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Department of Pharmacology, Kyung Hee University College of Oriental Medicine (H.-J.J., H.-J.N., H.-M.K.), 130-701 Seoul, South Korea; and College of Pharmacy, VestibuloCochlear Research Center of Wonkwang University (H.-J.J., H.-J.N., S.-H.H.), 570-749 Jeonbuk, South Korea
Address all correspondence and requests for reprints to: Dr. Hyung-Min Kim, Department of Pharmacology, Kyung Hee University College of Oriental Medicine, 1 Hoegi-Dong, Dongdaemun-Gu, 130-701 Seoul, South Korea. E-mail: hmkim{at}khu.ac.kr.
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Abstract |
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90.1% at 100 nM; P < 0.05). The MAPK p38 inhibitor SB203580 (20 µM) also inhibited the SCF-induced migration. The ability of SCF to enhance morphological alteration and filamentous actin formation was also abolished by treatment with dexamethasone. Dexamethasone inhibited SCF-induced p38 MAPK activation to near-basal levels and induced MAPK phosphatase-1 expression. In addition, SCF-induced inflammatory cytokine production was significantly inhibited by treatment with dexamethasone or SB203580 (P < 0.01). Our results show that dexamethasone potently regulates SCF-induced migration, p38 MAPK activation, and inflammatory cytokine production through the expression of MKP-1 protein in RPMCs. Such modulation may have functional consequences during dexamethasone treatment, especially mast cell-mediated allergic inflammation disorders. |
Introduction |
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Stem cell factor (SCF) is a crucial growth factor in mast cell biology. It regulates such diverse cellular functions as proliferation, differentiation, survival, adhesion, and release of inflammatory mediators (10). SCF acts as a mast cell chemotaxin (1). Furthermore, injection of SCF into the skin causes mast cell hyperplasia (11), indicating that SCF may be of importance for the recruitment of mast cells in vivo. SCF also induces the proinflammatory cytokines, including TNF
, IL-1ß, IL-6, IL-8, IL-16, and IL-18, from mast cells (12).
The MAPK family comprises at least six subsets: ERK1/ERK2, p38 kinase (p38 and p38-ß, -
, and -
), c-Jun NH2-terminal protein kinase (JNK), ERK5, ERK6, and ERK7 (13, 14, 15). MAPKs are believed to play a pivotal role in cell proliferation, apoptosis, differentiation, cytoskeleton remodeling, and the cell cycle (16, 17, 18, 19). SCF similarly activates all MAPK (20). Previously, Sundstrom et al. (21) reported that SCF induced a rapid and transient activation of ERK and p38 in mouse mast cells. Inhibition of p38 activity by SB203580 was paralleled by a marked reduction of migration toward SCF, whereas the effect of the ERK inhibitor was less pronounced.
In mammalian cells, inactivation of MAPK is achieved mainly by a family of dual specificity MAPK phosphatases (MKP) that are capable of targeting the two regulatory phosphorylation sites of these kinases (22). According to their patterns of transcriptional regulation and subcellular localization, these phosphatases can be roughly divided into two groups (22). The first group includes MKP-3/Pyst1, Pyst2, MKP-4, MKP-5, and M3/6, which are localized predominantly in the cytosol. The second group of enzymes includes MKP-1, MKP-2, phosphatase of activated cells-1, and B23, which are localized primarily in the nuclear compartment (23). Recently, Hutter et al. (24) reported that MKP-1 binds to p38 both in vivo and in vitro, and that this interaction enhances the catalytic activity of MKP-1.
Steroidal antiinflammatory drugs such as dexamethasone are effective in the treatment of allergic and inflammatory diseases. The local delivery of dexamethasone to tissues significantly decreases mast cell number by reducing the production of SCF (25), but this event is not fully defined. Recently, Engelbrecht et al. (26) reported that glucocorticoids induce rapid up-regulation of MKP-1.
In this study we investigated the SCF-dependent effects in vitro and the effect of dexamethasone on the migration, morphological change, filamentous actin (F-actin) formation, p38 activation, and inflammatory cytokines production of rat peritoneal mast cells (RPMCs). We also examined the effect of dexamethasone on MKP-1 expression. In addition, the effect of SB203580, a p38 inhibitor, was investigated to support the effect of dexamethasone through p38 inhibition.
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Materials and Methods |
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, purified antirat IL-6 and TNF, and biotin-conjugated antirat IL-6 and TNF were purchased from R&D Systems (Minneapolis, MN). Fetal bovine serum, MEM, ampicillin, and streptomycin were purchased from Life Technologies, Inc. (Grand Island, NY). Antibody against MKP-1, p38, and phosphorylated p38 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). N-7-Nitrobenz-2-oxa-1,3-diazol-4-phallacidin (NBD-phallacidin) was purchased from Molecular Probes (Eugene, OR). Kinase assay kit was purchased from Cell Signaling Technology, Inc. (Beverly, MA).
Animals
The original stock of male Wistar rats, weighing 200–300 g, were purchased from Dae-Han Experimental Animal Center (Taejeon, South Korea). The animals were housed 5–10/cage in laminar air flow room maintained at 22 ± 1 C and relative humidity of 55 ± 10% throughout the study. All protocols were approved by the institutional animal care and use committee of Wonkwang University College of Pharmacy.
Preparation of RPMCs
RPMCs were isolated as previously described (27). In brief, rats were anesthetized by ether and injected with 20 ml Tyrode buffer B (NaCl, glucose, NaHCO3, KCl, and NaH2PO4) containing 0.1% gelatin (Sigma-Aldrich Corp.) into the peritoneal cavity; the abdomen was gently massaged for about 90 sec. The peritoneal cavity was carefully opened, and the fluid containing peritoneal cells was aspirated by Pasteur pipette. Then the peritoneal cells were sedimented at 150 x g for 10 min at room temperature and resuspended in Tyrode buffer B. Mast cells were separated from the major components of rat peritoneal cells, i.e. macrophages and small lymphocytes, according to the method described by Yurt et al. (28). In brief, peritoneal cells suspended in 1 ml Tyrode buffer B were layered on 2 ml 22.5% (wt/vol) metrizamide (density, 1.120 g/ml; Sigma-Aldrich Corp.) and centrifuged at room temperature for 15 min at 400 x g. The cells remaining at the buffer-metrizamide interface were aspirated and discarded; the cells in the pellet were washed and resuspended in 1ml Tyrode buffer B containing calcium. Mast cells preparations were about 95% pure as assessed by toluidine blue staining. More than 97% of the cells were viable as judged by trypan blue uptake.
Cell culture
Purified RPMCs were maintained in MEM medium (Life Technologies, Inc.) with 10% fetal bovine serum (JRH Bioscience, Lenexa, KS) at 37 C under 5% CO2 in air. RPMCs (3 x 105 cells) were preincubated with dexamethasone or dimethylsulfoxide at 37 C for 1 h before the stimulation with SCF (50 ng/ml) for 6 h. The cells were separated from the released TNF and IL-6 by centrifugation at 400 x g for 5 min at 4 C.
Assessment of cell viability and altered morphology
At time zero and subsequent time points as indicated, cells were counted in a hemocytometer, and viability was assessed by trypan blue dye exclusion. To assess the percentage of cells showing characteristic morphological features, the cells were examined by phase contrast microscopy. Photomicrography was performed using Fuji film at x100 magnification.
Chemotaxis assay
SCF or the assay medium alone was applied into each well of four-well culture plates. After 10-mm tissue culture inserts (Nalge, Nunc International, Naperville, IL) were placed into each well, 5 x 104 RPMCs (500 µl) were added to each insert. The lower compartment of the well was separated from the cell suspension in the upper compartment with an 8-µm pore size polycarbonate membrane of the culture inserts. RPMCs were incubated for 4 h at 37 C in a humidified atmosphere flushed with 5% CO2 in air. After aspiration of nonadherent RPMCs in the upper compartment, cells adherent to the upper surface of the membrane were removed by scraping with a rubber blade. Migrated cells adherent to lower surface of the membrane were fixed with methanol for 5 min and stained with 0.5% toluidine blue. The membranes were mounted on glass slides by routine histological methods. The total number of mast cells that migrated across the membrane was counted under a light microscope.
F-Actin formation in RPMCs treated with SCF
Detection of polymerized actin (F-actin) was determined in RPMCs migrating toward the lower side of the membrane according to the method described by Pteiffer and Oliver (29). Briefly, RPMCs were preincubated with or without dexamethasone for 1 h and seeded into each culture insert for chemotaxis assay or into each well of six-well culture plates. After stimulation with SCF for 1 h, RPMCs were fixed with 3% paraformaldehyde/PBS for 1 h at room temperature, washed three times with PBS, and permeabilized with 1% Triton X-100/PBS for 15 min. The preparations were stained for 30 min with F-actin-specific probe and 1 U/ml NBD-phallacidin at room temperature. All specimens were examined with a confocal laser scanning microscope using an argon ion laser that is capable of excitation at 488 nm.
ELISA of IL-6 and TNF
Sandwich ELISA for IL-6 and TNF was carried out in duplicate in 96-well ELISA plates (Nunc) coated with 100-µl aliquots each of antirat IL-6 and TNF monoclonal antibodies (R&D Systems, Minneapolis, MN) at 1.0 µg/ml in PBS at pH 7.4 and was incubated overnight at 4 C. The plates were washed in PBS containing 0.05% Tween 20 (Sigma-Aldrich Corp.) and blocked with PBS containing 1% BSA, 5% sucrose, and 0.05% NaN3 for 1 h. After additional washes, sample were added and incubated at 37 C for 2 h. Recombinant IL-6 and TNF were diluted and used as a standard. Serial dilutions starting from 5 ng/ml were used to establish the standard curve. After 2-h incubation at 37 C, the wells were washed, and then biotinylated antirat IL-6 and TNF (0.2 µg/ml each) were added and again incubated at 37 C for 2 h. After washing the wells, streptavidin-peroxidase was added, and plates were incubated for 20 min at 37 C. Wells were again washed, and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) substrate was added. Color development was measured at 450 nm using an automated microplate ELISA reader. A standard curve was run on each assay plate using recombinant IL-6 and TNF in serial dilutions
Western blot analysis
Cell extracts were prepared using a detergent lysis procedure. Cells (5 x 106 cells) were scraped, washed once with PBS, and resuspended in lysis buffer. Samples were vortexed for lysis for a few seconds every 15 min at 4 C for 1 h and centrifuged at 15,000 x g for 5 min at 4 C. Supernatants were assayed. Samples were heated at 95 C for 5 min and briefly cooled on ice. After the centrifugation at 15,000 x g for 5 min, 50-µl aliquots were resolved by 12% SDS-PAGE. Resolved proteins were electrotransferred overnight to nitrocellulose membranes in 25 mM Tris (pH 8.5), 200 mM glycine, and 20% methanol at 25 V. Blots were blocked for at least 2 h with 1x PBS containing 0.05% Tween 20 (PBST) containing 10% nonfat dry milk. The phosphorylated p38 antibody (1:500 in PBST) was added and incubated for 1 h. Afterward, nitrocellulose membrane was washed five times for 15 min each time with PBST. For protein detection, the blot was incubated with antimouse secondary antibody conjugated with peroxidase for 40 min, followed by enhanced chemiluminescence detection.
MAPK assay
The MAPK assay was performed according to the manufacturer’s specification using a p38 MAPK assay kit (Cell Signaling Technology, Beverly, MA).
Immunofluorescence
Cells (8000 cells/ml) were washed with PBS, fixed with 3.7% paraformaldehyde for 30 min, and permeabilized with wash buffer (0.5% Triton X-100 and 0.01% sodium azide in PBS). Cells were blocked with wash buffer containing 5% BSA for 30 min and provided with primary Ab, anti-MKP-1 at a 1:500 dilution (Santa Cruz Biotechnology, Inc.). After washing, cells were incubated with secondary Ab (antirabbit, fluorescein isothiocyanate conjugated). After extensive washing, cover-slips were placed with polyvinyl alcohol/DABCO (Sigma-Aldrich Corp.) mounting medium and allowed to dry. Slides were scanned under fluorescence with an Olympus confocal microscope (New Hyde Park, NY).
Statistical analysis of data
The experiments shown are a summary of the data from at least three experiments and are presented as the mean ± SEM. Statistical significance of the data was determined using the independent t test; P < 0.05 was accepted as statistically significant.
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Results |
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). The maximum effective dose of SCF was 75 ng/ml. We chose a concentration of 50 ng/ml SCF for cell stimulation. SCF (50 ng/ml) was placed in the lower compartment, and then RPMCs were incubated for various times in the upper compartment. SCF significantly increased RPMC number, which migrated toward the lower surface of the polycarbonate membrane through 8-µm pores (P < 0.001, compared with medium alone without SCF) 3 and 4 h later. The maximum number of 214 cells was reached at 4 h (P < 0.001; Fig. 1B). RPMCs were still migrating 5 h later, but cells detached from the membrane toward the lower compartment were detected. This migration was significantly decreased by treatment with dexamethasone (P < 0.05; Fig. 1B). A dose-response study was performed at the time of SCF-induced maximal effects (at 4 h). Treatment with dexamethasone resulted in a dose-dependent inhibition of SCF- induced migration. The maximum inhibition occurred at 100 nM (Fig. 1C). The inhibitory effect of dexamethasone was abolished by the glucocorticoid receptor antagonist RU486. Figure 1D shows the migrated cells in Fig. 1B. Dimethylsulfoxide had no effect on mast cell migration (data not shown). We also examined the effect of SB203580 (10 and 20 µM) on SCF-induced cell migration at 4 h. SB203580 significantly inhibited the migration of RPMCs (Fig. 1C).
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, SCF (50 ng/ml) induced morphological alterations in about 80% of RPMCs after 4 d of culture, but the effect of SCF was mostly abolished by treatment with dexamethasone (100 nM). Cell toxicity by dexamethasone was not observed.
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). F-Actin levels were obtained from a single cell and evaluated as the fluorescence intensity (Fig. 3B).
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, the addition of 50 ng/ml SCF to RPMCs induced phosphorylation of p38 MAPK. Dexamethasone (100 nM) or SB203580 (20 µM) reduced the level of phosphorylated p38 (Fig. 4A) and the activity of p38 MAPK (dexamethasone, 10 and 100 nM; SB203580, 10 and 20 µM), but dexamethasone (10 nM) plus SB203580 (10 µM) had no effect on p38 MAPK activation (Fig. 4C) in SCF-stimulated RPMCs. The inhibitory effect of dexamethasone was abolished by the glucocorticoid receptor antagonist RU486. Protein levels were quantitated by densitometry (Fig. 4B; Amersham Pharmacia Biotech, Piscataway, NJ).
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). When RPMCs were stimulated with dexamethasone for 1 h, immunoreactive MKP-1 was increased by dexamethasone. In contrast, when cells were pretreated with RU486 for 30 min, immunoreactive MKP-1 was decreased (Fig. 5B).
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and IL-6 production and IL-6 production from RPMCs, the cells were treated with dexamethasone (100 nM) or SB203580 (20 µM) for 1 h before stimulation with SCF for 6 h. Culture supernatants were assayed for TNF and IL-6 protein levels by ELISA. As shown in Fig. 6, dexamethasone had no effect on cytokine production by itself, whereas SCF significantly enhanced TNF (0.33 ± 0.001 ng/ml; P < 0.01) and IL-6 (0.68 ± 0.12 ng/ml; P < 0.01) production compared with that in the medium control (0.22 ± 0.007 ng/ml for TNF and 0.10 ± 0.07 ng/ml for IL-6). This induction was significantly inhibited by treatment with dexamethasone (0.18 ± 0.002 ng/ml for TNF and 0.22 ± 0.09 ng/ml for IL-6; P < 0.01) or SB203580 (0.21 ± 0.003 ng/ml for TNF and 0.20 ± 0.01 ng/ml for IL-6; P < 0.01). Inhibition of TNF and IL-6 production by treatment with dexamethasone was 136 ± 2% and 80 ± 16%, respectively. However, cell viability was not affected (data not shown).
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Discussion |
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and IL-6 production in RPMCs. In addition, dexamethasone inhibited SCF-induced p38 MAPK activation via an increase in MKP-1 expression. Mast cell development is a complex process that results in the appearance of a phenotypically distinct population of mast cells at different anatomical sites (30). Connective tissue-type mast cells, such as those present in the peritoneal cavity and skin, represent a major mast cell population. SCF, the ligand for the receptor encoded by c-kit, is essential for the development of mast cells (31). We previously reported that SCF-induced morphological alteration of RPMC and mast cells is an important step for participation in adhesion to tissue (32). Previously, Irani et al. (33) reported that dexamethasone inhibits the development of mast cells from dispersed human fetal liver cells cultured in the presence of recombinant human SCF. Some groups have shown that dexamethasone inhibits adhesion of the IL-3-dependent mast cell line, MC/9, to NIH-3T3 fibroblasts, with an accompanying decrease in IL-3 receptor expression (34). These results, including our findings, suggest that dexamethasone might regulate the migratory process of mast cells after SCF stimulation.
Directed migration of mast cells toward a chemical gradient of specific chemoattractants locally produced in inflamed tissues is the first integrated event in the process of allergic and nonallergic inflammatory responses (35, 36). The localization of mast cell precursors to specific tissue sites and the accumulation of mast cells within the given tissue at an inflammatory response were induced by chemotactic factor, SCF (1). SCF stimulates specific receptors, c-Kit, on the cell surface that initiate several second messenger cascades; this action results in a change in F-actin distribution from azimuthal symmetry around the cell rim to concentrate in a particular region involved in migratory behavior (37). In this study we demonstrated that dexamethasone inhibited the SCF-induced migration of RPMCs and the distribution of F-actin. Local contact of glucocorticoids with dermal, respiratory, and intestinal tissues is reported to be associated with a decrease in mast cell number within these tissues (38, 39). Some reported that glucocorticoids decreased tissue mast cell number by reducing the production of SCF (25). Recently, Da Silva et al. (40) reported that glucocorticoid treatment inhibited expression of the mast cell growth factor SCF, but our findings provide new evidence that the decrease in tissue mast cell number caused by dexamethasone treatment is related to the inhibition of mast cell migration.
Binding of SCF to c-Kit activates different intracellular signaling components, including p38 MAPK (41). p38 MAPK activation by SCF is of major importance for cell migration toward SCF in general. We showed that p38 MAPK activation and activity were blocked when RPMCs were pretreated with dexamethasone or SB203580. Therefore, suppressing p38 MAPK signaling in mast cells may be a useful tool to reduce mast cell number in inflammatory conditions.
Kumar et al. (42) reported that SB203580 inhibits the activity, but not the activation, of p38 MAPK in TNF-, lipopolysaccharide-, sorbitol-, or IL-1ß-stimulated human monocytic THP.1 cells. Recently, however, some researchers reported that SB203580 effectively blocked p38 activation by insulin in C2C12 cells and LPS in macrophage cells (43, 44). In this study SB203580 inhibited the activation and activity of p38 by SCF in mast cells. Thus, a detailed analysis of the mechanism of action or the specificity of this inhibitor is indispensable to interpretation of our results for use in future studies.
MKP-1 was demonstrated to dephosphorylate and inactivate not only ERK, but also JNK and p38 MAPK (45, 46, 47). Kassel et al. (48) reported that MKP-1, as a specific glucocorticoid-regulated gene, plays an important role in mediation of the antiinflammatory action of glucocorticoid in mast cells. In this study we showed that MKP-1 expression was induced by dexamethasone. Therefore, our results suggest that dexamethasone inhibit p38 MAPK via the increase in MKP-1 expression.
Previously, Bokemeyer et al. (49) reported that the anisomycin-induced expression of MKP-1 was inhibited after preincubation with SB203580. In this study preincubation with dexamethasone (10 nM) plus SB203580 (10 µM) had no effect on p38 MAPK activation, but dexamethasone (10 nM) or SB203580 (10 µM) inhibited p38 activity, respectively. From this we can speculate that SB203580 inhibited dexamethasone-induced MKP-1 expression. However, dexamethasone could also modify other protein kinase activities, which is not ruled out by use of SB203580. At a concentration of 20 µM, SB203580 has been shown to also block protein kinase B phosphorylation (50). In addition, SB203580 may inhibit activating transcription factor-2 via some other signal transduction pathways, except for p38. Further investigation is necessary to clarify the interaction of dexamethasone and SB203580.
TNF is constitutively expressed cytokine in mast cells, and it is considered a major initiator of inflammation (51). TNF also regulated the expression of chemokines such as IL-8, macrophage inflammatory protein-1, and RANTES. IL-6 is an integral part of the inflammatory response to sepsis and endotoxemia (52). These cytokines can be generated in the mast cell and potentate inflammatory immune responses through the subsequent induction of other inflammatory mediators. These cytokines are also partially responsible for migration of Langerhans cells into the epidermis (53). Cumberbatch et al. (54) reported that dexamethasone impairs the de novo synthesis and/or release of these cytokines. We demonstrated that dexamethasone inhibited SCF-induced TNF and IL-6 production. These findings may contribute to understanding the antiinflammatory effect of dexamethasone.
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Footnotes |
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Abbreviations: F-actin, Filamentous actin; JNK, c-Jun NH2-terminal protein kinase; MKP-1, MAPK phosphatase-1; NBD, N-7-nitrobenz-2-oxa-1,3-diazol-4-phallacidin; PBST, PBS containing 0.05% Tween 20; RPMCs, rat peritoneal mast cells; SCF, stem cell factor.
Received January 24, 2003.
Accepted for publication May 19, 2003.
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RI cross linking to increased cell substrate adhesion in RBL-2H3 tumor mast cells. J Immunol 152:270–279[Abstract]
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