AJP - Lung AJP: Gastrointestinal and Liver Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 294: L505-L515, 2008. First published January 11, 2008; doi:10.1152/ajplung.00347.2007
1040-0605/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/3/L505    most recent
00347.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Coffman, L. G.
Right arrow Articles by Torti, S. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Coffman, L. G.
Right arrow Articles by Torti, S. V.

Cleavage of high-molecular-weight kininogen by elastase and tryptase is inhibited by ferritin

Lan G. Coffman,1 Julie C. Brown,2 David A. Johnson,5 Narayanan Parthasarathy,2 Ralph B. D'Agostino, Jr.,4,9 Mark O. Lively,2 Xiaoyang Hua,7 Stephen L. Tilley,7 Werner Muller-Esterl,6 Mark C. Willingham,3,9 Frank M. Torti,8,9 and Suzy V. Torti2,9

1Program in Molecular Medicine, Departments of 2Biochemistry, 3Pathology, and 4Public Health Sciences, Wake Forest University School of Medicine, Winston-Salem, North Carolina; 5Department of Biochemistry and Molecular Biology, J. H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee; 6Institute of Biochemistry II, University of Frankfurt Medical School, Frankfurt, Germany; 7Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of North Carolina at Chapel Hill, Chapel Hill; 8Department of Cancer Biology and 9Comprehensive Cancer Center, Wake Forest University School of Medicine, Winston-Salem, North Carolina

Submitted 23 August 2007 ; accepted in final form 10 January 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Ferritin is a protein principally known for its role in iron storage. We have previously shown that ferritin can bind high-molecular-weight kininogen (HK). Upon proteolytic cleavage by the protease kallikrein, HK releases the proinflammatory peptide bradykinin (BK) and other biologically active products, such as two-chain high-molecular-weight kininogen, HKa. At inflammatory sites, HK is oxidized, which renders it a poor substrate for kallikrein. However, oxidized HK remains a good substrate for elastase and tryptase, thereby providing an alternative cleavage mechanism for HK during inflammation. Here we report that ferritin can retard the cleavage of both native HK and oxidized HK by elastase and tryptase. Initial rates of cleavage were reduced 45–75% in the presence of ferritin. Ferritin is not a substrate for elastase or tryptase and does not interfere with the ability of either protease to digest a synthetic substrate, suggesting that ferritin may impede HK cleavage through direct interaction with HK. Immunoprecipitation and solid phase binding studies reveal that ferritin and HK bind directly with a Kd of 134 nM. To test whether ferritin regulates HK cleavage in vivo, we used THP-1 cells, a human monocyte/macrophage cell line that has been used to model pulmonary inflammatory cells. We observed that ferritin impedes the cleavage of HK by secretory proteases in stimulated macrophages. Furthermore, ferritin, HK, and elastase are all present in or on alveolar macrophages in a mouse model of pulmonary inflammation. Collectively, these results implicate ferritin in the modulation of HK cleavage at sites of inflammation.

inflammation; bradykinin

HIGH-MOLECULAR-WEIGHT KININOGEN (HK) is a 120-kDa plasma glycoprotein. HK, together with prekallikrein and factor XII, comprise the contact system of coagulation (9). HK also plays important roles distinct from the initiation of coagulation via interactions with platelets, neutrophils, monocytes, macrophages, and endothelial cells. Through these interactions, HK modulates cellular adhesion, leukocyte extravasation, and vascular permeability (11, 16, 17, 41). HK is the precursor to a number of bioactive molecules, including bradykinin (BK) and HKa. HKa has antiangiogenic properties at endothelial surfaces and affects adhesion of macrophages, neutrophils, and platelets to the endothelium (6, 7). BK is a well-known mediator of inflammation and has been implicated in disease processes including asthma, atherosclerosis, hypertension, and cancer (2, 12, 31, 43, 46). BK is one of the key activators of airway inflammation where it induces bronchoconstriction and increases in vascular permeability and mucus secretion and cholinergic and sensory nerve stimulation (3). Therefore, cleavage of HK to release BK is an important step in inflammation and contributes substantially to the pathogenesis of inflammatory diseases such as asthma.

Although the classic cleavage pathway of HK involves the activation of prekallikrein to kallikrein and the subsequent cleavage of HK to release BK (57), alternative mechanisms of HK cleavage have been reported (10, 47). In particular, the role of proteases such as elastase and tryptase in the local activation of HK at sites of inflammation has received attention (25). Elastase is a serine protease that degrades connective tissue components, facilitating the extravasation of leukocytes from the circulation into tissues. Elastase cleaves HK to produce E-kinin, a heptadeca-peptide that is subsequently cleaved to release BK (21). Kininogen has also been identified as a substrate of tryptase, a trypsin-like enzyme found in mast cell granules (30, 39, 42, 49). Tryptase cleaves within the light chain of HK and at sites flanking BK (28). Although tryptase directly cleaves HK to release BK, this activity is relatively minor (25). However, when a synthetic heptadeca-peptide containing BK (similar to E-kinin, the first product of elastase digestion of HK) is incubated with tryptase, the rate of BK release dramatically increases (25). Interestingly, codigestion of HK with the combination of elastase and tryptase produces BK at a rate similar to kallikrein (25). These results have led to the suggestion that elastase and tryptase digest HK sequentially, with elastase first cleaving HK to produce E-kinin, followed by tryptase cleavage of E-kinin to complete the release of BK (25).

This alternative pathway of HK cleavage and BK liberation may be important at sites of inflammation, particularly at epithelial surfaces such as the lung. In conditions such as asthma, for example, neutrophils or macrophages containing elastase and mast cells containing tryptase are activated concurrently, providing an environment in which these two proteases can target the same substrate. Furthermore, during inflammation, enzymes like myeloperoxidase (MPO), which colocalizes with HK at the endothelial cell surface, and reactive oxygen species are generated that can oxidize HK (1). Oxidized HK is a poor substrate of kallikrein yet remains a good substrate for elastase and tryptase (25). Thus, elastase and tryptase may represent a system for cleavage of oxidized HK at sites of inflammation (25).

Ferritin, a multi-subunit protein with a major role in iron storage, affects the dynamics of HK cleavage (34). Ferritin is composed of two subunits (L and H) that assemble into a 24-subunit complex (apoferritin) surrounding a ferrihydrite mineral core that can contain up to 4,500 iron atoms (holoferritin) (50). Serum ferritin levels can rise 10- to 100-fold during inflammation. Our laboratory has shown that ferritin binds to HK and inhibits the release of BK by kallikrein (34).

In this study, we demonstrate that ferritin inhibits the proteolytic cleavage of HK and oxidized HK by elastase and tryptase. Cleavage of HK by elastase and tryptase was retarded by ferritin when these proteases were used both separately and when they were used in combination. Immunohistochemical analysis revealed that ferritin, HK, and elastase colocalized in alveolar macrophages in a model of pulmonary inflammation. Because inflammation creates an environment in which elastase and tryptase may replace kallikrein as the main cleavage enzymes of oxidized HK, the ability of ferritin to impede the cleavage of HK by these two proteases suggests that ferritin may modulate inflammation in conditions such as asthma or other inflammatory processes in the lung.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. HK, human neutrophil elastase, MPO, and elastase substrate I, colorimetric (MeOSuc-Ala-Ala-Pro-Val-pNA) were purchased from EMD Biosciences, La Jolla, CA. Recombinant human lung β-tryptase was purchased from Promega. Human spleen ferritin was purchased from Scripps Laboratories. Horse spleen apoferritin, human plasma kallikrein, phorbol 12-myristate 13-acetate (PMA), porcine intestinal mucosal heparin, and the tryptase substrate N-CBZ-Gly-Pro-Arg-p-nitroanilide acetate were purchased from Sigma Aldrich. HRP-conjugated anti-ferritin antibody was purchased from Dako. Sheep anti-human HK antibody and donkey anti-sheep/goat antibody were purchased from The Binding Site. Porcine pancreatic elastase was a generous gift of Dr. Thomas Hollis (Wake Forest Univ. School of Medicine, Winston-Salem, NC). Monoclonal anti-HK antibodies directed against HK domain 1 (HKH4) and HK domain 6 (HKL16) have been described previously (18, 53). THP-1 human acute monocytic leukemic cells were a generous gift of Dr. Darren Seals (Wake Forest Univ. School of Medicine).

Elastase and tryptase independent digestion of HK with and without ferritin. HK was digested with either porcine pancreatic elastase, human neutrophil elastase, or recombinant human mast cell tryptase in the presence and absence of human spleen ferritin [porcine pancreatic elastase data are presented here; human neutrophil elastase produced similar results (data not shown)]. The molar ratio of elastase to HK and tryptase to HK was 1:200 and 1:600, respectively, and the molar ratio of HK to ferritin was 1:1. Reactions were conducted at an HK concentration of 50 µM at 25°C, pH 7.3, in 25 mM HEPES buffer. Heparin (0.1 U/µl) was added to the tryptase reactions to stabilize tryptase (28). Aliquots were removed at intervals, and the reaction stopped by boiling. Samples were subjected to 10% SDS-PAGE (unreduced conditions) and stained with Coomassie brilliant blue R250. Quantification of gel bands was performed using UNSCAN-IT software (Silk Scientific, Orem, UT). Percent cleaved HK was determined by dividing the integrated density of the cleaved band by the sum of the HK protein bands in the sample and multiplying by 100. Initial rates of cleavage were calculated by linear regression.

Elastase and tryptase codigestion of HK with and without ferritin. HK digestion with elastase and tryptase in the presence and absence of human spleen ferritin was conducted in the same manner as the tryptase digestion described above. The molar ratios of elastase, tryptase, and HK were 1:1:2,000, respectively.

Oxidation of HK. HK was oxidized as described (25). Briefly, HK in 20 mM HEPES buffer, pH 7.3, was incubated with a 100-molar excess of N-chlorosuccinimide at 37°C for 2 h. The reaction was quenched with 6 mM methionine at 37°C for 30 min. MPO oxidation of HK was based on the method of Yang et al. (56), using HK at a final concentration of 28 µg/ml, MPO at a final concentration of 10 µg/ml, and a total incubation time of 10 min. The reaction was quenched with 6 mM methionine at 37°C for 30 min.

Mass spectrometry analysis of HK. N-chlorosuccinimide and MPO-oxidized HK as well as native HK were subjected to SDS-PAGE analysis, stained with Coomassie blue, and destained with acetic acid/methanol. The protein bands were excised from the stained gels, and disulfide bonds were reduced with DTT and alkylated with iodoacetamide. Reduced and alkylated peptides were digested overnight at 37°C with trypsin. Peptides were separated by reverse phase chromatography and analyzed directly by electrospray ionization using a nanoelectrospray ionization source on a Bruker Esquire HCT ion trap mass spectrometer (27). MS/MS spectra were searched against the current release of the Mass Spectrometry Protein Sequence Data Base (obtained from the Imperial College London) using Mascot (36).

Ligand blot of oxidized HK. Oxidized HK (0.5 µg), HK, and low-molecular-weight kininogen (LK) were subjected to 10% SDS-PAGE and electroblotted onto three replicate nitrocellulose membranes. The three membranes were used for a ligand blot, a positive control anti-HK Western blot, and a negative control anti-ferritin Western blot. Ferritin binding to oxidized HK was analyzed by ligand blotting as previously described (51). The anti-HK Western blot was performed as follows: 1-h blocking with 5% (wt/vol) nonfat dry milk in TBST (25 mM Tris·HCl, pH 8.0, 125 mM NaCl, 0.1% Tween 20), 30-min blocking with 2% BSA, 2-h incubation with donkey anti-human HK, and 1-h incubation with mouse anti-donkey HRP conjugate, followed by visualization with ECL reagent (Amersham). The anti-ferritin Western blot was performed as follows: 1-h blocking with 5% (wt/vol) nonfat dry milk in PBS, 1-h incubation with anti-Ft HRP conjugate, and visualization via chemiluminescence (Pierce Pico-West).

Elastase digestion of control chromogenic substrate with and without apoferritin. Direct inhibition of elastase activity by ferritin was investigated by enzyme assays in the presence and absence of apoferritin using the chromogenic neutrophil elastase substrate MeOSuc-Ala-Ala-Pro-Val-p-nitroanilide at a concentration of 0.01 µM. Assays were performed in 0.5 M Tris, pH 8.8, at room temperature, and the rate of p-nitroaniline formation was measured at 405 nm every 5 min for 210 min. The same apoferritin-to-elastase ratio used in the elastase digestion of HK was used in the control assay (200:1) with an excess of substrate. Apoferritin, which behaved similarly to ferritin in HK binding capacity and HK digestion experiments (data not shown), was used instead of holoferritin due to iron's ability to interfere with spectrophotometric measurements.

Tryptase digestion of control chromogenic substrate with and without apoferritin. Direct inhibition of tryptase activity by ferritin was investigated by enzyme assays in the presence and absence of apoferritin using the chromogenic peptide substrate N-CBZ-Gly-Pro-Arg-p-nitroanilide acetate at a concentration of 5 nM. The procedure was based on a previously described assay (28). Assays were performed in 0.1 M HEPES, 10% glycerol wt/vol, 0.1 mg/ml heparin, and 0.02% NaN3, pH 7.5, at room temperature, and the rate of p-nitroaniline formation measured at 405 nm for 30 min.

Elastase and tryptase digestion of ferritin. To determine if ferritin is a substrate for either elastase or tryptase, ferritin was incubated with elastase and/or tryptase under the same conditions described above. Aliquots of each reaction as well as a control reaction without either protease were analyzed, in triplicate, on 10% SDS-PAGE (unreduced conditions), stained with Coomassie brilliant blue R250, and quantified as described above for cleavage by elastase.

Coimmunoprecipitation of HK and ferritin. HK and human spleen ferritin at a 1:1 molar ratio were incubated in a solution of 0.5 µg/µl BSA in PBS. The solution was precleared with 5 µl of protein A beads. Anti-HK (1 µl; HKH4 and HKL16) was added, and the solution was incubated at 25°C for 2 h with shaking. Protein A beads (30 µl) were added followed by a 1-h incubation. The beads were separated from the solution by centrifugation at 14,000 g for 1 min, and the supernatant was saved for Western blot analysis. The beads were washed three times with IMP buffer [50 mM Tris-acetate, pH 7.5, 150 mM NaCl, 1% (vol/vol) Triton X-100, 1% (wt/vol) deoxycholate, 0.1% (wt/vol) SDS, 0.02% (wt/vol) Na N3], SDS-PAGE loading buffer containing BME was added to the beads, and the eluted proteins were analyzed via Western blot with anti-HK and anti-Ft antibodies.

Plate assay to characterize HK binding to ferritin. HK and ferritin were biotinylated via sulpho-N-hydroxysuccinimido-long-chain-biotin (sulpho-NHS-LC-biotin) and the EZ-Link Sulpho-NHS-LC Biotinylation kit (Pierce) according to the manufacturer's instructions. The number of biotin molecules per HK was determined using the EZ biotin quantification kit (Pierce). To measure binding of HK to ferritin, we first calculated the amount of ferritin needed to coat the plate. Biotinylated ferritin in increasing concentrations was added to microtiter plates (Nalgene Nunc International, Rochester, NY), and the amount bound quantified by incubation with streptavidin-HRP and TMB substrate (3,3',5,5'-tetra-methylbenzidine; Pierce). Maximal specific binding (Bmax) was calculated as 7.45 nM (1 µg/100 µl per well) using Scatchard analysis. To perform the binding assay, plates were coated with human spleen ferritin (2.5 µg/100 µl per well) in PBS overnight at 4°C. The plates were washed with PBST (PBS + 0.1% Tween 20) and blocked with 2% BSA. Biotinylated HK at increasing concentrations was allowed to bind to the ferritin-coated wells for 2 h at 27°C with shaking. The wells were washed three times with PBST, treated with streptavidin-peroxidase for 30 min at 27°C, and incubated with TMB substrate (3,3',5,5'-tetra-methylbenzidine; Pierce), and the absorbance at 450 nm was measured. To measure nonspecific binding, control wells were coated with 2.5 µg/100 µl native HK, and biotinylated HK binding was analyzed in the same manner as above. Nonspecific binding was subtracted from total HK binding, and binding affinity was calculated using Scatchard analysis. To determine the dependence of Ft/HK binding on the presence of zinc, 10 µM ZnCl2 was added to each well along with biotinylated HK (38).

Cleavage of biotinylated HK by macrophage secretory proteases. THP-1 human acute monocytic leukemic cells were grown in RPMI media with 10% FBS, 1% penicillin/streptomycin, 1% L-glutamine, 0.05 mM 2-mercaptoethanol, and 1% MEM vitamin solution. HK was biotinylated as described above. THP-1 cells were seeded in triplicate in 96-well plates at a concentration of 3 x 105 cells/ml in a volume of 100 µl. The cells were stimulated with 100 ng/ml PMA. Biotinylated HK was added to each well at a final concentration of 4.5 µM with and without ferritin at a final concentration of 9 µM. Biotinylated HK was also added to THP-1 cells without PMA stimulation. The cells were incubated for 1 h at 37°C, 5% CO2. The media was removed and clarified, and SDS-PAGE loading buffer (10% glycerol, 62.5 mM Tris·HCl, pH 6.8, 2% wt/vol SDS, 2% 2-mercaptoethanol) was added to inhibit further proteolysis. The media was then subjected to 10% SDS-PAGE and electroblotted onto a nitrocellulose membrane for Western blot analysis using streptavidin-HRP followed by visualization with a chemiluminescent substrate (Pierce Pico-West). Full-length biotinylated HK was quantified by densitometry using UNSCAN-IT software (Silk Scientific), and HK cleavage was calculated as the fraction of remaining input HK following incubation with stimulated THP-1 cells. Media from stimulated THP-1 cells without added biotinylated HK was used as a negative control to ensure streptavidin-HRP specificity.

Colocalization of ferritin, HK, and elastase in mouse lung tissue. C57BL/6 mice were sensitized with an intraperitoneal injection of 20 µg of chicken egg ovalbumin (OVA; grade V, Sigma-Aldrich) emulsified in 200 µl of aluminum hydroxide (alum) adjuvant (Alhydrogel; Accurate Chemical & Scientific). Fourteen days later, mice were challenged with aerosolized 1% OVA for 30 min on three consecutive days in a whole body exposure chamber. Forty-eight hours following the last exposure, mice were euthanized, and lungs were removed and rapidly frozen in liquid nitrogen. Lungs were imbedded in optimized temperature cutting compound and cryosectioned to produce 8-µm-thick sections. Lungs were fixed in 10% formalin for 10 min and washed in PBS. Sections were blocked in 1% BSA/PBS and stained with 15 µg/ml of either goat anti-Ft, rabbit anti-HK, rat anti-elastase, or rat anti-CD68 antibodies for 1 h. For single stained sections, 10 µg/ml HRP-linked rabbit anti-goat, HRP-linked goat anti-rabbit, or HRP-linked goat anti-rat secondary antibody was added and incubated for 30 min followed by DAB (3,3'-diaminobenzidine tetrahydrochloride, Sigma) visualization and hematoxylin counterstaining. For fluorescent doubly stained sections, 10 µg/ml FITC-linked rabbit anti-goat and rhodamine-linked chicken anti-rabbit or rhodamine-linked goat anti-rat antibodies were added and incubated for 30 min. Stained sections were analyzed using a Zeiss AxioPlan 2 microscope with Carl Zeiss AxioCam HP and AxioVision 3.1 software.

Statistical analysis. Results are reported as the mean value and 95% confidence interval (CI). Two-way repeated measures ANOVA models were used to compare the ferritin- and non-ferritin-treated HK cleavage results over the time course of the experiment, with time and treatment being the two factors studied. P values are reported ({alpha} = 0.05).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Ferritin retards cleavage of HK by elastase. HK plays an important role in inflammation by serving as the precursor of both BK and HKa. These proteins can be released from HK through the combined activity of tryptase and elastase, proteases present in inflammatory cells. Cleavage of HK by elastase and tryptase provides an alternative pathway of HK cleavage distinct from the classic cleavage mechanism of HK by kallikrein (25). Since ferritin binds to HK (34, 51), we postulated that ferritin might control HK activity at these sites by retarding HK cleavage by elastase and tryptase.

To test this possibility, we first examined the ability of ferritin to retard the cleavage of HK by elastase. HK was incubated with elastase in the presence and absence of ferritin at a 1:1 (HK:ferritin) molar ratio. As seen in Fig. 1A, ferritin substantially retarded the digestion of HK by elastase at all time points. Overall, the two-way ANOVA model confirmed a significant ferritin inhibitory effect on elastase cleavage of HK (P = 0.0022). We also measured the initial rate of cleavage of HK in the absence and presence of ferritin. We observed that 0.47 pmol of HK was cleaved per minute (CI ± 0.15) in the absence of ferritin, whereas 0.11 pmol/min (CI ± 0.03) was cleaved in the presence of ferritin. This demonstrates a 75% decrease in initial cleavage rate when ferritin is present (P = 0.042) (Fig. 1D).


Figure 1
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 1. Cleavage of high-molecular-weight kininogen (HK) by elastase, tryptase, or the combination of elastase and tryptase with and without ferritin. Proteases were incubated with HK in the presence or absence of human spleen ferritin as described in MATERIALS AND METHODS. The percent cleavage over time was measured using densitometric quantification of the major bands in each well. A: elastase cleavage of HK with and without ferritin (1:200 elastase:HK molar ratio). B: tryptase cleavage of HK with and without ferritin (1:600 tryptase:HK molar ratio). C: elastase and tryptase combined cleavage of HK with and without ferritin (1:1:2,000 elastase:tryptase:HK molar ratio). Shown are the means and SD of 3 independent experiments. D: comparison of the initial rates of HK (or oxidized HK; oxHK) cleavage in the presence or absence of ferritin. Linear regression models of the first 3 time points from each digestion were used to determine the initial rates of cleavage (pmol/min) with ferritin and without ferritin. The initial rates of HK (or oxHK) cleavage were then standardized such that initial cleavage rates without ferritin are represented as 100%, whereas the initial cleavage rates with ferritin are reported as a percentage of the initial rate without ferritin. Shown are the means and SD of 3 independent experiments.

 
Ferritin retards cleavage of HK by tryptase. We then measured the ability of ferritin to inhibit cleavage of HK by tryptase. Comparing tryptase digestion of HK with and without ferritin revealed a statistically significant reduction in HK cleavage in the presence of ferritin at all time points (P = 0.0033, 2-way ANOVA) (Fig. 1B). Measurement of the initial rate of cleavage of HK in the absence and presence of ferritin revealed that 0.60 pmol was cleaved per minute (CI ± 0.07) in the absence of ferritin, whereas 0.33 pmol/min (CI ± 0.04) was cleaved in the presence of ferritin. This demonstrates a 45% decrease in initial cleavage rate when ferritin is present (P = 0.008) (Fig. 1D).

Ferritin retards elastase and tryptase codigestion of HK. Because combined proteolysis of HK by elastase and tryptase produces significant levels of bradykinin (25), we next examined the ability of ferritin to reduce HK cleavage by the concerted action of tryptase and elastase. As show in Fig. 1C, at each time point measured after 0 min, cleavage of HK by combined enzyme treatment in the presence of ferritin was significantly less than cleavage of HK without ferritin. The two-way ANOVA model demonstrated a significant ferritin inhibitory effect on elastase/tryptase cleavage of HK (P = 0.0069). Measurement of the initial rate of cleavage of HK in the absence and presence of ferritin revealed that 0.077 pmol were cleaved per minute (CI ± 0.02) in the absence of ferritin, whereas 0.034 pmol (CI ± 0.01) were cleaved in the presence of ferritin. This demonstrates a 55% decrease in initial cleavage rate when ferritin is present (P = 0.017) (Fig. 1D).

Higher levels of apoferritin enhance HK cleavage inhibition. To investigate the effect of increasing the molar ratio of ferritin to HK on the ferritin-dependent inhibition of HK cleavage, HK was incubated with elastase, tryptase, or elastase plus tryptase in the presence of apoferritin at 1:0, 1:1, 1:2, or 1:5 HK:apoferritin molar ratio. As seen in Table 1, substantial inhibition (19–64%) was attained at a 1:1 HK:apoferritin ratio, although additional inhibition could be attained by increasing the amount of apoferritin to a molar ratio of 1:2 or 1:5. For example, when ferritin and HK were present at equimolar ratios, ferritin inhibited HK cleavage by elastase by 64%; ferritin at a fivefold molar excess inhibited HK cleavage by elastase 84%.


View this table:
[in this window]
[in a new window]

  		Table 1. Percent inhibition of HK digestion by ferritin

 
Ferritin binds to and retards the digestion of oxidized HK. At inflammatory sites, HK is susceptible to oxidation due to the release of oxidative enzymes and molecules during the respiratory burst of leukocytes. Kallikrein, the protease that mediates cleavage of HK via the classic pathway, cannot cleave oxidized HK (25). However, the combination of elastase and tryptase can cleave oxidized HK, acting as an alternative pathway for HK cleavage at inflammatory sites (25). To test the effect of ferritin on this pathway, oxidized HK was produced via treatment with N-chlorosuccinimide (25) as described in MATERIALS AND METHODS. Oxidized HK was confirmed to be partially refractory to digestion with kallikrein: after 90 min, unoxidized HK was ~75% cleaved, whereas oxidized HK was ~25% cleaved at a molar ratio of kallikrein to HK of 1:600 (data not shown). To ensure physiological relevancy of the chemically oxidized HK, we compared N-chlorosuccinimide-oxidized HK with MPO-oxidized HK. MPO, a potent oxidative enzyme found within inflammatory cells, is released during the oxidative burst, and is a potential source for oxidized HK in vivo (1). MPO-oxidized HK was produced as described in MATERIALS AND METHODS and subjected to kallikrein digestion at the same molar ratio as the N-chlorosuccinimide-oxidized HK digestion. MPO-oxidized HK proved to be similarly refractory to kallikrein digestion, with a reduction in kallikrein cleavage from 98% to 51%.

The reduction in susceptibility to kallikrein digestion following HK oxidation has been attributed to the oxidation of key methionine residues (Met375 and Met379) proximal to the enzyme cleavage site (25, 33). To directly determine whether these residues were indeed oxidized following treatment with either N-chlorosuccinimide or MPO, we digested oxidized HK with trypsin and analyzed the oxidation state of these residues using mass spectroscopy. As shown in Fig. 2 and Supplemental Table 1 (supplemental data for this article is available online at the AJP-Lung web site), singly oxidized methionines (i.e., methionine sulfoxide at either position 375 or 379) were present in native HK as well as in oxidized HK (the fully unoxidized form of this peptide was not observed in any of the mass spectra collected). However, following oxidation by either method, only the doubly oxidized form (i.e., methionine sulfoxide at both position 375 and 379) was observed.


Figure 2
View larger version (23K):
[in this window]
[in a new window]

  		Fig. 2. Myeloperoxidase and N-chlorosuccinimide oxidation of HK. A: tryptic peptides analyzed by mass spectrometry in unoxidized HK and HK oxidized by myeloperoxidase and N-chlorosuccinimide. The locations of the 2 methionine residues susceptible to oxidation (Met375 and Met379) relative to the kallikrein cleavage site are also shown. B: tryptic peptides identified in untreated HK and HK oxidized by myeloperoxidase or N-chlorosuccinimide. The unoxidized peptide was not observed in either treated or untreated preparations. See Supplemental Table 1 for details of MS/MS analysis.

 
Next, we investigated whether ferritin, which binds to the light chain of native HK, also binds to oxidized HK (34). A ligand blot was performed to compare the binding of ferritin to native HK (positive control), LK (negative control), and oxidized HK. As seen in Fig. 3A, native HK bound ferritin, and LK did not, as previously reported (51). Oxidized HK retained its ability to bind ferritin (Fig. 3A). Control Western blots using anti-HK antibody showed equal bands at 120 kDa in the HK and oxidized HK lanes and a band at 75 kDa in the LK lane (Fig. 3B), as expected. Western blots with anti-ferritin antibody showed no bands even after long exposures, indicating that there was no nonspecific binding of anti-ferritin antibody to the blot (Fig. 3C).


Figure 3
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 3. Oxidized HK retains binding to ferritin. A: oxidized HK, native HK (positive control), and low-molecular-weight kininogen (LK; negative control) were subjected to SDS-PAGE and analyzed for ferritin binding activity by ligand blotting using a ferritin probe followed by HRP-anti-Ft antibody. B: Western blot using anti-HK antibody against oxidized HK, native HK, and LK. C: Western blot using anti-Ft antibody against oxidized HK, native HK, and LK. HK and oxidized HK are 120 kDa, whereas LK is 75 kDa.

 
Since ferritin retained the ability to bind oxidized HK, we then investigated the ability of ferritin to inhibit the cleavage of oxidized HK by elastase and tryptase. To accomplish this, we performed the same time course digestions as described above, using oxidized HK rather than native HK. As with native HK, ferritin retarded cleavage of oxidized HK by elastase alone, tryptase alone, and elastase/tryptase together (Fig. 4). The cleavage of oxidized HK in the presence of ferritin was significantly less than the cleavage of oxidized HK without ferritin at all time points after 10 min. As with the unoxidized HK reactions, ANOVA demonstrated a statistically significant ferritin inhibitory effect on elastase, tryptase, and elastase/tryptase cleavage of oxidized HK (P = 0.0025, P = 0.0042, P = 0.0007, respectively). Initial rates of cleavage of oxidized HK were also decreased in the presence of ferritin (Fig. 1D). Thus, ferritin retards the cleavage of both native and oxidized HK by elastase and tryptase.


Figure 4
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 4. Elastase, tryptase, or combined elastase/tryptase cleavage of oxidized HK with and without ferritin. Proteases were incubated with oxidized HK in the presence or absence of human spleen ferritin. The percent cleavage over time was measured using densitometric quantification of the major bands in each well. A: elastase cleavage of oxidized HK with and without ferritin (1:200 elastase:HK molar ratio). B: tryptase cleavage of oxidized HK with and without ferritin (1:600 tryptase:HK molar ratio). C: elastase and tryptase cleavage of oxidized HK with and without ferritin (1:1:2,000 elastase:tryptase:HK molar ratio). Shown are the means and SD of 3 independent experiments.

 
Ferritin does not inhibit the enzymatic activity of elastase or tryptase on a synthetic substrate. The possibility that ferritin might act by directly inhibiting the enzymatic activity of elastase or tryptase was examined using synthetic peptide substrates. Elastase cleavage of its chromogenic substrate (MeOSuc-Ala-Ala-Pro-Val-p-nitroanilide) was measured in the presence and absence of apoferritin. The cleavage of the chromogenic substrate over time, as measured by the production of p-nitroaniline by spectrometric analysis at 405 nm, was not significantly different in the presence and absence of ferritin (P > 0.05) (Fig. 5A). Similarly, tryptase activity on its chromogenic substrate (N-CBZ-Gly-Pro-Arg-p-nitroanilide acetate) was not significantly different over time in the presence and absence of apoferritin (P > 0.05) (Fig. 5B). These results demonstrate that ferritin has no direct inhibitory effect on the activity of elastase or tryptase.


Figure 5
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 5. Ferritin does not inhibit the enzymatic activity of elastase or tryptase. A: elastase was incubated with a standard chromogenic substrate (MeOSuc-Ala-Ala-Pro-Val-p-Nitroanilide) in the presence and absence of apoferritin. Absorbance at 405 nm was measured over the course of the digestion. Elastase to apoferritin molar ratio was 1:200. Shown are the means and SD of 3 independent experiments. B: tryptase was incubated with a standard chromogenic substrate (N-CBZ-Gly-Pro-Arg-p-nitroanilide acetate) in the presence and absence of apoferritin. p-Nitroanilide production was measured over the course of the digestion via spectrophotometric absorbance at 405 nm. Tryptase to apoferritin molar ratio was 1:600. Shown are the means and SD of 3 independent experiments. C: elastase (E), tryptase (T), or elastase + tryptase (ET) were incubated with ferritin at a 1:200, 1:600 or 1:1:2,000 molar ratio of protease to ferritin for 150 min. A control reaction containing no proteases (C) was run at the same time. The level of proteolysis was assessed via 10% SDS-PAGE analysis (unreduced conditions) followed by staining with Coomassie brilliant blue R250. Intact ferritin runs as a single band of ~480 kDa. This experiment was performed 3 times; a typical result is shown.

 
Ferritin is not a substrate for either elastase or tryptase. To rule out the possibility that ferritin was itself a substrate for elastase or tryptase and thus reduced the cleavage of HK by simple substrate competition, we incubated ferritin with elastase and/or tryptase at the same molar ratios as used in the elastase, tryptase, and elastase plus tryptase digestions of HK for a time course of 150 min. The ferritin was then subjected to SDS-PAGE and quantified by densitometry as described above for HK cleavage. Since in silico analysis (using the NIH UniProtKB/Swiss Prot program) demonstrated that ferritin subunits each contain 18–32 potential cleavage sites for both tryptase and elastase (25, 28), we anticipated that there would be a substantial reduction in intact ferritin, if ferritin were indeed a substrate for these enzymes. However, as shown in Fig. 5C, our results demonstrated no significant reduction in ferritin following treatment with elastase (P = 0.8), tryptase (P = 0.99), or elastase/tryptase (P = 0.75). These results indicate that within the limitations of our analysis, ferritin is not a substrate for either elastase or tryptase (Fig. 5C). Since ferritin does not directly inhibit elastase or tryptase and is not a substrate for either protease, these results suggest that ferritin impedes the cleavage of HK through direct interaction with HK.

Ferritin and HK form a complex in solution. We have previously demonstrated that ferritin binds to HK using a solid phase ligand blotting assay (34, 51). To examine the ability of ferritin to form a complex with HK in solution, we tested whether ferritin and HK would coimmunoprecipitate when incubated with anti-HK antibodies. Ferritin and HK were incubated at a 1:1 molar ratio in the presence of excess BSA, and anti-HK antibodies were added. As seen in Fig. 6, ferritin was efficiently coprecipitated with HK under these conditions, with virtually no ferritin detected in the supernatant fraction. Control experiments using a solution that contained only ferritin and BSA (no HK) demonstrated that there was no cross reaction between the anti-HK antibody or protein A bead and ferritin (Fig. 6). These results provide further evidence for the direct interaction of ferritin with HK.


Figure 6
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 6. Coimmunoprecipitation of ferritin and HK. Anti-HK antibodies and protein G beads were used to coimmunoprecipitate HK/ferritin complexes in solution. The bead fraction (containing the coimmunoprecipitated proteins) and the supernatant fraction (containing those proteins not immunoprecipitated) were analyzed via Western blotting with anti-HK and anti-Ft antibodies. Left (Ft, no HK): immunoprecipitation of a solution contained only ferritin, demonstrating no cross-reactivity between the anti-HK antibody and ferritin. Right (Ft + HK): immunoprecipitation of a solution containing ferritin and HK in an equimolar ratio. Sup, supernatant fraction; Bead, bead fraction.

 
Ferritin/HK binding characteristics. To assess the affinity of the ferritin/HK interaction, we utilized a plate binding assay in which human spleen ferritin was immobilized on a 96-well microtiter plate and incubated with increasing concentrations of biotinylated HK (Fig. 7A). After subtracting nonspecific binding, a Scatchard analysis was performed, yielding a Kd of 134 nM and a Bmax of 7.5 nM (Fig. 7B). Non-biotinylated HK, used as a competitor (10- to 100-fold excess), inhibited biotinylated HK binding to ferritin by 93–95%, thus verifying binding specificity (data not shown). By comparing the maximal amount of HK bound to ferritin and the amount of ferritin bound to the plate in this solid phase assay, we determined that ferritin and HK were present at a 1:1 stoichiometric ratio at saturation.


Figure 7
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 7. Binding of HK to immobilized ferritin. A: biotinylated HK (0–1 µM) was added to triplicate wells coated with ferritin. Nonspecific binding of biotinylated HK and/or biotinylated HK polymerization was assessed on HK-coated wells. Binding was detected by streptavidin-HRP and a peroxidase substrate. Reported are the means and SD of 3 independent experiments. B: Scatchard analysis of specific HK binding to ferritin.

 
Ferritin/HK binding is not zinc dependent. Zinc induces conformational changes in the structure of HK and is a necessary cofactor for HK binding to cellular surfaces (14, 19). To examine whether ferritin binding was also zinc dependent, plate binding studies were performed in the presence and absence of 10 µM ZnCl2, a physiologically relevant concentration used in cell binding assays (29). There was no difference in the binding curves with and without zinc (data not shown), indicating that HK can bind to ferritin in the absence of exogenous zinc.

Ferritin inhibits the cleavage of biotinylated HK by macrophage secretory proteases. To test whether ferritin modulates the cleavage of HK in an inflammatory cell model, we used THP-1 monocyte/macrophage cells (22). Biotinylated HK, in the presence or absence of a twofold molar excess of ferritin, was added exogenously to THP-1 cells. The cells were stimulated to differentiate and secrete elastase by the addition of PMA (4, 54). After 1 h, the media was collected, and the biotinylated HK was analyzed by Western blotting as described in MATERIALS AND METHODS. In the absence of ferritin, 78% (CI ± 10) of biotinylated HK was cleaved, whereas in the presence of ferritin, only 16% (CI ± 4.5) of biotinylated HK was cleaved (Fig. 8). The results indicate that activated macrophages are capable of secreting proteases that cleave HK, and that ferritin can inhibit this cleavage. This demonstrates that the binding of ferritin to HK has functional consequences in regulating HK cleavage in a cell culture model of inflammation.


Figure 8
View larger version (25K):
[in this window]
[in a new window]

  		Fig. 8. Cleavage of HK by stimulated macrophages with and without ferritin. Biotinylated HK, with and without ferritin, was added to PMA-stimulated THP-1 cells in triplicate. A: full-length biotinylated HK was assessed after 1 h of incubation via Western blot. Triplicate analyses (B) show quantification of HK cleavage by densitometric analysis. The percent of biotinylated HK cleaved was calculated using biotinylated HK incubated with unstimulated THP-1 cells as our no-cleavage comparison. Means and SD are shown.

 
Ferritin, HK, and elastase colocalize in mouse alveolar macrophages. To determine whether ferritin and HK colocalize at sites of inflammation in vivo, we used a mouse model of pulmonary inflammation. C57BL/6 mice were sensitized with OVA and subsequently challenged with aerosolized albumin, a protocol that induces lung inflammation, recruits inflammatory cells, and has been used extensively to model asthma (15). Lung tissue was cryosectioned, stained with anti-ferritin antibody, and counterstained with hematoxylin. As seen in Fig. 9A, this experiment revealed the presence of ferritin in or on infiltrating inflammatory cells that morphologically resemble macrophages. To determine whether these cells were also positive for HK, we performed simultaneous staining with fluorescent antibodies to ferritin (FITC/green) and HK (rhodamine/red). As seen in Fig. 9B, this experiment demonstrated that ferritin and HK colocalize in/on these cells (Fig. 9Bi). Costaining with antibodies to ferritin and elastase further revealed the colocalization of ferritin and elastase (Fig. 9Bii). Additional staining with anti-CD68, a macrophage-specific marker, confirmed that these cells were indeed alveolar macrophages (Fig. 9Biii). These results were seen in the majority of cells examined: 85% of CD68-positive macrophages were positive for ferritin, 90% of morphologically identified macrophages were positive for both elastase and ferritin, and 95% were positive for both HK and ferritin. Thus, ferritin, HK, and elastase are all present in alveolar macrophages recruited in response to an inflammatory stimulus.


Figure 9
View larger version (61K):
[in this window]
[in a new window]

  		Fig. 9. Colocalization of ferritin, HK, and elastase in mouse alveolar macrophages challenged with an inflammatory stimulus. A: frozen section of C57/BL6 ovalbumin-sensitized and challenged mouse lungs stained for ferritin, visualized with HRP-linked secondary antibody and a DAB-peroxidase substrate, and counterstained with hematoxylin (left) and a control section stained with DAB-peroxidase substrate and counterstained with hematoxylin (right). B: frozen sections of C57/BL6 ovalbumin-sensitized and challenged mouse lungs costained for ferritin (Ft) and HK (i), ferritin and elastase (EL) (ii), and ferritin and the macrophage marker CD68 (iii). The ferritin signal was visualized with a FITC-linked secondary antibody (middle) and HK; elastase and CD68 signals were visualized with a rhodamine-linked secondary antibody (right). Control sections were prepared by omitting primary antibodies and staining with FITC and rhodamine-linked secondary antibodies (iv). Left panels show the overlay of the 2 fluorochrome signals with a phase contrast image. Approximately 20–30 macrophages were observed per section. Arrows point to representative positive cells.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
HK and its cleavage products are essential components of numerous processes including coagulation, angiogenesis, leukocyte adhesion, and inflammation (7, 10). BK, a cleavage product of HK, plays a particularly important role in the progression of inflammatory diseases such as asthma (3). As a consequence, HK must be carefully regulated to control its inflammatory and hemostatic properties. This is in part attained through regulation of levels of HK. For example, increases in HK plasma levels have been observed in disease states such as allergic asthma, sepsis, atherosclerosis, arthritis, and cancer (13, 20, 23, 35). HK can also be regulated by controlling its cleavage into bioactive proteolytic products, including BK and HKa. Although tissue and plasma kallikrein are generally responsible for proteolytic digestion of HK, oxidation renders HK refractory to digestion with these enzymes (25). Therefore, the generation of kinins from HK by kallikrein may be compromised at inflammatory sites where the activity of respiratory burst enzymes, such as MPO, results in oxidation of HK. However, kinins can also be released from HK through the concerted action of tryptase and elastase, enzymes released at inflammatory sites that retain the ability to release kinins from oxidized HK (25). Elastase- and tryptase-dependent cleavage of HK may maintain production of vasoactive kinins from HK at sites of inflammation.

We previously reported that ferritin can bind to the light chain of HK and retard HK cleavage by kallikrein (34). In this study, we investigate whether ferritin affects the cleavage of HK by proteases released from inflammatory cells and explore the influence of HK oxidation on these events. We report that HK contains two methionines proximate to the kallikrein cleavage site (Met375 and Met379) that are susceptible to oxidation, and these are, in fact, oxidized following exposure to either chemical oxidants or MPO, an oxidative enzyme produced by inflammatory cells (Fig. 2 and Supplemental Table 1). Since HK oxidized by either of these methods loses its ability to be digested by kallikrein (see RESULTS) but retains the ability to be digested by elastase and tryptase (data not shown and Figs. 1 and 4), our data support the suggestion (25) that tryptase and elastase may play an important role in HK cleavage at inflammatory sites.

We observed that ferritin inhibits tryptase and elastase-mediated cleavage of both native and oxidized HK, when HK is exposed to these proteases individually or in concert (Figs. 1 and 4). Increasing the ratio of apoferritin to HK enhances the inhibitory effect of ferritin on HK cleavage, although ferritin exerts most of its inhibitory effects when present at concentrations equimolar to HK or in slight molar excess of HK (Table 1), consistent with our finding of a 1:1 Ft:HK stoichiometric binding relationship in the solid phase binding assay. Coimmunoprecipitation data confirm the ability of HK and ferritin to form a complex in solution (Fig. 6). Binding characteristics of the HK/ferritin interaction (Kd 134 nM) reveal a sufficient binding affinity to allow for a physiologically relevant HK/ferritin complex, especially at sites of inflammation where ferritin levels are elevated. These results are consistent with a model in which the direct binding of ferritin to HK alters the ability of tryptase and/or elastase to proteolyse HK. Furthermore, since binding of HK to ferritin is not affected by exogenous zinc and zinc induces conformational changes in HK (19), ferritin may effectively inhibit the digestion of HK in both conformations.

We propose that the inhibition of HK cleavage by ferritin, with its attendant reduction in proinflammatory HK cleavage products, may modulate the inflammatory response. We demonstrate that ferritin is able to reduce the cleavage of exogenously added HK in inflammatory macrophages (Fig. 8). Also consistent with this model is the temporal and spatial colocalization of ferritin, HK, and elastase that we observed in alveolar macrophages following an inflammatory stimulus (Fig. 9). We hypothesize that the interaction among these proteins occurs extracellularly, since the cleavage of HK occurs primarily on cell surfaces, although our results do not exclude the possibility that intracellular interactions among these proteins may also take place. Ferritin is a multi-subunit protein that is ubiquitously distributed in tissues and is also present in plasma (37). Both HK and ferritin have been localized to sites of inflammation, including atherosclerotic plaques (14, 26, 35, 52), rheumatic synovium (48), and asthmatic lung tissue (8). Alveolar macrophages in inflamed lungs accumulate ferritin, and stimulation with NO [a vasoactive molecule induced by BK (44)] leads to ferritin secretion from these cells (45). Alveolar macrophages and neutrophils also contain HK and have HK receptors, in addition to containing and secreting elastase (5, 16, 40). Thus, accumulation of neutrophils, macrophages, and mast cells, especially during lung inflammation, provides a spatial and temporal juxtaposition of HK, ferritin, elastase, and tryptase and creates a physiological context for their regulatory interactions.

While ferritin was able to retard the time course of HK cleavage by elastase and tryptase, digestion of HK was not abolished by ferritin. This implies that the role of ferritin may be to dampen rather than to abrogate the production of kinins at sites of inflammation. Ferritin may also serve to downregulate the cleavage of HK at the termination of the inflammatory response. In support of these hypotheses, the HK cleavage product, HKa, induces monocyte secretion of TNF{alpha} and IL-1, which are known to induce ferritin synthesis (24, 32, 55). In addition, the HK cleavage product BK induces endothelial cell release of NO, which in turn stimulates the release of ferritin from alveolar macrophages (44, 45). Thus, HK cleavage products may increase ferritin levels, which, in turn, serve to downregulate HK cleavage, constituting a negative feedback loop designed to control the amount of HK cleaved. Consistent with this interpretation, ferritin preferentially binds to partially cleaved forms of HK such as activated HK (HK that is partially cleaved but has not yet released BK) (34).


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by National Institutes of Health Grants R37-DK-42421 (F. M. Torti), R15-AI-45549 (D. A. Johnson), and R01-DK-71892 (S. V. Torti) and a predoctoral fellowship from the American Heart Association (L. G. Coffman). Mass spectroscopy was performed in the Biomolecular Resource Facility Protein Analysis Core Laboratory of the Comprehensive Cancer Center of Wake Forest University.

Present address of N. Parthasarathy: Bacteriology Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. V. Torti, Dept. of Biochemistry, Wake Forest Univ. Health Sciences, Winston-Salem, NC 27157 (e-mail: storti{at}wfubmc.edu)

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


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Astern JM, Pendergraft WF 3rd, Falk RJ, Jennette JC, Schmaier AH, Mahdi F, Preston GA. Myeloperoxidase interacts with endothelial cell-surface cytokeratin 1 and modulates bradykinin production by the plasma Kallikrein-Kinin system. Am J Pathol 171: 349–360, 2007.[Abstract/Free Full Text]
  2. Barnes PJ. Effect of bradykinin on airway function. Agents Actions Suppl 38: 432–438, 1992.[Medline]
  3. Barnes PJ, Chung KF, Page CP. Inflammatory mediators of asthma: an update. Pharmacol Rev 50: 515–596, 1998.[Abstract/Free Full Text]
  4. Campbell EJ, Silverman EK, Campbell MA. Elastase and cathepsin G of human monocytes. Quantification of cellular content, release in response to stimuli, and heterogeneity in elastase-mediated proteolytic activity. J Immunol 143: 2961–2968, 1989.[Abstract]
  5. Chao J, Simson JA, Chung P, Chen LM, Chao L. Regulation of kininogen gene expression and localization in the lung after monocrotaline-induced pulmonary hypertension in rats. Proc Soc Exp Biol Med 203: 243–250, 1993.[CrossRef][Medline]
  6. Chavakis T, Kanse SM, Pixley RA, May AE, Isordia-Salas I, Colman RW, Preissner KT. Regulation of leukocyte recruitment by polypeptides derived from high molecular weight kininogen. FASEB J 15: 2365–2376, 2001.[Abstract/Free Full Text]
  7. Chavakis T, Santoso S, Clemetson KJ, Sachs UJ, Isordia-Salas I, Pixley RA, Nawroth PP, Colman RW, Preissner KT. High molecular weight kininogen regulates platelet-leukocyte interactions by bridging Mac-1 and glycoprotein Ib. J Biol Chem 278: 45375–45381, 2003.[Abstract/Free Full Text]
  8. Christiansen SC, Proud D, Sarnoff RB, Juergens U, Cochrane CG, Zuraw BL. Elevation of tissue kallikrein and kinin in the airways of asthmatic subjects after endobronchial allergen challenge. Am Rev Respir Dis 145: 900–905, 1992.[Web of Science][Medline]
  9. Cochrane CG, Griffin JH. The biochemistry and pathophysiology of the contact system of plasma. Adv Immunol 33: 241–306, 1982.[Web of Science][Medline]
  10. Colman RW. Contact activation pathway: inflammatory, fibrinolytic, anticoagulation, antiadhesive, and antiangiogenic activities. In: Hemostasis and Thrombosis: Basic Principles and Clinical Practice, edited by Colman RW, Hirsh J, Marder VJ, Salzman EW. Philadelphia: Lippincott, 1998, p. 1567–1583.
  11. Colman RW, Schmaier AH. Contact system: a vascular biology modulator with anticoagulant, profibrinolytic, antiadhesive, and proinflammatory attributes. Blood 90: 3819–3843, 1997.[Free Full Text]
  12. Emanueli C, Madeddu P. Role of the kallikrein-kinin system in the maturation of cardiovascular phenotype. Am J Hypertens 12: 988–999, 1999.[CrossRef][Web of Science][Medline]
  13. Espinola RG, Uknis A, Sainz IM, Isordia-Salas I, Pixley R, DeLa Cadena R, Long W, Agelan A, Gaughan J, Adam A, Colman RW. A monoclonal antibody to high-molecular weight kininogen is therapeutic in a rodent model of reactive arthritis. Am J Pathol 165: 969–976, 2004.[Abstract/Free Full Text]
  14. Fernando AN, Fernando LP, Fukuda Y, Kaplan AP. Assembly, activation, and signaling by kinin-forming proteins on human vascular smooth muscle cells. Am J Physiol Heart Circ Physiol 289: H251–H257, 2005.[Abstract/Free Full Text]
  15. Gonzalo JA, Lloyd CM, Wen D, Albar JP, Wells TN, Proudfoot A, Martinez AC, Dorf M, Bjerke T, Coyle AJ, Gutierrez-Ramos JC. The coordinated action of CC chemokines in the lung orchestrates allergic inflammation and airway hyperresponsiveness. J Exp Med 188: 157–167, 1998.[Abstract/Free Full Text]
  16. Gustafson EJ, Schmaier AH, Wachtfogel YT, Kaufman N, Kucich U, Colman RW. Human neutrophils contain and bind high molecular weight kininogen. J Clin Invest 84: 28–35, 1989.[Web of Science][Medline]
  17. Gustafson EJ, Schutsky D, Knight LC, Schmaier AH. High molecular weight kininogen binds to unstimulated platelets. J Clin Invest 78: 310–318, 1986.[Web of Science][Medline]
  18. Hasan AA, Cines DB, Herwald H, Schmaier AH, Muller-Esterl W. Mapping the cell binding site on high molecular weight kininogen domain 5. J Biol Chem 270: 19256–19261, 1995.[Abstract/Free Full Text]
  19. Herwald H, Morgelin M, Svensson HG, Sjobring U. Zinc-dependent conformational changes in domain D5 of high molecular mass kininogen modulate contact activation. Eur J Biochem/FEBS 268: 396–404, 2001.[Web of Science][Medline]
  20. Hesselvik JF, Blomback M, Brodin B, Maller R. Coagulation, fibrinolysis, and kallikrein systems in sepsis: relation to outcome. Crit Care Med 17: 724–733, 1989.[Web of Science][Medline]
  21. Imamura T, Tanase S, Hayashi I, Potempa J, Kozik A, Travis J. Release of a new vascular permeability enhancing peptide from kininogens by human neutrophil elastase. Biochem Biophys Res Commun 294: 423–428, 2002.[CrossRef][Web of Science][Medline]
  22. Jagielo PJ, Quinn TJ, Qureshi N, Schwartz DA. Grain dust-induced lung inflammation is reduced by Rhodobacter sphaeroides diphosphoryl lipid A. Am J Physiol Lung Cell Mol Physiol 274: L26–L31, 1998.[Abstract/Free Full Text]
  23. Kalter ES, Daha MR, ten Cate JW, Verhoef J, Bouma BN. Activation and inhibition of Hageman factor-dependent pathways and the complement system in uncomplicated bacteremia or bacterial shock. J Infect Dis 151: 1019–1027, 1985.[Web of Science][Medline]
  24. Khan MM, Bradford HN, Isordia-Salas I, Liu Y, Wu Y, Espinola RG, Ghebrehiwet B, Colman RW. High-molecular-weight kininogen fragments stimulate the secretion of cytokines and chemokines through uPAR, Mac-1, and gC1qR in monocytes. Arterioscler Thromb Vasc Biol 26: 2260–2266, 2006.[Abstract/Free Full Text]
  25. Kozik A, Moore RB, Potempa J, Imamura T, Rapala-Kozik M, Travis J. A novel mechanism for bradykinin production at inflammatory sites. Diverse effects of a mixture of neutrophil elastase and mast cell tryptase versus tissue and plasma kallikreins on native and oxidized kininogens. J Biol Chem 273: 33224–33229, 1998.[Abstract/Free Full Text]
  26. Li W, Hellsten A, Xu LH, Zhuang DM, Jansson K, Brunk UT, Yuan XM. Foam cell death induced by 7beta-hydroxycholesterol is mediated by labile iron-driven oxidative injury: mechanisms underlying induction of ferritin in human atheroma. Free Radic Biol Med 39: 864–875, 2005.[CrossRef][Web of Science][Medline]
  27. Lin Z, Johnson LC, Weissbach H, Brot N, Lively MO, Lowther WT. Free methionine-(R)-sulfoxide reductase from Escherichia coli reveals a new GAF domain function. Proc Natl Acad Sci USA 104: 9597–9602, 2007.[Abstract/Free Full Text]
  28. Little SS, Johnson DA. Human mast cell tryptase isoforms: separation and examination of substrate-specificity differences. Biochem J 307: 341–346, 1995.[Web of Science][Medline]
  29. Mahdi F, Shariat-Madar Z, Schmaier AH. The relative priority of prekallikrein and factors XI/XIa assembly on cultured endothelial cells. J Biol Chem 278: 43983–43990, 2003.[Abstract/Free Full Text]
  30. Maier M, Spragg J, Schwartz LB. Inactivation of human high molecular weight kininogen by human mast cell tryptase. J Immunol 130: 2352–2356, 1983.[Abstract]
  31. McLean PG, Perretti M, Ahluwalia A. Kinin B(1) receptors and the cardiovascular system: regulation of expression and function. Cardiovasc Res 48: 194–210, 2000.[Abstract/Free Full Text]
  32. Miller LL, Miller SC, Torti SV, Tsuji Y, Torti FM. Iron-independent induction of ferritin H chain by tumor necrosis factor. Proc Natl Acad Sci USA 88: 4946–4950, 1991.[Abstract/Free Full Text]
  33. Nieziolek M, Kot M, Pyka K, Mak P, Kozik A. Properties of chemically oxidized kininogens. Acta Biochim Pol 50: 753–763, 2003.[Web of Science][Medline]
  34. Parthasarathy N, Torti SV, Torti FM. Ferritin binds to light chain of human H-kininogen and inhibits kallikrein-mediated bradykinin release. Biochem J 365: 279–286, 2002.[CrossRef][Web of Science][Medline]
  35. Peerschke EI, Minta JO, Zhou SZ, Bini A, Gotlieb A, Colman RW, Ghebrehiwet B. Expression of gC1q-R/p33 and its major ligands in human atherosclerotic lesions. Mol Immunol 41: 759–766, 2004.[CrossRef][Web of Science][Medline]
  36. Perkins DN, Pappin DJ, Creasy DM, Cottrell JS. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20: 3551–3567, 1999.[CrossRef][Web of Science][Medline]
  37. Ponka P, Beaumont C, Richardson DR. Function and regulation of transferrin and ferritin. Semin Hematol 35: 35–54, 1998.[Web of Science][Medline]
  38. Raab A, Kemme M. High-molecular-weight kininogen is a binding protein for tissue prokallikrein. FEBS Lett 467: 165–168, 2000.[CrossRef][Web of Science][Medline]
  39. Ren S, Lawson AE, Carr M, Baumgarten CM, Schwartz LB. Human tryptase fibrinogenolysis is optimal at acidic pH and generates anticoagulant fragments in the presence of the anti-tryptase monoclonal antibody B12. J Immunol 159: 3540–3548, 1997.[Abstract]
  40. Rodriguez RJ, White RR, Senior RM, Levine EA. Elastase release from human alveolar macrophages: comparison between smokers and nonsmokers. Science 198: 313–314, 1977.[Abstract/Free Full Text]
  41. Schmaier AH, Kuo A, Lundberg D, Murray S, Cines DB. The expression of high molecular weight kininogen on human umbilical vein endothelial cells. J Biol Chem 263: 16327–16333, 1988.[Abstract/Free Full Text]
  42. Schwartz LB, Bradford TR, Littman BH, Wintroub BU. The fibrinogenolytic activity of purified tryptase from human lung mast cells. J Immunol 135: 2762–2767, 1985.[Abstract]
  43. Sharma JN, Thani RB. Therapeutic prospects for bradykinin receptor agonists in the treatment of cardiovascular diseases. IDrugs 7: 926–934, 2004.[Web of Science][Medline]
  44. Skidgel RA, Stanisavljevic S, Erdos EG. Kinin- and angiotensin- converting enzyme (ACE) inhibitor-mediated nitric oxide production in endothelial cells. Biol Chem 387: 159–165, 2006.[CrossRef][Web of Science][Medline]
  45. Smith JJ, O'Brien-Ladner AR, Kaiser CR, Wesselius LJ. Effects of hypoxia and nitric oxide on ferritin content of alveolar cells. J Lab Clin Med 141: 309–317, 2003.[CrossRef][Web of Science][Medline]
  46. Stewart JM, Gera L, Chan DC, York EJ, Simkeviciene V, Bunn PA Jr, Taraseviciene-Stewart L. Combination cancer chemotherapy with one compound: pluripotent bradykinin antagonists. Peptides 26: 1288–1291, 2005.[CrossRef][Web of Science][Medline]
  47. Svensjo E, Batista PR, Brodskyn CI, Silva R, Lima AP, Schmitz V, Saraiva E, Pesquero JB, Mori MA, Muller-Esterl W, Scharfstein J. Interplay between parasite cysteine proteases and the host kinin system modulates microvascular leakage and macrophage infection by promastigotes of the Leishmania donovani complex. Microbes Infect 8: 206–200, 2005.[CrossRef][Web of Science][Medline]
  48. Telfer JF, Brock JH. Expression of ferritin, transferrin receptor, and non-specific resistance associated macrophage proteins 1 and 2 (Nramp1 and Nramp2) in the human rheumatoid synovium. Ann Rheum Dis 61: 741–744, 2002.[Abstract/Free Full Text]
  49. Thomas VA, Wheeless CJ, Stack MS, Johnson DA. Human mast cell tryptase fibrinogenolysis: kinetics, anticoagulation mechanism, and cell adhesion disruption. Biochemistry 37: 2291–2298, 1998.[CrossRef][Web of Science][Medline]
  50. Torti FM, Torti SV. Regulation of ferritin genes and protein. Blood 99: 3505–3516, 2002.[Free Full Text]
  51. Torti SV, Torti FM. Human H-kininogen is a ferritin-binding protein. J Biol Chem 273: 13630–13635, 1998.[Abstract/Free Full Text]
  52. Turi JL, Yang F, Garrick MD, Piantadosi CA, Ghio AJ. The iron cycle and oxidative stress in the lung. Free Radic Biol Med 36: 850–857, 2004.[CrossRef][Web of Science][Medline]
  53. Vogel R, Assfalg-Machleidt I, Esterl A, Machleidt W, Muller-Esterl W. Proteinase-sensitive regions in the heavy chain of low molecular weight kininogen map to the inter-domain junctions. J Biol Chem 263: 12661–12668, 1988.[Abstract/Free Full Text]
  54. Waki H, Yamauchi T, Kamon J, Kita S, Ito Y, Hada Y, Uchida S, Tsuchida A, Takekawa S, Kadowaki T. Generation of globular fragment of adiponectin by leukocyte elastase secreted by monocytic cell line THP-1. Endocrinology 146: 790–796, 2005.[Abstract/Free Full Text]
  55. Wei Y, Miller SC, Tsuji Y, Torti SV, Torti FM. Interleukin 1 induces ferritin heavy chain in human muscle cells. Biochem Biophys Res Commun 169: 289–296, 1990.[CrossRef][Web of Science][Medline]
  56. Yang C, Wang J, Krutchinsky AN, Chait BT, Morrisett JD, Smith CV. Selective oxidation in vitro by myeloperoxidase of the N-terminal amine in apolipoprotein B-100. J Lipid Res 42: 1891–1896, 2001.[Abstract/Free Full Text]
  57. Yarovaya GA, Blokhina TB, Neshkova Contact system EA. New concepts on activation mechanisms and bioregulatory functions. Biochemistry 67: 13–24, 2002.[Web of Science][Medline]



This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
L. G. Coffman, D. Parsonage, R. D'Agostino Jr., F. M. Torti, and S. V. Torti
Regulatory effects of ferritin on angiogenesis
PNAS, January 13, 2009; 106(2): 570 - 575.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/3/L505    most recent
00347.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Coffman, L. G.
Right arrow Articles by Torti, S. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Coffman, L. G.
Right arrow Articles by Torti, S. V.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.