Exp. Pathol. 1991; 42: 47-58 Gustav Fischer Verlag lena
Department of Pneumology and Allergology, Medical Academy, L6dt, Poland
Erythrocytes protect a-I-proteinase inhibitor from oxidative inactivation induced by chemicals, the myeloperoxidase-H20 r haiide system and stimulated polymorphonuclear leukocytes By D. NOWAK and G. PIASECKA With 6 figures Received: June 20, 1989; Accepted: August 4, 1989
Address for correspondence: DARIUSZ NOWAK, Department of Pneumology and Allergology, Medical Academy, Kopcinskiego 22, 90-153 L6dt, Poland Keywords: erythrocyte; alpha-I-proteinase inhibitor; myeloperoxidase-H2 0z-halide system; leukocyte; oxidants
Summary The oxidative inactivation of the ex-I-proteinase inhibitor (exlPI) is one of the mechanisms responsible for creating the elastase/antielastase imbalance during inflammation in the lower respiratory tract. Chronic supremacy of elastase may cause degradation of elastin fibers leading to the pulmonary emphysema. In this study we have investigated the effect of erythrocytes (RBC, concentrations 0.125 to 1.5 %) on the oxidative inactivation of exlPI by the phorbol myristate acetate-stimulated polymorphonuclear leukocytes (PMNL) and PMNL myeloperoxidase-H 20 2halide system. During exposure of exlPI to both systems in the presence ofRBC the significant protection (p < 0.001) of ex 1PI was found for all RBC concentrations, e. g. at RBC concentration of! % the elastase inhibitory capacity (EIC) ofa: 1PIincreased from 0 to 60 ± 6and88 ± 9% of the control value (untreated a: 1PI) , n = 5, respectively. The preincubation ofRBC with chloramine-T (1 mM), inhibition of RBC catalase or depletion of RBC reduced glutathione alone did not diminish the capacity of RBC to protect a:1PI. However, these treatments together completely deprivedRBC of their protective properties. Moreover, we have compared the decrease in the EIC of human blood and its plasma after incubation with H 20 2 (0.1 mM to 0.1 M) or chloramine-T (l f.tM to 1 mM). For the incubation with H 20 2 no decrease in blood EIC was found whereas in plasma the loss of EIC was already visible at a H20 2 concentration of 0.1 mM. Also for the incubation with chloramine-T the EIC of blood was more resistant to oxidant damage than EIC of plasma. It is suggested that RBC contaminations present in the phagocyte inflammatory infiltration in the lower airways may protect a:lPI from oxidative inactivation and thus indirectly diminish proteolytic lung injury related to inflammation.
Exp. Pathol. 42 (1991) 1
47
Introduction The physiological activity of the ex-I-proteinase inhibitor (ex IPI), a major neutrophil elastase inhibitor in the lower airways, is highly susceptible to oxidative inactivation (6). This process consisting in the oxidation to the sulfoxide form of the methionine thioether residue at the active elastase inhibitory site of ex 1PI (21) causes almost total reduction of its elastase inhibitory capacity (6) and can create elastasel antielastase imbalance in the lung tissue. Thus, the elastase supremacy induced may lead to degradation of elastin fibers and as a consequence of this to the development of pulmonary emphysema. Various exogenic oxidants present in tobacco smoke (13), ozone (22), chlorine (29), nitrogen dioxide (25) may inactivate exlPI in the lower airways. Another source of oxidants in lung, which is probably more important, are phagocytic cells: macrophages and polymorphonuclear leukocytes (PMNL). These cells release large amounts of reactive oxidants after stimulation, which can inactivate (dPI (9, 37) and may occur during inflammation in the lower airways. There are various antioxidants protecting exlPI from oxidative damage in plasma and lung tissue. Recent studies showed that ceruloplasmin inhibits oxidative inactivation of exPI by the human PMNL myeloperoxidase system, chloramine-T and cigarette smoke solution (15, 32). Another enzyme, methionine sulfoxide-peptide reductase, was able to reactivate previously oxidized exlPI by treatment with chemicals or with enzymatic systems (10). Moreover, catalase and superoxide dismutase protected exlPI from activated phagocytes (9, 19,37). It is well known that erythrocytes (RBC) contain antioxidant systems including the last two enzymes mentioned above and glutathione peroxidase (17), which are able to protect not only their own structures but also cells and substances surrounding them (1, 27, 35). RBC can act as a hydrogen peroxide scavenger and inhibit the following generation of the hydroxyl radical or hypochlorous acid (33, 36). In cigarette smokers, these cells have increased catalase activity and contain more glutathione, and it may be one of the links of defense against oxidants present in cigarette smoke (35). Therefore, we have investigated the effect of RBC on PMNL- and myeloperoxidase-H20zhalide system-mediated oxidative inactivation of exlPI in vitro in this study. We have also compared the decrease in the elastase inhibitory capacity of blood and matched plasma induced by chloramine-T and H20 2 . Material and Methods Reagents
Elastin-orcein, peroxidase from horseradish type II (200 UImg solid); o-dianisidine dihydrochloride (o-DD), phorbol 12-myristate 13-acetate (PMA) , 1-chloro-2,4-dinitrobenzene, 3amino-1,2,4-triazole, phenylmethylsulfonylfluoride (PMSF) were obtained from Sigma (St. Louis, MO, USA). D-glucose, dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), heparin (preservative-free), Triton X-I 00 were from BDH England. Porcine pancreatic elastase (100 UI mg) was purchased from Merck. All other reagents used were of analytical grade. Elastinorcein was washed once, resuspended in methanol and 200-I-lI-portions containing 2 mg elastin were placed into Eppendorf tubes, dried and stored at 4°C until assay. PMA (10 I-lg/ml) in DMSO was stored at -20°C. PMSF (0.2 M) was dissolved in isopropanol. Elastase solution (0.25 mgl ml in PBS) was prepared freshly before the assay. Isolation of PMNL
Human polymorphonuclear leukocytes (PMNL) were isolated from blood obtained from healthy nonsmoking donors according to standard procedures (8). PMNL were washed once and resuspended to 107 per ml in PBS (PH 7.4 containing 0.9mM CaCl2 and 0.5 mM MgClz) with addition of glucose 1 gil. The purity ofPMNL suspensions and viability as assessed by trypan blue were always above 97 % .
48
Exp. Pathol. 42 (1991) 1
Preparation and treatment of RBC RBC were isolated from one young healthy donor by blood centrifugation at 1000 X g for 5 min, removal of plasma and leukocytes, washing 3 times with PBS and resuspending in PBS to 10% hematocrit. RBC catalase was inhibited by incubating a 10% cell suspension with 50 mM 3amino-l ,2,4-triazo1e and 4.5 mM H20 2 (36). RBC-reduced glutatione (GSH) was depleted by incubation of these cells (10 % suspension) with 2 mM 1-chloro-2,4-dinitrobenzene (3). Various groups on the RBC surface, which can scavenge hypochlorous acid (HClO), were blocked by addition of chloramine-T (Ch-T) 1 mM to 10% RBC suspension. In all cases, RBC were incubated for 1 h at 37°C and washed 3 times in PBS following exposure to these inhibitors and immediately used in experiments as a 20% suspension.
Measurement of RBC catalase The catalase activity in untreated and treated RBC was assessed by spectrophotometric determination of the decomposition of H2 0 2 at 230 nm by placing 2 ml PBS in the reference cuvette and 2 ml 10 mM H2 0 2 solution in PBS in the sample cuvette. This solution gives an absorbance of 0.816. Then 1 ftl hemolysate ofthe 10% RBC suspension was added to the sample cuvette, mixed thoroughly and the absorbance decrease was recorded over 3 min at room temperature. The amounts of H 20 2 decomposed were calculated from changes of absorbance using coefficientE = 81 cm -1 M- 1 (18), and the catalase activity was expressed as a percentage of the activity presented in untreated cells. RBC hemolysate was obtained by mixing 200 ftllO % cell suspension with 2ft11O% TritonX-100 in PBS.
Preparation of cx1PI Partially purified cx1PI was obtained from serum after chromatography on carboxymethyl cellulose (CM celulose 52 Whatman 1.0meq/g) (36).10 ml serum was acidified to pH 6.4 with HCl, diluted to 20ml with 0.01 M phosphate buffer pH 6.4 and then placed on the carboxymethylcellulose column (2 X 22 cm) and eluted with the same buffer. Eluted protein containing cxlPI was concentrated by precipitation with (NH4hS04 (75% saturation, 4°C), dialyzed overnight against three changes of PBS, divided into 200 ftl portions and stored at -20°C. This preparation contained 2.56mg (dPI per m!.
Preparation of the crude extract of myeloperoxidase (MPO) 3.75 X 108 PMNL resuspended in 10 ml PBS containing 0.1 % Triton x-100 and 5 mM PMSF were sonicated (Ultrasonic disintegrator UD-20 Techpan, Poland) at O°C at 80% of maximal power for 3 min and then centrifuged for 15 min at 15000 X g at 4°C. All subsequent centrifugations were carried out under these conditions. After placing in an ice bath the supernatant was treated with solid (NH4hS04 to yield 50 % saturaion, incubated for 30 min and centrifuged. The residue was discarded and the supernatant again treated with solid (NH4hS04 to increase the concentration to 65 % of saturation, incubated for 30 min at 0 °C and centrifuged. The obtained precipitate was dissolved in PBS, dialyzed overnight against 3 changes of PBS, divided into 100 [il portions and stored at - 20°C. This procedure is a modification of the initial part of the method for myeloperoxidase isolation (2). This MPO extract contained 1.84 fig protein per 1 [iI, had no elastase activity and 1 fig of this protein decomposed 3.62nmoles H 20 2 during 1 min incubation under conditions as for measurement of catalase. 1 [A.g MPO when mixed with 1 ml PBS containing 80 [ig/ml o-DD, 134 [A.M H 20 2 and 0.8 mg/ml BSA catalyzed H 20 r mediated o-DD oxidation (increase in absorbance at 470 nm) and was equivalent to 3.4mU horseradish peroxidase. BSA was added to protect oxidized o-DD precipitation at pH 7.4. 4
Exp. Pathol. 42 (1991) 1
49
Oxidation of (XIPI a) - oxidation of (XIPI with chemicals Heparinized (3 Vlml) human venous blood and plasma were divided into 300!J.I portions, mixed with 30 !J.I freshly prepared Ch-T and H 20 2 solutions, final concentrations 1 !J.M to 1 mM and 0.1 mM to 0.1 M, respectively. Control samples received 30!J.I PBS. After 1 h incubation at 37°C samples were centrifuged and supernatants assayed for elastase inhibitory capacity (EIC). b) - oxidation of (X1PI by myeloperoxidase-H 20 2-halide system 80!J.1 PBS, 2!J.I (X1PI solution and 1 !J.I MPO extract were mixed and then 50!J.I 0.2 mM H20 Z solution in PBS were added and the samples were quickly cooled to 0 °C after 20 min of incubation at 37°C, and the reaction was stopped with 20 !J.l1 % sodium azide. The following control systems were included: 1) (X1PI in PBS, 2) (XPI and MPO, 3) (X1PI and HzO z alone. c) - oxidation of (X1PI by PMA-stimulated PMNL 80!J.I PBS and 2!J.I (XIPI were mixed with 50!J.I PMA (lOng/ml)-stimulated PMNL suspension (5 x 105 cells), incubated for 15 min at 37°C, cooled to 0 °C, mixed with 20!J.I 1 % sodium azide and then centrifuged for 1 min at 10,000 x g at 4 °C. The number of cells and the time of incubation of (XIPI were based on our previous work (28). Control systems contained: 1) (X 1PI in PBS, 2) (X1PI and unstimulated PMNL (PMA omitted), 3) unstimulated PMNL in PBS, 4) stimulated PMNL in PBS.
Effect of RBC on (XIPI oxidative inactivation Various volumes of 20% RBC suspension were added to the aforementioned reaction mixtures (b and c) before addition of H Z0 2 and PMA-activated PMNL. Final RBS concentrations were 0.125 to 1.50%. Samples were incubated and centrifuged under conditions as described above. Since RBC bound (XIPI to its outer membrane (5) and may cause EIC decrease in supernatants, appropriate control systems «(XIPI and RBC) were included for all investigated cell concentrations. Previous experiments showed that the content of DMSO in samples related to PMNL stimulation (solvent for PMA) did not induce RBS hemolysis.
Determination of the elastase inhibitory capacity of (Xl PI Assays of (X1PI inactivated with the MPO-H 20z-halide system and oxidants derived from PMA-stimulated PMNL (b and c) were performed by the elastin-orcein test (7) with some modifications (30). 150!J.I of each supernatant containing (X1PI were mixed with 20!J.I elastase solution, incubated for 20 min at 37°C and then mixed with 2 mg of elastin-orcein resuspended in 1.33 ml PBS. The samples were incubated for 16 h at 37°C with gentle stirring. Elastase standards were prepared by mixing 20!J.I elastase solution with 130!J.I PBS and 20 !J.I 1 % sodium azide. Previous experiments showed that MPO alone and HZ0 2 concentrations used had no effect on elastase activity. The undigested substrate was centrifuged off and absorbance was read at 590 nm. Results were compared with the straight line calibration curve obtained with completely digested different amounts of elastin-orcein. 5.12!J.g native (untreated) (X1PI inhibited 0.52 activity of 5 !J.g elastase and it was taken for EIC calculations as 100%. Appropriate amounts of plasma treated with chemicals (a) were mixed with PBS and 20 !tIl % sodium azide to give final volume 150 fll and then their EIC was measured as described above. The volume of added plasma was equal the volume of untreated plasma which under this conditions inhibited 0.5 elastase activity (determined in previous experiments for plasma from all 3 donors). In order to check if oxidants added to plasma may inhibit elastase activity, plasma was replaced by BSA in PBS (50 mg/ml) , incubated for 1 hwithCh-T(l mM)orHzO z (0.1 M) and mixed with elastase. In this case no inhibition of elastase was found. 50
Exp. Pathol. 42 (1991) 1
Other techniques Serum exlPI, ceruloplasmin, ex2-macroglobulin and transferrin concentrations were determined by radial immunodiffusion in commercially prepared plates (M -Partigen-ex 1-Antitrypsin, M-Partigen-Coeruloplasmin, Protein-Standard-Plasma, Behring Institut) and by rocket immunoelectrophoresis with rabbit antiserum to human transferrin and to ex-2-macroglobulin (Biomed, Warsaw, Poland) and Standard-Human-Serum (Behring Institut). Protein concentration was measured using the method of LOWRY et al. (23) with bovine serum albumin as a standard. All individual determinations were carried out in duplicates. Typical blood examinations (red blood count, haemoglobin, hematocrit) were done using Technicon Haematology System 1 (Technicon Chemicals Company, Belgium).
Statistical analysis Results are expressed as the mean value ± the standard deviation. Statistical analyses were performed using paired t-statistic by comparison of the results obtained with exlPI exposed to oxidants and results of corresponding experiments where exlPI was protected by RBC.
Results Fig. I shows the ratio between absorbance at 590 nm and the amount of elastin-orcein digested. The curve is rectilinear in the whole range investigated (0-6.2 mg). 0.92 mg elastinorcein (as calculated from the standard curve) was solubilized after 16 h incubation of 2 mg substrate with 5 flg elastase and this quantity of elastin was used for EIC determination. The time course of exlPI inactivation by MPO-H20 2-halide system is shown in fig. 2. The functional activity of exlPI decreased rapidly with time and already after 20 min incubation no elastase inhibition was noted. This time of incubation was chosen for further experiments. MPO alone and H20 2did not inactivate exlPI (data not shown) and it is in agreement with our previous observations (28) where H20 2 concentrations below 0.15 mM had no influence on exlPI activity. The changes of EIC of blood and plasma from 3 various donors after treatment with Ch-T and H20 2 are presented in figs. 3 and 4. Incubation of blood with all H 20 2 concentrations did not diminish its EIC (fig. 3). However, in the case of plasma moderate decrease in elastase inhibition was observed especially for H202 concentration 0.1 M (p < 0.1). When blood was incubated with Ch-T the EIC was not significantly impaired up to oxidant concentration 0.1 mM (fig. 4). lO-fold higher Ch-T concentration caused severe decline of blood EIC (p < 0.01), which was approximate to that found for Ch-T treated plasma. The characteristics of blood samples used in the aforementioned experiments, e.g. hematocrit, red blood count, concentrations of haemoglobin, ex1PI, ex-2macroglobulin, ceruloplasmin, transferrin are presented in table 1. RBC protected exlPI from oxidative inactivation by the MPO-H20 2-halide system and PMA-stimulated PMNL (table 2). Highly significant protection (p < 0.001) was observed at all RBC concentrations. When exlPI was inactivated by MPO-H20z-halide the EIC increased from 0% of control value (untreated exlPI) in the case without RBC to 88 ± 9 at RBC concentration 1%. However, protection of exlPI from PMA-activated PMNL was slightly lower, EIC increased from 0% to 63 ± 8 for RBC concentration 1.5 % and it may be due to moderate release of proteases (not only elastase) from cells during stimulation. These proteases could inactivate some part of exlPI. It should be mentioned that incubation of exlPI alone with RBC caused loss of antielastase activity. For example, when exlPI was incubated with RBC at concentration 1.5 %, the EIC decreased by 8 % (mean value from 5 experiments). This decrease is probably related to exlPI binding to outer erythrocyte membrane (5) and was taken in account in above calculations. In order to determine mechanisms of the RBC protective action on exlPI these cells were incubated with H20 2 and aminotriazole - catalase inhibitor, chlorodinitrobenzene - GSH-GSH peroxidase system inhibitor, Ch-T - blocker of groups scavenging HClO. The effect of this treatment on the decomposition of H20 2 by RBC is shown in fig. 5. Inhibition of RBC catalase diminished H20 2 decomposition by RBC to 59% activity of untreated cells. Ch-T and chlorodinitrobenzene had 4*
Exp. Pathol. 42 (1991) 1
51
I,ZO
1.00 e c
O.eo
0
0> U'I
I-
«
0.60
UJ
~
III
Il'
a
(J)
III
«
Of/J
OJ!)
E~TIN.ORCEIN
DIGESTED (mg)
Fig.l. The dependence of absorbance at 590 nm upon the amount of elastin-orcein digested. Different amounts of elastin-orcein were added to 1.5 ml PBS containing 50 f.lg elastase and 2 mM sodium azide. After complete elastin solubilization the absorbance was measured against PBS with addition of sodium azide. The correlation coefficient r, calculated for the experimental points was 0.999, p < 001.
toO
U/
80
~
l-
S w 60 u. 0
z
0
H
I-
ItO
H
III H
:r
z
H
Department of Pneumology and Allergology, Medical Academy, L6dt, Poland
Erythrocytes protect a-I-proteinase inhibitor from oxidative inactivation induced by chemicals, the myeloperoxidase-H20 r haiide system and stimulated polymorphonuclear leukocytes By D. NOWAK and G. PIASECKA With 6 figures Received: June 20, 1989; Accepted: August 4, 1989
Address for correspondence: DARIUSZ NOWAK, Department of Pneumology and Allergology, Medical Academy, Kopcinskiego 22, 90-153 L6dt, Poland Keywords: erythrocyte; alpha-I-proteinase inhibitor; myeloperoxidase-H2 0z-halide system; leukocyte; oxidants
Summary The oxidative inactivation of the ex-I-proteinase inhibitor (exlPI) is one of the mechanisms responsible for creating the elastase/antielastase imbalance during inflammation in the lower respiratory tract. Chronic supremacy of elastase may cause degradation of elastin fibers leading to the pulmonary emphysema. In this study we have investigated the effect of erythrocytes (RBC, concentrations 0.125 to 1.5 %) on the oxidative inactivation of exlPI by the phorbol myristate acetate-stimulated polymorphonuclear leukocytes (PMNL) and PMNL myeloperoxidase-H 20 2halide system. During exposure of exlPI to both systems in the presence ofRBC the significant protection (p < 0.001) of ex 1PI was found for all RBC concentrations, e. g. at RBC concentration of! % the elastase inhibitory capacity (EIC) ofa: 1PIincreased from 0 to 60 ± 6and88 ± 9% of the control value (untreated a: 1PI) , n = 5, respectively. The preincubation ofRBC with chloramine-T (1 mM), inhibition of RBC catalase or depletion of RBC reduced glutathione alone did not diminish the capacity of RBC to protect a:1PI. However, these treatments together completely deprivedRBC of their protective properties. Moreover, we have compared the decrease in the EIC of human blood and its plasma after incubation with H 20 2 (0.1 mM to 0.1 M) or chloramine-T (l f.tM to 1 mM). For the incubation with H 20 2 no decrease in blood EIC was found whereas in plasma the loss of EIC was already visible at a H20 2 concentration of 0.1 mM. Also for the incubation with chloramine-T the EIC of blood was more resistant to oxidant damage than EIC of plasma. It is suggested that RBC contaminations present in the phagocyte inflammatory infiltration in the lower airways may protect a:lPI from oxidative inactivation and thus indirectly diminish proteolytic lung injury related to inflammation.
Exp. Pathol. 42 (1991) 1
47
Introduction The physiological activity of the ex-I-proteinase inhibitor (ex IPI), a major neutrophil elastase inhibitor in the lower airways, is highly susceptible to oxidative inactivation (6). This process consisting in the oxidation to the sulfoxide form of the methionine thioether residue at the active elastase inhibitory site of ex 1PI (21) causes almost total reduction of its elastase inhibitory capacity (6) and can create elastasel antielastase imbalance in the lung tissue. Thus, the elastase supremacy induced may lead to degradation of elastin fibers and as a consequence of this to the development of pulmonary emphysema. Various exogenic oxidants present in tobacco smoke (13), ozone (22), chlorine (29), nitrogen dioxide (25) may inactivate exlPI in the lower airways. Another source of oxidants in lung, which is probably more important, are phagocytic cells: macrophages and polymorphonuclear leukocytes (PMNL). These cells release large amounts of reactive oxidants after stimulation, which can inactivate (dPI (9, 37) and may occur during inflammation in the lower airways. There are various antioxidants protecting exlPI from oxidative damage in plasma and lung tissue. Recent studies showed that ceruloplasmin inhibits oxidative inactivation of exPI by the human PMNL myeloperoxidase system, chloramine-T and cigarette smoke solution (15, 32). Another enzyme, methionine sulfoxide-peptide reductase, was able to reactivate previously oxidized exlPI by treatment with chemicals or with enzymatic systems (10). Moreover, catalase and superoxide dismutase protected exlPI from activated phagocytes (9, 19,37). It is well known that erythrocytes (RBC) contain antioxidant systems including the last two enzymes mentioned above and glutathione peroxidase (17), which are able to protect not only their own structures but also cells and substances surrounding them (1, 27, 35). RBC can act as a hydrogen peroxide scavenger and inhibit the following generation of the hydroxyl radical or hypochlorous acid (33, 36). In cigarette smokers, these cells have increased catalase activity and contain more glutathione, and it may be one of the links of defense against oxidants present in cigarette smoke (35). Therefore, we have investigated the effect of RBC on PMNL- and myeloperoxidase-H20zhalide system-mediated oxidative inactivation of exlPI in vitro in this study. We have also compared the decrease in the elastase inhibitory capacity of blood and matched plasma induced by chloramine-T and H20 2 . Material and Methods Reagents
Elastin-orcein, peroxidase from horseradish type II (200 UImg solid); o-dianisidine dihydrochloride (o-DD), phorbol 12-myristate 13-acetate (PMA) , 1-chloro-2,4-dinitrobenzene, 3amino-1,2,4-triazole, phenylmethylsulfonylfluoride (PMSF) were obtained from Sigma (St. Louis, MO, USA). D-glucose, dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), heparin (preservative-free), Triton X-I 00 were from BDH England. Porcine pancreatic elastase (100 UI mg) was purchased from Merck. All other reagents used were of analytical grade. Elastinorcein was washed once, resuspended in methanol and 200-I-lI-portions containing 2 mg elastin were placed into Eppendorf tubes, dried and stored at 4°C until assay. PMA (10 I-lg/ml) in DMSO was stored at -20°C. PMSF (0.2 M) was dissolved in isopropanol. Elastase solution (0.25 mgl ml in PBS) was prepared freshly before the assay. Isolation of PMNL
Human polymorphonuclear leukocytes (PMNL) were isolated from blood obtained from healthy nonsmoking donors according to standard procedures (8). PMNL were washed once and resuspended to 107 per ml in PBS (PH 7.4 containing 0.9mM CaCl2 and 0.5 mM MgClz) with addition of glucose 1 gil. The purity ofPMNL suspensions and viability as assessed by trypan blue were always above 97 % .
48
Exp. Pathol. 42 (1991) 1
Preparation and treatment of RBC RBC were isolated from one young healthy donor by blood centrifugation at 1000 X g for 5 min, removal of plasma and leukocytes, washing 3 times with PBS and resuspending in PBS to 10% hematocrit. RBC catalase was inhibited by incubating a 10% cell suspension with 50 mM 3amino-l ,2,4-triazo1e and 4.5 mM H20 2 (36). RBC-reduced glutatione (GSH) was depleted by incubation of these cells (10 % suspension) with 2 mM 1-chloro-2,4-dinitrobenzene (3). Various groups on the RBC surface, which can scavenge hypochlorous acid (HClO), were blocked by addition of chloramine-T (Ch-T) 1 mM to 10% RBC suspension. In all cases, RBC were incubated for 1 h at 37°C and washed 3 times in PBS following exposure to these inhibitors and immediately used in experiments as a 20% suspension.
Measurement of RBC catalase The catalase activity in untreated and treated RBC was assessed by spectrophotometric determination of the decomposition of H2 0 2 at 230 nm by placing 2 ml PBS in the reference cuvette and 2 ml 10 mM H2 0 2 solution in PBS in the sample cuvette. This solution gives an absorbance of 0.816. Then 1 ftl hemolysate ofthe 10% RBC suspension was added to the sample cuvette, mixed thoroughly and the absorbance decrease was recorded over 3 min at room temperature. The amounts of H 20 2 decomposed were calculated from changes of absorbance using coefficientE = 81 cm -1 M- 1 (18), and the catalase activity was expressed as a percentage of the activity presented in untreated cells. RBC hemolysate was obtained by mixing 200 ftllO % cell suspension with 2ft11O% TritonX-100 in PBS.
Preparation of cx1PI Partially purified cx1PI was obtained from serum after chromatography on carboxymethyl cellulose (CM celulose 52 Whatman 1.0meq/g) (36).10 ml serum was acidified to pH 6.4 with HCl, diluted to 20ml with 0.01 M phosphate buffer pH 6.4 and then placed on the carboxymethylcellulose column (2 X 22 cm) and eluted with the same buffer. Eluted protein containing cxlPI was concentrated by precipitation with (NH4hS04 (75% saturation, 4°C), dialyzed overnight against three changes of PBS, divided into 200 ftl portions and stored at -20°C. This preparation contained 2.56mg (dPI per m!.
Preparation of the crude extract of myeloperoxidase (MPO) 3.75 X 108 PMNL resuspended in 10 ml PBS containing 0.1 % Triton x-100 and 5 mM PMSF were sonicated (Ultrasonic disintegrator UD-20 Techpan, Poland) at O°C at 80% of maximal power for 3 min and then centrifuged for 15 min at 15000 X g at 4°C. All subsequent centrifugations were carried out under these conditions. After placing in an ice bath the supernatant was treated with solid (NH4hS04 to yield 50 % saturaion, incubated for 30 min and centrifuged. The residue was discarded and the supernatant again treated with solid (NH4hS04 to increase the concentration to 65 % of saturation, incubated for 30 min at 0 °C and centrifuged. The obtained precipitate was dissolved in PBS, dialyzed overnight against 3 changes of PBS, divided into 100 [il portions and stored at - 20°C. This procedure is a modification of the initial part of the method for myeloperoxidase isolation (2). This MPO extract contained 1.84 fig protein per 1 [iI, had no elastase activity and 1 fig of this protein decomposed 3.62nmoles H 20 2 during 1 min incubation under conditions as for measurement of catalase. 1 [A.g MPO when mixed with 1 ml PBS containing 80 [ig/ml o-DD, 134 [A.M H 20 2 and 0.8 mg/ml BSA catalyzed H 20 r mediated o-DD oxidation (increase in absorbance at 470 nm) and was equivalent to 3.4mU horseradish peroxidase. BSA was added to protect oxidized o-DD precipitation at pH 7.4. 4
Exp. Pathol. 42 (1991) 1
49
Oxidation of (XIPI a) - oxidation of (XIPI with chemicals Heparinized (3 Vlml) human venous blood and plasma were divided into 300!J.I portions, mixed with 30 !J.I freshly prepared Ch-T and H 20 2 solutions, final concentrations 1 !J.M to 1 mM and 0.1 mM to 0.1 M, respectively. Control samples received 30!J.I PBS. After 1 h incubation at 37°C samples were centrifuged and supernatants assayed for elastase inhibitory capacity (EIC). b) - oxidation of (X1PI by myeloperoxidase-H 20 2-halide system 80!J.1 PBS, 2!J.I (X1PI solution and 1 !J.I MPO extract were mixed and then 50!J.I 0.2 mM H20 Z solution in PBS were added and the samples were quickly cooled to 0 °C after 20 min of incubation at 37°C, and the reaction was stopped with 20 !J.l1 % sodium azide. The following control systems were included: 1) (X1PI in PBS, 2) (XPI and MPO, 3) (X1PI and HzO z alone. c) - oxidation of (X1PI by PMA-stimulated PMNL 80!J.I PBS and 2!J.I (XIPI were mixed with 50!J.I PMA (lOng/ml)-stimulated PMNL suspension (5 x 105 cells), incubated for 15 min at 37°C, cooled to 0 °C, mixed with 20!J.I 1 % sodium azide and then centrifuged for 1 min at 10,000 x g at 4 °C. The number of cells and the time of incubation of (XIPI were based on our previous work (28). Control systems contained: 1) (X 1PI in PBS, 2) (X1PI and unstimulated PMNL (PMA omitted), 3) unstimulated PMNL in PBS, 4) stimulated PMNL in PBS.
Effect of RBC on (XIPI oxidative inactivation Various volumes of 20% RBC suspension were added to the aforementioned reaction mixtures (b and c) before addition of H Z0 2 and PMA-activated PMNL. Final RBS concentrations were 0.125 to 1.50%. Samples were incubated and centrifuged under conditions as described above. Since RBC bound (XIPI to its outer membrane (5) and may cause EIC decrease in supernatants, appropriate control systems «(XIPI and RBC) were included for all investigated cell concentrations. Previous experiments showed that the content of DMSO in samples related to PMNL stimulation (solvent for PMA) did not induce RBS hemolysis.
Determination of the elastase inhibitory capacity of (Xl PI Assays of (X1PI inactivated with the MPO-H 20z-halide system and oxidants derived from PMA-stimulated PMNL (b and c) were performed by the elastin-orcein test (7) with some modifications (30). 150!J.I of each supernatant containing (X1PI were mixed with 20!J.I elastase solution, incubated for 20 min at 37°C and then mixed with 2 mg of elastin-orcein resuspended in 1.33 ml PBS. The samples were incubated for 16 h at 37°C with gentle stirring. Elastase standards were prepared by mixing 20!J.I elastase solution with 130!J.I PBS and 20 !J.I 1 % sodium azide. Previous experiments showed that MPO alone and HZ0 2 concentrations used had no effect on elastase activity. The undigested substrate was centrifuged off and absorbance was read at 590 nm. Results were compared with the straight line calibration curve obtained with completely digested different amounts of elastin-orcein. 5.12!J.g native (untreated) (X1PI inhibited 0.52 activity of 5 !J.g elastase and it was taken for EIC calculations as 100%. Appropriate amounts of plasma treated with chemicals (a) were mixed with PBS and 20 !tIl % sodium azide to give final volume 150 fll and then their EIC was measured as described above. The volume of added plasma was equal the volume of untreated plasma which under this conditions inhibited 0.5 elastase activity (determined in previous experiments for plasma from all 3 donors). In order to check if oxidants added to plasma may inhibit elastase activity, plasma was replaced by BSA in PBS (50 mg/ml) , incubated for 1 hwithCh-T(l mM)orHzO z (0.1 M) and mixed with elastase. In this case no inhibition of elastase was found. 50
Exp. Pathol. 42 (1991) 1
Other techniques Serum exlPI, ceruloplasmin, ex2-macroglobulin and transferrin concentrations were determined by radial immunodiffusion in commercially prepared plates (M -Partigen-ex 1-Antitrypsin, M-Partigen-Coeruloplasmin, Protein-Standard-Plasma, Behring Institut) and by rocket immunoelectrophoresis with rabbit antiserum to human transferrin and to ex-2-macroglobulin (Biomed, Warsaw, Poland) and Standard-Human-Serum (Behring Institut). Protein concentration was measured using the method of LOWRY et al. (23) with bovine serum albumin as a standard. All individual determinations were carried out in duplicates. Typical blood examinations (red blood count, haemoglobin, hematocrit) were done using Technicon Haematology System 1 (Technicon Chemicals Company, Belgium).
Statistical analysis Results are expressed as the mean value ± the standard deviation. Statistical analyses were performed using paired t-statistic by comparison of the results obtained with exlPI exposed to oxidants and results of corresponding experiments where exlPI was protected by RBC.
Results Fig. I shows the ratio between absorbance at 590 nm and the amount of elastin-orcein digested. The curve is rectilinear in the whole range investigated (0-6.2 mg). 0.92 mg elastinorcein (as calculated from the standard curve) was solubilized after 16 h incubation of 2 mg substrate with 5 flg elastase and this quantity of elastin was used for EIC determination. The time course of exlPI inactivation by MPO-H20 2-halide system is shown in fig. 2. The functional activity of exlPI decreased rapidly with time and already after 20 min incubation no elastase inhibition was noted. This time of incubation was chosen for further experiments. MPO alone and H20 2did not inactivate exlPI (data not shown) and it is in agreement with our previous observations (28) where H20 2 concentrations below 0.15 mM had no influence on exlPI activity. The changes of EIC of blood and plasma from 3 various donors after treatment with Ch-T and H20 2 are presented in figs. 3 and 4. Incubation of blood with all H 20 2 concentrations did not diminish its EIC (fig. 3). However, in the case of plasma moderate decrease in elastase inhibition was observed especially for H202 concentration 0.1 M (p < 0.1). When blood was incubated with Ch-T the EIC was not significantly impaired up to oxidant concentration 0.1 mM (fig. 4). lO-fold higher Ch-T concentration caused severe decline of blood EIC (p < 0.01), which was approximate to that found for Ch-T treated plasma. The characteristics of blood samples used in the aforementioned experiments, e.g. hematocrit, red blood count, concentrations of haemoglobin, ex1PI, ex-2macroglobulin, ceruloplasmin, transferrin are presented in table 1. RBC protected exlPI from oxidative inactivation by the MPO-H20 2-halide system and PMA-stimulated PMNL (table 2). Highly significant protection (p < 0.001) was observed at all RBC concentrations. When exlPI was inactivated by MPO-H20z-halide the EIC increased from 0% of control value (untreated exlPI) in the case without RBC to 88 ± 9 at RBC concentration 1%. However, protection of exlPI from PMA-activated PMNL was slightly lower, EIC increased from 0% to 63 ± 8 for RBC concentration 1.5 % and it may be due to moderate release of proteases (not only elastase) from cells during stimulation. These proteases could inactivate some part of exlPI. It should be mentioned that incubation of exlPI alone with RBC caused loss of antielastase activity. For example, when exlPI was incubated with RBC at concentration 1.5 %, the EIC decreased by 8 % (mean value from 5 experiments). This decrease is probably related to exlPI binding to outer erythrocyte membrane (5) and was taken in account in above calculations. In order to determine mechanisms of the RBC protective action on exlPI these cells were incubated with H20 2 and aminotriazole - catalase inhibitor, chlorodinitrobenzene - GSH-GSH peroxidase system inhibitor, Ch-T - blocker of groups scavenging HClO. The effect of this treatment on the decomposition of H20 2 by RBC is shown in fig. 5. Inhibition of RBC catalase diminished H20 2 decomposition by RBC to 59% activity of untreated cells. Ch-T and chlorodinitrobenzene had 4*
Exp. Pathol. 42 (1991) 1
51
I,ZO
1.00 e c
O.eo
0
0> U'I
I-
«
0.60
UJ
~
III
Il'
a
(J)
III
«
Of/J
OJ!)
E~TIN.ORCEIN
DIGESTED (mg)
Fig.l. The dependence of absorbance at 590 nm upon the amount of elastin-orcein digested. Different amounts of elastin-orcein were added to 1.5 ml PBS containing 50 f.lg elastase and 2 mM sodium azide. After complete elastin solubilization the absorbance was measured against PBS with addition of sodium azide. The correlation coefficient r, calculated for the experimental points was 0.999, p < 001.
toO
U/
80
~
l-
S w 60 u. 0
z
0
H
I-
ItO
H
III H
:r
z
H
