Mast Cell Tryptase in Mast Cell Granules Enhances MCP-1 and Interleukin-8 Production in Human Endothelial Cells
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动脉硬化血栓血管生物学 2005年第9期
From the Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan.
Correspondence to Akira Matsumori, MD, PhD, Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, 54 Kawaharacho Shogoin, Sakyo-ku, Kyoto, 606-8397, Japan. E-mail amat@kuhp.kyoto-u.ac.jp
Abstract
Objective— Recent studies have highlighted the pathogenetic importance of chronic inflammation in cardiovascular disorders such as congestive heart failure and atherosclerosis. Mast cells release a wide variety of immune mediators that may initiate inflammatory responses, whereas endothelial cells (ECs) play a prominent role in the pathogenesis of cardiovascular diseases by secreting cytokines. The purpose of this study was to clarify the role of mast cells as an activator of ECs.
Methods and Results— ECs harvested from human umbilical cord veins were stimulated with mast cell granules (MCGs) prepared from sonicated human leukemic mast cells. The supernatants and total RNA from cells were collected. Levels of interleukin (IL)-1?, tumor necrosis factor-, and granulocyte colony-stimulating factor remained unchanged up to 24 hours. In contrast, levels of monocyte chemoattractant protein-1 (MCP-1) and IL-8 increased significantly within 6 hours. Northern blot analysis revealed an increase in MCP-1 and IL-8 mRNA expression in MCG-treated ECs. Induction of these chemokines was attenuated by antitryptase neutralizing antibody. Furthermore, MCP-1 and IL-8 were induced in ECs by incubation with human mast cell tryptase, but not with chymase.
Conclusions— These results indicate that the production of MCP-1 and IL-8 in ECs was induced by MCG and amplified by tryptase.
The role of mast cells in the pathogenesis of cardiovascular disorders has been recently highlighted. However, the mechanism remains unclear. This study demonstrates that degranulation of mast cells causes chemokine production in endothelial cells. These observations suggest the link between mast cells and atherosclerosis via endothelial production of chemokine.
Key Words: chemokine ? endothelium ? mast cell ? tryptase
Introduction
Recent studies have showed that chronic infection, inflammation, and immunologic factors are closely associated with the development of certain cardiovascular disorders. Chronic inflammation increases the numbers of macrophages and T lymphocytes in atherosclerotic lesions.1 Progression of lesions may also be associated with increased plasma concentrations of C-reactive protein, a marker of inflammation thought to be an early sign of atherosclerosis.2 The immune response to viral infection may be a major source of dilated cardiomyopathy. The balance between inflammatory and antiinflammatory cytokines plays a pivotal role in the development of heart failure and of atherosclerotic lesions. Chemoattractant proteins (chemokines) are found in human atheromas,3,4 and mice lacking chemokines or their receptors are less prone to atherosclerosis.5–8 In addition, we have shown that the expression of monocyte chemoattractant protein-1 (MCP-1) is increased in the pressure-overloaded hypertrophied and failing heart.9
Mast cells are essential resident effector cells in the elicitation of the immune response, found in nearly all major organs, near blood vessels in particular.10 Recent studies have suggested that mast cells play a role in the progression of heart failure, atherosclerosis, and rupture of atheroma.11–13
Mast cells are generally perivascular and may regulate endothelial cell (EC) function. ECs are a major source of various bioactive molecules, including cytokines and chemokines. This study tested the hypothesis that degranulation of mast cells by certain stimuli may regulate the production of cytokines from ECs and participate in the development of heart failure and of atherosclerotic lesions.
Methods
Biochemicals and Other Materials
Medium199 (M-199), grade I-A heparin sodium salt from porcine intestinal mucosa, human tryptase solution, N-benzoyl-D,L-arginine-p-nitroanilide (BAPNA), and N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (SAAPP) were purchased from Sigma Chemical Co (St Louis, Mo). Recombinant human chymase was obtained from Teijin Ltd (Osaka, Japan), and SF-8257 was from Suntory Biomedical Research Ltd (Osaka, Japan). Fetal calf serum and trypsin/EDTA were obtained from Life Technologies Inc (Grand Island, NY). Specific enzyme-linked immunosorbent assay kits for interleukin (IL)-1?, tumor necrosis factor-, granulocyte colony-stimulating factor-, and granulocyte macrophage colony-stimulating factor were purchased from Otsuka Pharmaceutical Co (Tokushima, Japan), and kits for MCP-1 and IL-8 were from Toray Industries Inc (Tokyo, Japan). Neutralizing antibody against tryptase was purified as described previously.14,15
Cell Culture
ECs were isolated from human umbilical veins as described previously16 and cultured in M-199 supplemented with 20% heat-inactivated fetal calf serum, 90 μg/mL heparin, and antibiotics (penicillin, 50 U/mL; streptomycin, 50 μg/mL; and amphotericin B, 125 ng/mL). Cells at passages 3 or 4 were seeded on culture plates coated with 0.5% gelatin and incubated at 37°C in a humidified atmosphere of 5% CO2/95% air. After the monolayer had become confluent, the culture medium was changed to M-199 with 5% heat-inactivated fetal calf serum, and the cells were incubated overnight and mast cell granules (MCGs) were added. ECs were identified by their typical "cobblestone" appearance and staining factor VIII antigen by immunofluorescence. Human mast cell line 1 (HMC-1) (a kind gift of J. H. Butterfield, Mayo Clinic, Rochester, Minn) were cultured in Iscove’s modified Dulbecco’s medium (Life Technologies, Grand Island, NY) with 10% heat-inactivated fetal calf serum, 4 mmol/L L-glutamine, and antibiotics (penicillin, 50 U/mL; streptomycin, 50 μg/mL; and amphotericin B, 125 ng/mL) in a humidified atmosphere of 5% CO2/95% air. The cell number was adjusted to 5x106 cells/mL twice weekly by adding fresh medium.
Preparation of MCGs
MCGs were prepared as described previously.17 Briefly, under sterile conditions, MCGs were obtained by bath sonication in ice for 5 seconds from HMC-1 (5x106 cells/mL) suspended in culture medium. The sonicate was then microcentrifuged (5 minutes) at 4°C, and debris-free supernatants were aliquoted and stored at –80°C. This solution was used as MCG and then added to human umbilical vein endothelial cells (ECs) incubated in a 24-well plate. To confirm that enzyme activities exist in MCG preparation procedure, we measured tryptase and chymase activity as described previously.18 Tryptase activity was determined by its ability to cleave a synthetic substrate N-benzoyl-D,L-arginine-p-nitroanilide (BAPNA) 2 mmol/L in Tris-HCl 0.1 mol/L (pH 8.0) and glycerol 1mol/L at 410 nm. Chymase activity was determined spectrophotometrically (410 nm) by the rate of hydrolysis of N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (SAAPP) 0.7 mmol/L in NaCl 1.5mol/L and Tris 0.3mol/L (pH 8.0). Protease activity was expressed in milliunits per milliliter (mU/mL), in which 1 U of enzyme activity was defined as the amount degrading 1 μmol of substrate per minute at 25°C. Tryptase activity of MCG preparations used in the present study was 12.62 mU/mL, and chymase activity was 3.81 mU/mL.
Experimental Protocols
Measurement of Cytokine Production by Endothelial Cells
ECs, in a density of 500 cells/mm2, were incubated in complete medium and allowed to adhere in a 24-well plate for 48 hours. The medium was then changed to M-199 with 5% heat-inactivated fetal calf serum for a period of 24 hours. MCG were then added for up to 24 hours. The concentrations of IL-1?, tumor necrosis factor-, IL-8, MCP-1, granulocyte colony-stimulating factor, and granulocyte macrophage colony-stimulating factor in the supernatant were measured by enzyme-linked immunosorbent assay and compared with those from control cells exposed to the HMC-1 solution for the same length of time, however, without previous sonication.
RNA Isolation and Northern Blot Hybridization
Total RNA was isolated by the guanidinium thiocyanate-phenol-chloroform-isoamylalcohol procedure from ECs incubated with MCG for 0, 1, 6, or 24 hours, and Northern blot analysis was performed.19 Equal amounts of RNA were electrophoresed on a 1.2% agarose/formaldehyde gel and transferred to a nylon membrane (Hybond-N+; Amersham Corp, Bunckinghamshire, England) by standard procedures.20 The blots were sequentially hybridized with alpha-32P-dCTP–labeled cDNA probes for human MCP-1 and IL-8. After overnight hybridization, the membranes were washed with 2xSSPE/0.1% SDS at room temperature, 1xSSPE/0.1% SDS at 65°C, and 0.1xSSPE/0.1% SDS at 65°C. The blots were analyzed with a FUJIX bioimaging analyzer BAS 2000 (Fujix, Tokyo, Japan) and normalized to the corresponding 18S rRNA level.
Roles of Tryptase and Chymase on MCG-Induced Gene Expression and Production of MCP-1 and IL-8
In a series of experiments, antitryptase neutralizing antibody, nonimmunized control IgG, or the selective chymase inhibitor SF-8257 was added to the culture medium 1 hour before MCG stimulation to study the role of tryptase and chymase in MCG-induced production of MCP-1 and IL-8. The antitryptase antibody was used in concentrations of 0.001 to 10 μg/mL, control IgG in 10 μg/mL, and SF8257 in concentrations from 10–7 to 10–5 mol. SF8257 was confirmed to be effective in a concentration of 10–5 mol.
Effects of Tryptase and Chymase on MCP-1 and IL-8 Production
ECs, in a density of 500 cells/mm2, were incubated in complete medium and allowed to adhere in a 24-well plate for 48 hours. The medium was then changed to M-199 with 5% heat-inactivated fetal calf serum for 24 hours. Tryptase in concentrations of 0.3 to 3 μg/mL or chymase in concentrations of 0.3 to 3 μg/mL was then added and the cells were cultured for up to 24 hours.
Statistical Analyses
Values are presented as mean±SEM. Results were analyzed by unpaired Student t test or by analysis of variance. P<0.05 was considered significant.
Results
MCG-Induced Cytokine Release
Figure 1 shows the mean levels of cytokines measured by enzyme-linked immunosorbent assay in 3 separate experiments. The levels of IL-1?, tumor necrosis factor-, granulocyte colony-stimulating factor, and granulocyte macrophage colony-stimulating factor were not changed by MCG. In contrast, levels of MCP-1 and IL-8 were increased significantly by the addition of MCG when compared with control cultures. The MCP-1 and IL-8 levels from ECs stimulated by MCG for 24 hours were, respectively, 1.6±0.1-fold (P<0.05) and 16.4±0.3-fold (P<0.05) higher than those from cells kept static for 24 hours. The increased production of MCP-1 and IL-8 in ECs by MCG was apparent within 6 hours (Figure 2).
Figure 1. Effect of MCGs on the production of cytokines by ECs. Human umbilical vascular ECs (HUVECs) were cultured in medium 199 in the presence or absence of MCGs for 24 hours and the concentration of each cytokine in the supernatants was measured. Values are mean±SEM (n=3). *P<0.05 vs controls.
Figure 2. Time-dependent effect of MCGs on the production of MCP-1 and IL-8. HUVECs were cultured in complete medium 199 in the presence or absence of MCG for 0, 1, 6, and 24 hours, and the concentration of MCP-1 or IL-8 in the supernatants was measured. Values are mean±SEM (n=3). *P<0.05 vs controls.
Effects of MCG on Gene Expression of MCP-1 and IL-8
Figure 3 is a representative Northern blot showing the time course of mRNA levels of MCP-1 and IL-8. The MCP-1 mRNA level increased within 1 hour, peaked at 6 hours, and remained significantly increased at 24 hours. Densitometric analysis of the MCP-1 mRNA level normalized to the 18S rRNA showed a 2.2±0.3-fold increase in MCG-stimulated ECs at 6 hour compared with static controls. IL-8 mRNA level was also increased at 1 hour, peaked at 6 hours, and then returned toward baseline. Densitometric analysis of the IL-8 mRNA level normalized to the 18S rRNA showed a 2.4±0.4-fold increase in MCG-stimulated ECs exposed to MCG for 6 hours compared with static controls. MCP-1 and IL-8 mRNA levels of the controls unstimulated by MCG showed no significant change within 6 hour (Figure I, available online at http://atvb.ahajournals.org).
Figure 3. Time-dependent effect of MCGs on gene expression of MCP-1 and IL-8. Total RNAs were isolated from ECs incubated with MCGs for 0, 1, 6, or 24 hours, and Northern blot analysis was performed. The blots were sequentially hybridized with cDNA probes for human MCP-1 and IL-8. Corresponding 18S rRNA bands are shown as internal controls. Values are mean±SEM (n=3). *P<0.05 vs static controls.
Roles of Tryptase and Chymase on MCG-Induced Gene Expression and Production of MCP-1 and IL-8
Because mast cells contain a variety of mediators, including proteases, proteoglycans, and histamine, which could stimulate ECs to produce chemokines, an attempt was made to identify the factor that caused the production of MCP-1 and IL-8. Treatment of ECs with polyclonal antibody against tryptase at 1 μg/mL inhibited the MCG-induced production of MCP-1 and IL-8 (Figure 4A). Northern blot analysis showed that tryptase inactivation by antitryptase antibody, 1 μg/mL, inhibited the MCG-induced gene expression of MCP-1x48±16%, and of IL-8x74±19% (P<0.05 versus untreated controls; Figure 4B; Figure II, available online at http://atvb.ahajournals.org). The same concentrations of nonimmune polyclonal IgG altered neither the MCG-induced gene expression nor protein production of MCP-1 and IL-8 by MCG. Furthermore, selective inhibition of chymase by SF-8257, 10–5 mol did not decrease the MCG-induced production of MCP-1 and IL-8 (Figure 4C). These results suggest that tryptase, but not chymase, plays an essential role in the MCG-induced gene expression of MCP-1 and IL-8.
Figure 4. Roles of tryptase and chymase on MCG-induced gene expression and production of MCP-1 and IL-8. ECs were pretreated with neutralizing tryptase antibody (0.001 to 10 μg/mL) (A) and SF-8257 (10–7 to 10–5 mol/L) (C), a chymase inhibitor, each for 1 hour before exposure of ECs to MCGs for 24 hours in analyzing protein levels and for 6 hours in examining gene expressions (B). Values are mean±SEM (n=3). *P<0.05 vs controls. Corresponding 18S rRNA bands are shown as internal controls.
Effects of Tryptase and Chymase on MCP-1 and IL-8 Production
Tryptase significantly increased the productions of MCP-1 and IL-8 in ECs within 6 hours of exposure (Figure 5A). There were dose-dependent releases of MCP-1 and IL-8 from ECs over a range of tryptase concentrations. In contrast, chymase did not modify the induction of MCP-1 and IL-8 (Figure 5B).
Figure 5. Time- and dose-dependent effect of proteases on the production of MCP-1 and IL-8. ECs were incubated in complete medium allowed to adhere for 48 hours, and then medium was changed to medium 199 with 5% heat-inactivated fetal calf serum for a period of 24 hours. Tryptase at concentrations of 0.3 to 3 μg/mL or chymase at concentrations of 0.3 to 3 μg/mL was then added and the cells were cultured for up to 24 hours. Values are mean±SEM (n=3). *P<0.05 vs controls.
Discussion
This study indicates that MCGs induce the production by human ECs of MCP-1, a potent chemoattractant for monocytes, and of IL-8, a potent chemoattractant for neutrophils, T lymphocytes, and eosinophils. These effects were mediated by human mast cell tryptase. The role of mast cells in the pathogenesis of cardiovascular disorders, heart failure and atherosclerosis in particular, has been recently highlighted. The number of mast cells is increased in the failing human heart11 and in recently infarcted rat myocardium,21 along with an increase in their mediators such as tryptase and histamine. We have previously shown that MCGs cause apoptosis of cardiomyocytes and proliferation of cardiac fibroblasts in vitro.12 In addition, in infarct-related coronary arteries, the number of degranulated mast cells in the adventitia backing ruptured plaques is increased.13 These observations suggest that mast cells and mast cell tryptase are activated in failing hearts and in atherosclerotic lesions.
Chemokines act mainly on neutrophils, monocytes, lymphocytes, and eosinophils, and play a pivotal role in the immune system.22–26 The study of chemokines has recently expanded to fields well beyond immunology, and it has become evident that they play key roles in cardiovascular diseases.27,28 MCP-1, a chemotactic factor for monocytes and one of the C-C chemokines, is believed to be among the important molecules involved in atherogenesis5 and heart failure.9 We have previously reported that plasma levels of MCP-1 are also increased in patients with acute myocardial infarction29 and have shown that antibody against MCP-1 reduces myocardial infarct size in a rat ischemia/reperfusion model.30 These data suggest that MCP-1, as an inflammatory mediator, plays a pivotal role in cardiac inflammatory responses in acute myocardial infarction. IL-8, a chemotactic factor for neutrophils and T lymphocytes, which belongs to the C-X-C chemokine family, may also play important roles in atherogenesis31 and myocardial ischemia/reperfusion injury.32
Several studies have reported an interaction between ECs and mast cells. ECs regulate the survival and development of mast cells,33 and mast cell tryptase stimulates the production of IL-8 and the expression of IL-1? gene of ECs.34 No longer regarded simply as a passive barrier separating the blood and surrounding tissue, ECs are now recognized as key players in the process of inflammation by producing various biomolecules, including cytokines and chemokines. Mast cells are localized at the adventitia in atherosclerotic coronary arteries. Adventitial inflammation is recognized as an important promoting factor of atherogenesis and the progression of arteriosclerosis.35 Pro-inflammatory effects of adventitial mast cells on ECs seen in this study suggest that the mast cells can play a role in atherosclerosis by stimulating ECs as effector cells. Moreover, mast cell tryptase released from adventitia may influence the function of remote intimal ECs via vasa vasorum circulation. Our study is the first to show that the degranulation of mast cells causes the production of MCP-1 in ECs. This observation may explain the link between mast cells and the development of atherosclerosis and heart failure via the production of chemokine by ECs.
MCGs contain a wide variety of inflammatory mediators, the release of which depends on the stimulus. These mediators include histamine, cytokines, and proteases, which have various effects on neighboring cells.36 Tryptase, chymase, cathepsin G, and carboxypeptidase are the major proteases contained in MCGs.37 Tryptase plays a role as is a growth factor for a number of cell types, including fibroblasts, epithelial cells, and smooth muscle cells.38 It may also be implicated in angiogenesis, because it induces tubular formation of ECs.39
Chymase is closely associated with cardiovascular disorders. It is activated in pressure-overloaded hearts, is able to convert angiotensin I to angiotensin II independently of the angiotensin-converting enzyme, and plays a major role in the formation of angiotensin II.40 Other proteases are known to have functions, which remain to be fully clarified.
The mechanism of chemokine production by ECs stimulated with mast cell tryptase is unclear. One possible mechanism involves activation of protease-activated receptors (PARs). Tryptase or thrombin cleaves the amino-terminal extracellular extension of the intact and inactivated receptor, exposing the amino terminus, which then functions as a receptor agonist, binding to a region of the receptor and activating it. Four subtypes of PAR have been cloned. The thrombin receptor (PAR-1) is expressed on ECs but does not appear to be activated by tryptase. PAR-2 is also expressed on ECs, and it may be activated by tryptase.41 The effect of human mast cell tryptase on ECs inducing the production of chemokine may be mediated by this receptor. PAR-3 and PAR-4 can also be cleaved by thrombin.42 However, little is known about the relationship between tryptase and these receptors.
In summary, we found that MCGs upregulate the secretion and gene expression of MCP-1 and IL-8 in human ECs. Human mast cell tryptase, but not chymase, is involved in the MCG-induced production of these chemokines. These results suggest that mast cells contribute to the pathogenesis of cardiovascular disorders, for instance by stimulating EC production of chemokines and enhancing local inflammation.
Acknowledgments
This work was supported by a research grant from the Ministry of Health and Welfare of Japan and a grant-in-aid for general scientific research from the Ministry of Education, Science, Sports, and Culture of Japan.
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Correspondence to Akira Matsumori, MD, PhD, Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, 54 Kawaharacho Shogoin, Sakyo-ku, Kyoto, 606-8397, Japan. E-mail amat@kuhp.kyoto-u.ac.jp
Abstract
Objective— Recent studies have highlighted the pathogenetic importance of chronic inflammation in cardiovascular disorders such as congestive heart failure and atherosclerosis. Mast cells release a wide variety of immune mediators that may initiate inflammatory responses, whereas endothelial cells (ECs) play a prominent role in the pathogenesis of cardiovascular diseases by secreting cytokines. The purpose of this study was to clarify the role of mast cells as an activator of ECs.
Methods and Results— ECs harvested from human umbilical cord veins were stimulated with mast cell granules (MCGs) prepared from sonicated human leukemic mast cells. The supernatants and total RNA from cells were collected. Levels of interleukin (IL)-1?, tumor necrosis factor-, and granulocyte colony-stimulating factor remained unchanged up to 24 hours. In contrast, levels of monocyte chemoattractant protein-1 (MCP-1) and IL-8 increased significantly within 6 hours. Northern blot analysis revealed an increase in MCP-1 and IL-8 mRNA expression in MCG-treated ECs. Induction of these chemokines was attenuated by antitryptase neutralizing antibody. Furthermore, MCP-1 and IL-8 were induced in ECs by incubation with human mast cell tryptase, but not with chymase.
Conclusions— These results indicate that the production of MCP-1 and IL-8 in ECs was induced by MCG and amplified by tryptase.
The role of mast cells in the pathogenesis of cardiovascular disorders has been recently highlighted. However, the mechanism remains unclear. This study demonstrates that degranulation of mast cells causes chemokine production in endothelial cells. These observations suggest the link between mast cells and atherosclerosis via endothelial production of chemokine.
Key Words: chemokine ? endothelium ? mast cell ? tryptase
Introduction
Recent studies have showed that chronic infection, inflammation, and immunologic factors are closely associated with the development of certain cardiovascular disorders. Chronic inflammation increases the numbers of macrophages and T lymphocytes in atherosclerotic lesions.1 Progression of lesions may also be associated with increased plasma concentrations of C-reactive protein, a marker of inflammation thought to be an early sign of atherosclerosis.2 The immune response to viral infection may be a major source of dilated cardiomyopathy. The balance between inflammatory and antiinflammatory cytokines plays a pivotal role in the development of heart failure and of atherosclerotic lesions. Chemoattractant proteins (chemokines) are found in human atheromas,3,4 and mice lacking chemokines or their receptors are less prone to atherosclerosis.5–8 In addition, we have shown that the expression of monocyte chemoattractant protein-1 (MCP-1) is increased in the pressure-overloaded hypertrophied and failing heart.9
Mast cells are essential resident effector cells in the elicitation of the immune response, found in nearly all major organs, near blood vessels in particular.10 Recent studies have suggested that mast cells play a role in the progression of heart failure, atherosclerosis, and rupture of atheroma.11–13
Mast cells are generally perivascular and may regulate endothelial cell (EC) function. ECs are a major source of various bioactive molecules, including cytokines and chemokines. This study tested the hypothesis that degranulation of mast cells by certain stimuli may regulate the production of cytokines from ECs and participate in the development of heart failure and of atherosclerotic lesions.
Methods
Biochemicals and Other Materials
Medium199 (M-199), grade I-A heparin sodium salt from porcine intestinal mucosa, human tryptase solution, N-benzoyl-D,L-arginine-p-nitroanilide (BAPNA), and N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (SAAPP) were purchased from Sigma Chemical Co (St Louis, Mo). Recombinant human chymase was obtained from Teijin Ltd (Osaka, Japan), and SF-8257 was from Suntory Biomedical Research Ltd (Osaka, Japan). Fetal calf serum and trypsin/EDTA were obtained from Life Technologies Inc (Grand Island, NY). Specific enzyme-linked immunosorbent assay kits for interleukin (IL)-1?, tumor necrosis factor-, granulocyte colony-stimulating factor-, and granulocyte macrophage colony-stimulating factor were purchased from Otsuka Pharmaceutical Co (Tokushima, Japan), and kits for MCP-1 and IL-8 were from Toray Industries Inc (Tokyo, Japan). Neutralizing antibody against tryptase was purified as described previously.14,15
Cell Culture
ECs were isolated from human umbilical veins as described previously16 and cultured in M-199 supplemented with 20% heat-inactivated fetal calf serum, 90 μg/mL heparin, and antibiotics (penicillin, 50 U/mL; streptomycin, 50 μg/mL; and amphotericin B, 125 ng/mL). Cells at passages 3 or 4 were seeded on culture plates coated with 0.5% gelatin and incubated at 37°C in a humidified atmosphere of 5% CO2/95% air. After the monolayer had become confluent, the culture medium was changed to M-199 with 5% heat-inactivated fetal calf serum, and the cells were incubated overnight and mast cell granules (MCGs) were added. ECs were identified by their typical "cobblestone" appearance and staining factor VIII antigen by immunofluorescence. Human mast cell line 1 (HMC-1) (a kind gift of J. H. Butterfield, Mayo Clinic, Rochester, Minn) were cultured in Iscove’s modified Dulbecco’s medium (Life Technologies, Grand Island, NY) with 10% heat-inactivated fetal calf serum, 4 mmol/L L-glutamine, and antibiotics (penicillin, 50 U/mL; streptomycin, 50 μg/mL; and amphotericin B, 125 ng/mL) in a humidified atmosphere of 5% CO2/95% air. The cell number was adjusted to 5x106 cells/mL twice weekly by adding fresh medium.
Preparation of MCGs
MCGs were prepared as described previously.17 Briefly, under sterile conditions, MCGs were obtained by bath sonication in ice for 5 seconds from HMC-1 (5x106 cells/mL) suspended in culture medium. The sonicate was then microcentrifuged (5 minutes) at 4°C, and debris-free supernatants were aliquoted and stored at –80°C. This solution was used as MCG and then added to human umbilical vein endothelial cells (ECs) incubated in a 24-well plate. To confirm that enzyme activities exist in MCG preparation procedure, we measured tryptase and chymase activity as described previously.18 Tryptase activity was determined by its ability to cleave a synthetic substrate N-benzoyl-D,L-arginine-p-nitroanilide (BAPNA) 2 mmol/L in Tris-HCl 0.1 mol/L (pH 8.0) and glycerol 1mol/L at 410 nm. Chymase activity was determined spectrophotometrically (410 nm) by the rate of hydrolysis of N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (SAAPP) 0.7 mmol/L in NaCl 1.5mol/L and Tris 0.3mol/L (pH 8.0). Protease activity was expressed in milliunits per milliliter (mU/mL), in which 1 U of enzyme activity was defined as the amount degrading 1 μmol of substrate per minute at 25°C. Tryptase activity of MCG preparations used in the present study was 12.62 mU/mL, and chymase activity was 3.81 mU/mL.
Experimental Protocols
Measurement of Cytokine Production by Endothelial Cells
ECs, in a density of 500 cells/mm2, were incubated in complete medium and allowed to adhere in a 24-well plate for 48 hours. The medium was then changed to M-199 with 5% heat-inactivated fetal calf serum for a period of 24 hours. MCG were then added for up to 24 hours. The concentrations of IL-1?, tumor necrosis factor-, IL-8, MCP-1, granulocyte colony-stimulating factor, and granulocyte macrophage colony-stimulating factor in the supernatant were measured by enzyme-linked immunosorbent assay and compared with those from control cells exposed to the HMC-1 solution for the same length of time, however, without previous sonication.
RNA Isolation and Northern Blot Hybridization
Total RNA was isolated by the guanidinium thiocyanate-phenol-chloroform-isoamylalcohol procedure from ECs incubated with MCG for 0, 1, 6, or 24 hours, and Northern blot analysis was performed.19 Equal amounts of RNA were electrophoresed on a 1.2% agarose/formaldehyde gel and transferred to a nylon membrane (Hybond-N+; Amersham Corp, Bunckinghamshire, England) by standard procedures.20 The blots were sequentially hybridized with alpha-32P-dCTP–labeled cDNA probes for human MCP-1 and IL-8. After overnight hybridization, the membranes were washed with 2xSSPE/0.1% SDS at room temperature, 1xSSPE/0.1% SDS at 65°C, and 0.1xSSPE/0.1% SDS at 65°C. The blots were analyzed with a FUJIX bioimaging analyzer BAS 2000 (Fujix, Tokyo, Japan) and normalized to the corresponding 18S rRNA level.
Roles of Tryptase and Chymase on MCG-Induced Gene Expression and Production of MCP-1 and IL-8
In a series of experiments, antitryptase neutralizing antibody, nonimmunized control IgG, or the selective chymase inhibitor SF-8257 was added to the culture medium 1 hour before MCG stimulation to study the role of tryptase and chymase in MCG-induced production of MCP-1 and IL-8. The antitryptase antibody was used in concentrations of 0.001 to 10 μg/mL, control IgG in 10 μg/mL, and SF8257 in concentrations from 10–7 to 10–5 mol. SF8257 was confirmed to be effective in a concentration of 10–5 mol.
Effects of Tryptase and Chymase on MCP-1 and IL-8 Production
ECs, in a density of 500 cells/mm2, were incubated in complete medium and allowed to adhere in a 24-well plate for 48 hours. The medium was then changed to M-199 with 5% heat-inactivated fetal calf serum for 24 hours. Tryptase in concentrations of 0.3 to 3 μg/mL or chymase in concentrations of 0.3 to 3 μg/mL was then added and the cells were cultured for up to 24 hours.
Statistical Analyses
Values are presented as mean±SEM. Results were analyzed by unpaired Student t test or by analysis of variance. P<0.05 was considered significant.
Results
MCG-Induced Cytokine Release
Figure 1 shows the mean levels of cytokines measured by enzyme-linked immunosorbent assay in 3 separate experiments. The levels of IL-1?, tumor necrosis factor-, granulocyte colony-stimulating factor, and granulocyte macrophage colony-stimulating factor were not changed by MCG. In contrast, levels of MCP-1 and IL-8 were increased significantly by the addition of MCG when compared with control cultures. The MCP-1 and IL-8 levels from ECs stimulated by MCG for 24 hours were, respectively, 1.6±0.1-fold (P<0.05) and 16.4±0.3-fold (P<0.05) higher than those from cells kept static for 24 hours. The increased production of MCP-1 and IL-8 in ECs by MCG was apparent within 6 hours (Figure 2).
Figure 1. Effect of MCGs on the production of cytokines by ECs. Human umbilical vascular ECs (HUVECs) were cultured in medium 199 in the presence or absence of MCGs for 24 hours and the concentration of each cytokine in the supernatants was measured. Values are mean±SEM (n=3). *P<0.05 vs controls.
Figure 2. Time-dependent effect of MCGs on the production of MCP-1 and IL-8. HUVECs were cultured in complete medium 199 in the presence or absence of MCG for 0, 1, 6, and 24 hours, and the concentration of MCP-1 or IL-8 in the supernatants was measured. Values are mean±SEM (n=3). *P<0.05 vs controls.
Effects of MCG on Gene Expression of MCP-1 and IL-8
Figure 3 is a representative Northern blot showing the time course of mRNA levels of MCP-1 and IL-8. The MCP-1 mRNA level increased within 1 hour, peaked at 6 hours, and remained significantly increased at 24 hours. Densitometric analysis of the MCP-1 mRNA level normalized to the 18S rRNA showed a 2.2±0.3-fold increase in MCG-stimulated ECs at 6 hour compared with static controls. IL-8 mRNA level was also increased at 1 hour, peaked at 6 hours, and then returned toward baseline. Densitometric analysis of the IL-8 mRNA level normalized to the 18S rRNA showed a 2.4±0.4-fold increase in MCG-stimulated ECs exposed to MCG for 6 hours compared with static controls. MCP-1 and IL-8 mRNA levels of the controls unstimulated by MCG showed no significant change within 6 hour (Figure I, available online at http://atvb.ahajournals.org).
Figure 3. Time-dependent effect of MCGs on gene expression of MCP-1 and IL-8. Total RNAs were isolated from ECs incubated with MCGs for 0, 1, 6, or 24 hours, and Northern blot analysis was performed. The blots were sequentially hybridized with cDNA probes for human MCP-1 and IL-8. Corresponding 18S rRNA bands are shown as internal controls. Values are mean±SEM (n=3). *P<0.05 vs static controls.
Roles of Tryptase and Chymase on MCG-Induced Gene Expression and Production of MCP-1 and IL-8
Because mast cells contain a variety of mediators, including proteases, proteoglycans, and histamine, which could stimulate ECs to produce chemokines, an attempt was made to identify the factor that caused the production of MCP-1 and IL-8. Treatment of ECs with polyclonal antibody against tryptase at 1 μg/mL inhibited the MCG-induced production of MCP-1 and IL-8 (Figure 4A). Northern blot analysis showed that tryptase inactivation by antitryptase antibody, 1 μg/mL, inhibited the MCG-induced gene expression of MCP-1x48±16%, and of IL-8x74±19% (P<0.05 versus untreated controls; Figure 4B; Figure II, available online at http://atvb.ahajournals.org). The same concentrations of nonimmune polyclonal IgG altered neither the MCG-induced gene expression nor protein production of MCP-1 and IL-8 by MCG. Furthermore, selective inhibition of chymase by SF-8257, 10–5 mol did not decrease the MCG-induced production of MCP-1 and IL-8 (Figure 4C). These results suggest that tryptase, but not chymase, plays an essential role in the MCG-induced gene expression of MCP-1 and IL-8.
Figure 4. Roles of tryptase and chymase on MCG-induced gene expression and production of MCP-1 and IL-8. ECs were pretreated with neutralizing tryptase antibody (0.001 to 10 μg/mL) (A) and SF-8257 (10–7 to 10–5 mol/L) (C), a chymase inhibitor, each for 1 hour before exposure of ECs to MCGs for 24 hours in analyzing protein levels and for 6 hours in examining gene expressions (B). Values are mean±SEM (n=3). *P<0.05 vs controls. Corresponding 18S rRNA bands are shown as internal controls.
Effects of Tryptase and Chymase on MCP-1 and IL-8 Production
Tryptase significantly increased the productions of MCP-1 and IL-8 in ECs within 6 hours of exposure (Figure 5A). There were dose-dependent releases of MCP-1 and IL-8 from ECs over a range of tryptase concentrations. In contrast, chymase did not modify the induction of MCP-1 and IL-8 (Figure 5B).
Figure 5. Time- and dose-dependent effect of proteases on the production of MCP-1 and IL-8. ECs were incubated in complete medium allowed to adhere for 48 hours, and then medium was changed to medium 199 with 5% heat-inactivated fetal calf serum for a period of 24 hours. Tryptase at concentrations of 0.3 to 3 μg/mL or chymase at concentrations of 0.3 to 3 μg/mL was then added and the cells were cultured for up to 24 hours. Values are mean±SEM (n=3). *P<0.05 vs controls.
Discussion
This study indicates that MCGs induce the production by human ECs of MCP-1, a potent chemoattractant for monocytes, and of IL-8, a potent chemoattractant for neutrophils, T lymphocytes, and eosinophils. These effects were mediated by human mast cell tryptase. The role of mast cells in the pathogenesis of cardiovascular disorders, heart failure and atherosclerosis in particular, has been recently highlighted. The number of mast cells is increased in the failing human heart11 and in recently infarcted rat myocardium,21 along with an increase in their mediators such as tryptase and histamine. We have previously shown that MCGs cause apoptosis of cardiomyocytes and proliferation of cardiac fibroblasts in vitro.12 In addition, in infarct-related coronary arteries, the number of degranulated mast cells in the adventitia backing ruptured plaques is increased.13 These observations suggest that mast cells and mast cell tryptase are activated in failing hearts and in atherosclerotic lesions.
Chemokines act mainly on neutrophils, monocytes, lymphocytes, and eosinophils, and play a pivotal role in the immune system.22–26 The study of chemokines has recently expanded to fields well beyond immunology, and it has become evident that they play key roles in cardiovascular diseases.27,28 MCP-1, a chemotactic factor for monocytes and one of the C-C chemokines, is believed to be among the important molecules involved in atherogenesis5 and heart failure.9 We have previously reported that plasma levels of MCP-1 are also increased in patients with acute myocardial infarction29 and have shown that antibody against MCP-1 reduces myocardial infarct size in a rat ischemia/reperfusion model.30 These data suggest that MCP-1, as an inflammatory mediator, plays a pivotal role in cardiac inflammatory responses in acute myocardial infarction. IL-8, a chemotactic factor for neutrophils and T lymphocytes, which belongs to the C-X-C chemokine family, may also play important roles in atherogenesis31 and myocardial ischemia/reperfusion injury.32
Several studies have reported an interaction between ECs and mast cells. ECs regulate the survival and development of mast cells,33 and mast cell tryptase stimulates the production of IL-8 and the expression of IL-1? gene of ECs.34 No longer regarded simply as a passive barrier separating the blood and surrounding tissue, ECs are now recognized as key players in the process of inflammation by producing various biomolecules, including cytokines and chemokines. Mast cells are localized at the adventitia in atherosclerotic coronary arteries. Adventitial inflammation is recognized as an important promoting factor of atherogenesis and the progression of arteriosclerosis.35 Pro-inflammatory effects of adventitial mast cells on ECs seen in this study suggest that the mast cells can play a role in atherosclerosis by stimulating ECs as effector cells. Moreover, mast cell tryptase released from adventitia may influence the function of remote intimal ECs via vasa vasorum circulation. Our study is the first to show that the degranulation of mast cells causes the production of MCP-1 in ECs. This observation may explain the link between mast cells and the development of atherosclerosis and heart failure via the production of chemokine by ECs.
MCGs contain a wide variety of inflammatory mediators, the release of which depends on the stimulus. These mediators include histamine, cytokines, and proteases, which have various effects on neighboring cells.36 Tryptase, chymase, cathepsin G, and carboxypeptidase are the major proteases contained in MCGs.37 Tryptase plays a role as is a growth factor for a number of cell types, including fibroblasts, epithelial cells, and smooth muscle cells.38 It may also be implicated in angiogenesis, because it induces tubular formation of ECs.39
Chymase is closely associated with cardiovascular disorders. It is activated in pressure-overloaded hearts, is able to convert angiotensin I to angiotensin II independently of the angiotensin-converting enzyme, and plays a major role in the formation of angiotensin II.40 Other proteases are known to have functions, which remain to be fully clarified.
The mechanism of chemokine production by ECs stimulated with mast cell tryptase is unclear. One possible mechanism involves activation of protease-activated receptors (PARs). Tryptase or thrombin cleaves the amino-terminal extracellular extension of the intact and inactivated receptor, exposing the amino terminus, which then functions as a receptor agonist, binding to a region of the receptor and activating it. Four subtypes of PAR have been cloned. The thrombin receptor (PAR-1) is expressed on ECs but does not appear to be activated by tryptase. PAR-2 is also expressed on ECs, and it may be activated by tryptase.41 The effect of human mast cell tryptase on ECs inducing the production of chemokine may be mediated by this receptor. PAR-3 and PAR-4 can also be cleaved by thrombin.42 However, little is known about the relationship between tryptase and these receptors.
In summary, we found that MCGs upregulate the secretion and gene expression of MCP-1 and IL-8 in human ECs. Human mast cell tryptase, but not chymase, is involved in the MCG-induced production of these chemokines. These results suggest that mast cells contribute to the pathogenesis of cardiovascular disorders, for instance by stimulating EC production of chemokines and enhancing local inflammation.
Acknowledgments
This work was supported by a research grant from the Ministry of Health and Welfare of Japan and a grant-in-aid for general scientific research from the Ministry of Education, Science, Sports, and Culture of Japan.
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