HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pang, L. Articles by Knox, A. J. Search for Related Content PubMed PubMed Citation Articles by Pang, L. Articles by Knox, A. J.

Mast Cell -Tryptase Selectively Cleaves Eotaxin and RANTES and Abrogates Their Eosinophil Chemotactic Activities¹

Division of Respiratory Medicine, City Hospital, University of Nottingham, Nottingham, United Kingdom

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Recent studies have shown that a lack of eosinophils in asthmatic airway smooth muscle (ASM) bundles in contrast to the large number of mast cells is a key feature of asthma. We hypothesized that this is caused by -tryptase, the predominant mast cell-specific protease, abrogating the eosinophil chemotactic activities of ASM cell-derived eosinophil chemoattractants such as eotaxin and RANTES. We studied the effect of -tryptase on the immunoreactivities of human ASM cell-derived and recombinant eotaxin and other recombinant chemokines that are known to be produced by human ASM cells. We report in this study that purified -tryptase markedly reduced the immunoreactivity of human ASM cell-derived and recombinant eotaxin, but had no effect on eotaxin mRNA expression. The effect was mimicked by recombinant human -tryptase in the presence of heparin and was reversed by heat inactivation and the protease inhibitor leupeptin, suggesting that the proteolytic activity of tryptase is required. -Tryptase also exerted similar effects on recombinant RANTES, but not on the other chemokines and cytokines that were screened. Furthermore, a chemotaxis assay revealed that recombinant eotaxin and RANTES induced eosinophil migration concentration-dependently, which was abrogated by pretreatment of these chemokines with -tryptase. Another mast cell protease chymase also markedly reduced the immunoreactivity of eotaxin, but had no effect on RANTES and other chemokines and did not affect the influence of -tryptase on RANTES. These findings suggest that mast cell -tryptase selectively cleaves ASM-derived eotaxin and RANTES and abrogates their chemotactic activities, thus providing an explanation for the eosinophil paucity in asthmatic ASM bundles.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Accumulation of inflammatory cells, such as occurs in airway tissues, has been regarded as a pathological feature of asthma. Recent studies have demonstrated that mast cells are the predominant inflammatory cells in airway smooth muscle (ASM)³ bundles in asthmatic subjects; in contrast, there are almost no T lymphocytes or eosinophils (1, 2). In fact, the significantly increased numbers of mast cells within the ASM bundles in bronchial biopsies in subjects with asthma, but virtually none in patients with eosinophilic bronchitis or normal subjects, is the only immunopathological feature that is different between asthmatic subjects and patients with eosinophilic bronchitis despite the fact that they have the same degree of eosinophilic airway inflammation (1). These observations have cast doubt on the importance of eosinophilic inflammation in asthma and suggest that the disordered airway physiology in asthma is due to mast cell smooth muscle myositis.

Mast cells differ from other inflammatory cells in the lung by their production and release of exceptionally large amounts of enzymatically active proteases, such as tryptases and chymase. There are three groups of known human tryptases: -, -, and - tryptase, or transmembrane tryptases (3, 4). -Tryptase comprises as much as 25% of the total mast cell protein and is stored in mast cell secretory granules as enzymatically active tetramers in a complex with heparin, which facilitates the conversion of mature -tryptase monomers to tetramers and stabilizes them (5). -Tryptase is the main type of tryptase released during mast cell degranulation and isolated from normal human lung tissues. Because of this, the enzymatically active lung tryptase preparations as well as -tryptase produced recombinantly will be subsequently referred to as tryptase. Although there are few known soluble substrates that are cleaved by tryptase (4), tryptase cleaves and activates the protease-activated receptor-2 and thereby initiates a variety of processes contributing to chronic inflammation and tissue remodeling. Chymase is another major protease stored in high amounts within the secretory granules of mast cells. According to the presence of tryptase and chymase in the granules, mast cells can be divided into two subtypes, mast cells containing only tryptase (MC_T) and mast cells containing both tryptase and chymase (MC_TC). It has been demonstrated that the mast cells localized in asthmatic ASM bundles are MC_TC (1).

The fact that mast cells are localized in the ASM layer in asthma indicates that ASM cells are able to recruit and retain mast cells in asthma. We and others have demonstrated that human ASM cells secrete a range of chemokines, including eotaxin (6), MCP-1 (7), IL-8 (8), stem cell factor (SCF) (9), RANTES (10), GM-CSF (11), and TGF-1 (12). The constitutive and stimulated production of SCF by human ASM cells, in particular, may play a key role in the recruitment, survival, and differentiation of mast cells. These effects may then be amplified by mast cell products. Indeed, a recent study shows that tryptase-stimulated human ASM cells may attract mast cells though the release of TGF-1 in addition to SCF (12). Tryptase is also an extremely potent mitogen for ASM cells (13), and in contrast, chymase profoundly inhibits mitogen-induced ASM cell proliferation (14). These observations strongly suggest that there are complex interactions between ASM cells and mast cells.

Inflammatory cells are recruited to local tissues by a complex network of chemokines and other chemotactic factors. Eotaxin is a potent eosinophil chemoattractant, acting selectively through the CCR3. RANTES is also a potent chemoattractant for eosinophils (15), memory T cells, and monocytes (16). Proteolysis is one means by which chemokine activity can be modulated. For example, RANTES, MCP-2, eotaxin, and IFN--inducible protein 10 are substrates of dipeptidyl peptidase IV (CD26), a leukocyte activation marker and a serine protease (17), and truncated RANTES molecule exerts different activities from those of RANTES because of its altered receptor specificity (17). Eotaxin cleaved at the N terminus by CD26 also shows a loss of its chemotactic potency (18). Furthermore, proteases produced by the adult hookworm Necator americanus cleave eotaxin, but not IL-8 or eotaxin-2, resulting in a loss of its immunoreactivity as well as its chemotactic activity (19).

Human ASM cells are a rich source of eotaxin (6, 20) and RANTES (10). However, despite the facts that strong signals of eotaxin mRNA and immunoreactivity are observed in vivo in smooth muscle in asthmatic airways (20) and that RANTES mRNA is elevated in airways of patients with asthma (21), there is an eosinophil paucity in asthmatic ASM bundles in contrast to the large number of mast cells (1). Because mast cells present in asthmatic airways are in a chronically activated secretory state (22), we hypothesize that tryptase secreted from microlocalized mast cells may reduce the chemotactic capability of ASM cell-derived eosinophil chemoattractants, eotaxin and RANTES in particular, either through inhibition of their expression or through cleavage of preformed chemokines. In this study we showed that among the chemokines and cytokines screened, eotaxin and RANTES were selectively cleaved by tryptase, which resulted in the loss of their eosinophil chemotactic potency. These findings thus suggest that the selective cleavage of eotaxin and RANTES by mast cell tryptase contributes to the eosinophil paucity seen in asthmatic ASM bundles.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Materials

Purified human lung tryptase was obtained as a liquid containing 50 mM NaOAc, 1 M NaCl, and 0.05 mM heparin (pH 5.0) with 0.01% NaN₃ from Europa Bioproducts (sp. act., 12,870 mU/mg protein using N-benzyl-D,L-arginine-pNA as substrate); purified human skin chymase was obtained from Merck Biosciences (sp. act., 30,000 mU/mg protein using N-benzoyl-L-tyrosine-ethyl ester as substrate); recombinant human lung -tryptase (rh-tryptase) was purchased from Promega (sp. act., 1000 U/mg protein using N-CBZ-L-lysine thiobenzyl ester as substrate); FCS was obtained from Seralab; ELISA kits for human eotaxin, RANTES, GM-CSF, MCP-1, IL-8, TGF-1, vascular endothelial growth factor (VEGF), Abs against eotaxin and eotaxin-2, and human eosinophil enrichment column kit were purchased from R&D Systems; the Hemacolor rapid staining kit was obtained from Merck; the RNeasy minikit was purchased from Qiagen; Moloney murine leukemia virus reverse transcriptase was obtained from Invitrogen Life Technologies; recombinant human VEGF₁₂₁ and RANTES were obtained from PeproTech; recombinant human eotaxin-1, eotaxin-2, IL-8, GM-CSF, MCP-1, TGF-1, RPMI 1640 medium, recombinant human TNF-, ReadyMix Taq PCR mix, leupeptin, soybean trypsin inhibitor (SBTI), heparin, and other unspecified chemicals were all purchased from Sigma-Aldrich.

Cell culture

Primary cultures of human ASM cells were established from explants obtained from four postmortem individuals with no history of respiratory diseases and no evidence of airway abnormalities, as previously reported (23). Cells at passages 5–6 depict the immunohistochemical and morphological characteristics of typical ASM cells (23) and were used in this study. This protocol was approved by the Nottingham City Hospital research ethics committee. The effect of tryptase on ASM cells was compared with those of its vehicle and heat inactivation was conducted by heating tryptase at 56°C for 60 min.

Cytokine and chemokine assays

The levels of cytokines and chemokines were measured by ELISA as previously reported (6, 24). The sensitivity of the ELISA kits in our hands was at least 5 pg/ml, consistent with the manufacturer’s specifications. According to the kit insert, there was no significant cross-reactivity or interference with other human cytokines and chemokines.

RT-PCR

Confluent human ASM cells in six-well plates were serum starved for 24 h. After treatment, total RNA was extracted using the RNeasy minikit and transcribed into cDNA using Moloney murine leukemia virus reverse transcriptase. The primers for eotaxin and the internal control GAPDH cDNA amplification and PCR conditions were described previously (24).

Western blotting

To detect the cleavage of eotaxin and eotaxin-2 by tryptase, 40 ng of recombinant human eotaxin or eotaxin-2 in 5 µl of serum-free medium was mixed with 30 mU of tryptase in 25 µl of serum-free medium (1 ng chemokine:0.75 mU tryptase) or medium alone and incubated at 37°C for the times indicated. The samples were then analyzed by Western blotting using specific Abs as described previously (23).

Isolation of human eosinophils

Peripheral venous blood was obtained from volunteers with mild to moderate eosinophilia. Eosinophils were isolated with a human eosinophil enrichment column kit according to the manufacturer’s instructions (R&D Systems). Briefly, blood was mixed with RBC gradient solution and left for 30–45 min to allow RBC sedimentation. The top plasma layer of cells was added on top of the polymorphonuclear cell (PMN) separation medium. After centrifugation at 500 x g for 30 min, the cell pellet containing PMNs was collected. The remaining RBCs were lysed by hypotonic wash. Up to 2 x 10⁸ PMNs were incubated with 1 ml of the Ab mixture for 20 min, washed with column buffer, then applied to the eosinophil selection column and incubated for 10 min. The eosinophils were finally eluted with column buffer (>97% purity) and resuspended in RPMI 1640 medium containing 1% serum. This protocol was approved by Nottingham University Medical School research ethics committee, and all human participants gave written informed consent.

Chemotaxis assay

The migration of eosinophils in response to eotaxin and RANTES in vitro was assessed using 5-µm pore size polycarbonate membrane Transwell inserts in 24-well culture plates (Corning). Medium alone (0.6 ml) or medium containing recombinant chemokines with or without neutralizing Ab or tryptase was added into the wells. Purified eosinophils (1 x 10⁵) in 0.1 ml of RPMI 1640 containing 1% serum were placed into the inserts, which were then placed into the wells. The plates were incubated at 37°C and 5% CO₂ for 90 min. Eosinophils that migrated through the filter into the lower chamber were collected, resuspended, and counted. Chemotaxis was expressed as a migration index (the ratio of migrated cell numbers from experimental groups divided by those from control groups) (25, 26).

Statistical analysis

Data were expressed as the mean ± SE. Statistical analysis was performed with GraphPad PRISM (version 4). Unpaired two-tailed Student’s t test was used to determine the significant differences between the means; p < 0.05 was accepted as statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Tryptase reduces the immunoreactivity of human ASM cell-derived eotaxin without affecting its mRNA expression

To examine the effect of tryptase on ASM cell-derived eotaxin immunoreactivity, cells were treated with or without tryptase for 24, 48, and 72 h. Eotaxin was released from ASM cells constitutively, but treatment with tryptase at all concentrations markedly reduced eotaxin levels in the medium (Fig. 1A). To test the effect of tryptase on TNF--induced immunoreactive eotaxin, cells were pretreated with or without tryptase for 30 min, then incubated in the presence or the absence of TNF- for 24 h. TNF- markedly increased eotaxin levels in the medium compared with control cells, but the increase was concentration-dependently abolished by tryptase (Fig. 1B). Heat inactivation of tryptase (30 mU/ml) completely reversed the effect of tryptase (Fig. 1B), suggesting a requirement for an intact catalytic site in this process.

View larger version (18K): [in this window] [in a new window]   FIGURE 1. Effect of tryptase on human ASM cell-derived eotaxin immunoreactivity and mRNA expression. A, ASM cells in 24-well plates were treated with increasing concentrations of tryptase for the times indicated. B, ASM cells were pretreated with or without increasing concentrations of tryptase or heat-inactivated tryptase for 30 min before incubation with TNF- for 24 h. Eotaxin levels in the medium were measured by ELISA. Each point represents the mean ± SE of two experiments performed in triplicate. C, ASM cells were pretreated with or without tryptase for 30 min before incubation with TNF- for the times indicated. mRNA levels of eotaxin and GAPDH were measured by RT-PCR. Results are representative of three independent experiments with similar results. D, ASM cells in 90-mm dishes were treated with TNF- for 24 h. Cell-free medium was collected, and aliquots were incubated at 37°C with or without increasing concentrations of tryptase or heat-inactivated tryptase for 16 h. Eotaxin levels in the medium were measured by ELISA. Each point represents the mean ± SE of two experiments performed in triplicate.

To confirm this, the effect of tryptase on the immunoreactivity of preformed eotaxin was assessed. Medium from TNF--treated ASM cells (10 ng/ml, 24 h) was incubated at 37°C for 16 h in the presence or the absence of tryptase, and the remaining immunoreactive eotaxin was measured by ELISA. As shown in Fig. 1D, incubation of the medium in the absence of tryptase resulted in a small loss of eotaxin measurable by ELISA compared with the control (without incubation). However, incubation in the presence of tryptase led to a concentration-dependent reduction and eventual abolishment of immunoreactive eotaxin, which was reversed by heat inactivation of tryptase, suggesting that proteolysis was responsible for the loss of immunoreactivity of preformed eotaxin.

Tryptase reduces the immunoreactivity of recombinant eotaxin

To examine the effect of tryptase on the immunoreactivity of recombinant eotaxin, recombinant human eotaxin (500 pg/ml) was incubated with increasing concentrations of tryptase at 37°C for 16 h. As shown in Fig. 2A, a small loss of immunoreactive eotaxin was observed after incubation in the absence of tryptase, whereas tryptase reduced immunoreactive eotaxin in a concentration-dependent manner. A marked reduction was observed at a concentration as low as 0.007 mU/ml, and complete abolishment was achieved with 7.5 mU/ml tryptase. To assess the effect of tryptase (7.5 mU/ml), recombinant human eotaxin at increasing concentrations was incubated at 37°C for 16 h in the presence or the absence of tryptase. As shown in Fig. 2B, tryptase (7.5 mU/ml) abolished eotaxin immunoreactivity regardless of its concentration. To explore the time course of the tryptase effect, recombinant human eotaxin (1000 pg/ml) was incubated at 37°C in the presence or the absence of tryptase for up to 15 min. Compared with the small natural loss of eotaxin immunoreactivity in the absence of tryptase (Fig. 2C), a marked loss of eotaxin immunoreactivity occurred immediately after the addition of tryptase to the reaction, and no measurable eotaxin immunoreactivity was detected after 2-min incubation.

View larger version (19K): [in this window] [in a new window]   FIGURE 2. Effect of tryptase on the immunoreactivity of recombinant human eotaxin and eotaxin-2. A, Recombinant human eotaxin (500 ng/ml) was incubated at 37°C with or without increasing concentrations of tryptase for 16 h. B, Recombinant human eotaxin at increasing concentrations was incubated at 37°C with or without tryptase (7.5 mU/ml) for 16 h. C, Recombinant human eotaxin (1000 ng/ml) was incubated at 37°C with or without tryptase (7.5 mU/ml) for the times indicated. Immunoreactive eotaxin in the reactions was then measured by ELISA. Each point represents data from a single experiment. D, Recombinant human eotaxin and eotaxin-2 (both 40 ng) were incubated at 37°C with or without tryptase (30 mU) or heat-inactivated tryptase for the times indicated. Immunoreactive eotaxin and eotaxin-2 in the reactions were then analyzed by Western blotting. Results are representative of two independent experiments with similar results.

Recombinant human tryptase also reduces eotaxin immunoreactivity, and leupeptin reverses the effect

To test whether rh-tryptase could exert a similar effect on eotaxin immunoreactivity as purified tryptase, recombinant eotaxin (500 pg/ml) was incubated with increasing concentrations of rh-tryptase (0.029–7.5 mU/ml) in the absence or the presence of heparin for 16 h, and the remaining immunoreactive eotaxin was then measured by ELISA. In the absence of heparin, rh-tryptase had no effect on eotaxin immunoreactivity. Heparin alone (29.14 nM) also had no effect. However, in the presence of heparin, rh-tryptase concentration-dependently reduced eotaxin immunoreactivity (Fig. 3A).

View larger version (25K): [in this window] [in a new window]   FIGURE 3. Effect of rh-tryptase on the immunoreactivity of recombinant human eotaxin and effect of protease inhibitors on tryptase cleavage of eotaxin. A, Recombinant human eotaxin was incubated at 37°C with increasing concentrations of rh-tryptase in the absence or the presence of heparin (ratio of heparin:rh-tryptase, 1:0.148 in molar concentration; concentration for heparin alone, 29.14 nM, equivalent to heparin concentration with 7.5 mU/ml tryptase). B, Tryptase (0.469 mU/ml) and rh-tryptase (7.5 mU/ml) were preincubated with or without the protease inhibitors, leupeptin and SBTI (both 50 µM), respectively, at 37°C for 30 min before incubation with recombinant eotaxin (500 pg/ml) for 16 h. Immunoreactive eotaxin in the reactions was then measured by ELISA. Each point represents data from a single experiment.

Tryptase reduces the immunoreactivity of RANTES, but not the other chemokines and cytokines screened

To determine whether tryptase could also cleave other chemokines and cytokines that are known to be produced by human ASM cells, recombinant human RANTES, GM-CSF, MCP-1, IL-8, TGF-1 and VEGF (all 31.25–1000 pg/ml) were incubated at 37°C in the presence or the absence of tryptase for 16 h, and the remaining immunoreactive chemokines and cytokines were measured by ELISA. In the absence of tryptase, the immunoreactivities of all tested chemokines and cytokines remained almost unaltered after incubation; however, in the presence of tryptase, the immunoreactivity of RANTES was markedly reduced (Fig. 4A), whereas the immunoreactivities of the others were not changed compared with the control (without tryptase; Fig. 4, B–F). These results indicate that the cleavage of eotaxin and RANTES by tryptase is selective.

View larger version (26K): [in this window] [in a new window]   FIGURE 4. Effect of tryptase on the immunoreactivities of other chemokines and cytokines. Increasing concentrations of recombinant human RANTES (A), GM-CFS (B), MCP-1 (C), IL-8 (D), TGF1 (E), and VEGF (F) were incubated at 37°C with or without tryptase for 16 h. Immunoreactive chemokines and cytokines in the reactions were then measured by ELISA. Each point represents data from a single experiment.

Two functional studies were conducted to determine whether the cleavage of eotaxin and RANTES by tryptase could result in consequent loss of their functional activities. As shown in Fig. 5A, TNF- markedly increased immunoreactive MCP-1 from human ASM cells after incubation for 16 h, and the effect was significantly inhibited by recombinant human eotaxin in a concentration-dependent manner. However, the inhibition was completely reversed when eotaxin was preincubated with tryptase at 37°C for 30 min, despite the fact that tryptase on its own had no effect on TNF--induced MCP-1 immunoreactivity. Both recombinant human eotaxin and RANTES induced human eosinophil migration in a concentration-dependent manner (Fig. 5, B and C). Tryptase on its own had no effect on eosinophil migration, but pretreatment of eotaxin and RANTES with tryptase at 37°C for 30 min abrogated their eosinophil chemotactic activities (Fig. 5, B and C). The chemotactic effect of eotaxin was also abolished by the anti-eotaxin neutralizing Ab (Fig. 5B). Collectively, these results indicate that tryptase cleavage of eotaxin and RANTES results in the loss of their immunoreactivities as well as their functional activities.

View larger version (22K): [in this window] [in a new window]   FIGURE 5. Effects of tryptase on the functional activities of eotaxin and RANTES. A, Recombinant human eotaxin was preincubated at 37°C with or without tryptase for 30 min. ASM cells in 24-well plates were treated with or without the above-mentioned eotaxin for 30 min before incubation with TNF- for 16 h. MCP-1 levels in the medium were measured by ELISA. Each point represents the mean ± SE of two experiments performed in triplicate. *, p < 0.05; ***, p < 0.001 (compared with TNF- alone). Recombinant human eotaxin (B) and RANTES (C) were incubated at 37°C with or without anti-eotaxin neutralizing Ab or tryptase, respectively, for 30 min. Human eosinophils were purified, an eosinophil chemotaxis assay was conducted, and the migration index was calculated as described in Materials and Methods. Each point represents the mean ± SE of two experiments performed in triplicate.

Because the mast cells microlocalized in asthmatic ASM bundles are mainly MC_TC (1), and chymase may exert different or even opposite effects from tryptase (14), the effect of purified chymase, either alone or in combination with tryptase, on the immunoreactivities of recombinant chemokines was studied. Incubation of eotaxin (31.25–1000 pg/ml) with chymase (7.5 mU/ml) at 37°C for 16 h resulted in an almost complete loss of immunoreactive eotaxin, in a similar manner as that of tryptase (7.5 mU/ml) alone and tryptase plus chymase (Fig. 6A). However, chymase had no effect on RANTES immunoreactivity or tryptase-induced loss of immunoreactive RANTES (Fig. 6B). Chymase, either alone or in combination with tryptase, also had no effect on the immunoreactivities of IL-8 (Fig. 6C) and MCP-1 (Fig. 6D). These results indicate that chymase has a similar proteolytic effect on eotaxin as tryptase, but does not cleave the other chemokines screened or alter the cleavage of RANTES by tryptase.

View larger version (21K): [in this window] [in a new window]   FIGURE 6. Effects of tryptase and chymase on the immunoreactivities of chemokines. Increasing concentrations of recombinant human eotaxin (A), RANTES (B), IL-8 (C), and MCP-1 (D) were incubated at 37°C with or without tryptase, chymase, or both for 16 h. Immunoreactive chemokines in the reactions were then measured by ELISA. Each point represents data from a single experiment.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

Tryptase has been previously shown to cleave certain extracellular substrates, including vasoactive intestinal peptide and calcitonin gene-related peptide (28), 72-kDa gelatinase, fibronectin (29), and prostromelysin (30). More recently, it has been established that tryptase is a potent growth factor for a number of cell types, including fibroblasts (31), epithelial cells (32), and ASM cells (13). It is of particular importance to note that tryptase can stimulate the release of chemokine IL-8 from epithelial cells (32), endothelial cells (33), and ASM cells (our unpublished observation), suggesting that tryptase may regulate the release of chemokines and subsequently the migration of inflammatory cells. In this study we found that tryptase reduced the release of immunoreactive eotaxin from ASM cells due to the proteolysis of preformed eotaxin, but not the inhibition of its mRNA expression. The loss of eotaxin immunoreactivity was accompanied by the loss of its functional activities, such as the inhibition of TNF--induced immunoreactive MCP-1 from ASM cells and eosinophil chemotaxis. A similar loss of RANTES immunoreactivity and chemotactic function was observed with tryptase as a result of its proteolytic activity. However, tryptase had no proteolytic effect on other chemokines and cytokines that are known to be produced by human ASM cells and to play a role in orchestrating airway inflammation, including MCP-1 (7), IL-8 (8), GM-CSF (11), RANTES (10), VEGF (34), and TGF-1 (12). Tryptase also had no effect on eotaxin-2, which is not expressed in human ASM cells (35). It is interesting to note that another major mast cell protease chymase also selectively cleaved eotaxin, but had no effect on other chemokines screened in this study and did not alter the effect of tryptase on eotaxin and RANTES. The selective cleavage of eotaxin and RANTES is the first report that mast cell tryptase can regulate chemokines and their chemotactic activities through its proteolytic activity and adds to the complexity of the interactions between ASM cells and mast cells.

We also demonstrated that the cleavage of eotaxin by purified tryptase was due to the proteolytic activity of tryptase, rather than any other contaminating proteases or heparin by studies showing that 1) eotaxin cleavage was achieved with very low concentrations of tryptase (as low as 0.007 mU/ml); 2) rh-tryptase also cleaved eotaxin in the presence of heparin, whereas heparin alone had no effect; and 3) the cleavage of eotaxin by both tryptase and rh-tryptase was markedly reversed by the protease inhibitor leupeptin (inhibits tryptase), but not SBTI (inhibits proteases other than tryptase).

There seems to be a dissociation between the strong eotaxin immunoreactivity (20) and the eosinophil paucity in asthmatic ASM (1). However, the eotaxin immunoreactivity detected by immunohistochemistry (20) probably represents only functionally inactive eotaxin inside ASM cells, because eotaxin secreted outside ASM cells can be cleaved quickly by tryptase released from microlocalized mast cells and subsequently loses its immunoreactivity as well as its eosinophil chemotactic activity. The same applies to RANTES. Therefore, even if there is strong eotaxin and RANTES expression in asthmatic ASM, eosinophil paucity can still be observed in asthmatic ASM bundles because it mainly consists of the chemokines inside the cells. However, in addition to eotaxin and RANTES, there are other chemokines that may contribute to eosinophil migration into asthmatic ASM bundles and it is difficult to mimic the microenvironment of asthmatic ASM cells in in vitro experiments. Additional studies with asthmatic ASM are needed to verify the role of tryptase in eosinophil paucity in asthmatic ASM bundles.

Eotaxin is a selective ligand for the chemokine receptor CCR3, whereas RANTES binds to CCR3 as well as CCR1, CCR5, and CCR9. CCR3 is expressed in eosinophils (36), basophils (37), Th2 cells (38), and MC_TC (39). Both eotaxin and RANTES exert chemotactic activities on mast cells through CCR3 (39). Tryptase cleavage of these chemokines may therefore provide a feedback regulation to prevent the excessive mast cell infiltration into human ASM bundle. However, there may be other, more important ASM cell-derived chemokines than eotaxin and RANTES that contribute to mast cell migration into in ASM. Indeed, a recent study has demonstrated that tryptase-stimulated human ASM cells attract mast cells through the release of TGF-1 in addition to SCF, and that both factors are localized to ASM in asthmatic airways (11). Another recent study has shown that the chemokine CXCL10 (IFN--inducible protein 10) is expressed preferentially by asthmatic ASM cells, and its receptor CXCR3 is most abundantly expressed on mast cells in asthmatic ASM, suggesting that interactions between ASM-derived CXCL10 and mast cell-expressed CXCR3 may play a key role in mast cell migration into the ASM bundles in asthma (40). Tryptase cleavage of eotaxin and RANTES may therefore not have an impact on mast cell microlocalization in asthmatic ASM.

Eosinophils have long been regarded as the fundamental effector cells in the pathogenesis of asthma. However, treatment with anti-IL-5 mAbs has no clinical benefit in asthma despite the profound reduction in circulating and airway lumen eosinophils (41, 42). The lack of efficacy of anti-IL-5 therapy and the lack of eosinophils in asthmatic ASM bundles in contrast to the large number of mast cells have cast serious doubt on the accepted role of eosinophils in asthma. Our current findings show that the selective cleavage of the preformed eosinophil chemokines, eotaxin and RANTES, from human ASM cells by tryptase from microlocalized mast cells contributes to the eosinophil paucity seen in asthmatic ASM bundles and support the new concept that mast cell smooth muscle myositis, rather than eosinophilic airway inflammation, is associated with airway dysfunction in asthma.

	Acknowledgments

 
We thank Dr. Colin Clelland for providing us with specimens of human trachea.

	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 the Wellcome Trust, Asthma U.K., and Novartis.

² Address correspondence and reprint requests to Dr. Linhua Pang, Division of Respiratory Medicine, Clinical Sciences Building, City Hospital, University of Nottingham, Hucknall Road, Nottingham NG5 1PB, U.K. E-mail address: linhua.pang{at}nottingham.ac.uk

³ Abbreviations used in this paper: ASM, airway smooth muscle; MC_T, mast cell containing only tryptase; MC_TC, mast cell containing both tryptase and chymase; PMN, polymorphonuclear cell; rh-tryptase, recombinant human -tryptase; SBTI, soybean trypsin inhibitor; SCF, stem cell factor; VEGF, vascular endothelial growth factor.

Received for publication July 28, 2005. Accepted for publication January 9, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Brightling, C. E., P. Bradding, F. A. Symon, S. T. Holgate, A. J. Wardlaw, I. D. Pavord. 2002. Mast cell infiltration of airway smooth muscle in asthma. N. Engl. J. Med. 346: 1699-1703. [Abstract/Free Full Text]
2. Carroll, N. G., S. Mutavdzic, A. J. James. 2002. Distribution and degranulation of airway mast cells in normal and asthmatic subjects. Eur. Respir. J. 19: 879-885. [Abstract/Free Full Text]
3. Pallaoro, M., M. S. Fejzo, L. Shayesteh, J. L. Blount, G. H. Caughey. 1999. Characterization of genes encoding known and novel human mast cell tryptases on chromosome 16p13.3. J. Biol. Chem. 274: 3355-3362. [Abstract/Free Full Text]
4. Little, S. S., D. A. Johnson. 1995. Human mast cell tryptase isoforms: separation and examination of substrate-specificity differences. Biochem. J. 307: 341-346. [Medline]
5. Fukuoka, Y., L. B. Schwartz. 2004. Human -tryptase: detection and characterization of the active monomer and prevention of tetramer reconstitution by protease inhibitors. Biochemistry 43: 10757-10764. [Medline]
6. Pang, L., A. J. Knox. 2001. Regulation of TNF-induced eotaxin release from cultured human airway smooth muscle cells by ₂-agonists and corticosteroids. FASEB J. 15: 261-269. [Abstract/Free Full Text]
7. Pype, J. L., L. J. Dupont, P. Menten, E. Van Coillie, G. Opdenakker, J. Van Damme, K. F. Chung, M. G. Demedts, G. M. Verleden. 1999. Expression of monocyte chemotactic protein (MCP)-1, MCP-2, and MCP-3 by human airway smooth-muscle cells: modulation by corticosteroids and T-helper 2 cytokines. Am. J. Respir. Cell Mol. Biol. 21: 528-536. [Abstract/Free Full Text]
8. Pang, L., A. J. Knox. 1998. Bradykinin stimulates IL-8 production in cultured human airway smooth muscle cells: role of cyclooxygenase products. J. Immunol. 161: 2509-2515. [Abstract/Free Full Text]
9. Kassel, O., F. Schmidlin, C. Duvernelle, B. Gasser, G. Massard, N. Frossard. 1999. Human bronchial smooth muscle cells in culture produce stem cell factor. Eur. Respir. J. 13: 951-954. [Abstract]
10. Ammit, A. J., A. L. Lazaar, C. Irani, G. M. O’Neill, N. D. Gordon, Y. Amrani, R. B. Penn, R. A. Panettieri, Jr. 2002. Tumor necrosis factor--induced secretion of RANTES and interleukin-6 from human airway smooth muscle cells: modulation by glucocorticoids and -agonists. Am. J. Respir. Cell Mol. Biol. 26: 465-474. [Abstract/Free Full Text]
11. Saunders, M. A., J. A. Mitchell, P. M. Seldon, M. H. Yacoub, P. J. Barnes, M. A. Giembycz, M. G. Belvisi. 1997. Release of granulocyte-macrophage colony stimulating factor by human cultured airway smooth muscle cells: suppression by dexamethasone. Br. J. Pharmacol. 120: 545-546.
12. Berger, P., P. O. Girodet, H. Begueret, O. Ousova, D. W. Perng, R. Marthan, A. F. Walls, J. M. Tunon De Lara. 2003. Tryptase-stimulated human airway smooth muscle cells induce cytokine synthesis and mast cell chemotaxis. FASEB J. 17: 2139-2141. [Abstract/Free Full Text]
13. Brown, J. K., C. A. Jones, L. A. Rooney, G. H. Caughey, I. P. Hall. 2002. Tryptase’s potent mitogenic effects in human airway smooth muscle cells are via nonproteolytic actions. Am. J. Physiol. 282: L197-L206.
14. Lazaar, A. L., M. I. Plotnick, U. Kucich., I. Crichton, S. Lotfi, S. F. Das, S. Kane, J. Rosenbloom, R. A. Panettieri, Jr, N. M. Schechter, et al 2002. Mast cell chymase modifies cell-matrix interactions and inhibits mitogen-induced proliferation of human airway smooth muscle cells. J. Immunol. 169: 1014-1020. [Abstract/Free Full Text]
15. Kameyoshi, Y., A. Dorschner, A. I. Mallet, E. Christophers, J. M. Schroder. 1992. Cytokine RANTES released by thrombin-stimulated platelets is a potent attractant for human eosinophils. J. Exp. Med. 176: 587-592. [Abstract/Free Full Text]
16. Schall, T. J., K. Bacon, K. J. Toy, D. V. Goedded. 1990. Selective attraction of monocytes and T lymphocytes of the memory phenotype by cytokine RANTES. Nature 347: 669-671. [Medline]
17. Oravecz, T., M. Pall, G. Roderiquez, M. D. Gorrell, M. Ditto, N. Y. Nguyen, R. Boykins, E. Unsworth, M. A. Norcross. 1997. Regulation of the receptor specificity and function of the chemokine RANTES (regulated on activation, normal T cell expressed and secreted) by dipeptidyl peptidase IV (CD26)-mediated cleavage. J. Exp. Med. 186: 1865-1872. [Abstract/Free Full Text]
18. Struyf, S., P. Proost, P. Schols, E. De Clercq, G. Opdenakker, J. P. Lenaerts, M. Detheux, M. Parmentier, I. De Meester, S. Scharpé, et al 1999. CD26/dipeptidyl-peptidase IV down-regulates the eosinophil chemotactic potency, but not the anti-HIV activity of human eotaxin by affecting its interaction with CC chemokine receptor 3. J. Immunol. 162: 4903-4909. [Abstract/Free Full Text]
19. Culley, F. J., A. Brown, D. M. Conroy, I. Sabroe, D. I. Pritchard, T. J. Williams. 2000. Eotaxin is specifically cleaved by hookworm metalloproteases preventing its action in vitro and in vivo. J. Immunol. 165: 6447-6453. [Abstract/Free Full Text]
20. Ghaffar, O., Q. Hamid, P. M. Renzi, Z. Allakhverdi, S. Molet, J. C. Hogg, S. A. Shore, A. D. Luster, B. Lamkhioued. 1999. Constitutive and cytokine-stimulated expression of eotaxin by human airway smooth muscle cells. Am. J. Respir. Crit. Care Med. 159: 1933-1942. [Abstract/Free Full Text]
21. Berkman, N., V. L. Krishnan, T. Gilbey, R. Newton, B. O’Connor, P. J. Barnes, K. F. Chung. 1996. Expression of RANTES mRNA and protein in airways of patients with mild asthma. Am. J. Respir. Crit. Care Med. 154: 1804-1811. [Abstract]
22. Broide, D. H., G. J. Gleich, A. J. Cuomo, D. A. Coburn, E. C. Federman, L. B. Schwartz, S. I. Wasserman. 1991. Evidence of ongoing mast cell and eosinophil degranulation in symptomatic asthma airway. J. Allergy Clin. Immunol. 88: 637-648. [Medline]
23. Pang, L., A. J. Knox. 1997. Effect of interleukin-1, tumour necrosis factor- and interferon-r on the induction of cyclo-oxygenase-2 in cultured human airway smooth muscle cells. Br. J. Pharmacol. 121: 579-587.
24. Nie, M., L. Corbett, A. J. Knox, L. Pang. 2005. Differential regulation of chemokine expression by peroxisome proliferator-activated receptor agonists: interactions with glucocorticoids and ₂-agonists. J. Biol. Chem. 280: 2550-2561. [Abstract/Free Full Text]
25. Ferreira, H. H. A., M. L. S. Lodo, A. R. Martins, L. Kandratavicius, A. F. Salaroli, N. Conran, E. Antunes, G. De Nucci. 2002. Expression of nitric oxide synthases and in vitro migration of eosinophils from allergic rhinitis subjects. Eur. J. Pharmacol. 442: 155-162. [Medline]
26. Borchers, M. T., T. Ansay, R. DeSalle, B. L. Daugherty, H. Shen, M. Metzger, N. A. Lee, J. J. Lee. 2002. In vitro assessment of chemokine receptor-ligand interactions mediating mouse eosinophil migration. J. Leukocyte Biol. 71: 1033-1041. [Abstract/Free Full Text]
27. He, S. H., P. Chen, H. Q. Chen. 2003. Modulation of enzymatic activity of human mast cell tryptase and chymase by protease inhibitors. Acta Pharmacol. Sin. 24: 923-929. [Medline]
28. Tam, E. K., G. H. Caughey. 1990. Degradation of airway neuropeptides by human lung tryptase. Am. J. Respir. Cell Mol. Biol. 3: 27-32. [Medline]
29. Lohi, J., I. Harvima, J. Keski-Oja. 1992. Pericellular substrates of human mast cell tryptase: 72,000 dalton gelatinase and fibronectin. J. Cell. Biochem. 50: 337-349. [Medline]
30. Gruber, B. L., M. J. Marchese, K. Suziki, L. B. Schwartz, Y. Okada, H. Nagase, N. S. Ramamurthy. 1989. Synovial procollagenase activation by human mast cell tryptase: dependence upon matrix metalloproteinase 3 activation. J. Clin. Invest. 84: 1657-1662. [Medline]
31. Ruoss, S. J., T. Hartmann, G. H. Caughey. 1991. Mast cell tryptase is a mitogen for cultured fibroblasts. J. Clin. Invest. 88: 493-499. [Medline]
32. Cairns, J. A., A. F. Walls. 1996. Mast cell tryptase is a mitogen for epithelial cells: stimulation of IL-8 production and intercellular adhesion molecule-1 expression. J. Immunol. 156: 275-283. [Abstract]
33. Compton, S. J., J. A. Cairns, S. T. Holgate, A. F. Walls. 1998. The role of mast cell tryptase in regulating endothelial cell proliferation, cytokine release, and adhesion molecule expression: tryptase induces expression of mRNA for IL-1 and IL-8 and stimulates the selective release of IL-8 from human umbilical vein endothelial cells. J. Immunol. 161: 1939-1946. [Abstract/Free Full Text]
34. Knox, A. J., L. Corbett, J. Stocks, E. Holland, Y. M. Zhu, L. Pang. 2001. Human airway smooth muscle cells secrete vascular endothelial growth factor: up-regulation by bradykinin via a protein kinase C and prostanoid-dependent mechanism. FASEB J. 15: 2480-2488. [Abstract/Free Full Text]
35. Zuyderduyn, S., P. S. Hiemstra, K. F. Rabe. 2004. TGF- differentially regulates Th2 cytokine-induced eotaxin and eotaxin-3 release by human airway smooth muscle cells. J. Allergy Clin. Immunol. 114: 791-798. [Medline]
36. Ponath, P. D., S. Qin, D. J. Ringler, I. Clark-Lewis, J. Wang, N. Kassam, H. Smith, X. Shi, J. A. Gonzalo, W. Newman, et al 1996. Cloning of the human eosinophil chemoattractant, eotaxin: expression, receptor binding and functional properties suggest a mechanism for the selective recruitment of eosinophils. J. Clin. Invest. 97: 604-612. [Medline]
37. Uguccioni, M., C. R. Mackay, B. Ochensberger, P. Loetscher, S. Rhis, G. J. LaRosa, P. Rao, P. D. Ponath, M. Baggiolini, C. A. Dahinden. 1997. High expression of the chemokine receptor CCR3 in human blood basophils: role in activation by eotaxin, MCP-4 and other chemokines. J. Clin. Invest. 100: 1137-1143. [Medline]
38. Sallusto, F., C. R. Mackay, A. Lanzavecchia. 1997. Selective expression of the eotaxin receptor CCR3 by human T helper 2 cells. Science 277: 2005-2007. [Abstract/Free Full Text]
39. Romagnani, P., A. De Paulis, C. Beltrame, F. Annunziato, V. Dente, E. Maggi, S. Romagnani, G. Marone. 1999. Tryptase-chymase double-positive human mast cells express the eotaxin receptor CCR3 and are attracted by CCR3-binding chemokines. Am. J. Pathol. 155: 1195-1204. [Abstract/Free Full Text]
40. Brightling, C. E., A. J. Ammit, D. Kaur, J. L. Black, A. J. Wardlaw, J. M. Hughes, P. Bradding. 2005. The CXCL10/CXCR3 axis mediates human lung mast cell migration to asthmatic airway smooth muscle. Am. J. Respir. Crit. Care Med. 171: 1103-1108. [Abstract/Free Full Text]
41. Leckie, M. J., A. T. Brinke, J. Khan, Z. Diamant, B. J. O’Connor, C. M. Walls, A. K. Mathur, H. C. Cowley, K. F. Chung, R. Djukanovic, et al 2000. Effects of an interleukin-5 blocking monoclonal antibody on eosinophils, airway hyper-responsiveness, and the late asthmatic response. Lancet 356: 2144-2148. [Medline]
42. Kips, J. C., B. J. O’Connor, S. J. Langley, A. Woodcock, H. A. Kerstjens, D. S. Postma, M. Danzig, F. Cuss, R. A. Pauwels. 2003. Effect of SCH55700, a humanized anti-human interleukin-5 antibody, in severe persistent asthma: a pilot study. Am. J. Respir. Crit. Care Med. 167: 1655-1659. [Abstract/Free Full Text]

This article has been cited by other articles:

				C. S. Mullan, M. Riley, D. Clarke, A. Tatler, A. Sutcliffe, A. J. Knox, and L. Pang {beta}-Tryptase Regulates IL-8 Expression in Airway Smooth Muscle Cells by a PAR-2-Independent Mechanism Am. J. Respir. Cell Mol. Biol., May 1, 2008; 38(5): 600 - 608. [Abstract] [Full Text] [PDF]

				S. Siddiqui, F. Hollins, S. Saha, and C. E. Brightling Inflammatory cell microlocalisation and airway dysfunction: cause and effect? Eur. Respir. J., December 1, 2007; 30(6): 1043 - 1056. [Abstract] [Full Text] [PDF]

				J. I. Ellyard, L. Simson, A. Bezos, K. Johnston, C. Freeman, and C. R. Parish Eotaxin Selectively Binds Heparin: AN INTERACTION THAT PROTECTS EOTAXIN FROM PROTEOLYSIS AND POTENTIATES CHEMOTACTIC ACTIVITY IN VIVO J. Biol. Chem., May 18, 2007; 282(20): 15238 - 15247. [Abstract] [Full Text] [PDF]

				J. Chhabra, Y-Z. Li, H. Alkhouri, A. E. Blake, Q. Ge, C. L. Armour, and J. M. Hughes Histamine and tryptase modulate asthmatic airway smooth muscle GM-CSF and RANTES release Eur. Respir. J., May 1, 2007; 29(5): 861 - 870. [Abstract] [Full Text] [PDF]

				N. Henderson, L. J. Markwick, S. R. Elshaw, A. M. Freyer, A. J. Knox, and S. R. Johnson Collagen I and thrombin activate MMP-2 by MMP-14-dependent and -independent pathways: implications for airway smooth muscle migration Am J Physiol Lung Cell Mol Physiol, April 1, 2007; 292(4): L1030 - L1038. [Abstract] [Full Text] [PDF]

				D. Kaur, R. Saunders, P. Berger, S. Siddiqui, L. Woodman, A. Wardlaw, P. Bradding, and C. E. Brightling Airway Smooth Muscle and Mast Cell-derived CC Chemokine Ligand 19 Mediate Airway Smooth Muscle Migration in Asthma Am. J. Respir. Crit. Care Med., December 1, 2006; 174(11): 1179 - 1188. [Abstract] [Full Text] [PDF]

				V. Chan, J. K. Burgess, J. C. Ratoff, B. J. O'Connor, A. Greenough, T. H. Lee, and S. J. Hirst Extracellular Matrix Regulates Enhanced Eotaxin Expression in Asthmatic Airway Smooth Muscle Cells Am. J. Respir. Crit. Care Med., August 15, 2006; 174(4): 379 - 385. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pang, L. Articles by Knox, A. J. Search for Related Content PubMed PubMed Citation Articles by Pang, L. Articles by Knox, A. J.