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Tryptase-stimulated human airway smooth muscle cells induce cytokine synthesis and mast cell chemotaxis¹

PATRICK BERGER^*,, PIERRE-OLIVIER GIRODET^*, HUGUES BEGUERET^*, OLGA OUSOVA^*, DIAHN-WARNG PERNG, ROGER MARTHAN^*, ANDREW F. WALLS and J. MANUEL TUNON DE LARA^*,,2

^* Laboratoire de Physiologie Cellulaire Respiratoire, INSERM E0356, Université Victor Ségalen Bordeaux 2, 33076 Bordeaux, France;
Immunopharmacology Group, Southampton General Hospital, Southampton, SO16 6YD, UK; and
Service des Maladies Respiratoires, Hôpital Haut Lévêque, CHU de Bordeaux, 33604 Pessac, France

²Correspondence: Laboratoire de Physiologie Cellulaire Respiratoire, INSERM E0356 Université, Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France. E-mail: manuel.tunondelara{at}u-bordeaux2.fr

SPECIFIC AIMS

Mast cell-derived mediators alter a variety of functions of human airway smooth muscle, and recent reports have demonstrated the infiltration of smooth muscle bundles by mast cells in the asthmatic disease. The aims of the present study were to investigate the ability of human smooth muscle cells to exert a chemotactic activity for mast cells, the mechanisms involved, and the potential consequences of such activity.

PRINCIPAL FINDINGS

1. Tryptase-stimulated HASMC induce mast cell chemotaxis
Supernatants from tryptase-stimulated HASMC induced a significant chemotaxis of cells of the HMC-1 mast cell line when compared with supernatants from unstimulated HASMC. Challenge with purified human lung tryptase resulted in a migration that was maximal after 72 h (Fig. 1 A). Heat inactivation of tryptase (leading to a 92.3±4.1% loss in activity) abolished this effect. A similar effect and time course were observed after activation of PAR-2 by the synthetic agonist peptide SLIGKV-NH₂. The peptide with the reverse sequence LSIGKV-NH₂, which fails to activate PAR-2, had no effect on the chemotactic activity of HASMC over the same range of concentrations (Fig. 1A ). Since tryptase by itself has been shown to stimulate mast cell degranulation, we checked for tryptase activity in the stimulated HASMC supernatants. We found a strong decrease in tryptase activity had occurred within 72 h, from 30 mU/mL to 1.6 ± 0.1 mU/mL and 6.4 ± 2.5 mU/mL for purified and recombinant tryptase, respectively (Fig. 1B ). Addition of the protease inhibitor leupeptin led to a decrease of 83.2 ± 0.8% and 92.4 ± 1.9% in the activity of purified and recombinant tryptase, respectively, and abolished the effect on cell migration induced by both forms of tryptase (Fig. 1C ). An intact catalytic site is thus necessary for this chemotactic effect since heat inactivation of tryptase and enzymatic inhibition by leupeptin both abolished the effect induced by tryptase. Regarding transduction mechanisms, we found that preincubation of HASMC with pertussis toxin (50 ng/mL) or ERK inhibitors (U0126 at 10 µM, PD98059 at 50 µM) abolished tryptase- and SLIGKV-NH₂-induced mast chemotaxis. These results suggest the involvement of pertussis toxin-sensitive G-protein and the subsequent activation of MAP kinase cascade.

View larger version (30K): [in this window] [in a new window]   Figure 1. Effect of HASMC supernatants on HMC-1 chemotaxis. A) Cell migration toward supernatants from stimulated HASMC was calculated as percentage migration toward supernatants from unstimulated HASMC. The x-axis represents the incubation time of HASMC with heat-inactivated tryptase (open circle), LSIGKV 10-4 M (open square), purified tryptase 30 mU/mL (gray circle), or SLIGKV-NH2 10-4 M (black square). B) Tryptase activity was assessed in the HASMC supernatant stimulated for 0 to 96 h by heparin (open square), heat-inactivated tryptase (open circle), purified lung tryptase (gray circle), or recombinant ß II tryptase (black circle) 30 mU/mL. C) HASMC were stimulated for 72 h with heparin (Hep), recombinant tryptase from 3.75 to 30 mU/mL (r.T black bars), r.T inhibited by 100 µM leupeptin (white bar), purified lung tryptase 30 mU/mL (p.T gray bar), p.T inhibited by 100 µM leupeptin (white bar), 100 µM leupeptin alone (white bar), SLIGKV-NH2 10-4 M alone (black bar), or SLIGKV-NH2 in the presence of 100 µM leupeptin (white bar). Data are mean ± SE of 6 independent experiments. Results were compared using the paired Student’s t test (*P <0.05).

2. Mast cell chemotaxis is due to HASMC production of TGF-ß1 and SCF
Mast cell migration induced by supernatants of tryptase- or SLIGKV-NH₂-stimulated HASMC, was significantly inhibited by blocking antibodies directed against TGF-ß1 or SCF, but not by antibody against RANTES. The effect of supernatants from TNF--stimulated HASMC, used as positive control, was inhibited by anti-TGF-ß1 or anti-RANTES antibodies. TGF-ß1 and SCF proteins were measured using ELISA in the supernatant of HASMC activated by tryptase or SLIGKV. Maximal secretion of both cytokines occurred after 72 h, which also corresponded to the time of maximal chemotactic effect. A comparison of amounts of cytokine detected in supernatants with concentrations needed for mast cell chemotaxis suggests that TGF-ß1 plays a central role. The maximal concentration of SCF did not exceed 1 ng/mL in the supernatant of stimulated HASMC, whereas significant mast cell migration required 30 ng/mL of that cytokine. By contrast, the level of TGF-ß1 secretion was close to that inducing mast cell chemotaxis (i.e., 0.1–1 ng/mL). Using real-time RT-PCR, we measured the production of mRNA for TGF-ß1, SCF, and RANTES by stimulated cells. Compared with the levels for unstimulated cells, production of mRNA by tryptase or SLIGKV-NH₂-stimulated HASMC was not significantly higher for TGF-ß1, SCF, or RANTES, indicating that tryptase influences the production of TGF-ß1 protein at a post-transcriptional level.

3. Mast cell infiltration is correlated with TGF-ß1 expression in airway smooth muscle from asthmatic patients
Using immunohistochemistry, we analyzed mast cell infiltration and expression of TGF-ß1 in the bronchial mucosa from asthmatic patients and control subjects (Fig. 2 A, B). The number of mast cells within the airway tissue in persistent asthmatic patients was higher than that in tissue from intermittent asthmatic or control subjects. Immunostaining intensity for anti-LAP or anti-TGF-ß1 was significantly higher within the smooth muscle layer from persistent asthmatic patients than that measured in intermittent asthmatic subjects or controls. This increase in expression did not reach significance in the other layers of the bronchial wall (epithelium and submucosa). The number of mast cells within the airway smooth muscle of persistent and intermittent asthmatic patients was positively correlated with the expression of either TGF-ß1 latency associated protein (LAP, Fig. 2C )or TGF-ß1 (Fig. 2D ). No significant correlation was found with the two other bronchial layers defined. Smooth muscle cells from persistent asthmatic or control patients also expressed TGF-ß1 at the mRNA level as assessed by in situ hybridization.

View larger version (96K): [in this window] [in a new window]   Figure 2. Relationship of mast cell numbers and TGF-ß1 expression in the smooth muscle layer from asthmatic patients. Representative sections of human bronchial tissue obtained from a control smoking subject (A) and an intermittent asthmatic patient (B) observed at x200 (1, 3, 5) or x400 (2, 4, 6) magnification. Serial sections were stained with anti-human tryptase (1, 2), anti-human LAP (3, 4), or anti-human TGF-ß1 (5, 6). Relationship between the number of mast cells within the smooth muscle layer and LAP (C) or TGF-ß1 staining intensity (D) in smokers (open symbols) and asthmatic patients (filled symbols, from persistent and intermittent asthmatic groups) are shown. r represents the Spearman correlation coefficient.

CONCLUSIONS AND SIGNIFICANCE

Our results demonstrate that upon activation by tryptase or proinflammatory cytokines, HASMC are able to attract mast cells through the production of functionally active TGF-ß1 and, to a lesser extent, of SCF. These results provide a mechanistic basis for understanding recent findings concerning smooth muscle infiltration by mast cells in asthma. These results also reveal an autoactivation loop involving mast cells and smooth muscle cells.

We found that TGF-ß1 was a major chemoattractant for mast cells that is synthesized and secreted by HASMC. mRNA for TGF-ß1 is constitutively produced by unstimulated HASMC, but tryptase does not alter the production of mRNA. The increase in TGFß-1 production is thus due to an effect of tryptase at a post-transcriptional level involving activation of the MAP kinase cascade. All of these tryptase-related effects were mimicked by the activation of PAR-2, which is highly expressed by HASMC. The secreted TGF-ß1 was functionally active, as supernatants from stimulated HASMC induced a mast cell chemotaxis in vitro. These results are relevant to those previously obtained in vivo in asthmatic subjects. Such patients have higher levels of tryptase and TGF-ß1 in the bronchoalveolar lavage fluid, and levels may be further increased after allergen challenge. In our study, we paid special attention to TGF-ß1 expression in the smooth muscle layer from asthmatic patients and found that the smooth muscle itself expresses high levels of TGF-ß1. This overexpression was closely related to mast cell infiltration of smooth muscle, suggesting that TGF-ß1 may indeed stimulate mast cell accumulation in vivo. Our results obtained both in vitro and ex vivo clearly demonstrate that tryptase is a key factor for mast cell chemotaxis. Tryptase is the major product of mast cells and in turn induces a mast cell attraction through the secretion of TGF-ß1 by HASMC. This agrees with the ability of HASMC to secrete high levels of TGF-ß1 and with the potent mast cell chemotactic properties ascribed to TGF-ß1. Tryptase should thus be considered as the major link in an autoactivation loop involving HASMC, TGF-ß1, and mast cells. Products of such activation may play an important role in the remodeling process observed in asthma. Besides its chemoattractant effect, TGF-ß1 induces collagen production by fibroblasts or HASMC. TGF-ß1 has also been implicated in other lung diseases associated with fibrogenic processes. Similarly, tryptase can enhance fibroblast collagen production directly and may contribute to the remodeling process through stimulating an increase in smooth muscle mass. The autoactivation loop involving mast cells and airway smooth muscle could represent a promising target for preventive and/or curative strategy against airway remodeling in asthma.

View larger version (32K): [in this window] [in a new window]   Figure 3. Schematic diagram. Autoactivation loop that involves (1) tryptase release upon mast cell degranulation, (2) enzymatic cleavage of PAR-2 and subsequent pertussis toxin-sensitive G-protein activation, (3) activation of ERK pathway of MAPK cascade, (4) target mRNA translation, (5) TGF-ß1 and SCF protein synthesis and excretion, inducing (6) the recruitment of mast cells. These recruited mast cells (7) can in turn perpetuate the autoactivation loop.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0041fje; doi: 10.1096/fj.03-0041fje

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