© 1990 S. K arger A G. Basel 0020-5915/90/0933-0133 S 2.75/0
Int Arch Allergy Appl Immunol 1990;93:133-138
Neutral Endopeptidase Modulates Substance P-Induced Activation of Human Neutrophils Ilsuo Iwamoto, Akira Kimura, Hiroomi Yamazaki, Noriaki Nakagawa. Hisao Tomioka, Sho Yoshida Second Department of Internal Medicine, Chiba University School of Medicine, Chiba, Japan
Abstract. Neutral endopeptidase (NEP; EC 3.4.24.11) is well recognized as a regulatory peptidase for sub stance P(SP)-induced responses in various tissues. To determine whether NEP regulates SP-induced activation of human neutrophils, we examined the effect of the NEP inhibitor phosphoramidon on SP-induced superox ide generation and chemotaxis in human blood neutrophils. SP (10~6-10~4 M) induced superoxide generation and chemotaxis in the neutrophils dose dependently. The NEP inhibitor enhanced the SP-induced responses. Thus, phosphoramidon (10'6 M) shifted the dose-response curves of SP-induced superoxide generation and chemotaxis of the neutrophils to the left by 0.5-0.6 log. Phosphoramidon prevented the hydrolysis of SP by the neutrophils, the NEP activity of the neutrophils being assessed as 125 ± 13 pmol of SP/min/106 cells. The Nterminal peptide SPi_9 (up to 3 x 10"4 M), which was a major degrading product by NEP of the neutrophils, did not activate the neutrophils. We conclude that NEP modulates SP-induced activation of human neutrophils.
Substance P (SP), an undecapeptide being present in the sensory neurones [1,2], has been shown to be a major mediator for inducing neurogenic inflamma tion [3, 4]. Thus, SP increases vascular permeability [5] and activates neutrophils [6-8]. SP induces the respir atory burst, chemotaxis, and enzyme release in hu man neutrophils [7, 8]. However, the mechanism that regulates SP-induced activation of the neutrophils is unknown. One possible mechanism for decreasing SP-induced responses is enzymatic inactivation of SP by peptidases [9, 10]. Among those peptidases, neutral endopeptidase (NEP; enkephalinase, EC 3.4.24.11) is one of the enzymes that most efficiently cleaves and inactivates SP [ 11-14], NEP is known to be present on the cell surface in many tissues including human neu trophils [11-13, 15-19], Furthermore, we have re cently shown that NEP inhibitors increase SP-in duced vascular permeability [20], thus suggesting that NEP modulates the SP-induced response.
The purpose of this study was to determine whether NEP modulates SP-induced activation of hu man neutrophils. For this purpose, we examined the effect of the NEP inhibitor on SP-induced superoxide generation and chemotaxis in human blood neutro phils. We also examined the potency of the major de grading peptide of SP by NEP of the neutrophils in these responses.
Materials and Methods Materials SP, SP,.,, SP6_n, luminol, dextran (molecular weight = 200,000), bovine serum albumin (BSA), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and superoxide dismutase (SOD) were purchased from Sigma Chemical Co. (St. Louis, Mo.). Met5-enkephalin, phosphoramidon, and bestatin were purchased from Peptide Institute (Osaka, Japan). Captopril was obtained from Squibb Pharmaceutical Co. (Princeton, N.J.). Ficoll-Paque solution was obtained from Pharmacia Fine Chemicals (Uppsala, Sweden). Hanks" balanced salt solution (HBSS) was obtained from Gibco (Grand Island, N.Y.).
Downloaded by: King's College London 137.73.144.138 - 12/11/2017 2:40:57 PM
Introduction
Iwamoto/Kimura/Yamazaki/Nakagawa/Tomioka/Yoshida
Protocol To determine whether NEP of human blood neutrophils effi ciently hydrolyses SP, we examined the hydrolysis of SP by the neu trophils in the absence or presence of an NEP inhibitor phosphoramidon and then compared it to that of Met5-enkephalin, which is one of the best substrates for N EP [ 12, 13]. To determine whether NEP modulates SP-induced activation of human neutrophils, we examined the effect of the NEP inhibitor phosphoramidon on SP-induced superoxide generation and chemotaxis of the neutrophils. To determine whether SP|_9, the major degrading product of SP by NEP of the neutrophils, activates human neutrophils, we ex amined the potencies of SP, SP,.,, and SP6_n (a C-terminal peptide of SP) in inducing superoxide generation and chemotaxis of the neutrophils.
of purified neutrophils ( lx lO 6) were incubated with luminol (0.1 mM) at 37°C for 5 min in a plastic cuvette. Then the reaction was initiated by adding SP (10-7- 10”4 M). The chemiluminescence was continuously measured for absorbance at 440 nm with a spectrofluorimeter equipped with a magnetic stirrer under the thermostatted cuvette holder (Unicorder U-228; Nippon Electric Co., To kyo, Japan). Each reaction was performed in duplicate. The SOD control reaction that contained 50 pg/ml SOD was also performed in a similar manner. Superoxide generation is expressed as an arbi trary unit (total response minus SOD control response). In one series of experiments, we examined the effect of the NEP inhibitor phosphoramidon (1 pM) on the response by adding the inhibitor or HBSS as a control 5 min before the initiation of the reaction. In an other series of experiments, we examined the potencies oftSP,., (10~7-10~4 M) and SP,.,, (10- 7- 10-4 M) in inducing the response.
Isolation o f Human Blood Neutrophils Neutrophils were isolated from heparinized venous blood of normal subjects by Ficoll-Paque density gradient centrifugation as described by Boyum [21]. Briefly, after erythrocytes were sedi mented in 3% dextran at 20°C for 30 min, the leukocyte-rich plasma was collected and centrifuged at 300 g for 10 min. The cell pellet was resuspended in HBSS. The cell suspension was then overlayered on Ficoll-Paque solution (specific gravity 1.077 g/ml) and was centrifuged at 400 g for 30 min at 20°C. The cells recovered from the bottom of the Ficoll-Paque layer were used as neutrophils (purity >96%, n = 32) after the contaminating erythrocytes were lysed with 165 mM ammonium chloride (pH 7.4). The neutrophils were washed in cold (4°C) H BSS three times and suspended in cold HBSS containing 0.1% BSA and 10 mM HEPES (pH 7.4).
Chemotaxis Chemotaxis of the neutrophils was measured as described by Boyden [24]. Two hundred microliters of SP (10~7- 10-4 M) in H BSS containing 1% BSA and 10 mM HEPES was put in the lower com partment of the chemotactic chamber and 200 pi of purified neutro phils (1 x lOVml) in HBSS containing 1% BSA and 10 m M HEPES was added to the upper compartment. A nitrocellulose filter 5 pm pore size, 180 pm thick; Sartorius Co., FRG) was placed between the two compartments. The chambers were kept at 37°C for 3 h. ^ t the end of the incubation, the filters were washed with cold HBSS, fixed with ethanol, and stained with hematoxylin. The number of neutrophils which reached the surface of the filter was counted in ten high-power fields for triplicate filters. In one series of experi ments, we examined the effect of the NEP inhibitor phosphoram idon (1 pM) on the response by incubating the neutrophils with the inhibitor or with HBSS as a control. In another series of experi ments, we also examined the potencies of SP,., (10-7—10_J M) and SP6_,, ( 10~7- 1(T4 M) in inducing the response.
Hydrolysis ofSP by NEP o f Neutrophils Hydrolysis of SP by NEP of the neutrophils was measured in the absence or presence of the NEP inhibitor phosphoramidon (1 gM) [22], The neutrophils (1 x 106) were incubated with 10"5 MSP at 37 °C for 20 min in 0.2 ml of HBSS containing 10 mM HEPES, 0.1 mM bestatin (an inhibitor against aminopeptidases), and lOpM captopril (an inhibitor against kininase II). The reaction was stopped by immediate centrifugation at 4°C and the following ad dition of 20 pi of 5% trifluoroacetic acid to the supernatants. Hy drolysis of Met5-enkephalin by NEP of the neutrophils was mea sured in a similar manner using 10"4 M Met'-enkephalin as a sub strate. Hydrolysis of SP and Met5-enkephalin was monitored by a high performance liquid chromatography (HPLC; Waters 600 E system with model 484 multiwavelength detector; Millipore Co., Milford, Mass.) using p Bondasphere C-18 reverse phase column (Waters). Twenty-five microliter aliquots of each reaction was injected onto the column and eluted with a linear gradient of increasing concen trations of acetonitrile (0-60%)/0.1% trifluoroacetic acid mixed into H:O/0.1% trifluoroacetic acid over 40 min at a flow rate of 1 ml/min. The peptide products were detected at 214 nm and were identified by amino acid analysis following the hydrolysis in 6 N HC1 at 110°C for 24 h [23], The amounts of SP and Met'-enkephalin were calculated by the integrated peak areas compared to those of known amounts of authentic SP and Mets-enkephalin. Superoxide Generation Superoxide (O,-) generation of the neutrophils was determined as SOD-inhibitable chemiluminescence using luminol. Briefly, I ml
Data Analysis Data are summarized as mean ±SD. The statistical analysis of the results was performed by the analysis of variance using Fisher’s least significant difference test for multiple comparisons.
Results Hydrolysis ofSPby NEP o f Human Neutrophils NEP of human blood neutrophils efficiently hy drolyzed SP. When SP (I0-5 M) was incubated with the neutrophils (5 x 106/ml) at 37°C for 20 min, SP in the incubation medium was decreased and six degrad ing products appeared (fig. lb). The phosphoramidon-sensitive SP degrading activity was 125x13 pmol/min/106cells (mean ± SD, n = 6 experiments in duplicate) (fig. 1). In contrast, the phosphoramidonsensitive Met5-enkephalin degrading activity was 92 ± 11 pmol/min/106 cells (n = 8 experiments). The amino acid analysis of the six degrading products re vealed to be SP|_6, SPj_7, SPi_9, SP7.9, SP8.9, and SP10-11 (fig- lb, 2). From the results, NEP of the neu-
Downloaded by: King's College London 137.73.144.138 - 12/11/2017 2:40:57 PM
134
Neutral Endopeptidase and Substance P-Induced Neutrophil Activation
135
Absorbance (214 nm)
Fig. 1. HPLC analysis of hydrolysis ofS P by NEP of human blood neutrophils. SP (IO' 5 M) was incubated with human blood neutrophils (5 x IOVml) at 37°C for 20 min in the presence (a) or absence (b) of the NEP in hibitor phosphoramidon (1 pAF). Peaks were identified by amino acid analysis (1 = SPU6, 2 = SPlo-ii» 3 = SPs_9, 4 = SPi_7, 5 = SP7.9, and 6 = SP,.,).
Effect o f NEP Inhibitor on S P-Induced Neutrophil Activation The NEP inhibitor enhanced SP-induced activa tion of human blood neutrophils. SP (10-6—10-4 M) induced the superoxide (CL") generation in the neu trophils dose dependently (fig. 3). The NEP inhibitor phosphoramidon (1 pM) increased the SP-induced superoxide generation of the neutrophils and thus shifted the dose-response curve to the left by 0.5-0.6 log (p
Int Arch Allergy Appl Immunol 1990;93:133-138
Neutral Endopeptidase Modulates Substance P-Induced Activation of Human Neutrophils Ilsuo Iwamoto, Akira Kimura, Hiroomi Yamazaki, Noriaki Nakagawa. Hisao Tomioka, Sho Yoshida Second Department of Internal Medicine, Chiba University School of Medicine, Chiba, Japan
Abstract. Neutral endopeptidase (NEP; EC 3.4.24.11) is well recognized as a regulatory peptidase for sub stance P(SP)-induced responses in various tissues. To determine whether NEP regulates SP-induced activation of human neutrophils, we examined the effect of the NEP inhibitor phosphoramidon on SP-induced superox ide generation and chemotaxis in human blood neutrophils. SP (10~6-10~4 M) induced superoxide generation and chemotaxis in the neutrophils dose dependently. The NEP inhibitor enhanced the SP-induced responses. Thus, phosphoramidon (10'6 M) shifted the dose-response curves of SP-induced superoxide generation and chemotaxis of the neutrophils to the left by 0.5-0.6 log. Phosphoramidon prevented the hydrolysis of SP by the neutrophils, the NEP activity of the neutrophils being assessed as 125 ± 13 pmol of SP/min/106 cells. The Nterminal peptide SPi_9 (up to 3 x 10"4 M), which was a major degrading product by NEP of the neutrophils, did not activate the neutrophils. We conclude that NEP modulates SP-induced activation of human neutrophils.
Substance P (SP), an undecapeptide being present in the sensory neurones [1,2], has been shown to be a major mediator for inducing neurogenic inflamma tion [3, 4]. Thus, SP increases vascular permeability [5] and activates neutrophils [6-8]. SP induces the respir atory burst, chemotaxis, and enzyme release in hu man neutrophils [7, 8]. However, the mechanism that regulates SP-induced activation of the neutrophils is unknown. One possible mechanism for decreasing SP-induced responses is enzymatic inactivation of SP by peptidases [9, 10]. Among those peptidases, neutral endopeptidase (NEP; enkephalinase, EC 3.4.24.11) is one of the enzymes that most efficiently cleaves and inactivates SP [ 11-14], NEP is known to be present on the cell surface in many tissues including human neu trophils [11-13, 15-19], Furthermore, we have re cently shown that NEP inhibitors increase SP-in duced vascular permeability [20], thus suggesting that NEP modulates the SP-induced response.
The purpose of this study was to determine whether NEP modulates SP-induced activation of hu man neutrophils. For this purpose, we examined the effect of the NEP inhibitor on SP-induced superoxide generation and chemotaxis in human blood neutro phils. We also examined the potency of the major de grading peptide of SP by NEP of the neutrophils in these responses.
Materials and Methods Materials SP, SP,.,, SP6_n, luminol, dextran (molecular weight = 200,000), bovine serum albumin (BSA), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and superoxide dismutase (SOD) were purchased from Sigma Chemical Co. (St. Louis, Mo.). Met5-enkephalin, phosphoramidon, and bestatin were purchased from Peptide Institute (Osaka, Japan). Captopril was obtained from Squibb Pharmaceutical Co. (Princeton, N.J.). Ficoll-Paque solution was obtained from Pharmacia Fine Chemicals (Uppsala, Sweden). Hanks" balanced salt solution (HBSS) was obtained from Gibco (Grand Island, N.Y.).
Downloaded by: King's College London 137.73.144.138 - 12/11/2017 2:40:57 PM
Introduction
Iwamoto/Kimura/Yamazaki/Nakagawa/Tomioka/Yoshida
Protocol To determine whether NEP of human blood neutrophils effi ciently hydrolyses SP, we examined the hydrolysis of SP by the neu trophils in the absence or presence of an NEP inhibitor phosphoramidon and then compared it to that of Met5-enkephalin, which is one of the best substrates for N EP [ 12, 13]. To determine whether NEP modulates SP-induced activation of human neutrophils, we examined the effect of the NEP inhibitor phosphoramidon on SP-induced superoxide generation and chemotaxis of the neutrophils. To determine whether SP|_9, the major degrading product of SP by NEP of the neutrophils, activates human neutrophils, we ex amined the potencies of SP, SP,.,, and SP6_n (a C-terminal peptide of SP) in inducing superoxide generation and chemotaxis of the neutrophils.
of purified neutrophils ( lx lO 6) were incubated with luminol (0.1 mM) at 37°C for 5 min in a plastic cuvette. Then the reaction was initiated by adding SP (10-7- 10”4 M). The chemiluminescence was continuously measured for absorbance at 440 nm with a spectrofluorimeter equipped with a magnetic stirrer under the thermostatted cuvette holder (Unicorder U-228; Nippon Electric Co., To kyo, Japan). Each reaction was performed in duplicate. The SOD control reaction that contained 50 pg/ml SOD was also performed in a similar manner. Superoxide generation is expressed as an arbi trary unit (total response minus SOD control response). In one series of experiments, we examined the effect of the NEP inhibitor phosphoramidon (1 pM) on the response by adding the inhibitor or HBSS as a control 5 min before the initiation of the reaction. In an other series of experiments, we examined the potencies oftSP,., (10~7-10~4 M) and SP,.,, (10- 7- 10-4 M) in inducing the response.
Isolation o f Human Blood Neutrophils Neutrophils were isolated from heparinized venous blood of normal subjects by Ficoll-Paque density gradient centrifugation as described by Boyum [21]. Briefly, after erythrocytes were sedi mented in 3% dextran at 20°C for 30 min, the leukocyte-rich plasma was collected and centrifuged at 300 g for 10 min. The cell pellet was resuspended in HBSS. The cell suspension was then overlayered on Ficoll-Paque solution (specific gravity 1.077 g/ml) and was centrifuged at 400 g for 30 min at 20°C. The cells recovered from the bottom of the Ficoll-Paque layer were used as neutrophils (purity >96%, n = 32) after the contaminating erythrocytes were lysed with 165 mM ammonium chloride (pH 7.4). The neutrophils were washed in cold (4°C) H BSS three times and suspended in cold HBSS containing 0.1% BSA and 10 mM HEPES (pH 7.4).
Chemotaxis Chemotaxis of the neutrophils was measured as described by Boyden [24]. Two hundred microliters of SP (10~7- 10-4 M) in H BSS containing 1% BSA and 10 mM HEPES was put in the lower com partment of the chemotactic chamber and 200 pi of purified neutro phils (1 x lOVml) in HBSS containing 1% BSA and 10 m M HEPES was added to the upper compartment. A nitrocellulose filter 5 pm pore size, 180 pm thick; Sartorius Co., FRG) was placed between the two compartments. The chambers were kept at 37°C for 3 h. ^ t the end of the incubation, the filters were washed with cold HBSS, fixed with ethanol, and stained with hematoxylin. The number of neutrophils which reached the surface of the filter was counted in ten high-power fields for triplicate filters. In one series of experi ments, we examined the effect of the NEP inhibitor phosphoram idon (1 pM) on the response by incubating the neutrophils with the inhibitor or with HBSS as a control. In another series of experi ments, we also examined the potencies of SP,., (10-7—10_J M) and SP6_,, ( 10~7- 1(T4 M) in inducing the response.
Hydrolysis ofSP by NEP o f Neutrophils Hydrolysis of SP by NEP of the neutrophils was measured in the absence or presence of the NEP inhibitor phosphoramidon (1 gM) [22], The neutrophils (1 x 106) were incubated with 10"5 MSP at 37 °C for 20 min in 0.2 ml of HBSS containing 10 mM HEPES, 0.1 mM bestatin (an inhibitor against aminopeptidases), and lOpM captopril (an inhibitor against kininase II). The reaction was stopped by immediate centrifugation at 4°C and the following ad dition of 20 pi of 5% trifluoroacetic acid to the supernatants. Hy drolysis of Met5-enkephalin by NEP of the neutrophils was mea sured in a similar manner using 10"4 M Met'-enkephalin as a sub strate. Hydrolysis of SP and Met5-enkephalin was monitored by a high performance liquid chromatography (HPLC; Waters 600 E system with model 484 multiwavelength detector; Millipore Co., Milford, Mass.) using p Bondasphere C-18 reverse phase column (Waters). Twenty-five microliter aliquots of each reaction was injected onto the column and eluted with a linear gradient of increasing concen trations of acetonitrile (0-60%)/0.1% trifluoroacetic acid mixed into H:O/0.1% trifluoroacetic acid over 40 min at a flow rate of 1 ml/min. The peptide products were detected at 214 nm and were identified by amino acid analysis following the hydrolysis in 6 N HC1 at 110°C for 24 h [23], The amounts of SP and Met'-enkephalin were calculated by the integrated peak areas compared to those of known amounts of authentic SP and Mets-enkephalin. Superoxide Generation Superoxide (O,-) generation of the neutrophils was determined as SOD-inhibitable chemiluminescence using luminol. Briefly, I ml
Data Analysis Data are summarized as mean ±SD. The statistical analysis of the results was performed by the analysis of variance using Fisher’s least significant difference test for multiple comparisons.
Results Hydrolysis ofSPby NEP o f Human Neutrophils NEP of human blood neutrophils efficiently hy drolyzed SP. When SP (I0-5 M) was incubated with the neutrophils (5 x 106/ml) at 37°C for 20 min, SP in the incubation medium was decreased and six degrad ing products appeared (fig. lb). The phosphoramidon-sensitive SP degrading activity was 125x13 pmol/min/106cells (mean ± SD, n = 6 experiments in duplicate) (fig. 1). In contrast, the phosphoramidonsensitive Met5-enkephalin degrading activity was 92 ± 11 pmol/min/106 cells (n = 8 experiments). The amino acid analysis of the six degrading products re vealed to be SP|_6, SPj_7, SPi_9, SP7.9, SP8.9, and SP10-11 (fig- lb, 2). From the results, NEP of the neu-
Downloaded by: King's College London 137.73.144.138 - 12/11/2017 2:40:57 PM
134
Neutral Endopeptidase and Substance P-Induced Neutrophil Activation
135
Absorbance (214 nm)
Fig. 1. HPLC analysis of hydrolysis ofS P by NEP of human blood neutrophils. SP (IO' 5 M) was incubated with human blood neutrophils (5 x IOVml) at 37°C for 20 min in the presence (a) or absence (b) of the NEP in hibitor phosphoramidon (1 pAF). Peaks were identified by amino acid analysis (1 = SPU6, 2 = SPlo-ii» 3 = SPs_9, 4 = SPi_7, 5 = SP7.9, and 6 = SP,.,).
Effect o f NEP Inhibitor on S P-Induced Neutrophil Activation The NEP inhibitor enhanced SP-induced activa tion of human blood neutrophils. SP (10-6—10-4 M) induced the superoxide (CL") generation in the neu trophils dose dependently (fig. 3). The NEP inhibitor phosphoramidon (1 pM) increased the SP-induced superoxide generation of the neutrophils and thus shifted the dose-response curve to the left by 0.5-0.6 log (p
