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The Journal of Immunology, 2006, 177: 8315-8319.
Copyright © 2006 by The American Association of Immunologists, Inc.

Human Mast Cell Chymase Cleaves Pro-IL-18 and Generates a Novel and Biologically Active IL-18 Fragment1

Youichi Omoto*,§, Kazuya Tokime*,§, Keiichi Yamanaka*,§, Koji Habe*, Tatsuhiko Morioka*, Ichiro Kurokawa*, Hiroko Tsutsui{dagger},§, Kiyofumi Yamanishi{ddagger},§, Kenji Nakanishi{dagger},§ and Hitoshi Mizutani2,*,§

* Department of Dermatology, Mie University, Graduate School of Medicine, Tsu, Mie, Japan; {dagger} Department of Immunology and Medical Zoology and {ddagger} Dermatology, Hyogo College of Medicine, Nishinomiya, Japan; and § Core Research of Evolutional Science and Technology, Japan Science and Technology Agency, Kawaguchi, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Increased release of IL-18 in the skin causes atopic dermatitis (AD)-like skin lesions, suggesting a role of IL-18 in the pathogenesis of AD. Caspase-1 is a well-known activator of IL-18, but caspase-1 knockout mice still have biologically active IL-18. Normal human keratinocyte constitutively produces pro-IL-18, but it is unable to activate it, suggesting the existence of an alternative pathway for IL-18 in the skin. Dermal accumulation of mast cells is commonly observed in AD patients and in experimental mouse models of AD. Connective tissue mast cells contain high amounts of chymase and tryptase in their cytoplasmic granules. In the present study, we demonstrated that activation of IL-18 is a novel function of human mast cell chymase. Human mast cell chymase rapidly cleaves recombinant pro-IL-18 at 56-phenylalanine and produces a biologically active IL-18 fragment that is smaller than any other reported IL-18-derived species. The human mast cell chymase and the novel IL-18-derived active peptide may be novel therapeutic targets in AD- and IL-18-associated diseases


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Interleukin-18 is a proinflammatory cytokine that belongs to the IL-1 cytokine family (1). Two mutant mice (2, 3, 4) that show enhanced production and release of the mature form of IL-18 (mat-IL-18)3 spontaneously develop atopic dermatitis (AD)-like chronic itchy dermatitis associated with increased serum level of IgE. The serum levels of IL-18 are elevated in patients with AD (5), and thereby IL-18 is considered to play an important role in the pathogenesis of AD. Like other members of the IL-1 family, IL-18 is released in an inactive form and then is activated by specific enzymes (6). Caspase-1/IL-1beta-converting enzyme is a well-known activator of IL-18 (7, 8). Unlike monocytic cells, keratinocytes and fibroblasts lack functional caspase-1 (9, 10); therefore, they produce and store only the inactive form of IL-18, precursor IL-18 (pro-IL-18), in their cytoplasm. The release of the mature form of IL-18 is increased in the skin lesions of active dermatitis, but caspase-1 knockout mice still have biologically active forms of IL-18 (11), suggesting the existence of an alternative pathway for IL-18 activation.

Dermal infiltration of mast cells is common in AD and allergic skin diseases, but the precise role of mast cells in these diseases is not completely understood. Degranulation of mast cells upon Ag-specific or nonspecific stimulation releases several inflammatory mediators, including histamine, chemical factors, and enzymes. Two lineages of mast cells have been described: 1) tryptase-positive mucosa-associated mast cells and 2) chymase- and tryptase-positive connective tissue-associated mast cells (12). Infiltration of connective tissue-associated mast cells is predominant in AD lesions. In the present study, we evaluated the role of human mast cell chymase in the activation of human recombinant pro-IL-18 (rpro-IL-18). The results showed that human mast cell chymase rapidly cleaves recombinant pro-IL-18, producing a novel biologically active form of IL-18.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

Recombinant human mast cell chymase and anti-chymase neutralizing Ab (13) were provided by Teijin. MatIL-18 was purchased from MBL, and the KG-1 cell line was from the American Type Culture Collection (CCL-246). The cells were cultured in RPMI 1640 medium (Nikken Bio Medical Laboratories) supplemented with 10 mM L-glutamine, 24 mM NaHCO3, 100 U/ml penicillin, and 100 µg/ml streptomycin. All restriction endonucleases and TaqDNA polymerase were purchased from Nippon Gene and Ni-NTA His-trap affinity column from Qiagen. Inhibitors of human antitrypsin and antichymotrypsin and FBS were purchased from Sigma-Aldrich, and porcine heparin from Mochida. Mouse anti-human IL-18 mAb and ELISA kit of human IL-18 were purchased from MBL. Rabbit anti-mouse IgG alkaline phosphatase-conjugated polyclonal Ab and enhanced Western Blue stabilized substrate were from Promega. ELISA kit of human IFN-{gamma} was from BioSource International.

Design of IL-18 gene construct

Human pro-IL-18 is a 24.2-kDa protein composed of 193 aa. A human pro-IL-18 cDNA (nucleotides 1–579; GenBank/EBI Data Bank accession number AF077611) was amplified by RT-PCR from RNA purified from human PBMC using the following primers: 5'-CGGGATCCATGGCTGCTGAACCAGTAGAAGA-3' and 5'-CGGGATCCCTAGTCTTCGC TTTGAACAGTGAAC-3'.

For subcloning of the fragments, the pQE-30 vector (Qiagen) and the amplified cDNA were digested with BamHI and then treated with bacterial alkaline phosphatase to prevent self-ligation. The reaction was conducted using a mixture containing 3 µl of 10x alkaline phosphatase buffer, 5 µl of bacterial alkaline phosphatase (2 U/µl), 20 µl of DNA fragments, and 2 µl of water (total reaction volume of 30 µl) at 37°C for 30 min. The vector fragments were purified using the Gene Clean kit (Qiagen). As final step, purified PCR fragments were ligated into the pQE-30 vector generating the pQE-30-pro-IL-18 and the resultant clones were bidirectionally sequenced.

Expression

Transformation of competent Escherichia coli strain M-15 (Qiagen) with pQE-30-pro-IL-18 was conducted according to the manufacturer’s instructions. The rpro-IL-18 expressed using this vector contains hexahistidine tag at the N-terminal end. Protein expression was performed as follows: each transformed colony was incubated overnight in 50 ml of lysogeny broth medium containing 0.5 g of tryptone, 0.25 g of yeast extract, 0.5 g of NaCl, and 5 mg of ampicillin. After overnight incubation, 950 ml of the same medium was added to incubated medium and then kept shaking at 37°C until obtaining an absorbance of 0.5 at 600 nm. Protein production was then induced by the addition of isopropyl-beta-D-thiogalactopyranoside at a final concentration of 1 mM. Before adding isopropyl-beta-D-thiogalactopyranoside, 1 ml of each culture was transferred into a sterile test tube and incubated separately until the next step. After 8 h, 10 ml of each culture was used for expression analysis using SDS-PAGE.

Purification

After centrifugation (5000 x g for 20 min at 4°C), bacterial cell pellets were resuspended in 20 ml of lysis buffer (PBS (pH 7.4) containing 100 mM imidazole). After thawing, the cells were completely lysed for 10 min followed by aggressive sonication for 10 s at 50-s intervals in ice water bath and centrifugation (15,000 rpm) at 4°C for 30 min. The clear lysate was filtered through a 0.22-µm filter (Millipore), and then the filtrate was applied onto a Ni-NTA His-trap affinity column. The column was then washed with 5 ml of lysis buffer. A total of 5 ml of elution buffer (PBS (pH 7.4) with 300 mM imidazole) was applied onto a Ni-NTA column, and elutes were collected.

Immunoblotting

All samples were diluted in SDS sample buffer and boiled at 95°C for 5 min. SDS-PAGE was performed on a 15% polyacrylamide slab gel containing 0.1% SDS under reducing conditions (9). Resolved proteins were transferred to a nitrocellulose membrane (BioScience) with a semidry transblot system (Bio-Rad). The membrane was then blocked with 5% nonfat dry milk in 1x PBS containing 0.05% Tween 20 for 5 min and incubated with 1 µg/ml mouse anti-human IL-18 pAb. The membrane was washed four times with 1x PBS containing 0.05% Tween 20 and then incubated with 1 µg/ml polyclonal rabbit IgG anti-mouse alkaline phosphatase-conjugated Ab for 1 h. After final washing, the blot was developed using Western Blue substrate (Promega).

Protease reaction

Digestion of purified human rpro-IL-18 by chymase was performed in 1x PBS (pH 7.4) supplemented with 10% glycerol, at an enzyme/substrate (w/w) ratio of 1:1000 for various time intervals from 0 to 120 min at 37°C. Cleavage of mat-IL-18 (100 ng) by chymase (1 ng) was performed for 60 min under the same conditions. The effect of heparin on chymase-mediated cleavage was evaluated under the same conditions by adding 1, 10, 100, or 1000 U/L heparin. Inhibition by anti-chymase or nonspecific Ab was performed at an enzyme/substrate (w/w) ratio of 1:3000. Cleavage of purified human rpro-IL-18 (1 µg) by caspase-1 (1 U) was performed in 1x PBS (pH 7.4) with 10% glycerol for 60 min at 37°C. The reactions were stopped by adding inhibitors or boiling. The samples were boiled in 2% SDS containing 2-ME and analyzed by SDS-PAGE.

Amino acid sequence

Briefly, rpro-IL-18 and proteins cleaved by chymase or caspase-1 were transferred onto Immobilon P membranes (Millipore). The proteins were then visualized by Coomassie brilliant blue (CBB) staining and excised for direct N-terminal sequencing. Amino acid sequences of the N-terminal portions of cleaved fragments of IL-18 were determined using an automated protein sequencing system Procise cLC (Applied Biosystems).

Bioassay

The bioactivity of IL-18 was assessed by the ability of IL-18 to induce IFN-{gamma} secretion from the myelomonocytic cell line KG-1. A total of 3 x 106/ml KG-1 cells was suspended in RPMI 1640 medium containing 10% FBS, and 100 µl of resuspended medium was seeded in a 96-well microplate. The same volume of sample was added to the wells and incubated for 24 h. The concentration of IFN-{gamma} in the cell culture supernatant was measured by a specific ELISA. A standard curve was prepared using human mat-IL-18 purchased from MBL.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Grade of rpro-IL-18 purity and fragments released after treatment with human caspase-1

Eluate from the Ni-NTA His-trap affinity column was electrophoresed and stained with CBB. Purified rpro-IL-18 was recognized as a clear 25-kDa single band (Fig. 1A). The purity was >95% with CBB and silver staining (data not shown). The rpro-IL-18 was cleaved into 18- and 7-kDa fragments after treatment with human caspase-1 for 60 min. The purified rpro-IL-18 was confirmed as a single band by immunoblotting using specific anti-human IL-18 mAb (Fig. 1B). The IL-18 species cleaved by caspase-1 had the same molecular size as mat-IL-18.


Figure 1
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  		FIGURE 1. Activation of rpro-IL-18 by caspase-1. A, Samples were electrophoresed and stained with CBB. Purified rpro-IL-18 can be recognized as a clear band (lane 1). rpro-IL-18 was cleaved into an 18-kDa mature form after treatment with human caspase-1 (lane 2). B, Samples were electrophoresed and stained with anti-human IL-18 mAb. The rpro-IL-18 was confirmed by immunoblotting using specific anti-L-18 mAb (lane 1). Cleavage of rpro-IL-18 by caspase-1 released a fragment (lane 2) with the same molecular size as mat-IL-18 (lane 3).

 
Cleavage of rpro-IL-18 by chymase

Recombinant human mast cell chymase cleaved rpro-IL-18 into two major bands with different molecular weights: 20 kDa (p20) and 16 kDa (p16) (Fig. 2A). The size of these molecules was not identical with that reported so far for mat-IL-18. The p20 fragment was produced within 5 min, but it was rapidly degraded with just a scarce amount being detectable after 30 min. On the other hand, the p16 fragment, which was smaller than mat-IL-18, gradually increased over time but remained uncleaved. This chymase-mediated cleavage was completely inhibited by pretreatment with either antitrypsin inhibitor (100 ng) or antichymotrypsin inhibitor (100 ng). MatIL-18 was resistant to chymase. The cleavage was also clearly blocked by neutralizing anti-chymase Ab (Fig. 2B). The production of p16 was unaffected by heparin.


Figure 2
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  		FIGURE 2. Rpro-IL-18 activation by chymase. A, Human mast cell chymase cleaved rpro-IL-18 into two bands with different m.w. Lanes 2–7, A band of 20 kDa (p20) and band of 16 kDa (p16). Lane 1, rpro-IL-18 alone; lane 2, rpro-IL-18 plus chymase at 0 min; lane 3, rpro-IL-18 plus chymase at 5 min; lane 4, rpro-IL-18 plus chymase at 10 min; lane 5, rpro-IL-18 plus chymase at 30 min; lane 6, rpro-IL-18 plus chymase at 60 min; lane 7, rpro-IL-18 plus chymase at 120 min; lane 8, mat-IL-18; lane 9, mat-IL-18 plus chymase at 60 min; lane 10, antitrypsin (AT)-treated chymase plus rpro-IL-18 at 60 min; lane 11, mantitchymorypsin (ACT)-treated chymase plus rpro-IL-18 at 60 min; lane 12, chymase. The p20 fragment was produced within 5 min (lane 2); however, it was degraded after 30 min (lane 5) and became undetectable after 120 min (lane 7). The p16 fragment was a little smaller than mat-IL-18 showing gradual increase in density. This chymase-mediated degradation was completely inhibited by pretreatment with antitrypsin or antichymotrypsin (lanes 10 and 11). MatIL-18 was resistant to chymase. B, Inhibition of chymase by neutralizing Ab. Lane 1, rpro-IL-18; lane 2, rpIL-18 plus chymase; lane 3, rpro-IL-18 plus nonspecific Ab-treated chymase; lane 4, rpro-IL-18 plus neutralizing anti-chymase Ab-treated chymase. The cleavage was blocked by anti-chymase neutralizing Ab. C, The effect of heparin on pro-IL-18 and chymase. Heparin did not affect the cleavage of pro-IL-18 by chymase at 60 min. Lane 1, rpro-IL-18; lane 2, rpro-IL-18 alone at 60 min; lane 3, rpro-IL-18 plus chymase; lane 4, rpro-IL-18 plus chymase plus heparin 1000 U/L; lane 5, rpro-IL-18 plus chymase plus heparin 100 U/L; lane 6, rpro-IL-18 plus chymase plus heparin 10 U/L; lane 7, rpro-IL-18 plus chymase plus heparin 1 U/L; lane 8, mat-IL-18; lane 9, rpro-IL-18 plus heparin 1000 U/L; lane 10, chymase plus heparin 1000 U/L; lane 11, heparin 1000 U/L; lane 12, chymase.

 
Cleavage sites and amino acid sequences of IL-18

As shown in Fig. 3, chymase cleaves rpro-IL-18 at two positions. The N-terminal residue sequence of the 16-kDa fragment released after cleavage by chymase was I-D-Q-G-N, indicating that chymase mainly cleaves rpro-IL-18 between Phe56-Ile57. The C-terminal residue sequence of the 20-kDa fragment released after cleavage by chymase was N-K-M-Q-F. The sequence of the N-terminal residue of the 5-kDa fragment was E-S-S-S-Y, suggesting that the minor cleavage site of chymase on rpro-IL-18 is Phe151-Glu152. These are novel IL-18-derived species different from IL-18 mature forms released after cleavage by caspase-1, caspase-3, or proteinase-3. We also analyzed rpro-IL-18 and 18-kDa species formed after cleavage by caspase-1. The N-terminal amino acid sequence of rpro-IL-18 and 18-kDa fragment was M-R-G-S-H and Y-F-G-K-L after cleavage by caspase-1, respectively. These results were consistent with the pQE-30-pro-IL-18 vector and the well-known mat-IL-18. Fig. 3 also shows the fragments released after cleavage by caspase-1, caspase-3, and proteinase-3.


Figure 3
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  		FIGURE 3. Cleavage sites and amino acid sequence of IL-18. This is the complete sequence of rpro-IL-18 containing hexahistidine tag sequence with 12 amino acids at the N-terminal end; hexahistidine tag sequence is indicated in bold. The known cleavage sites of caspase-1 and caspase-3 are indicated by solid arrows. The N-terminal residue of mat-IL-18 that is activated by caspase-1 is Tyr36. Putative proteinase-3-cleavage site is indicated by dotted arrow. The N-terminal amino acid residue of p16 was Ile57 and the C-terminal amino acid residue of p20 was Glu151; this cleavage site is different from the reported cleavage sites of caspase-1, caspase-3, or proteinase-3. P16 molecule is indicated by a straight line and p20 by a dotted line. The novel cleavage sites by chymase are indicated by solid arrow. Numbering is from the N-terminal methionine of pro-IL-18.

 
Fragment of rpro-IL-18 released after cleavage by chymase is biologically active

Biological activity was analyzed by the ability of the fragments to induce IFN-{gamma} production in KG-1 cells. The concentration of IFN-{gamma} in the culture medium was measured using a specific ELISA. The relative levels of biological activity were calculated based on the concentration of mat-IL-18 in the supernatant, and then the values were log transformed (Fig. 4). Caspase-1-treated rpro-IL-18 containing cleaved fragments of mat-IL-18 showed 100% activity of the standard rmat-IL-18. Chymase-treated rpro-IL-18 containing p16 fragments showed ~20% of the biological activity of mat-IL-18. Chymase itself, or rpro-IL-18 incubated with antitrypsin- or antichymotrypsin-treated chymase showed no biological activity (Fig. 4).


Figure 4
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  		FIGURE 4. Biological activity of IL-18 fragments released after cleavage by chymase. The biological activity was evaluated by the ability of IL-18 fragments to induce production of IFN-{gamma} in KG-1 cells. Caspase-1-treated rpro-IL-18 showed 100% activity of the standard recombinant mat-IL-18. Chymase-treated rpro-IL-18 (p16) showed ~20% of the biological activity of mat-IL-18. Chymase itself, antitripsin, or antichymotripsin showed no biological activity. Rpro-IL-18 cleaved by chymase pretreated with specific protease inhibitors exerts no biological activity.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This is the first report demonstrating that human mast cell chymase cleaves rpro-IL-18 at Phe56-Ile57 releasing new IL-18 species, which are able to induce secretion of IFN-{gamma} in KG-1 cells (9, 13). Recombinant human mast cell chymase cleaved rpro-IL-18 into two different fragments with molecular weights of p20 and p16. The p20 fragment was rapidly produced, but it was unstable and subsequently degraded. On the other hand, the p16 was resistant to degradation and was biological activity. Chymase was unable to cleave recombinant mat-IL-18. Both mat-IL-18 and rpro-IL-18 contain Phe56 and Phe151 residues, and therefore they are potent substrates for chymase. It is likely that once pro-IL-18 is cleaved at Asp35 or Phe56, the molecular conformation of the protein changes forming stable three-dimensional structures. The proposed three-dimensional structure of IL-18 localizes the Phe56 and Phe151 residues inside the folded mat-IL-18 (14, 15). It is well known that chymase prefers substrates with P1 Phe or Tyr16; thus, the cleavage sites and the proteolytic reactions observed in the present study are consistent with the specificity of chymase. It is also known that chymase is active at neutral pH or alkaline conditions (16). The specificity of chymase reaction was confirmed by experiments showing complete blockade by neutralizing Ab and specific inhibitors. Heparin may bind chymase in vivo, and thus it may affect the effect of chymase on pro-IL-18 (17, 18). However, the addition of heparin in the reaction exerted no effect on chymase-mediated cleavage of pro-IL-18 (Fig. 2C).

Several other enzymes have been reported to cleave IL-18 and/or IL-1beta. Accumulated lines of evidence confirmed that caspase-1 is the authentic activator of IL-18 and IL-1beta with the ability of cleaving them intracellularly (Ref. 19 and Fig. 3). Caspase-3, another member of the caspase family, degrades pro-IL-18 into biologically inactive products (Ref. 19 and Fig. 3). Proteinase-3, a serine protease stored in the granules of neutrophils and macrophages, can also cleave IL-18 and IL-1beta and likely exerts its converting action outside the cells as seen in chymase (Fig. 3) (20, 21). Additional fragments of IL-18 smaller than mat-IL-18 have been reported but enzymes that cleave them remain unknown (22). Other proteases, such as neutrophil elastase and cathepsin G, were reported to cleave IL-1beta (23, 24), but whether they can cleave IL-18 is unknown.

IL-1beta and IL-18 can be activated by other undefined enzymes. Fas-ligand is a protein that binds to FasR and induces apoptosis. MatIL-18 is secreted from Fas-expressing macrophages after stimulation with Fas-ligand, but this secretion is inhibited by caspase-inhibitors (11). Fas-ligand stimulates neutrophils to release mature IL-1beta and induces neutrophil apoptosis in a caspase-1-independent manner (25). Caspase-1 is not indispensable for the release of mature IL-1beta in caspase-1-deficient mice with candidiasis (26). In patients with sepsis, a different mechanism regulates IL-18 secretion because caspase inhibitors exert no influence on IL-18 release (27). In vitro, the production of IFN-{gamma} induced by LPS or zymogen, but not that induced by Con A, is impaired in spleen cells from caspase-1-deficient mice (28). These findings indicate the existence of multiple alternative pathways for pro-IL-18 activation other than caspase-1.

As reported previously, IL-18, particularly in conjunction with IL-3, can activate mast cells to release histamine (29). It is conceivable that IL-18 also induces the release of chymase from mast cells and that the released chymase in turn activates IL-18. This vicious cycle between mast cell degranulation and IL-18 activation might play a critical role in the pathogenesis of several mast cell-associated inflammatory diseases. Indeed, several diseases have been reported to be associated with mast cell activation and elevated IL-18 concentrations. For example, elevated plasma level of IL-18 has been reported in patients with myocardial infarction (30, 31), cardiac chymase has been involved in heart failure and fibrosis, and chymase inhibitors were found to be protective in several cardiac disorders (32). Increased infiltration of mast cells around blood vessels and nerves of the CNS (33) has been reported in association with elevated concentrations of IL-18 in patients with multiple sclerosis (34). Increased infiltration of mast cells and enhanced expression of IL-18 have been also reported in the salivary glands of patients with Sjogren’s syndrome (35, 36). Mast cells have been also found to play a critical role in the pathogenesis of arthritis in an experimental mouse model, and it is known that the serum and the intraarticular levels of IL-18 are increased in patients with rheumatoid arthritis (37). These observations suggest that IL-18 is implicated in the pathogenesis of chronic inflammatory diseases of multiple organs.

Our present results showed that, in vitro, the biological activity of chymase-cleaved IL-18 was ~20% of that of mat-IL-18. However, this does not mean that they have low activity in vivo. Several carrier proteins, such as IgM present in blood or intercellular fluids, have been shown to serve as stabilizers of mat-IL-18 (22); IL-18 fragments released after cleavage by chymase might form complex with these carrier proteins to exert stronger effect in vivo.

We have demonstrated previously that skin-derived IL-18 causes AD-like skin lesions in caspase-1 and IL-18-transgenic mice (2). Release of mat-IL-18 from epidermis causes dermatitis associated with marked mastocytosis (38). These observations suggest the pivotal role of IL-18 in dermatitis. Mast cell is known as an itching inducer because it is a source of histamine. Chymase is a specific enzyme of connective tissue mast cells; however, its biological relevance in Th2 type inflammation is unclear. Our present results clearly showed that chymase from the skin-associated mast cells may contribute to the development of IL-18-mediated atopic inflammation by promoting pro-IL-18 activation. Scratching, which is a well-known aggravating factor of AD, damages the epidermis and induces mast cell degranulation. Injured keratinocytes and skin resident cells release pro-IL-18, which is then activated by the simultaneous release of mast chymase creating a vicious cycle that further worsens skin inflammation. Caspase-1 is a well-known convertase of IL-18 produced by monocytes; however, accumulation of monocytes at sites of AD lesions is not significant. Itching in AD lesions markedly promotes the release of mast cell chymase, which in turn may increase the activation of IL-18 in the skin. Overall, the results of the present study suggest that mast cell chymase and the novel IL-18 active fragment may be novel therapeutic targets for AD and IL-18-associated diseases.


   Acknowledgments
 
We thank Yuichi Kunori from Teijin for providing the human mast cell chymase and the anti-chymase neutralizing Ab. We also thank Dr. Esteban C. Gabazza, Mie University, Mie, Japan, for reviewing the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict 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 supported by Core Research of Evolutional Science and Technology, Japan Science and Technology Corp. Back

2 Address correspondence and reprint requests to Dr. Hitoshi Mizutani, Department of Dermatology, Mie University, Graduate School of Medicine, Tsu, Mie 514-8507, Japan. E-mail address: h-mizuta{at}clin.medic.mie-u.ac.jp Back

3 Abbreviations used in this paper: mat-IL-18, mature IL-18; AD, atopic dermatitis; pro-IL-18, precursor IL-18; rpro-IL-18, recombinant precursor IL-18; CBB, Coomassie brilliant blue. Back

Received for publication June 7, 2006. Accepted for publication October 2, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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