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Originally published online as doi:10.1189/jlb.0704400 on March 30, 2005

Published online before print March 30, 2005
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(Journal of Leukocyte Biology. 2005;78:239-247.)
© 2005 by Society for Leukocyte Biology

Synergistic effect of SCF and TNF-{alpha} on the up-regulation of cell-surface expression of ICAM-1 on human leukemic mast cell line (HMC)-1 cells

Chi-Man Tsang, Chun-Kwok Wong, Wai-Ki Ip and Christopher Wai-Kei Lam1

Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, N.T., Hong Kong

1 Correspondence: Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, N.T., Hong Kong, China. E-mail: waikeilam{at}cuhk.edu.hk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Intercellular adhesion molecule-1 (ICAM-1) has been shown to play crucial roles in mast cell interaction with other inflammatory cells and recruitment into the inflamed tissue. In the present study, human mast cell line-1 (HMC-1) was stimulated with different cytokines including stem cell factor (SCF), tumor necrosis factor {alpha} (TNF-{alpha}), interleukin (IL)-13, IL-18, and IL-25. Cell-surface expression of ICAM-1 was assessed by flow cytometry. To elucidate the intracellular signal transduction regulating the ICAM-1 expression, phosphorylated extracellular signal-regulated kinase (ERK), phosphorylated p38 mitogen-activated protein kinase (MAPK), and nuclear factor (NF)-{kappa}B translocation were assessed by enzyme-linked immunosorbent assay. Results showed that SCF, TNF-{alpha}, and IL-13 but not IL-18 and IL-25 could up-regulate the surface expression of ICAM-1 on HMC-1 cells. A synergistic effect of SCF and TNF-{alpha} on ICAM-1 expression was demonstrated. This synergistic effect was shown to be dose-dependently enhanced by SCF but not TNF-{alpha}. Results indicated that SCF activated ERK, and TNF-{alpha} activated the p38 MAPK and NF-{kappa}B pathway. Selective inhibitor of ERK, PD098059, and c-kit inhibitors, STI571 and PP1, suppressed the combined SCF and TNF-{alpha}-induced ICAM-1 expression. BAY117082 but not SB203580, which are the inhibitors of NF-{kappa}B and p38 MAPK, respectively, suppressed the TNF-{alpha}-induced ICAM-1 expression. Therefore, SCF and TNF-{alpha} acted through ERK and the NF-{kappa}B pathway to regulate the ICAM-1 expression and elicited the synergistic effect. In conclusion, our results provide insight for cross-talk between different signaling pathways that can help in understanding the fine control of adhesion molecule expression under the concerted effects of cytokines.

Key Words: adhesion molecule • MAPK • NF-{kappa}B


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mast cells are the central effector cells in type 1 hypersensitivity. Upon activation, mast cells undergo degranulation to release an array of mediators including histamine, leukotrienes, and prostaglandins leading to allergic symptoms [1 ]. They have been recognized for their crucial functions in T helper cell type 2 (Th2)-mediated inflammation such as allergy and parasitic infestations [2 ]. Recently, there is strong evidence that they are also involved in natural and acquired immunity [2 ] and Th1-mediated inflammation such as inflammatory bowel disease [3 ] and rheumatoid arthritis [4 ]. All the mast cell-mediated inflammations are characterized by an accumulation of mast cells in the inflammatory sites [5 ]. Apart from the release of inflammatory mediators, mast cells also play their multiple roles in many inflammatory disorders by releasing cytokines and chemokines for autocrine and paracrine activation of the inflamed tissue [6 , 7 ].

Therefore, the regulation of recruitment of mast cells and also their intercellular interaction and cell-extracellular matrix adhesion have essential importance in the mechanism of inflammation [8 ]. However, the underlying principles in the mechanisms of migration and the distribution of immature and mature mast cells in the inflammation are still poorly defined [5 ]. The interaction of mast cells with other inflammatory cells is also largely not clarified.

Intercellular adhesion molecule-1 (ICAM-1) belongs to the immunoglobulin (Ig) superfamily cellular adhesion molecules, which is a 80- to 114-kDa calcium-independent transmembrane glycoprotein [9 ]. It can bind to lymphocyte function-associated antigen-1 (LFA-1), macrophage antigen-1, fibrinogen, hyaluronan, and CD43 on leukocytes [10 ]. It is the cell adhesion molecule that is important in the regulation of immune cells [11 ]. The up-regulation of ICAM-1 expression contributes to the accumulation of leukocytes [11 ] and facilitates cell contact-dependent regulation of immune cells in inflamed tissues [12 ]. ICAM-1 induces allergic response by mediating mast cell accumulation into inflammatory sites [13 ]. Profound down-regulation of ICAM-1 therefore leads to the elimination of the immediate-type hypersensitivity and the subsequent inhibition of the late-phase reaction [14 ]. It is also suggested that activated T cells mediate mast cell degranulation via ICAM-1-LFA-1 interaction [15 ]. Therefore, the study of the ICAM-1 expression under the stimulation of different cytokines can help to elucidate the mechanism of mast cell recruitment and interaction with other inflammatory cells, epithelial cells, and fibroblasts.

In the present study, the effects of stem cell factor (SCF), tumor necrosis factor {alpha} (TNF-{alpha}), interleukin (IL)-13, IL-18, and IL-25 on the expression of ICAM-1 in human mast cell line-1 (HMC-1) were studied. SCF is a cytokine that is important for the survival, proliferation, and differentiation of mast cells [16 ]. It is also a potent mast cell chemotactic factor to stimulate mast cell adhesion to connective tissue matrix [17 ]. TNF-{alpha} is a proinflammatory cytokine that can up-regulate the ICAM-1 in many cell types such as endothelial cells [18 ]. It is shown to be involved in the recruitment of mast cells in Th1-mediated inflammations [19 ]. IL-13, IL-18, and IL-25 have also been demonstrated to play crucial roles in allergic inflammation [20 , 21 ].

To further investigate how the cytokines direct the downstream regulation of ICAM-1 expression, activation of several intracellular signaling molecules, including mitogen-activated protein kinases (MAPK) and nuclear factor (NF)-{kappa}B, was studied. They can act as the key regulators in coordinating genes that control immune responses [22 , 23 ]. The extracellular signal-regulated kinase (ERK) pathway and the p38 MAPK pathway, which are primary members of MAPK signaling, transduce growth and differentiation signals and mediate inflammatory and stress responses [24 ]. The constitutive activation of NF-{kappa}B pathways has been shown to correlate with inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease, and allergic asthma [25 ]. Intracellular signaling molecules can therefore conduct pathological consequences and have become important therapeutic targets [26 ]. As inhibition of these pathways may have implications for treating inflammatory diseases, specific inhibitors that can suppress the activation of different signaling pathways, including PD98059 for ERK, SB203580 for p38 MAPK, BAY117082 for NF-{kappa}B, SP600125 for c-Jun N-terminal kinase (JNK), and AG490 for Janus kinase (JAK) pathway, were also used in the present study to investigate the expression of ICAM-1 on HMC-1 under the stimulation of cytokines.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Human recombinant SCF, TNF-{alpha}, and IL-13 were obtained from PeproTech (Rocky Hill, NJ). Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human ICAM-1 monoclonal antibody (mAb) and its corresponding fluorescein-conjugated IgG1 isotype were purchased from R&D Systems (Minneapolis, MN). Signaling inhibitors PD98059, SP600125, AG490, BAY117082, and PP1 were purchased from Calbiochem (San Diego, CA). STI 571, a c-kit inhibitor, was obtained from Novartis Pharma (Basel, Switzerland). Rabbit anti-human inhibitor of {kappa}B (I{kappa}B)-{alpha}(Ser 32) and rabbit anti-human phospho-p38 MAPK(Thr180/Tyr182) antibodies were purchased from Cell Signaling Technology (Beverly, MA).

Cell cultures
HMC-1 cells were a generous gift from Dr. Joseph H. Butterfield of the Mayo Clinic (Rochester, MN). These cells were maintained in suspension culture at a density between 3 and 7 x 105 cells/ml in Iscove’s medium supplemented with 10% (vol/vol) fetal bovine serum (Gibco Laboratories, Grand Island, NY) and 1.2 mM {alpha}-thioglycerol (Sigma Chemical Co., St. Louis, MO). They were kept under a humidified atmosphere with 5% CO2 at 37°C.

Endotoxin-free solution
Cell culture medium, which was free of detectable lipopolysaccharide (LPS; <0.1 EU/mL), was purchased from Gibco Laboratories. All other solutions were prepared using pyrogen-free water and sterile polypropylene plasticware. No solution contained detectable LPS, as determined by the Limulus amoebocyte lyase assay (sensitivity limit, 12 pg/ml, Associate of Cape Cod, Woods Hole, MA).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay
HMC-1 cells (2x105 cells/0.2 ml) were inoculated into a 96-well plate. Various inhibitors at serial concentrations were added to the cells. After 48 h incubation, MTT (50 µg, Sigma Chemical Co.) was added to each well and incubated for 2 h. Viable cells took up MTT and reduced it into dark blue, water-insoluble formazan by mitochondrial dehydrogenase, which reflected the normal function of mitochondria and cell viability. The cells were then lysed with dimethyl sulfoxide (DMSO; 200 µl) to yield the color solution. The absorbance at an optical density of 550 nm was measured to quantify the viable cells.

Measurement of phosphorylated ERK and phosphorylated p38 MAPK concentration
The concentrations of phosphorylated ERK and phosphorylated p38 MAPK in cell lysate of HMC-1 cells were quantitated by enzyme-linked immunosorbent assay (ELISA) using the reagent kits of Assay Designs (Ann Arbor, MI).

Detection of NF-{kappa}B activity
Nuclear proteins of HMC-1 cells were extracted with NE-PERTM nuclear and cytoplasmic extraction reagents (Pierce Chemical Co., Rockford, IL) for the determination of NF-{kappa}B activity. Nuclear extracts were subjected to a test for NF-{kappa}B protein/NF-{kappa}B oligonucleotide binding using the MercuryTM TransFactor NF-{kappa}B p50 kit (BD Biosciences Clontech Corp., San Jose, CA).

Western blot analysis
HMC-1 cells (1x106), after the preceding treatment, were washed with phosphate-buffered saline (PBS) and lysed in 0.15 ml radio immunoprecipitation assay cell lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2 mM sodium orthovanadate, 20 mM sodium pyrophosphate, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and 1x protease inhibitors, Assay Designs]. An equal amount of proteins was subjected to SDS-10% polyacrylamide gel electrophoresis before blotting onto a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech, Piscataway, NJ). The membrane was blocked with 5% skimmed milk in Tris-buffered saline with 0.05% Tween 20, pH 7.6, for 1 h at room temperature and was probed with primary rabbit anti-human I{kappa}B-{alpha} or phosphor-p38 MAPK antibody (Cell Signaling Technology) at 4°C overnight. After washing, membranes were incubated with secondary donkey anti-rabbit antibody coupled to horseradish peroxidase (Amersham Pharmacia Biotech) for 1 h at room temperature. Antibody-antigen complexes were then detected using an enhanced chemiluminescence detection system, according to the manufacturer’s instructions (Amersham Pharmacia Biotech).

Flow cytometry of cell-surface expression of ICAM-1
HMC-1 cells (5x105 cells/0.5 ml), after the preceding treatment, were harvested and resuspended with cold PBS supplemented with 0.5% bovine serum albumin (BSA). After blocking with 2% human pooled serum for 20 min at 4°C and washed with PBS supplemented with 0.5% BSA, cells were incubated with FITC-conjugated mouse anti-human ICAM-1 mAb antibody or fluorescein-conjugated mouse IgG1 isotype for 30 min at 4°C in the dark. After washing, cells were finally resuspended in 1% paraformaldehyde in 1x PBS as fixative. Cell-surface expression of ICAM-1 was then analyzed by flow cytometry (FACSCalibur, BD Biosciences) in terms of mean fluorescence intensity (MFI).

Statistical analysis
Data in figures were presented as histograms plus SD or as mean ± SD in curves. Differences between groups were assessed by the nonparametric Mann-Whitney rank-sum test. A probability of P < 0.05 was considered significantly different. All analyses were performed using the statistical software GraphPad Prism for Windows, Version 3.00 (GraphPad Software, San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
SCF, TNF-{alpha}, and IL-13 up-regulated ICAM-1 expression on HMC-1 cells
To generate a profile of cytokines that may regulate ICAM-1 expression, the effect of SCF (50 ng/ml), TNF-{alpha} (20 ng/ml), IL-13 (20 ng/ml), IL-18 (20 ng/ml), and IL-25 (50 ng/ml) was examined after 48 h incubation with the HMC-1 cells. The optimal dose and incubation time of the above cytokines had been determined previously for a significant effect (data not shown). Compared with controls (Fig. 1A ), a slight increase in ICAM-1 expression by SCF (Fig. 1B) but a significant up-regulation of ICAM-1 expression by TNF-{alpha} and IL-13 (P<0.05, Fig. 1C and 1D ) were observed. However, IL-18 and IL-25 showed no significant effect on ICAM-1 expresssion (Fig. 1E and 1F) .



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  		Figure 1. Effects of SCF, TNF-{alpha}, IL-13, IL-18, and IL-25 on ICAM-1 expression on HMC-1 cells (1x106 cells/ml), which were incubated for 48 h (A) without any cytokines or with (B) SCF (50 ng/ml), (C) TNF-{alpha} (20 ng/ml), (D) IL-13 (20 ng/ml), (E) IL-18 (20 ng/ml), or (F) IL-25 (50 ng/ml). Staining of HMC-1 cells with mAb IgG1 isotype control antibody (dotted line) and mAb against ICAM-1 (solid line) was determined by flow cytometry. Data are presented as histograms of relative cell counts with fluorescence intensity. Each histogram represents 1 x 104 cells. These are representative figures from five independent experiments with similar results. Vertical bars indicate cutoff for peak of ICAM-1 staining with control medium.

 
Synergistic up-regulation of ICAM-1 expression in combined treatment of SCF and TNF-{alpha} was dose-dependently enhanced by SCF
Figure 2 shows that the combined treatment of SCF and TNF-{alpha} resulted in a synergistic up-regulation of ICAM-1 expression. However, the combined treatment of SCF and IL-13 was found to have only an additional effect on ICAM-1 expression (Fig. 2) . To investigate whether the synergistic effect was dose-dependent with SCF and TNF-{alpha}, serial concentrations of SCF and TNF-{alpha} were used in the combined treatment. As shown in Figure 3 , a fixed concentration of SCF (50 ng/ml) with serial concentrations of TNF-{alpha} (5–70 ng/ml) showed that the synergistic effect could not be enhanced dose-dependently by TNF-{alpha}. Conversely, the synergistic effect could be enhanced further when the SCF concentration increased from 10 ng/ml to 100 ng/ml in combination with fixed TNF-{alpha} concentration (20 ng/ml).



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  		Figure 2. Effects of SCF, TNF-{alpha}, IL-13, SCF + IL-13, and SCF + TNF-{alpha} on the ICAM-1 expression on HMC-1 cells (1x106 cells/ml), which were incubated for 48 h without or with SCF (50 ng/ml), TNF-{alpha} (20 ng/ml), and IL-13 (20 ng/ml). Percentage increase of ICAM-1 expression was calculated as: (MFI after cytokine treatment–MFI without treatment)/MFI without treatment x 100%. Results are presented as mean + SD of seven independent experiments. Mann-Whitney rank-sum test was used to assess the difference of percent increase between single-treatment control groups and combined treatment group. *, P < 0.05; **, P < 0.01.

 


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  		Figure 3. Effects of various concentrations of SCF and TNF-{alpha} on the expression of ICAM-1 on HMC-1 cells (1x106 cells/ml), which were treated with SCF (10–100 ng/ml) and TNF-{alpha} (5–70 ng/ml) for 48 h. In the combined treatment, a fixed concentration of SCF (S; 50 ng/ml) or TNF-{alpha} (T; 20 ng/ml) was used with a serial concentration of TNF-{alpha} or SCF. *, P< 0.05, for the combined treatment of SCF (100 ng/ml) + TNF-{alpha} (20 ng/ml) compared with the combined treatment of SCF (10 ng/ml) + TNF-{alpha} (20 ng/ml), assessed by Mann-Whitney rank-sum test. No significant difference was found between the combined treatments of TNF-{alpha} (5–70 ng/ml) and SCF (50 ng/ml). Results are expressed as mean + SD of five independent experiments.

 
SCF and TNF-{alpha} activated ERK, p38 MAPK, and NF-{kappa}B, respectively
Total cellular protein extracted from HMC-1 cells after cytokine stimulation was used for the determination of phosphorylated p38 MAPK and phosphorylated ERK. Figure 4A and 4B , shows that SCF could activate the ERK but not the p38 MAPK pathway that peaked at 30 min and declined afterwards. It was also observed that TNF-{alpha} did not cause any phosphorylation of the ERK but caused activation of p38 MAPK, reaching peak level at 15 min, and declined afterwards.



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  		Figure 4. Effects of SCF, TNF-{alpha}, and SCF + TNF-{alpha} on the activation of ERK, p38 MAPK, and NF-{kappa}B activity of HMC-1 cells (1x106 cells/ml), which were stimulated for various time-periods with SCF (50 ng/ml), TNF-{alpha} (20 ng/ml), and combined treatment of SCF + TNF-{alpha}. Extracted total cellular protein (10 ng) from treated and nontreated (control) HMC-1 cells after 5, 15, 30, and 120 min was used for the ELISA of (A) phosphorylated ERK and (B) phosphorylated p38 MAPK. (C and D) Extracted total cellular protein (30 µg) after 15 min and 2 h treatment was used for the detection of phosphorylated p38 MAPK and I{kappa}B-{alpha}, respectively, by Western blot analysis. Representative blot is shown from triplicate experiments with essentially identical results. (E) Extracted nuclear protein (30 µg) after treatment for 1, 2, 4, 7, 12, and 18 h was used for the ELISA of NF-{kappa}B binding to NF-{kappa}B oligonucleotide. Results are expressed as mean ± SD of six independent experiments.

 
As shown in Figure 4C and 4D , results of Western blot showed that the treatment of TNF-{alpha} could induce the phosphorylation of p38 MAPK and the degradation of IkB-{alpha}, thereby releasing the NF-{kappa}B to translocate into the nucleus.

ELISA of NF-{kappa}B protein/NF-{kappa}B oligonucleotide binding (Fig. 4E) showed that the peak level of nuclear-translocated NF-{kappa}B protein occurred at 7 h after treatment of TNF-{alpha} or combined treatment of TNF-{alpha} and SCF. Afterwards, the level of NF-{kappa}B protein-binding declined but was found to be higher in the combined treatment of SCF and TNF-{alpha} than that of treatment of TNF-{alpha} at 18 h.

PD98059 and BAY117082 suppressed the combined treatment of SCF and TNF-{alpha}, and TNF-{alpha} induced ICAM-1 expression, respectively
The cytotoxicity of different inhibitors for different signaling pathways of HMC-1 cells was first determined. The dose at which at least 80% cells were viable was used as the optimal dosage of the inhibitors. As shown in Figure 5 , the optimal dosages were found to be 50, 20, 70, 2, and 100 µM for PD98059, SB203580, BAY117082, SP600125, and AG490, respectively. DMSO (1 µl/ml) was used as vehicle for inhibitors, except SB203580, as it did not have a significant cytotoxic effect on HMC-1 cells (Fig. 5F) .



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  		Figure 5. Effects of PD98059, BAY117082, SB203580, AG490, and SP600125 on the viability of HMC-1 cells, which were incubated with (A) PD98059, (B) BAY117082, (C) SB203580, (D) AG490, (E) SP600125, and (F) DMSO for 48 h, and the viability was assessed by MTT test. DMSO was used as a vehicle for inhibitors PD98059, BAY117082, AG490, and SP600125. Results are expressed as mean ± SD of triplicate experiments.

 
Figure 6 shows that ERK inhibitor PD98059 and NF-{kappa}B inhibitor BAY117082 could significantly suppress the combined effect of SCF- and TNF-{alpha}-induced ICAM-1 expression (P<0.01 and P<0.05, respectively). PD98059 and BAY117082 had no effect on TNF-{alpha}- and SCF-induced ICAM-1 expression, respectively (P>0.05). BAY117082 could also significantly suppress the effect of TNF-{alpha}-induced ICAM-1 expression (P<0.05). Combined treatment of PD98059 and BAY117082 could significantly suppress the ICAM-1 expression induced by the combined treatment of SCF and TNF-{alpha} (P<0.005). SB203580 showed no effect on any SCF- or TNF-{alpha}-induced ICAM-1 expression on HMC-1 cells (P>0.05). SP600125 and AG490 also had no significant effect on any treatment (data not shown).



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  		Figure 6. Effects of PD98059, BAY117082, and SB203580 on the SCF- and TNF-{alpha}-induced ICAM-1 expression on HMC-1 cells (1x106 cells/ml), which were treated without or with PD98059 (50 µM), BAY117082 (70 µM), and SB203580 (20 µM) for 1 h followed by stimulation with SCF, TNF-{alpha}, and SCF + TNF-{alpha} for 48 h. The ICAM-1 expression was detected by flow cytometry using mAb IgG1 isotype control antibody and mAb against ICAM-1. Results are expressed as MFI, and Mann-Whitney rank-sum test was used to assess the difference between the inhibitor-treated group and the noninhibitor-treated group under the same cytokine activation. Results are expressed as mean + SD of nine experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.005.

 
PD98059 and BAY117082 suppressed the SCF-induced ERK activity and TNF-{alpha}-induced NF-{kappa}B activity, respectively
Figure 7A shows that PD98059 could significantly suppress the SCF-induced ERK activity (P<0.001), and BAY117082 could significantly decrease the TNF-{alpha}-induced translocation of NF-{kappa}B into the nucleus (P<0.001, Fig. 7B ).



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  		Figure 7. Effects of PD98059 and BAY117082 on the inhibition of ERK and NF-{kappa}B activities. HMC-1 (1x106 cells/ml) were pretreated with or without PD98059 (50 µM) and BAY117082 (70 µM) for 1 h followed by stimulation with SCF, TNF-{alpha}, and SCF + TNF-{alpha}. (A) Phosphorylated ERK was measured after treatment for 30 min. (B) Nuclear NF-{kappa}B protein was extracted after the treatment for 7 h. Mann-Whitney rank-sum test was used to assess the difference between the inhibitor-treated group and noninhibitor-treated group under the same cytokine stimulation. (C) Nuclear NF-{kappa}B protein was extracted after the treatment for 2, 7, and 18 h. Mann-Whitney rank-sum test was used to assess the difference among all the treatments with the same time-point. Results are expressed as mean + SD of quadruplicate experiments. *, P < 0.05; ***, P < 0.001.

 
PD98059 suppressed the enhanced NF-{kappa}B activity after the combined treatment of SCF and TNF-{alpha} for 18 h
The enhanced NF-{kappa}B activity upon the combined treatment of SCF and TNF-{alpha} for 18 h could be significantly suppressed by PD98059 (P<0.05, Fig. 7C ). No significant effect was found in the shorter period (2 and 7 h) of treatment (P>0.05).

ST1 571 and PP1 suppressed the ICAM-1 expression under the treatment of TNF-{alpha} and combined treatment of SCF and TNF-{alpha}
To investigate if the autophosphorylation of the c-kit receptor had any effect on the ICAM-1 expression, STI571 and PP1, which are selective inhibitors of c-kit receptor tyrosine kinase and c-kit src tryrosine kinase, respectively, were used. Figure 8 shows that they could significantly suppress the ICAM-1 expression induced by treatment of TNF-{alpha} and combined treatment of SCF and TNF-{alpha} (P<0.05 and P<0.01, respectively).



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  		Figure 8. Effect of STI571 and PP1 on the SCF- and TNF-{alpha}-induced ICAM-1 expression on HMC-1 cells, which were treated with or without STI571 (0.1 µM) and PP1 (5 µM) for 1 h followed by cytokine stimulation for 48 h. Mann-Whitney rank-sum test was used to assess the difference between the inhibitor-treated group and noninhibitor-treated group under the same cytokine stimulation. Results are expressed as mean + SD of six experiments. *, P < 0.05; **, P< 0.01.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Adhesion molecule ICAM-1 plays a crucial role in mast cell recruitment to the inflamed tissue and interaction with other inflammatory cells [15 ]. In fact, it has been targeted for treating different inflammatory diseases [27 , 28 ], e.g., Cronh’s disease and ulcerative colitis [29 ], in which mast cell hyperplasia and degranulation are observed [30 ]. Antisense ICAM-1 has actually been administrated for treating patients with other inflammatory diseases [31 ].

In the present study, we adopted HMC-1 [32 ] as the in vitro mast cell model to study the cytokine effects on human mast cell. This is because mast cells are not normally found in human blood circulation, and culture of mast cells from peripheral blood or inflamed tissues is difficult and time-consuming [33 ]. HMC-1 cells possess many characteristics of human mast cells including the expression of c-kit receptors (SCF receptors) and the production of inflammatory mediators such as histamine, tryptase, leukotrienes, and prostaglandins [34 ].

We found that TNF-{alpha} and IL-13 potently increased the cell-surface expression of ICAM-1 (Fig. 1) , and SCF could only slightly increase its expression. Our results on the effects of TNF-{alpha} and IL-13 on ICAM-1 therefore concur with those of a previous study [13 ]. The observation of no up-regulation effect of SCF on ICAM-1 expression in this study was probably a result of the shorter treatment time (24 h) used [13 ]. It has been proposed by Wedi et al. [13 ] that mast cells could self-amplify their adhesive properties via the increased synthesis of ICAM-1 stimulated by TNF-{alpha}, IL-4, and interferon-{gamma} [14 ]. In view of our results regarding the effect of SCF and IL-13, which were also synthesized by mast cells [35 ], we may conclude from our study of HMC-1 that mast cells possess the autocrine function of self-regulating their cell-surface expression of ICAM-1.

This is the first report of an additive effect of SCF and IL-13 and a synergistic effect of SCF and TNF-{alpha} on the ICAM-1 cell-surface expression. Besides, we also found that the synergistic effect was dose-dependent on SCF but not TNF-{alpha}, suggesting that it was mainly a result of the effects of SCF. To understand the mechanisms of this synergistic effect, the intracellular signaling pathways involving cytokine stimulations were investigated.

Intracellular signal transduction is a highly interactive network composed of various types of protein kinases and other messenger cascades [36 , 37 ]. Its complexity allows the fine control and integration of the signal transduced, which can elicit the precise and diversified cellular responses upon different extracellular stimulations [38 ]. We demonstrated that p38 MAPK and NF-{kappa}B were activated by TNF-{alpha}, and ERK was activated by SCF (Fig. 4) . NF-{kappa}B is a pivotal regulator of proinflammatory gene expression and induces transcription of proinflammatory cytokines, chemokines, and adhesion molecules [23 ]. It is highly activated at inflammation tissues in various diseases, including allergic asthma, by enhancing the recruitment of inflammatory cells and production of proinflammatory cytokines [39 ]. Inhibition of NF-{kappa}B activity is effective at controlling inflammatory diseases in several animal models [40 ]. p38 MAPK, another important regulator in inflammation [41 ], can regulate the cellular degranulation, chemotaxis, adhesion molecules, and expression of regulated on activation, normal T cell expressed and secreted and granulocyte macrophage-colony stimulating factor [37 , 42 ]. It was shown that the NF-{kappa}B inhibitor BAY117082 but not the p38 MAPK inhibitor SB203580 could down-regulate TNF-{alpha}-induced ICAM-1 expression on HMC-1 cells (Fig. 6) , which is similar to the results in our previous report of eosinophils [43 , 44 ]. Together, we conclude that TNF-{alpha}-induced surface ICAM-1 expression through the NF-{kappa}B pathway is independent of the TNF-{alpha}-activated p38 MAPK on HMC-1 cells.

ERK activation is essential for cell proliferation and provides an integrated response by activating many gene transcriptions via transcription factors, chromatin phosphorylation, and increasing nucleotide synthesis [38 ]. We demonstrated that SCF could activate ERK pathway (Fig. 4A) and enhance the ICAM-1 expression (Fig. 1) . It has been documented that HMC-1 has a mutation in the c-kit (SCF) receptor, which is constitutively phosphorylated on tyrosine residues and associated with phosphatidylinositol-3 kinase, but does not lead to a constitutive phosphorylation of ERK and Akt [45 ]. Nevertheless, the c-kit receptor could still receive stimulation from exogenous SCF, and we also showed that SCF could be an activator of HMC-1 by the activation of the ERK pathway. PD98059, the specific inhibitor of the ERK pathway, suppressed the combined treatment of the SCF- and TNF-{alpha}-induced synergistic effect of ICAM-1 expression (Fig. 6) and also the SCF-mediated acitivation of ERK (Fig. 7) . Consequently, SCF may act through the ERK pathway, at least in part, to enhance the TNF-{alpha}-induced ICAM-1 expression of HMC-1 cells dose-dependently.

In fact, Jiang et al. [46 , 47 ] demonstrated a temporal control of NF-{kappa}B activation by ERK in rat vascular smooth muscle cells. ERK was shown to enhance the persistent but not the transient activation of NF-{kappa}B [47 ]. In concurrence with these findings, our results indicated that combined treatment of SCF and TNF could prolong the activation of NF-{kappa}B when treatment time was up to 18 h (Fig. 4E) , and PD98059 could suppress the enhanced activation (Fig. 7C) . Conversely, there is increasing evidence showing that MAPK, e.g., p38 MAPK, is required for NF-{kappa}B-dependent gene expression [48 ] and cross-talk between discrete intracellular signaling pathways [37 ]. As a result, it is quite possible that ERK can cross-talk with the NF-{kappa}B upon combined treatment of SCF and TNF on ICAM-1 expression to elicit the synergistic effects. However, a further experiment is required to confirm this type of regulatory mechanisms for the discrete response of mast cells.

To address the concern that HMC-1 has a constitutively phosphorylated c-kit receptor, ST1571 (imatinib mesilate), an inhibitor of c-kit receptor tyrosine kinase [49 ], and pyrazolo-pyrimidine compound PP1, an inhibitor of Src tyrosine kinase, which have been shown to block SCF receptor c-kit activation and SCF-mediated activation of ERK selectively [50 , 51 ], were used to determine if the autophosphorylation posed any effect on the ICAM-1 expression. Figure 8 shows that STI571 (0.1 µM) and PP1 (5 µM) could significantly inhibit the combined treatment of SCF- and TNF-induced ICAM-1 expression. This further indicated that the combined effect of the SCF- and TNF-{alpha}-induced c-kit mediated activation of Src kinase, and ERK plays important roles for the synergistic effect on ICAM-1 expression on HMC-1 cells. This is because STI571 and PP1 could reduce cell viability of HMC-1 cells [50 , 52 ], at least partly, via the inhibition of ERK. It may account for the suppressive effect of ST1571 and PP1 on TNF-{alpha}-induced ICAM-1 expression. Moreover, our results showed that the inhibitors of the JNK and JAK pathway, i.e., SP600125 and AG-490, respectively, had no effect in SCF- or TNF-{alpha}-induced ICAM-1 expression, thereby indicating that there was no involvement of the JNK and JAK pathway in ICAM-1 expression in HMC-1 cells.

In conclusion, ICAM-1-mediated mast cell recruitment and adhesion are under fine and complicated regulation according to different physiological conditions and the interactive effect of cytokines. Our results provide insight for cross-talk between different signaling cascades and a better rationale for the design of drug therapy for mast cell-mediated diseases.


   ACKNOWLEDGEMENTS
 
The study was supported by RGC Earmarked Research Grant, Hong Kong (CHUK 4473/03M), and a donation from GreaterChina Technology Group Limited.

Received July 15, 2004; revised January 13, 2005; accepted January 25, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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