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The Journal of Immunology, 2009, 182, 7906 -7915
Copyright © 2009 by The American Association of Immunologists, Inc.
doi:10.4049/jimmunol.0803624
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Neutrophil-Derived Proteinase 3 Induces Kallikrein-Independent Release of a Novel Vasoactive Kinin1,2

Robin Kahn*, Thomas Hellmark{dagger}, L. M. Fredrik Leeb-Lundberg{ddagger}, Nasrin Akbari*, Mihail Todiras§, Tor Olofsson, Jörgen Wieslander||, Anders Christensson#, Kerstin Westman{dagger}, Michael Bader§, Werner Müller-Esterl** and Diana Karpman3,*

* Department of Pediatrics, Clinical Sciences Lund, {dagger} Department of Nephrology, Clinical Sciences Lund, {ddagger} Unit of Drug Target Discovery, Division of Cellular and Molecular Pharmacology, Department of Experimental Medical Science, Lund University, Lund, Sweden; § Max-Delbrück-Center for Molecular Medicine, Berlin-Buch, Germany; Division of Hematology and Transfusion Medicine, Department of Laboratory Medicine, Lund University, Lund, Sweden; || Wieslab, Lund, Sweden; # Department of Nephrology and Transplantation, Clinical Sciences Malmö, Lund University, Lund, Sweden; and ** Institute of Biochemistry II, University Hospital, Frankfurt, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The kinin-forming pathway is activated on endothelial cells and neutrophils when high-molecular weight kininogen (HK) is cleaved by plasma kallikrein liberating bradykinin, a potent mediator of inflammation. Kinins are released during inflammatory conditions such as vasculitis, associated with neutrophil influx around blood vessels. Some patients with vasculitis have elevated plasma levels of neutrophil-derived proteinase 3 (PR3) and anti-PR3 Abs. This study investigated if neutrophil-derived PR3 could induce activation of the kinin pathway. PR3 incubated with HK, or a synthetic peptide derived from HK, induced breakdown and release of a novel tridecapeptide termed PR3-kinin, NH2-MKRPPGFSPFRSS-COOH, consisting of bradykinin with two additional amino acids on each terminus. The reaction was specific and inhibited by anti-PR3 and {alpha}1-antitrypsin. Recombinant wild-type PR3 incubated with HK induced HK breakdown, whereas mutated PR3, lacking enzymatic activity, did not. PR3-kinin bound to and activated human kinin B1 receptors, but did not bind to B2 receptors, expressed by transfected HEK293 cells in vitro. In human plasma PR3-kinin was further processed to the B2 receptor agonist bradykinin. PR3-kinin exerted a hypotensive effect in vivo through both B1 and B2 receptors as demonstrated using wild-type and B1 overexpressing rats as well as wild-type and B2 receptor knockout mice. Neutrophil extracts from vasculitis patients and healthy controls contained comparable amounts of PR3 and induced HK proteolysis, an effect that was abolished when PR3 was immunoadsorbed. Neutrophil-derived PR3 can proteolyze HK and liberate PR3-kinin, thereby initiating kallikrein-independent activation of the kinin pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In vivo activation of the kinin pathway occurs when the circulating plasma prekallikrein-high-molecular weight kininogen (HK)4 complex binds to receptors present on endothelial cells and neutrophils. Proteolytic cleavage of plasma prekallikrein results in the generation of active plasma kallikrein that in turn cleaves HK and liberates bradykinin (1). Bradykinin binds to B2 receptors constitutively expressed on endothelial cells and is degraded by carboxypeptidases to des-Arg9-bradykinin, which binds to B1 receptors induced during inflammation (2, 3). Binding of bradykinin to its receptor results in capillary leakage, inflammation, pain, and local reduction of blood pressure. Bradykinin also stimulates intimal hypertrophy and proliferation of smooth muscle cells during endothelial damage (1). The kinin system is activated during several conditions associated with systemic inflammation such as vasculitis (4), sepsis (1), and colitis (5).

Neutrophil extravasation into inflamed tissues with degranulation and release of proteolytic enzymes, such as proteinase 3 (PR3) (6), is one of the major features of systemic inflammatory conditions such as vasculitides. PR3 is encoded by a gene located on chromosome 19p13.3 consisting of five exons. The catalytic site of PR3 consists of His44, Asp91, and Ser176, located on exons two, three and five, respectively. In resting neutrophils, PR3 is stored in the azurophilic and specific granules as well as secretory vesicles, or it is expressed on the plasma membrane. The acidic environment of the azurophilic granules keeps PR3 in a conformationally inactive form. Upon cell activation degranulation and translocation to an environment with neutral pH enables the protease to become enzymatically active (reviewed in Ref. 6). When neutrophils are activated, membrane-bound PR3 is up-regulated (7) and a subset of patients with vasculitis has more PR3 on their neutrophil membranes as well as higher levels of PR3 in plasma than do controls (8). PR3 degrades extracellular matrix proteins, kills microbes (6), and has the ability to cleave and inactivate C1 inhibitor (9). PR3 binds to endothelial cells (10) and induces apoptosis (11) and IL-8 production and secretion from these cells (6). The physiological inhibitor of PR3 in plasma is {alpha}1-antitrypsin ({alpha}1-AT) (12).

In this study we investigated vasculitis as a model for systemic inflammation with activation of the kinin pathway (4). Vasculitis is a systemic condition manifested by inflammation in and around vessel walls with neutrophil infiltration accompanied by perturbed vessel patency and secondary tissue damage. Many different organs may be involved, such as the kidneys, lungs, skin, intestines, and joints. Patients exhibit various symptoms such as renal dysfunction, purpura, abdominal pain, and joint pain and swelling (13, 14, 15). Severe vasculitis is often associated with the presence of anti-neutrophil cytoplasmic Abs (ANCA) directed toward granular components of neutrophils, predominantly PR3 or myeloperoxidase (6).

The components of the activated kinin system may contribute to the inflammatory process during vasculitis (4). As neutrophil infiltrates are a cardinal pathological feature of vasculitis, and activated neutrophils express and secrete PR3, the present study aimed to investigate if PR3 from neutrophils could cleave HK and liberate bradykinin or bradykinin-like peptides in a kallikrein-independent manner, establishing a novel kinin-forming pathway that may operate during systemic inflammation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
PR3-induced HK proteolysis

To investigate if PR3 could proteolyze HK, purified HK (100 µg/ml, corresponding to the plasma concentration (1); Enzyme Research Laboratories) was incubated for 20 min at 37°C with PR3 (Wieslab) at a final concentration of 1 or 10 µg/ml (5x and 50x the concentration in plasma from vasculitis patients (16, 17)) or with active plasma kallikrein (Enzyme Research Laboratories) at a final concentration of 16.3 µg/ml, as a positive control. Recombinant PR3 (10 µg/ml, final concentration), wild-type, or enzymatically inactivate PR3 (18, 19) were incubated with HK (100 µg/ml, final concentration) for 20 min at 37°C. Specificity was tested by preincubating PR3 with polyclonal rabbit anti-human PR3 (Wieslab) at 67 µg/ml (1:1 molar ratio) for 30 min at 37°C before adding HK. The anti-PR3 Ab did not cross-react with plasma kallikrein (0.5 µg), tissue kallikrein (KLK1; Abnova), or kallikreins (KLKs) 4, 5, and 8 (a gift from Maria Brattsand, Department of Public Health and Clinical Medicine, Umeå University, Sweden), all of which were run on a 10% SDS-PAGE gel (0.5 µg of kallikrein per row) and electroblotted. The immunoblot was compared with a protein gel exposed to SilverSnap stain (Thermo Scientific).

PR3 (1 and 10 µg/ml, final concentration) was also incubated with {alpha}1-AT (1.8 mg/ml, final concentration, corresponding to the known plasma concentration (20), gift from Dr. Sabina Janciauskiene, Lund University, Sweden), known to rapidly inhibit PR3 activity in plasma (12), for 5 min at 37°C and then added to HK (100 µg/ml, final concentration) for 20 min at 37°C. PR3 (10 µg/ml, final concentration) was incubated with normal platelet-poor plasma (obtained as in Ref. 4), concentration of HK {approx}80 µg/ml (1) for 90 min at 37°C. To investigate if PR3 liberates kinins from HK (by ELISA), purified HK (5 µg/ml) was incubated for 20 min with PR3 at a final concentration of 1 or 10 µg/ml at 37°C or with plasma kallikrein at a final concentration of 16.3 µg/ml, as the positive control. Samples were frozen at –20°C (for immunoblotting) and at –80°C (for assay by ELISA).

Detection of HK proteolysis by immunoblotting

Proteolytic processing of HK was assayed by immunoblotting as previously described (4). Samples containing HK were diluted 1/10 and samples containing plasma were diluted 1/100. The samples were run on a SDS-10% PAGE gel, electroblotted for 2 h, and membranes were incubated with a primary Ab against HK. Three different primary Abs (4) were used: antiserum AS88 (from sheep) against purified HK (1/5000) (21), mAb HKH8 (from mouse) directed to domain D2 (1/1000) (22), and mAb HKL22 directed to domain D6H (1/500) (22). Appropriate secondary Abs were applied, including goat anti-mouse/HRP (1/3000), goat anti-rabbit/HRP (1/3000; both Abs from Dako), and donkey anti-sheep/HRP (1/3000; ICN Biomedicals). The positive controls were either HK incubated with plasma kallikrein or platelet-poor plasma incubated with dextran sulfate (75 µmol/L; Sigma-Aldrich) in the presence of ZnCl2 (2 mmol/L; Sigma-Aldrich) (4). The negative controls were HK incubated with PBS (Medicago) or plasma with PBS. The signal was detected by chemiluminescence.

Bradykinin ELISA

Samples were analyzed for bradykinin release using the MARKIT-M bradykinin-ELISA kit (Dainippon Pharmaceutical) as per the manufacturer and as described (4). To determine the optimal time for incubation of PR3 with purified HK, purified HK (5 µg/ml) was incubated with PR3 (1 and 10 µg/ml) for 1, 5, 10, 20, 30, 45, 60, and 90 min. Liberation of kinins peaked at 20 min and declined toward 90 min. The length of incubation of PR3 with purified HK was therefore set at 20 min.

Mass spectrometry

For analysis by MALDI, HK (400 µg/ml, final concentration) was incubated with PR3 (100 µg/ml, final concentration) or with plasma kallikrein (326 µg/ml, final concentration) at 37°C for 20 min. Trichloroacetic acid (18.5 mg/ml, final concentration; Dainippon Pharmaceutical) was added to the samples, after which samples were centrifuged at 1300 x g for 10 min and analyzed directly. An aliquot of 0.5 µl was applied to a MALDI target slide surface and allowed to dry. One-half microliter of matrix solution (2.5 mg/ml of {alpha}-cyano-4-hydroxy-cinnamic acid in 50% acetonitrile containing 0.05% trifluoroacetic acid) and another 0.5 µl of the sample were added and dried before being used for analysis on a MALDI-TOF mass spectrometer (MALDI micro MXT; Waters, Manchester, U.K.).

In separate experiments synthetic PR3-kinin (position 361–374 in HK, MKRPPGFSPFRSS; final concentration, 0.1 mg/ml; MedProbe) was incubated with platelet-poor plasma, plasma kallikrein (final concentration, 163 µg/ml), or PBS for 30 s or 1, 5, or 10 min. Additionally, PR3-kinin was incubated for 10 min with platelet-poor plasma, which was preincubated for 20 min with carboxypeptidase inhibitors: {epsilon}-amino-n-caproic acid (EACA; Kebo Lab), which inhibits carboxypeptidase B; 2-guanidinoethylmercaptosuccinic acid (GEMSA; Calbiochem), which inhibits carboxypeptidase B-like processing enzyme; and Plummer’s inhibitor (Calbiochem), which inhibits carboxypeptidase N. PR3 was also separately incubated with platelet-poor plasma preincubated for 60 min with Complete Mini (Roche), a protease inhibitor cocktail that inhibits serine, cysteine, and metalloproteases, as well as calpains. The samples were analyzed by MALDI as above.

To analyze the amino acid sequence of released kinins and rule out the possibility of contaminating proteases (such as plasma kallikrein) in purified HK, a peptide of 28 aa residues covering the sequence of the human HK domain 4 (D4; position 354–381) was synthesized (MedProbe): NH2-PLGMISLMKRPPGFSPFRSSRIGEIKEE-COOH, referred to as synthetic HK D4. This synthetic HK D4 consists of bradykinin (underlined) with an additional 9 and 10 residues at the N terminus and C terminus, respectively, and the amino acid sequence matches parts of the D4 domain identical in both HK and low-molecular weight kininogen. Synthetic HK D4 (at a final concentration of 9 mg/ml) was incubated with PR3 at a final concentration of 13 or 130 µg/ml or with plasma kallikrein (the positive control) at a final concentration of 163 µg/ml at 37°C for 20 min and analyzed by MALDI (as above) or by electrospray ionization (ESI) with a QToF Ultima API (Waters) coupled to a CapLC HPLC. The digests were separated on reversed-phase analytical column (Atlantis; C18, 75 x 150 mm, 3 mm, 100 Å; Waters) using a 60 min linear gradient. Dynamic data acquisition scanning was used over the mass range m/z from 400 to 1600 for MS (MALDI) and from 50 to 1800 for MS/MS (ESI). Only spectra from ions with charge state 2 and 3 were acquired. Database searches were done using the known sequence of HK.

Competition binding assays

HEK293 cells (American Type Culture Collection), stably transfected with the human kinin B1 receptor and B2 receptor, were cultured as described (23). Radioligand binding with 1 nM [3H]des-Arg10-kallidin (77.5 Ci/mmol) and 1 nM [3H]bradykinin (90.0 Ci/mmol) (PerkinElmer) was conducted as previously described (3). Binding assays were conducted on ice in duplicates, and nonspecific binding was defined as the amount of radiolabeled ligand bound in the presence of 1 µM unlabeled des-Arg10-kallidin or bradykinin. The IC50 was determined from the dose-dependent displacement of 1 nM [3H]des-Arg10-kallidin (for the B1 receptor) or 1 nM [3H]bradykinin (for the B2 receptor) by PR3-kinin. Binding data were analyzed using Prism (GraphPad Software).

Kinin receptor activation quantified by phosphoinositide (PI) hydrolysis

HEK293 cells were seeded in 6-well dishes and labeled with 1 µCi/ml [3H]myo-inositol (PerkinElmer) in DMEM/10% FBS for 20–24 h at 37°C. The cells were then washed four times in DMEM, and further incubated in DMEM for 1 h at 37°C. This was followed by incubation in the presence or absence of PR3-kinin in DMEM supplemented with 50 mM LiCl for 30 min at 37°C. The cells were then lysed with 0.1 M formic acid for 20 min at 4°C and centrifuged at 16,100 x g for 5 min at 4°C. The supernatants were added to anion exchange columns, which were washed twice with a low salt solution (60 mM ammonium formate, 5 mM sodium borate). Inositol phosphates were then eluted with a high salt solution (1 M ammonium formate, 0.1 M formic acid) and radioactivity was quantified in a Beckman LS6000 liquid scintillation counter (Beckman Coulter).

In vivo experiments using wild-type and B2 receptor knockout mice

Adult (12 wk old) male C57BL/6 wild-type and kinin B2 receptor knockout mice maintained on the same background (24) were used as previously described (25). Blood pressure and heart rate were recorded with a transducer (model 1050.1; AD Instruments) that was connected to a computer system for data acquisition and analysis (Chart 5.5.5 PowerLab; AD Instruments) in freely moving mice. Bradykinin (Bachem) and PR3-kinin were given as a bolus dose (1 µl/10 g of body weight) in cumulative concentrations (0.1–100 µg/kg) at 45- to 60-min intervals.

In vivo experiments using wild-type and B1 receptor overexpressing rats

Twelve-week-old male rats (350–400 g) from colonies of Hannover Sprague-Dawley rats and heterozygous TGR-Tie2B1 transgenic rats on the Hannover Sprague-Dawley background were maintained at the Max-Delbrück-Center. The generation of TGR-Tie2B1 rats overexpressing the B1 receptor exclusively in the endothelium and the experimental procedures were previously described (26). Substances were given in 1 µl per 100 g of body weight at the following doses: 0.1 µg/kg des-Arg10-kallidin (Bachem); 1 and 10 µg/kg bradykinin and PR3-kinin. Icatibant (HOE 140, 100 µg/kg; Sigma-Aldrich) was administered i.v. 10 min before administration of bradykinin or PR3-kinin. Blood pressure was measured as previously described (26).

All experimental procedures in mice and rats were performed in accordance with the guidelines for the humane use of laboratory animals as approved by the local ethical committee of Max-Delbrück-Center for Molecular Medicine, Berlin.

Subjects

Healthy adult controls (n = 13, 8 women, 5 men) and patients with vasculitis (n = 10, 3 adults, 7 children) participated in the study. Vasculitis was defined according to Chapel Hill nomenclature (27). Neither the patients nor the controls were receiving any medications at the time of sampling, and all patient samples were taken at the onset of vasculitis symptoms. Patients were admitted to the Department of Nephrology, University Hospital, Malmö, Sweden, between September and October 2002 (n = 3, patients 1–3) or the Department of Pediatrics, Section of Pediatric Nephrology, University Hospital, Lund, Sweden, between June 1999 and September 2001 (n = 7, patients 4–10). Patients 4–10 have been previously described (4). The clinical and laboratory aspects of all patients are summarized in Table I. Informed consent was obtained from all participants and the study was approved by the Ethics Committee of the Medical Faculty of Lund University.


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  		Table I. Clinical and laboratory features of patients at time of sampling

 
Blood samples

Venous blood from patients and controls were collected in 2.7-ml plastic tubes (BD Diagnostics) containing 0.129 M sodium citrate. Plasma was obtained by centrifugation as previously described (4). Whole blood for neutrophil isolation was obtained in 15-ml plastic tubes (Sarstedt) with sodium heparin (150 USP, Venoject; Terumo Europe).

Evidence for HK proteolysis and bradykinin release in patients and controls

Criteria for inclusion of patients in this study were the presence of HK proteolysis and bradykinin release in plasma samples. HK proteolysis and bradykinin release were analyzed in patients and controls as previously described (4) and are presented in Table I. Plasma samples were available from 10 vasculitis patients (pediatric and adult) and 12 adult controls. Samples from patients were taken at the onset of symptoms. All patient samples exhibited HK proteolysis and bradykinin release in plasma samples with a median of 406 pg/ml (range, 81–1908 pg/ml) (Table I), whereas the controls did not exhibit any HK proteolysis and their bradykinin levels were considerably lower, with a median of 86 pg/ml (range, 0–425 pg/ml).

Neutrophil purification

Neutrophils were purified from five patients and five controls (of which four controls also contributed plasma samples). Neutrophils were isolated from whole blood with Polymorphprep (Axis-Shield) or NIM (Cardinal Associates) as per the manufacturers’ instructions. The neutrophils were resuspended at a concentration of 1 x 106 cells/ml in a buffer containing 5.6 mM D -glucose, 127 mM NaCl, 5.4 mM KCl, 1.2 mM KH2PO4, 0.8 mM MgSO4, 10 mM HEPES, and 1.8 mM CaCl2 (28). The cells were identified as leukocytes by flow cytometry using anti-CD45:FITC, and the neutrophil population was identified using three separate Abs, anti-CD11b:RPE, anti-CD18:FITC, and anti-CD16:FITC. The lymphocyte population was identified using anti-CD3:FITC, anti-CD19:FITC, and a platelet population was ruled out using anti-CD41:RPE (all Abs from Dako; 5 µl of Ab to 50 µl of cells). The cells isolated consisted only of leukocytes, of which at least 90% were neutrophils.

Preparation of neutrophil extracts

The neutrophils were incubated for 30 min with or without 0.05% Triton X-100 (Merck) on ice to induce cell lysis and release of the cell content to the medium. The cell extracts were then centrifuged at 14,000 x g for 1 min. These extracts contain cell lysates and membranes. No IgG was detected in the neutrophil extracts from patients or controls by immunoblotting using rabbit anti-human IgG-HRP Ab (Dako). The supernatants were transferred into plastic tubes and frozen at –80°C until analyzed.

Immunoadsorption of PR3

A CNBr-activated Sepharose 4B gel (Amersham Biosciences) was coupled with the mAb 4A3 against PR3 (29) (0.4 mg/ml, 1.2 mg to 0.5 g Sepharose gel). Neutrophil extracts from patients and controls were incubated individually with the Sepharose gel for 3 h at room temperature and the flow-through was collected. The 4A3 Ab does not react with other neutrophil proteases, such as elastase and cathepsin G (29, 30), or with plasma kallikrein, tissue kallikrein (KLK1), or KLKs 4, 5, and 8 (method as described above for the rabbit anti-human PR3 Ab). Levels of PR3 were measured in neutrophil extracts by ELISA, as previously described (31). The efficiency of immunoadsorption was determined in this manner.

Neutrophil extract-induced HK proteolysis

Four variants of neutrophil extracts were tested: Triton X-100-treated neutrophil extract, Triton X-100-treated neutrophil extract in which PR3 was immunoadsorbed, untreated neutrophil extract, and untreated neutrophil extract in which PR3 was immunoadsorbed. The neutrophil extracts were added to purified HK (5 µg/ml for ELISA and 100 µg/ml for immunoblotting) at volume proportions of 1:9 (neutrophil extract/HK). As a control, the neutrophil buffer without neutrophils was combined with purified HK. The samples were incubated for 20 min at 37°C and assayed as above.

Statistics

The Mann-Whitney U test was used for comparing the difference between the levels of kinins released from HK by two concentrations of PR3 and the negative control and for comparison of differences in kinin levels between neutrophil extracts from the patients and controls and of differences in PR3 levels in the neutrophil extracts between the patients and the controls. The Wilcoxon signed ranks test was used for comparison of differences in kinin release from HK by the neutrophil extracts between the four experimental groups. A p value of ≤0.05 was considered significant. SPSS version 14 was used for the statistical analyses.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
PR3 proteolysis of HK resembles the physiological breakdown pattern

HK consists of six domains representing the H chain, the bradykinin sequence, and the L chain (Fig. 1) (1). In vitro degradation of HK in plasma by dextran sulfate results in three fragments visible by immunoblotting using the polyclonal AS88 Ab, which recognizes all domains of HK including the H chain (63 kDa, domains 1–3), the L chain (58 kDa, domains 5 and 6), and a breakdown product of the L chain (45 kDa, domain 6; Fig. 1, lanes 1 and 2) (4). These three fragments resemble the physiological breakdown pattern in plasma seen during kinin system activation in vasculitis (Fig. 1, lane 3) (4). Plasma kallikrein degrades purified HK (1, 32) into two fragments (63 and 45 kDa; Fig. 1, lane 5). When HK was incubated with PR3, three bands were detected (Fig. 1, lane 6), corresponding to the bands seen in plasma. mAbs against domains 2 or 6 identified these bands as the H chain, L chain, and the breakdown product of the L chain (Fig. 1, lanes 7 and 8) (4). A fourth band at ~70–75 kDa was also identified, which may represent a breakdown product of HK.


Figure 1
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  		FIGURE 1. Breakdown of HK in plasma and in purified form by PR3 and plasma kallikrein. Lanes 1–6, Immunoblots detected with AS88, a polyclonal Ab recognizing all of the domains of HK and its breakdown products. Lane 1, Control plasma, showing two bands at 116 and 63–66 kDa corresponding to HK and its H chain and low-molecular weight kininogen (4 ). Lane 2, Normal plasma with dextran sulfate inducing kinin pathway activation showing three bands at 63–66, 58, and 45 kDa, corresponding to H chain, L chain, and degradation of the L chain. Lane 3, Representative plasma from an adult vasculitis patient, showing HK proteolysis. Lane 4, Purified HK (100 µg/ml). Lane 5, Purified HK incubated with plasma kallikrein (16.3 µg/ml) inducing breakdown of HK, showing bands at 63–66 and 45 kDa. Lane 6, Purified HK with PR3 (10 µg/ml) inducing breakdown of HK, showing four bands, corresponding to an unidentified breakdown product (70 kDa), H chain (63 kDa), L chain (58 kDa), and a degradation product of the L chain (45 kDa). Lane 7, Detected with HKH 8, mAb recognizing domain 2 (H chain): purified HK incubated with PR3 (10 µg/ml) inducing breakdown of HK, showing one band at 63 kDa corresponding to the H chain. Lane 8, Detected with HKL 22, mAb recognizing domain 6 (L chain and degradation of L chain): purified HK incubated with PR3 (10 µg/ml) inducing breakdown of HK, showing three bands at 70, 58, and 45 kDa corresponding to an unidentified breakdown product, L chain, and degradation of L chain. D1–D6, domains 1–6 of high-molecular weight kininogen.

 
These results indicate that the PR3-induced proteolysis pattern of HK resembles HK breakdown in plasma and is similar, but not identical, to the pattern produced by the known physiological processing enzyme, plasma kallikrein.

PR3 proteolyzes HK in a dose-dependent and specific manner

Incubation of purified HK (Fig. 2A, lane 2) with PR3 resulted in a dose-dependent breakdown of HK (Fig. 2A, lanes 3 and 4) and release of kinins (Fig. 2A, lanes 3 and 4). Incubation of HK with recombinant wild-type PR3 resulted in partial degradation of HK (Fig. 2B, lane 3), whereas no degradation was seen when HK was incubated with recombinant enzymatically inactive PR3 (Fig. 2B, lane 4). Preincubation of PR3 with the anti-PR3 Ab could partially inhibit the PR3-induced proteolysis of HK (Fig. 2C, lanes 2 and 3).


Figure 2
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  		FIGURE 2. PR3 induced HK degradation. A, PR3 induces breakdown of HK. Lane 1, Immunoblot showing purified HK (100 µg/ml) incubated with plasma kallikrein (16.3 µg/ml), inducing total HK proteolysis. Lane 2, Purified HK (100 µg/ml). Lane 3, HK incubated with PR3 (10 µg/ml), inducing almost total breakdown of HK. Lane 4, HK incubated with PR3 (1 µg/ml), inducing partial breakdown of HK. Samples were run on the same gel. The experiment was repeated five times with reproducible results. One representative experiment is shown. The ELISA results showing bradykinin levels (in pg/ml, median (range)) are depicted under the immunoblot figure. Purified HK (5 µg/ml) was incubated with PR3 (1 and 10 µg/ml). ELISA experiment was repeated eight times. Differences in bradykinin release after incubation with PR3 were significant when comparing the two different PR3 concentrations individually with the negative control as well as when comparing the two different PR3 concentrations with each other (p = 0.001). B, HK proteolysis of PR3 was dependent on enzymatic activity. Lane 1, Purified HK (100 µg/ml). Lane 2, HK incubated with PR3 (10 µg/ml), inducing breakdown of HK. Lane 3, HK incubated with recombinant PR3 (10 µg/ml), inducing partial breakdown of HK. Lane 4, HK incubated with recombinant mutated PR3 (10 µg/ml), resulting in no breakdown of HK. The experiments were repeated three times and one representative gel is shown. C, Specificity of PR3-induced proteolysis. Lane 1, Purified HK (100 µg/ml). Lane 2, HK incubated with PR3 (10 µg/ml), inducing breakdown of HK. Lane 3, PR3 preincubated with an anti-PR3 Ab (67 µg/ml) and then incubated with HK. The Ab could partially inhibit HK proteolysis. The experiment was repeated three times and the lanes were run on the same gel. D, {alpha}1-Antitrypsin inhibits PR3-induced breakdown of HK. Lane 1, Purified HK (100 µg/ml). Lane 2, PR3 (10 µg/ml) incubated with HK, inducing total breakdown of HK. Lane 3, PR3 incubated with {alpha}1-antitrypsin (1.8 mg/ml) before combining with HK resulted in total inhibition of PR3-induced breakdown of HK. The samples were run on the same gel. The experiment was repeated four times.

 
{alpha}1-AT is the principal inhibitor of PR3 in plasma (12). Incubation of {alpha}1-AT with PR3 before adding HK inhibited PR3-induced HK proteolysis (Fig. 2D, lanes 2 and 3). Purified PR3 was added to normal plasma to investigate whether the proteolytic activity of PR3 was functional in normal plasma, and no proteolysis of HK was detected (data not shown). The results indicate that PR3 proteolysis of HK is inhibited by {alpha}1-AT, and this most probably accounts for the inhibition of proteolysis in plasma.

PR3 proteolysis of native HK and of synthetic HK D4 liberates a tridecapeptide containing the full bradykinin sequence

To identify potential kinins liberated from HK by PR3, PR3 was incubated with HK, which resulted in the liberation of a peptide with a molecular mass of 1492.7 Da (analyzed by MALDI). To rule out the possibility of contamination with plasma kallikrein, PR3 was incubated with a synthetic HK D4 (28-aa-long peptide, containing the 9-aa-long bradykinin sequence and its flanking regions), which showed release of a peptide with the same molecular mass. The amino acid sequence of this peptide was determined by MS/MS and found to be a novel tridecapeptide, NH2-MKRPPGFSPFRSS-COOH, referred to as PR3-kinin. Incubation of HK or synthetic HK D4 with plasma kallikrein, as a positive control, led to the release of bradykinin (1059.4 Da), as expected, but not PR3-kinin (data not shown).

PR3-kinin binds to and activates the human B1 receptor but does not bind to the B2 receptor

PR3-kinin, at concentrations of 10–10 to 10–6 M, was added in a competition binding assay with radiolabeled ligands [3H]des-Arg10-kallidin (B1 receptor ligand) or [3H]bradykinin (B2 receptor ligand) to HEK293 cells stably expressing the human B1 or B2 receptors (23). PR3-kinin bound with a relatively high affinity to the human B1 receptor (IC50 value of 6.3 ± 0.4 x 10–9 M, Fig. 3A), whereas it did not interact appreciably with the human B2 receptor (IC50 value at >1 x 10–6 M, Fig. 3A).


Figure 3
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  		FIGURE 3. PR3-kinin binds to and activates the human B1 receptor but not the B2 receptor. A, PR3-kinin binding to bradykinin receptors. PR3-kinin competition with [3H]des-Arg10-kallidin binding (•) for human kinin B1 receptors and [3H]BK binding ({circ}) for kinin B2 receptors. PR3-kinin binds to the kinin B1 receptor with relatively high affinity whereas it does not interact appreciably with the kinin B2 receptor. B, PR3-kinin-stimulated PI hydrolysis via the B1 receptor. HEK293 cells stably expressing B1 receptors were labeled with [3H]myo-inositol and then assayed for PI hydrolysis in the presence of increasing concentrations of PR3-kinin. The data are expressed as percentage of maximum, which refers to the stimulation with 1 µM des-Arg10-kallidin and are presented as means ± SEM from three separate experiments with each data point done in duplicate.

 
Cells stably expressing the B1 receptor were radiolabelled with [3H]myo-inositol and incubated with increasing concentrations of PR3-kinin. Data show that PR3-kinin induced PI hydrolysis in a dose-dependent manner (Fig. 3B). The results indicate that PR3-kinin binds to and activates the B1 receptor in vitro.

PR3 exerts a hypotensive effect in vivo through both B1 and B2 receptors

PR3-kinin and bradykinin were administered i.v. at increasing concentrations to wild-type or B2 receptor knockout mice. PR3-kinin induced an instantaneous dose-dependent hypotensive effect equivalent to the effect of bradykinin (Fig. 4A). The effect was mediated via B2 receptors, as administration of PR3-kinin or bradykinin to B2 receptor knockout mice had no effect on their blood pressure (Fig. 4A) and expression of the B1 receptor is negligible under these experimental conditions. Taken together, the in vitro and in vivo results point to further processing of PR3-kinin into active derivative(s) capable of B2 receptor stimulation.


Figure 4
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  		FIGURE 4. In vivo administration of PR3-kinin stimulates both the B1 and the B2 receptor. A, Wild-type (WT) and B2 receptor knockout (B2KO) mice were injected with cumulative doses of PR3-kinin or bradykinin (0.1, 1, 10, 50, 100 µg/kg) and the drop in mean arterial blood pressure (delta MAP, in mmHg) was measured. The hypotensive effect of PR3-kinin was equivalent to that of bradykinin. The effect was mediated via the B2 receptor as B2KO mice did not respond to PR3-kinin or bradykinin. B, Wild-type rats (WT, Sprague-Dawley) and transgenic rats overexpressing B1 receptors (TGR-Tie2B1) were injected with two different concentrations of PR3-kinin or bradykinin (1 and 10 µg/kg) and the drop in mean arterial blood pressure (delta MAP, in mmHg) was measured. At 10 µg/kg PR3-kinin was more efficient than bradykinin in lowering the blood pressure in the TGR-Tie2B1 rats, suggesting that PR3-kinin induces potent B1 receptor activity. C, As in B, WT and TGR-Tie2B1 rats were injected with PR3-kinin or bradykinin (10 µg/kg). The specific effect on B2 receptors was demonstrated by blocking with HOE-140 (HOE) and the drop in mean arterial blood pressure (delta MAP, in mmHg) was measured. PR3-kinin had a more potent hypotensive effect in TGR-Tie2B1 rats than bradykinin and the effect was only partially abolished by HOE-140, suggesting a B1 receptor-mediated effect. As a positive control, des-Arg10-kallidin (0.1 µg/kg, a B1 receptor agonist) was administered to both WT and TGR-Tie2B1 rats.

 
Transgenic rats TGR-Tie2B1, which overexpress the B1 receptor in the endothelium, and wild-type Hannover Sprague-Dawley rats were investigated for the in vivo effect of PR3-kinin on B1 receptors. The hypotensive effect was studied after i.v. injection of PR3-kinin and bradykinin at two concentrations. PR3-kinin exerted a dose-dependent hypotensive effect in TGR-Tie2B1 rats, which was greater than the effect of bradykinin, suggesting that the effect was partially mediated via binding to the B1 receptor (Fig. 4B). In wild-type rats the effects of PR3-kinin and bradykinin were similar (Fig. 4B). Pretreatment of the TGR-Tie2B1 rats with HOE-140, a selective B2 receptor antagonist, almost totally abrogated the effect of bradykinin (Fig. 4C). On the other hand, the hypotensive effect of PR3-kinin was only partially inhibited by HOE-140 (Fig. 4C), indicating that the effect was partially mediated via B1 receptor activity. As a positive control, the selective B1 receptor agonist des-Arg10-kallidin was administered. The results indicate that administration of PR3-kinin i.v. triggers a blood pressure drop mediated via both B1 (directly) and B2 receptors (presumably after processing).

PR3-kinin is processed to bradykinin and des-Arg-bradykinin in plasma

As PR3-kinin bound primarily to B1 receptors in vitro but exerted an immediate effect via both B1 and B2 receptors in vivo, we investigated if PR3-kinin was processed in contact with plasma. PR3-kinin incubated with plasma resulted in processing of PR3-kinin to peptides with the molecular mass of bradykinin (1059.4 Da), des-Arg-bradykinin (903.3 Da), bradykinin-Ser-Ser (1233.7 Da), Met-Lys-bradykinin (1318.4 Da), and Lys-bradykinin-Ser-Ser (1361.7 Da), as analyzed by MALDI (data not shown). Processing commenced at ~5 min and after 10 min PR3-kinin was completely converted. Plasma alone did not contain any detectable bradykinin-like peptides. When PR3-kinin was incubated with plasma that was preincubated with the three carboxypeptidase inhibitors separately (EACA, GEMSA, and Plummer’s inhibitor), the processing of PR3-kinin was not inhibited, although no generation of des-Arg-bradykinin was detected (data not shown). In contrast, when PR3-kinin was incubated with plasma preincubated with Complete Mini there was a marked reduction in the enzymatic processing of PR3-kinin (data not shown). Taken together, these results indicate that the enzyme(s) responsible for the proteolytic processing of PR3-kinin in plasma could be serine, cysteine, metalloproteases, or calpains, but not carboxypeptidase B or N.

PR3-kinin is enzymatically processed to bradykinin and des-Arg-bradykinin by plasma kallikrein in vitro

PR3-kinin incubated with plasma kallikrein resulted in rapid (within 30 s) processing of PR3-kinin to peptides with the molecular mass of bradykinin, des-Arg-bradykinin, and Met-Lys-bradykinin, as analyzed by MALDI (data not shown). This breakdown would not be expected to account for the processing of PR3-kinin detected in normal plasma, as plasma kallikrein is not present in normal plasma but circulates in inactive prekallikrein form (1).

PR3 in neutrophil extracts induces HK proteolysis

Neutrophil extracts were obtained from five of the vasculitis patients (nos. 1, 2, 3, 6, and 7, four with severe ANCA-positive vasculitis) and five controls and examined to investigate if PR3 was involved in HK proteolysis and if differences in this respect could be noted between samples from patients and controls. The neutrophil extracts were incubated with purified HK. Neutrophil extracts from patients treated with Triton X-100 induced breakdown of HK into four bands resembling the physiological pattern of proteolysis (Fig. 5, lane 2). Neutrophil extracts from controls induced a similar breakdown of HK (data not shown). Untreated neutrophil extracts (in the absence of Triton X-100) from both patients and controls induced minimal breakdown of HK (Fig. 5, lane 4) when compared with HK incubated with the neutrophil buffer in the absence of neutrophil extracts (Fig. 5, lane 1).


Figure 5
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  		FIGURE 5. PR3 in neutrophil extracts induced breakdown of HK. Lane 1, HK (100 µg/ml) with neutrophil buffer without neutrophils. Lane 2, Triton X-100-treated neutrophil extracts from a patient (no. 1) incubated with HK, resulting in breakdown of HK. Lane 3, Triton X-100-treated neutrophil extract from the same patient, in which PR3 had been immunoadsorbed, incubated with HK, resulting in partial breakdown of HK. Lane 4, Untreated neutrophil extracts from the same patient incubated with HK, resulting in almost no breakdown. Lane 5, Untreated neutrophil extract from the same patient, in which PR3 had been immunoadsorbed, incubated with HK, resulting in almost no breakdown of HK. The gel shown is representative for all tested patients (n = 5) and controls (n = 5). No difference in the breakdown pattern of HK was observed between the patients and the controls. The samples were run on the same gel. Results of the ELISA for bradykinin levels (in pg/ml, median (range)) are shown in appropriate lanes under the immunoblot figure. Neutrophil extracts were incubated with HK (5 µg/ml). No differences in the levels of bradykinin between the patients and the controls were observed, although a tendency to higher bradykinin levels was noted when incubating the patients’ Triton X-100-treated neutrophil extracts with HK.

 
When proteinase 3 was immunoadsorbed from the neutrophil extracts the proteolysis was markedly reduced (Fig. 5, lanes 3 and 5). No difference was noted between patients and controls with regard to the breakdown pattern induced by neutrophil extracts before and after immunoadsorption. Taken together, these results indicate that PR3-mediated release of PR3-kinin may occur under physiological conditions, and that this proteolytic process could be enhanced under pathophysiological conditions.

PR3 in neutrophil extracts induces release of bradykinin-like peptides from HK

Neutrophils extracts from vasculitis patients (nos. 1, 2, 3, 6, and 7) and controls (n = 5) treated with Triton X-100 induced release of bradykinin or bradykinin-like peptide(s) from HK (Fig. 5, lane 2), whereas untreated neutrophil extracts from the same patients and controls did not (p = 0.002; Fig. 5, lane 4). Bradykinin levels did not differ significantly between HK-incubated neutrophil extracts from patients and controls. When proteinase 3 was immunoadsorbed from the neutrophil extract of patients and controls the release of bradykinin or bradykinin-like peptide(s) was significantly reduced (Fig. 5, lane 3).

PR3 levels were measured in Triton X-100-treated neutrophil extracts from the five vasculitis patients and the controls. No differences were found in the levels between the patients (median, 3.9 µg/ml; range 2.7–7.2 µg/ml) and the controls (median, 4.8 µg/ml; range, 1.6–7.3 µg/ml; p = 0.69). The results indicate that PR3 in neutrophil extracts leads to the breakdown of HK and subsequent release of bradykinin or bradykinin-like peptide(s), and no differences in this respect were noted in neutrophils from patients and controls.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study presents a novel mechanism of kinin pathway activation in which HK breakdown by PR3 was demonstrated in the fluid phase, in the absence of endothelial cells or neutrophils. More importantly, release of a novel kinin, termed PR3-kinin, occurred in the absence of plasma kallikrein, the known physiological enzymatic activator of this system. PR3-kinin bound to and activated the human B1 receptor but did not bind to the human B2 receptor in vitro. PR3-kinin was as potent as the B2 agonist bradykinin in lowering blood pressure in wild-type mice, an effect that was absent in B2 knockout mice. PR3-kinin was more potent than bradykinin in B1 receptor overexpressing TGR-Tie2B1 rats, suggesting that the in vivo hypotensive effects were exerted via both B1 and B2 receptors. These results indicate that PR3-kinin is a B1 receptor agonist and that it is converted in vivo to a B2 receptor agonist. This novel kinin-generating pathway is controlled by {alpha}1-AT and would thus circumvent the established inhibitory mechanisms dominated by C1 inhibitor. We envisage that during inflammatory conditions, in which activated neutrophils infiltrate extravascular spaces, neutrophil-derived PR3 in membrane-bound or secreted form could generate PR3-kinin in a kallikrein-independent manner and that in tissues where {alpha}1-AT levels are presumably low, and/or {alpha}1-AT activity is compromised due to oxidation of a crucial methionine residue (33), liberation of kinins could proceed in a relatively uninhibited manner.

The results suggest that PR3-kinin can exert an effect both via the B1 receptor (directly) and the B2 receptor (after processing to bradykinin). In plasma, PR3-kinin is processed to bradykinin and des-Arg-bradykinin within 5–10 min. Although our analysis has not unequivocally assigned the lacking Arg residue to the C terminus of bradykinin, des-Arg9-bradykinin, which binds to B1 receptors, may have been generated, whereas des-Arg1-bradykinin does not bind to B1 and B2 receptors (2). The effect of PR3-kinin in vivo on both B1 and B2 receptors was immediate, suggesting that conversion of PR3-kinin to the form active on B2 receptors occurs very rapidly in vivo. Murine B2 receptors differ pharmacologically from human B2 receptors in their response to antagonists (2, 34). Thus, species-specific properties may also contribute to the effect of PR3-kinin in mice.

Plasma kallikrein was also capable of proteolytic cleavage of PR3-kinin to bradykinin in vitro. In normal plasma active kallikrein would not be present (1). However, on activated neutrophils and endothelial cells, where plasma kallikrein is active (35), such proteolytic breakdown of PR3-kinin could potentially occur in vivo.

Previous studies have shown that stimulated neutrophils induced the generation of biologically active kinins from HK (36, 37, 38), whereas nonstimulated neutrophils did not hydrolyze HK (39). Neutrophil-derived tissue kallikrein participated in kinin release as the reaction was blocked by antihuman urinary kallikrein but not by antineutrophil elastase Ab (36). The generated kinins were analyzed by HPLC and radioimmunoassay and shown to have similar retention times to bradykinin and Met-Lys-bradykinin. The biological activity was mediated by kinin B2 receptors (36). We have taken these results further and demonstrated, using Triton X-100 lyzed neutrophils, that PR3, in a dose-dependent, specific, and physiological manner, is almost exclusively responsible for HK proteolysis and that the product of this reaction is a novel kinin peptide that we have termed PR3-kinin. This tridecapeptide corresponds to Met-Lys-bradykinin with two additional serine residues at the C terminus. By studying the reaction of purified PR3 (in native and recombinant form) with HK and synthetic HK D4, we could rule out the participation of contaminating enzymes, such as elastase or plasma kallikrein, in these proteolytic processes. Furthermore, the Abs used to immunoadsorb PR3 from neutrophil extracts, and to block PR3 activity in specificity assays, have been shown to be specific for PR3 and do not cross-react with other proteins in neutrophil azurophilic granules (29) or with kallikreins, as demonstrated in this study.

In addition to PR3, other neutrophil proteases, such as elastase and cathepsins, have been shown to cleave HK and liberate peptides with bradykinin-like properties (40, 41, 42, 43). Neutrophil elastase releases a peptide from HK termed E-kinin SLMKRPPGFSPFRSSRI (the bradykinin sequence is underlined). E-kinin and two synthetic E-kinin-like peptides (SLMKRPPGFSPFRSS and SLMKRPPGFSPFR) are longer than bradykinin and PR3-kinin and are capable of lowering blood pressure in an animal model (41).

The preferential amino acid sequence for proteinase 3 cleavage consists of hydrophobic amino acids at the P1, P3, and P4 positions, negatively charged amino acids at P2 and P2', and positively charged amino acids at P1' and P3'. Although this represents the ideal sequence, other amino acids may be found at the cleavage sites (44, 45). The cleavage sites on HK do not fully match the consensus sequence established for PR3 (46) (Fig. 6); however, our results clearly show that PR3 releases PR3-kinin from HK, allowing cleavage at nonconsensus sites.


Figure 6
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  		FIGURE 6. HK sequence cleaved by PR3. The PR3-kinin sequence is marked in gray. h, Hydrophobic; neg, negative; pos, positive; h-phil, hydrophilic; n, neutral.

 
Patients with vasculitides exhibit activation of the kinin system as demonstrated by degradation of HK and elevated plasma levels of bradykinin (4). In a previous study we showed that patients with both mild (Henoch Schönlein purpura) and severe (Wegener’s granulomatosis) vasculitis had kinin system activation whereas controls did not (4). In the present study not all patients had ANCA-related vasculitis. Patients with ANCA-associated vasculitis had higher levels of PR3 in plasma than did controls (16, 17) and the expression of PR3 on the membrane of leukocytes correlates with disease activity in patients with ANCA-associated vasculitis (Wegener’s granulomatosis) (8). However, as presented in the present study, even neutrophil extracts from healthy controls contained PR3 capable of cleaving HK. We did not detect differences in PR3 levels or activity in neutrophil extracts from patients or controls. Thus, PR3-induced activation of the kinin pathway may also occur at low PR3 levels under physiological conditions; however, it may be abundant at sites of massive inflammation and neutrophil accumulation around vessels.

We have shown a novel function for PR3 in inflammation, namely generation of the vasoactive kinin PR3-kinin. The typical pathological finding in vasculitis consists of neutrophil infiltrates in and around vessels and in tissues. Activated neutrophils infiltrating affected tissues secrete granular components including PR3. This could create a microenvironment rich in cytokine-activated neutrophils that express PR3 on their membrane and secrete PR3 into the blood vessels and tissues. PR3-induced activation of the kinin pathway could occur at these sites and possibly in secluded environments that do not allow {alpha}1-AT entry. Thus PR3-kinin is formed and binds to the B1 receptor followed by capillary leakage (2). This will allow exposure to plasma enzymes that rapidly convert PR3-kinin into the active B2 agonist bradykinin, further enhancing the inflammatory response.


   Acknowledgments
 
The authors thank the Swegene Centre for Integrative Biology at Lund University and especially Peter James, Liselotte Andersson, and Mats Mågård for their excellent assistance with mass spectrometry. We also thank Caroline Sandén for her excellent assistance with the bradykinin receptor phosphoinositide hydrolysis assay as well as Lena Gunnarson for her excellent technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was presented in part in poster form at the 13th Congress of the International Pediatric Nephrology Association, August 29 to September 2, 2004, Adelaide, Australia, and the 2nd International Conference on Exploring the Future of Vascular and Inflammatory Mediators, Kinin 2007, May 30 to June 2, 2007, Berlin, Germany. Back

2 This study was supported by grants from the Swedish Research Council (K2007-64X-14008-07-3), the fund for Renal Research, Crown Princess Lovisa’s Society for Child Care, the Sven Jerring Foundation, Konung Gustaf V:s 80-årsfond, Fanny Ekdahl’s Foundation (all to D.K.) and Queen Silvia’s Jubilee fond (to R.K.) and the Swedish Research Council (grant 15057; to L.M.F.L.L.). Diana Karpman is the recipient of a clinical-experimental research fellowship from the Royal Swedish Academy of Sciences. Back

3 Address correspondence and reprint requests to Dr. Diana Karpman, Department of Pediatrics, Lund University, 22185 Lund, Sweden. E-mail address: Diana.Karpman{at}med.lu.se Back

4 Abbreviations used in this paper: HK, high-molecular weight kininogen; PR3, proteinase 3; {alpha}1-AT, {alpha}1-antitrypsin; ANCA, anti-neutrophil cytoplasmic Abs; KLKs, kallikreins; EACA, {epsilon}-amino-n-caproic acid; GEMSA, 2-guanidinoethylmercaptosuccinic acid; ESI, electrospray ionization; PI, phosphoinositide. Back

Received for publication October 31, 2008. Accepted for publication April 9, 2009.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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