounced than that produced by the same supernatants on H-kininogen (Figs. 3 and 4) .

The effect produced by fMLP supernatants on H- and L-kininogens was even more dramatic than that produced by silica–IgG and thrombin supernatants on the same molecules (Figs. 3 and 4) . After 2 min of incubation with supernatants, the whole H-kininogen molecule was transformed to proteins of 58, 50, and 44 kDa, which were also rapidly hydrolyzed after 20 min into small polypeptides similar to those generated by silica–IgG supernatants (Fig. 3) . The formation of these small mass polypeptides from L-kininogen also occurred more rapidly with fMLP supernatants than with those obtained after using the other two stimuli (Fig. 4) . In contrast, control experiments performed with supernatants obtained from nonstimulated neutrophils did not produce significant changes in the molecular mass of H- and L-kininogens (not shown). The progressive, time-dependent increase in low molecular mass polypeptides, observed after incubation of the various supernatants with both kininogens, was confirmed when the intensity of most representative bands was estimated after 0, 5, and 45 min of incubation (Figs. 3B and 4B) .

Effect of antineutrophil elastase and anti-human urinary kallikrein antibodies on the hydrolysis produced by supernatants of fMLP-degranulated neutrophils on L-kininogen
As supernatants obtained from neutrophils stimulated with fMLP were very effective to produce a rapid hydrolysis of both kininogens and as tissue kallikrein preferentially releases the kinin moiety from L-kininogen, we used these supernatants to test the inhibitory effect that antielastase and anti-human urinary kallikrein antibodies may have on the hydrolysis of this kinin-containing substrate. When the antiurinary kallikrein antibody was used, the hydrolysis of the kininogen molecule was retarded in time, and the formation of the low molecular weight polypeptides, previously detected in the absence of antibody, was less evident (Fig. 5 ). The use of an antielastase antibody allowed us to clearly detect the release of the 62-kDa heavy chain from the 68-kDa intact kininogen molecule in a similar manner to that produced by purified human urinary kallikrein (Fig. 5) . This result was supported by our observation that fMLP supernatants contain two kallikrein immunoreactive bands of 58 and 43 kDa, and silica–IgG supernatants had an extra immunoreactive band of 39 kDa (Fig. 6 ). In contrast, immunoblots did not reveal any immunoreactivity after stimulation of neutrophils with thrombin (Fig. 6) .



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  		Figure 5. Effect of an excess of anti-human urinary kallikrein and antineutrophil elastase antibodies on the hydrolysis of L-kininogen by a fMLP supernatant. The hydrolysis pattern produced by human urinary kallikrein (HUK) on this molecule is shown as a comparison. The release of L-kininogen heavy chain (62 kDa) by HUK and fMLP supernatant in the presence of an excess of antielastase IgG is seen. Gels are representative of three independent experiments (n=3).

 


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  		Figure 6. Identification of immunoreactive kallikrein in fMLP supernatants. Each supernatant (35 µL) was run on 12.5% SDS-PAGE, and proteins were transferred to a nitrocellulose membrane. An antibody to human urinary kallikrein was used at a 1:500 dilution. Lane 1, Purified human urinary kallikrein; lanes 2–4 contain an equal amount of fMLP, silica–IgG, and thrombin supernatants. The blot is representative of two independent experiments (n=2).

 
Kinin release by neutrophil supernatants
All stimuli used to induce neutrophil degranulation produced supernatants that hydrolyzed both kininogens, leading to the generation of immunoreactive kinin peptides. Nevertheless, the amount of immunoreactive kinins generated from L-kininogen was higher than that released from H-kininogen (Table 1 ). In fact, the amount of immunoreactive kinins detected by RIA was between 6 and 50 times higher with the low molecular weight substrate than with H-kininogen, being the major difference recorded when fMLP supernatants were used (29.1±1.9 vs. 0.6±0.01 ng/ml; P<0.0001; Table 1 ).


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  		Table 1. Kinin Generation by Proteases Released in vitro from Stimulated Human Neutrophils

 
Our next step was to separate the released kinin peptides from L-kininogen by HPLC and then confirm the authenticity of the isolated molecules by RIA. For this purpose, the incubation of supernatants with purified L-kininogen was performed in the absence of the kininase inhibitors used above, as these inhibitors caused great interference with the separation procedure. Under these conditions, several peaks were observed, but at least two of them had a retention time similar to the pure standards, bradykinin, and Met-Lys-bradykinin (Fig. 7 ). Moreover, when these fractions were collected and subsequently immunoreactive kinins measured by RIA, there was a complete coincidence between the peaks identified by HPLC and kinin immunoreactivity being the amount of bradykinin almost double that of Met-Lys-bradykinin (Fig. 8 ). Other peaks collected and used as controls did not reveal any measurable immunoreactivity.



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  		Figure 7. Identification of kinin peptides by HPLC. L-kininogen was incubated with supernatant in the absence of kininase inhibitors. After precipitation in acid ethanol, the peptides present in the incubates were separated by HPLC. Retention time of separated peptides was compared with that produced by synthetic bradykinin (BK), Lys-BK, and Met-Lys-BK. The experiment is a representative one of peptides formed by thrombin supernatants on L-kininogen.

 


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  		Figure 8. Identification of HPLC-separated peptides by RIA. After separation by HPLC, fractions were run on a kinin immunoassay, which demonstrated the kinin nature of two fractions that exhibited a retention time similar to that of bradykinin and Met-Lys-bradykinin (cf. with Fig. 7 ).

 
Bioactivity of neutrophil-generated kinins
The fractions collected from HPLC and already assayed by RIA were then tested for biological activity on the hamster cheek-pouch microcirculation and on arterial blood pressure. The hamster cheek-pouch preparation provided us with a well-recognized method to assess the changes induced by the isolated kinin fractions on vascular permeability in vivo. Neither fluorochrome i.v. injection nor bradykinin topical application modified systemic blood pressure as monitored through a femoral artery cannula. Topical application of bradykinin did not promote an immediate extravasation of tracer but induced macromolecular leakage of FITC–Dx150, 2–4 min after the onset of peptide application (10–6 and 10–7 M), and was visualized as a bright spot spreading from postcapillary venules into the extravascular space (Fig. 9 ). When neutrophil-generated kinins were tested, they induced a microvessel leakage that was similar to that produced by the commercial bradykinin (Fig. 9) . Moreover, the increase in vascular permeability induced by the HPLC fractions was blocked by the topical preapplication of the kinin B2 receptor antagonist HOE140 (Fig. 9) . Unrelated HPLC fractions that did not contain immunoreactive kinins did not produce any effect on vascular permeability (not shown).



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  		Figure 9. Increase of venular permeability in the hamster cheek-pouch microcirculation. (A) The HPLC fractions containing immunoreactive kinins shown in Figure 7 were added (300 ng immunoreactive peptides, as determined by RIA) to the hamster cheek pouch in the presence of enalapril (10 µg/min, i.v.). The increase in vascular permeability is seen as an extravasation of FITC–Dx150. (B) Identical experiment performed in a cheek pouch pretreated with the kinin B2 receptor antagonist HOE140. (C) Increase in venular permeability induced by 200 ng commercial bradykinin.

 
In addition, neutrophil-generated kinins were injected through the jugular vein, and its effect on arterial blood pressure was recorded. Injection of neutrophil-generated kinins (250 ng immunoreactive kinins measured by RIA in the corresponding HPLC fraction) provoked a reduction in blood pressure in a magnitude that was similar to that evoked by 60 ng commercial bradykinin (Fig. 10 ). The transient hypotension induced by neutrophil-released kinins was inhibited by the injection of HOE140 previous to the injection of peptides (Fig. 10) . Other unrelated HPLC fractions used as controls did not produce significant changes in the hamster arterial blood pressure (not shown).



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  		Figure 10. Arterial blood-pressure record. Commercial bradykinin and neutrophil-formed kinins separated by HPLC and measured by RIA were injected through the jugular vein of an anesthetized hamster treated with enalapril as in Figure 9 . About 250 ng neutrophil-formed kinins induce a reduction in blood pressure that is similar to that evoked by 60 ng commercial bradykinin.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The mechanisms involved in the release of neutrophil neutral kininogenases, including kallikrein, have not been clarified yet. Our results show that stimuli commonly used to stimulate the release of neutrophil enzymes such as elastase and myeloperoxidase are effective also in delivering some of the kininogen-hydrolyzing enzymes into the incubation media. Furthermore, when the supernatants containing the released proteases came into contact with human kininogens, they produced a complete hydrolysis of both substrates into small polypeptides that ranged from 20 to less than 10 kDa in molecular mass. During the first 10 min of incubation, the supernatants acting on H-kininogen produced major bands of 60, 55, and 40 kDa in molecular mass, which thereafter, were almost completely metabolized into smaller molecular weight fragments. Conversely, L-kininogen was transiently transformed by supernatants from silica–IgG-stimulated cells into a major band of 62 kDa, indicating that the hydrolysis of the molecule included the release of its heavy chain. This observation was confirmed when the effect of fMLP supernatants was assessed in the presence of antielastase antibodies, suggesting that not only the heavy chain of low molecular weight kininogen had been released, but also, the kinin molecule may have been formed at that time by the action of one or more neutrophil kininogenases. In favor of the probable participation of neutrophil-tissue kallikrein in this release are the facts that the 62-kDa band was not observed when an anti-human urinary kallikrein antibody was added to the incubation mixture and that immunoblots showed the release of immunoreactive kallikrein into fMLP and silica–IgG supernatants. This initial observation was later confirmed by our RIA measurements, revealing the presence of kinin-immunoreactive peptides in the incubation media. These assays revealed that the amount of kinin-immunoreactive peptides formed was between six and 50 times higher using L- than with H-kininogen. Moreover, the immunoreactive peptides displayed, on HPLC, identical retention times of the commercial standards, bradykinin and Met-Lys-bradykinin. Finally, the neutrophil-generated kinin peptides, separated by HPLC and assessed its nature by RIA, were able to increase venular permeability to macromolecules in vivo and produce a transient hypotension that was mediated by kinin B2 receptors. This observation indicates that over the time-course of the experiments, only kinin B2 agonists appeared to have been generated. It has recently been reported that neutrophil elastase can hydrolyze H-kininogen to form a 17-amino acids peptide that can enhance vascular permeability and induce hypotension in a magnitude comparable with that produced by bradykinin [20 ]. Our study shows that the hydrolysis pattern produced by the neutrophil-released enzymes on H-kininogen was similar to that provoked by purified neutrophil elastase [32 ], as in addition to these small-size polypeptides, the supernatants generated three major bands of 60, 55, and 40 kDa. Additional studies have shown that kinin activity can be generated from L-kininogen by the synergistic action of neutrophil elastase and plasma kallikrein [33 ]; their results indicate that elastase would act first on L-kininogen to form a kinin-containing fragment, from which the kinin molecule is released by plasma kallikrein [33 ]. Nevertheless, in our study, the release of the 62-kDa L-kininogen heavy chain was observed even in the presence of an antineutrophil-elastase antibody. This observation indicates that there was a selective hydrolysis of the kininogen molecule and the release of the kinin domain by a kininogenase that probably resembled neutrophil kallikrein. The fact that the 62-kDa heavy chain of L-kininogen was easily observed in the presence of antielastase antibody is in agreement with the recent finding, indicating that digestion of H- and L-kininogens by human neutrophil elastase renders them essentially unsusceptible to further hydrolysis by human tissue kallikrein (urinary-type) [34 ]. However, it is important to take into account that this result was obtained using only purified enzymes and not the whole pool of enzymes that are released when neutrophils are challenged by inflammatory stimuli.

A decade ago, Cohen et al. [12 ] reported the release of tissue kallikrein from neutrophil leukocytes stimulated with thrombin using the chromogenic substrate, H-D-Val-Leu-Arg-pNA, and Western blot analysis. Four immunoreactive bands that ranged from 32 to 45 kDa were observed, and autoradiographic studies of the proteins, secreted by the thrombin-challenged neutrophils, revealed the incorporation of [35S]-methionine in a 35-kDa kallikrein-immunoreactive band [12 ]. Our results show that neutrophils stimulated with silica–IgG and fMLP released into the incubation media three immunoreactive bands of 58, 43, and 39 kDa molecular weights with kallikrein immunoreactivity. However, we did not find any immunoreactivity in supernatants of thrombin-stimulated neutrophils, indicating that the amount of kallikrein contained in these samples was probably lower than the detection level provided by the technique. In accord with this idea is the fact that large volumes of thrombin supernatants (80 µl vs. 5 µl) were required to hydrolyze both kininogen molecules. Almost identical kallikrein-immunoreactive bands have been reported in the bronchoalveolar lavage fluid of asthmatic subjects [23 ]. In fact, two molecules of 42 and 28 kDa displayed kininogenase activity and released immunoreactive kinins from purified H-kininogen, whereas a 52-kDa polypeptide was found to be inactive [23 ]. Our immunocytochemical analysis supports the occurrence of kallikrein in supernatants of degranulated neutrophils, as kallikrein immunoreactivity together with that for cathepsin G, elastase, and myeloperoxidase activity were significantly reduced after incubation of cells with the various stimuli as compared with the nonstimulated cells. It is interesting that the study of Williams et al. [35 ] has shown that neutrophils obtained from synovial fluid of patients with rheumatoid arthritis contain reduced tissue-kallikrein levels and have a loss in the kinin moiety residing in the surface-bound kininogen in comparison with circulating cells from healthy individuals.

Neutrophils are attracted to an inflammatory site by chemotactic factors formed in the very early phase of inflammation. Chemoattractants initiate migration by causing neutrophils to marginate and adhere to the endothelium of venules before diapedesis through endothelial cell gaps. Although the mechanism(s) by which neutrophils cause formation of endothelial cell gaps and migrate through them remain undetermined, it does seem likely that neutrophils secrete substances that affect the permeability of the blood vessel wall. Previous studies have shown that thrombin causes the release of various enzymes from human neutrophils, including elastase, at the moment at which they attach to the endothelium [36 , 37 ]. The presence of all kinin-forming components, namely kininogenases (tissue kallikrein and plasma prekallikrein) and kininogens, on the circulating neutrophil surface suggests that these cells may activate, under certain circumstances, a novel mechanism for their diapedesis between endothelial cell gaps. Our present results indicate that neutrophil kininogenases, released in vitro after degranulation, are able to form bioactive kinin peptides when they are incubated with the purified precursors.

Together, these findings raise important questions regarding the role of neutral kininogenases, including kallikrein, which may be released from neutrophils during inflammation, and suggest that kinin generation could be one of the mechanisms by which these cells may control vascular permeability.


   ACKNOWLEDGEMENTS
 
Grants 91/961 and 8980002 from FONDECYT, Chile, supported this work. We record our deepest gratitude and appreciation for the conceptual support and advice given by Dr. Luis Norambuena for this research project. The authors thank Prof. Alvin Schmaier and Dr. Yong Ping Jiang of the Department of Internal Medicine and Pathology of the University of Michigan, Ann Arbor, for their kind gift of purified H- and L-kininogens (human high and low molecular weight kininogens). We also thank Aventis Pharma Deutschland GmbH (Frankfurt, Germany) for providing us the HOE140 kinin B2 receptor antagonist.

Received November 7, 2003; accepted November 24, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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