Improved Method for Quantification of Tissue PMN Accumulation Measured by Myeloperoxidase Activity
CECILIASCHIERWAGEN, ANN-CHRISTIN BYLUND-FELLENIUS,AND CLAESLUNDBERG
Myeloperoxidase (MPO) was used as a marker enzyme for measuring polymorphonuclear leukocyte (PMN) accumulation in tissue samples. The MPO recovery from kidney, liver, myocardium, skeletal muscle (iliopsoas), and skin was measured, and a variation of 5%-100% was found, depending on the tissue analyzed. The recovery could be improved to 100% in all tissues by incubation of the tissue samples at 60°C for 2 hr. This improved method was used to measure PMN accumulation in rabbit myocardium after regional ischemia and reperfusion. The MPO activity increased fivefold in the occluded area as compared with intact myocardium. Treatment with IB4, a monoclonal antibody recognizing the leukocyte adhesion molecule Mac-l, decreased the MPO activity in the occluded area almost to the level in intact myocardium.
Key Words: Myocardial mutase
ABBREVIATIONS
Inflammation, Ischemia, Monoclonal antibodies, Myeloperoxidase, infarction, Polymorphonuclear leukocytes, Rabbits, Superoxide dis-
USED
C5a, complement product 5a; DMSO, dimethylsulfoxide; fMLF, formyl-methIFN-gamma, interferon-gamma; IL-l, interleukin-1; ionyl-leucyl-phenylalanine; LTB4, leukotriene B4; MPO, myeloperoxidase; PMN, polymorphonuclear leukocyte; r-h SOD, recombinant human superoxide dismutase; TMB, tetramethylbenzidine; TNF, tumor necrosis factor.
INTRODUCTION Accumulation of polymorphonuclear leukocytes (PMNs) is a characteristic event in the early stage of an acute inflammatory reaction. In the infarcted myocardium, which is one example of an acute inflammato~ reaction, PMNs accumulate within minutes to hours after coronary occlusion (Engler et al., 1986; Mullane et al., 1984). Although PMNs play an important role in the repair process, they may also contribute to destruction of potentially viable tissue. Observations have indicated, e.g.,
From the Depa~ment of Pharmaceutical Pharmacology (C. S.), Biomedical Center, Uppsala University, Sweden, and Department of inflammation Research (C. S., A.-C. B.-F., C. L.), Pharmacia LEO Therapeutics AB, Uppsala, Sweden Address reprint requests to: Claes Lundberg, Biomedical Research, Pharmacia LEO Therapeutics AB, S-751 82 Uppsala, Sweden. Received July 1989; revised and accepted September 1989. 179 Journal of Pharmacological Methods 0 1990
23,173-X16
(1990)
flsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY ICKllO
0160~5402/90/$3.50
180
C. Schierwagen et al. that accumulation of PMNs contributes to reperfusion-induced myocardial damage (Engler et al., 1983, Mullane et al., 1984; Romson et al., 1983; Simpson et al., 1988). To evaluate the role of PMNs in inflammatory diseases, it is necessary to be able to assess their accumulation in tissue samples with accuracy. Histologic techniques have been tried for this purpose (Romson et al., 1983; Simpson et al., 1988), but they have the disadvantage of being time-consuming, and quantification of the accumulation is difficult with these methods. In other studies, PMNs have been labeled with a radioactive isotope, but this has another major limitation-the method requires the cells to be taken out and labeled in vitro and then returned to the circulation, a procedure that could cause activation of the cells (Lundberg and Arfors, 1983; Romson et al., 1982). A frequently employed method is to use myeloperoxidase (MPO) as a marker enzyme for measuring PMN accumulation (Bradley et al., 1982; Crisham et al., 1986; Krawisz et al., 1984; Lundberg and Arfors, 1983; Mullane et al., 1985). This enzyme is essential for the oxygen-dependent bactericidal system of PMNs (Klebanoff, 1971). In man, the MPO content per neutrophil is greater than 5% of the dry weight (Schultz and Kaminker, 1962). Mononuclear leukocytes are known to have a low content of MPO, and this enzyme is therefore suitable as a marker for PMNs, as proposed by Bradley et al. (1982). However, MPO recovery from various tissues is known to be poor, and in many studies in which MPO has been used as a marker enzyme, the PMN accumulation has thus probably been underestimated (Ormrod et al., 1987). In this study, we have introduced a novel approach to improve the recovery of MPO in several tissues, for example the myocardium, thereby circumventing the problem with enzyme inhibition by the tissue. The improved method was applied for determination of MPO activity in rabbit hearts subjected to ischemia and reperfusion and treated with superoxide dismutase or with IB4, a monoclonal antibody directed toward the leukocyte adhesion complex, Mac-l. MATERIALS
AND METHODS
MPO Assay MPO activity was assayed by measuring the HZOz-dependent oxidation of 3,3’,5,5’-tetramethylbenzidine (TMB) (Suzuki et al., 1983). In its oxidized form, TMB has a blue color, which was measured spectrophotometrically at 650 nm. The reaction mixture for analysis consisted of 25 PL tissue sample, 25 FL TMB (final concentration 0.16 mM; Sigma, St. Louis, MO, USA) dissolved in dimethylsulfoxide (DMSO) and 200 FL H202 (final concentration 0.24 mM; Merck, Darmstadt, West Germany) diluted in 0.08 M phosphate buffer pH 5.4. The reaction was performed in a 96-well microtiter plate. The mixture was incubated for 5 min at 37°C and stopped with 25 FL bovine catalase (final concentration 13.6 pg/mL; Boehringer Mannheim GmbH, West Germany). To ensure linearity of the reaction during this time period, MPO standards (human leukocyte, myeloperoxidase, 0.004-0.5 U/mL; the Green Corporation, Osaka, Japan) were included in each assay. One unit of MPO activity was defined as the amount of enzyme reducing 1 pmol peroxide/min.
Improved Method for Measuring PMN Accumulation
MPO Recovery Rabbit tissue samples, 500 mg each, from the kidney, liver, myocardium, skeletal muscle (iliopsoas), and skin were excised and frozen (-20°C). The samples were thawed at room temperature and homogenized in 0.05 M potassium phosphate buffer, pH 6.0, containing 0.5% hexadecyltrimethylammonium bromide (HTAB; Fluka Chemic AC, Buchs, Switzerland) at 0°C for 15 sec. After centrifugation (1700 x g, 30 min, 4”C), 1 mL of the supernatant was removed and heated to temperatures ranging between 4 and 60°C for periods of 0 to 24 hr on a thermostated waterbath. The supernatant was again centrifuged (10,000 x g, 5 min, 4°C) and thereafter mixed with a known amount of MPO (=“exogenous”), and assayed for enzyme activity as described above.
INDUCTION
OF MYOCARDIAL
ISCHEMIA/REPERFUSION
IN RABBITS
The rabbit model for coronary artery occlusion followed by reperfusion described in detail by Downey et al. (1987) was used. Briefly, rabbits were anesthetized with pentobarbital (20 mg/kg), a tracheostomy was performed, and the animals were ventilated with a respirator. The blood pressure in the carotid artery was recorded. The heart was exposed by a left thoracotomy, and a diagonal branch of the left coronary artery was occluded for 45 min. The ischemic tissue was then reperfused by release of the ligature. Treatment was given by IV injection into the ear vein 10 min before reperfusion. After 3 hr of reperfusion, the heart was excised for MPO determination. One biopsy specimen was taken within the occluded area of the left ventricle affected (=occluded area), and another was obtained from the right ventricle (= nonoccluded area). The animals were divided into three groups and treated as follows: 1. Saline only (n = 6) 2. Recombinant human superoxide dismutase (r-h SOD), 15 mg/kg (Pharmacia AB, Uppsala, Sweden) (n = 6) 3. IB4, 2 mg/kg (from Dr. Samuel Wright, Rockefeller University, USA) (n = 6) In addition, a fourth group of normal, nonoperated, untreated animals (n = 6) was included (4). PMN accumulation in rabbit myocardium from the above four groups was determined by a modification of the method of Grisham et al. (1986), using MPO as a marker enzyme. Myocardial tissue (500 mg) was homogenized in 10 mL of 0.02 M potassium phosphate buffer, pH 7.4. One milliliter of the homogenate was centrifuged at 10,000 x g for 5 min at 4°C. The first supernatant was discarded. The pellet was then resuspended in 1 mL of 0.05 M potassium phosphate buffer (pH 6.0) containing 0.5% HTAB. The suspension was frozen and thawed once, sonicated in a Branson sonifier cell disruptor, incubated at 60°C for 2 hr in a water-bath, and then centrifuged once again at 10,000 x g for 5 min. The supernatant was assayed for MPO activity as described above.
181
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C. Schietwagen et al. Statistics
Data are presented as mean values + SD. For calculation of statistical significance, Student’s unpaired t test was used. In the incubation experiments using different temperatures (Figure I), statistical differences were evaluated using one-way analysis of variance (ANOVA) with a multiple range testing according to the method of least significant differences. A p value of ~0.05 was considered significant and is denoted in the figures with an asterisk. RESULTS MPO Recovery The recovery of a known amount of exogenously added MPO from myocardial homogenate after incubation of the homogenate at different temperatures and time periods was studied. As shown in Figure 1, the MPO recovery was only 65% when incubated at 4°C for 2 hr. However, after incubation of the homogenates at 60°C for 2 hr almost 100% of the exogenously added MPO was recovered. Incubation of the myocardial homogenates at 60°C for 2-24 hr resulted in complete MPO recovery. Pure MPO enzyme incubated at the same temperature and for the same length of time as the homogenates showed a slight decrease in activity with time, and had lost 2% of its activity at 2 hr and 17% at 24 hr, From these results, it was concluded that after incubation of tissue homogenates for 2 hr at 6O”C, the recovery was optimal, and this procedure was used in all subsequent experiments.
100
0 0
20
60
40 Temperature
(C “)
FIGURE 1. Recovery of exogenously added MPO from myocardial tissue incubated for 2 hr at temperatures ranging between 4 and 60°C. Mean + SD, n = 6 (at 6O”C, n = 12). Asterisks indicate a significant difference (p < 0.05) as compared to the 4°C value.
Improved Method for Measuring PMN Accumulation
Figure 2 shows the recovery of exogenously added MPO from homogenates of kidney, liver, myocardial, skeletal muscle tissue, and skin tissue. Only 5% of the exogenously added MPO was recovered from kidney homogenates at 4°C. However, after 2 hr of heating at 6O”C, the recovery was close to 100%. The MPO recovery from liver and skeletal muscle after a 2-hr incubation at 4°C was also low, but it increased to 100% after 2-hr of heating at 60°C. The MPO recovery from skin tissue was already 100% prior to heat incubation. MPO Activity in lschemic/Reperfused
Myocardium
MPO determination after incubation at 60°C for 2 hr was performed to quantify the PMN accumulation in ischemic-reperfused rabbit myocardium. After ligation of the left coronary artery for 45 min and reperfusion for 3 hr, the MPO activity was measured in a sample from the occluded area (the left ventricle) and the nonoceluded area in the right ventricle. For comparison, corresponding measurements were made in normal nonoperated animals. The results are presented in Figure 3. Myocardial tissue within the occluded area showed a fivefold increase in MPO activity as compared with intact myocardium from normal, nonoperated and untreated animals (p < 0.05). The effects of treatment with IB4, a monoclonal antibody recognizing the leukocyte adhesion molecule Mac-l, and r-hSOD (recombinant human superoxide dismutase), a superoxide radical scavenger administred IV prior to reperfusion of the ischemic myocardium, were also studied. Pretreatment with IB4 significantly decreased (p < 0.05) the myocardial MPO activity by 80%, but r-hSOD had no significant effect (Figure 3).
*
0 degr. H Kidney
Liver
Myocardium
Iliopsoas
60 degr.
Skin
FIGURE 2. Recovery of exogenously added MPO from kidney (n = 5), liver (n = 5), myocardium (n = 12), iliopsoas (n = 5), and skin (n = 5). The left and right bars represent MPO recovery before and after heat incubation (6O”C, 2 hr), respectively. Mean + SD. A significant increase (p < 6.05) in MPO recovery after heat incubation is indicated in the figure by an asterisk.
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non-occluded
q occluded
Control
r-hSOD
IB4
area
area
Intact Myocardium
FIGURE 3. Myocardial MPO activity in the nonischemic area (left bar) and ischemic area (right bar) in the four groups of animals: control (II = 6), r-hSOD-treated (n = 6) and IB4treated (n = 6) animals, and normal nonoperated untreated animals (intact myocardium) (n = 6). A significant increase (p < 0.05) in MPO activity was observed in the ischemic area of the control animals as compared with the same area of intact myocardium from nonoperated animals. IB4 treated animals showed a significant (p < 0.05) reduction in MPO activity in the ischemic area as compared with control treated animals (see asterisk). Mean 2 SD.
DISCUSSION
We have here demonstrated that the recovery of MPO activity in various tissues can be increased to 100% by the procedure of incubating the tissue homogenates at 60°C for 2 hr. The applicability of the method was illustrated by a fivefold increase in the MPO activity in infarcted myocardium, which could be prevented by pretreatment of the animals with the monoclonal antibody IB4. MPO is an enzyme that is located exclusively in PMNs and is therefore frequently used as a marker for quantifying PMN accumulation (Bradley et al., 1982; Grisham et al., 1986; Krawisz et al., 1984; Lundberg and Arfors, 1983; Mullane et al., 1985). For one of the substrates, TMB, an optimal final concentration of 0.16 mM was found to give an adequate color reaction without precipitation (data not shown). The optimal final concentration for the other substrate, H202, was found to be 0.24 mM and the pH-optimum was around 5.0 (data not shown). These results are in accordance with data published previously by Suzuki et al. (1983). The inhibition of exogenously added MPO activity by various tissues, and the large differences in MPO recovery between different organs found in this study, confirm previously reported findings (Ormrod et al., 1987). Only 5% of the MPO activity added to a kidney homogenate that had not been heat-inactivated could be recovered. In the liver, myocardium, and skeletal muscle the corresponding figures
Improved Method for Measuring PMN Accumulation
were 50,70, and 80% respectively. In the skin, on the other hand, 100% of the added MPO activity was recovered. MPO is an unusually heat-stable enzyme, and here we have utilized this specific feature of the enzyme as a tool to eliminate the activity of other factors that interfere with the MPO activity assay. The MPO recovery was 100% in all the tissues studied after incubation of their homogenates at 60°C for 224 hr, demonstrating that the factors interfering with the MPO enzyme were effectively eliminated by this procedure. Possible deleterious effects of this treatment on the MPO enzyme itself were also studied. A 17% decrease in the MPO activity was observed after 24 hr of incubation at 60°C. After incubation for periods of up to 6 hr, however, there was no appreciable loss of activity. An incubation time of 2 hr at 60°C can therefore be recommended for the tissues studied here. Measurement of MPO activity in tissue samples is complicated not only by the presence of factors inhibiting or masking the enzyme activity as discussed above, but also by the presence of other peroxidases, e.g., hemoglobin peroxidase, that can interfere with the assay by consuming H202 (Marklund, 1979). This problem can be circumvented by a two-step homogenization procedure as described by Grisham et al. (1986). In principle, the tissue is homogenized in a hypotonic buffer to achieve complete lysis of the cells, with the MPO-containing granule remaining intact. After centrifugation to sediment the granule, the supernatant, containing less than 5% of the total MPO activity, is discarded (Grisham et al., 1986). A detergent HTAB is then added to obtain lysis of the PMN granule and release of the MPO enzyme. The technique described earlier was used for measuring PMN accumulation in infarcted myocardium with use of MPO as a marker enzyme, followed by heat incubation of the final supernatant. A fivefold increase in MPO activity was found in the occluded area of the rabbit heart subjected to 45 min of ischemia and 3 hr of reperfusion, as compared with intact myocardial tissue. This is in accordance with the findings by Mullane et al. (1985) in the dog after 90 min of ischemia and 3 hr of reperfusion. In our study, pretreatment with r-hSOD had no significant effect on the MPO activity in the ischemic-reperfused myocardium. In intestinal ischemia in cats, however, treatment with SOD, a radical scavenger, decreased the mucosal MPO activity (Grisham et al., 1986). Animals treated with the monoclonal antibody IB4 showed significantly lower MPO activity in the occluded area than untreated animals. Earlier studies have shown that mabs recognizing Mac-l on the PMN cell surface membrane effectively reduce PMN accumulation in various in vivo inflammatory models (Arfors et al., 1987; Hernandez et al., 1987; Price et al., 1987; Simpson et al., 1988). In conclusion, we have demonstrated that heat incubation of tissue samples eliminates the problem of incomplete MPO recovery in myocardial, kidney, liver, and skeletal muscle tissue. Using this technique, PMN accumulation-e.g., in myocardial infarction-can be accurately measured with the use of MPO as a marker enzyme. This improved technique should provide a useful tool in future studies of acute inflammation. The authors are grateful to Ms. lnger Asberg for performing Catharina Wollert for preparation of the manuscript.
the myocardial infarction and to Mrs.
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C. Schierwagen et al.
REFERENCES Arfors KE, Lundberg C, Lindbom L, Lundberg K, Beatty PC, Harlan JM (1987) A monoclonal antibody to the membrane glycoprotein complex CD18 inhibits polymorphonuclear leukocyte accumulation and plasma leakage in vivo. Blood 69:338-340. Bradley PP, Priebat DA, Christensen RD, Rothstein G (1982) Measurement of cutaneous inflammation: Estimation of neutrophil content with an enzyme marker. / invest Dermato/78:206-209. Downey JM, Miura T, Eddy LJ, Chambers DE, Mellert T, Hearse DJ, Yellon DM (1987) Xanthine oxidase is not a source of free radicals in the ischemic rabbit heart. / MO/ Cell Cardio/19:10531060. Engler RL, Dahlgren MD, Peterson MA, Dobbs A, Schmid-Schonbein CW (1986) Accumulation of polymorphonuclear leukocytes during 3-h experimental myocardial ischemia. Am I fhysiol 251:93-100. Engler RL, Schmid-Schonbein GW, Pavelec RS (1983) Leukocyte capillary plugging in myocardial ischemia and reperfusion in the dog. Am 1 Pathol 111:98-111. Grisham MB, Hernandez LA, Cranger DN (1986) Xanthine oxidase and neutrophil infiltration in intestinal ischemia. Am / fhysiol251:567-574. Hernandez LA, Grisham MB, Twohig B, Arfors KE, Harlan JM, Granger DN (1987) Role of neutrophils in ischemia/reperfusion-induced microvascular injury. Am / Physio/253:699-703. Klebanoff SJ (1971) Intraleukocytic microbicidal defects. Ann Rev Med 22:39-62. Krawisz JE, Sharon P, Stenson WF (1984) Quantitative assay for acute intestinal inflammation based on myeloperoxidase activity Assessment of inflammation in rat and hamster models. Castroenterology 87:1344-1350. Lundberg C, Arfors KE (1983) Polymorphonuclear leukocyte accumulation in inflammatory dermal sites as measured by 5’Cr-labeled cells and myeloperoxidase. Inflammation 7~247-255. Marklund S (1979) A simple specific method for the determination of the hemoglobin content of tissue homogenates. C/in Chim Acta 92:229-234.
Mullane KM, Kraemer R, Smith B (1985) Myeloperoxidase activity as a quantitative assessment of neutrophil infiltration into ischemic myocardium. / Pharmacol Methods 14:157-167. Mullane KM, Read N, Salmon JA, Moncada S (1984) Role of leukocytes in acute myocardial infarction in anaesthetized dogs: relationships to myocardial salvage by antiinflammatory drugs. 1 Pharmacol Exp Ther 288:510-522. Nourshargh S, Hellewell PC, Rampart M, Jose PI, Harlan JM, Williams TJ (1987) Accumulation of polymorphonuclear leukocytes in allergic inflammation and inhibition by MOAB 60.3. Br I Pharmaco/90:56. Ormrod DJ, Harrison CL, MillerTE (1987) Inhibition of neutrophil myeloperoxidase activity by selected tissues. / Pharmacol Methods 18:137-142. Price TH, Beatty PG, Corpuz SR (1987) In vivo inhibition of neutrophil function in the rabbit using monoclonal antibody to CD18. J lmmunol 139:4174-4177. Romson JL, Hook BG, Kunkel SL, Abrams CD, Schork MA, Lucchesi BR (1983) Reduction of the extent of ischemic myocardial injury by neutrophil depletion in the dog. Circulation 67:10161023. Romson JL, Hook BG, Rigot VH, Schork MA, Swanson DP, Lucchesi BR (1982) The effect of ibuprofen on accumulation of indium-Ill-labelled platelets and leukocytes in experimental myocardial infarctions. Circulation 66:1002-1011. Schultz J, Kaminker K (1962) Myeloperoxidase of the leukocyte of normal human blood. I Content and localization. Arch Biochem Biophys 96:465467. Simpson PJ, Todd Ill RF, Fantone JC, Mickelson JK, Griffin JD, Lucchesi BR (1988) Reduction of experimental canine myocardial reperfusion injury by a monoclonal antibody (anti-MO 1, antiCDllb) that inhibits leukocyte adhesion. / C/in invest 81:624-629. Suzuki K, Ota H, Sasagawa S, Sakatani T, Fujikura T (1983) Assay method for myeloperoxidase in human polymorphonuclear leukocytes. Anal Biochem 132:345-352.
CECILIASCHIERWAGEN, ANN-CHRISTIN BYLUND-FELLENIUS,AND CLAESLUNDBERG
Myeloperoxidase (MPO) was used as a marker enzyme for measuring polymorphonuclear leukocyte (PMN) accumulation in tissue samples. The MPO recovery from kidney, liver, myocardium, skeletal muscle (iliopsoas), and skin was measured, and a variation of 5%-100% was found, depending on the tissue analyzed. The recovery could be improved to 100% in all tissues by incubation of the tissue samples at 60°C for 2 hr. This improved method was used to measure PMN accumulation in rabbit myocardium after regional ischemia and reperfusion. The MPO activity increased fivefold in the occluded area as compared with intact myocardium. Treatment with IB4, a monoclonal antibody recognizing the leukocyte adhesion molecule Mac-l, decreased the MPO activity in the occluded area almost to the level in intact myocardium.
Key Words: Myocardial mutase
ABBREVIATIONS
Inflammation, Ischemia, Monoclonal antibodies, Myeloperoxidase, infarction, Polymorphonuclear leukocytes, Rabbits, Superoxide dis-
USED
C5a, complement product 5a; DMSO, dimethylsulfoxide; fMLF, formyl-methIFN-gamma, interferon-gamma; IL-l, interleukin-1; ionyl-leucyl-phenylalanine; LTB4, leukotriene B4; MPO, myeloperoxidase; PMN, polymorphonuclear leukocyte; r-h SOD, recombinant human superoxide dismutase; TMB, tetramethylbenzidine; TNF, tumor necrosis factor.
INTRODUCTION Accumulation of polymorphonuclear leukocytes (PMNs) is a characteristic event in the early stage of an acute inflammatory reaction. In the infarcted myocardium, which is one example of an acute inflammato~ reaction, PMNs accumulate within minutes to hours after coronary occlusion (Engler et al., 1986; Mullane et al., 1984). Although PMNs play an important role in the repair process, they may also contribute to destruction of potentially viable tissue. Observations have indicated, e.g.,
From the Depa~ment of Pharmaceutical Pharmacology (C. S.), Biomedical Center, Uppsala University, Sweden, and Department of inflammation Research (C. S., A.-C. B.-F., C. L.), Pharmacia LEO Therapeutics AB, Uppsala, Sweden Address reprint requests to: Claes Lundberg, Biomedical Research, Pharmacia LEO Therapeutics AB, S-751 82 Uppsala, Sweden. Received July 1989; revised and accepted September 1989. 179 Journal of Pharmacological Methods 0 1990
23,173-X16
(1990)
flsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY ICKllO
0160~5402/90/$3.50
180
C. Schierwagen et al. that accumulation of PMNs contributes to reperfusion-induced myocardial damage (Engler et al., 1983, Mullane et al., 1984; Romson et al., 1983; Simpson et al., 1988). To evaluate the role of PMNs in inflammatory diseases, it is necessary to be able to assess their accumulation in tissue samples with accuracy. Histologic techniques have been tried for this purpose (Romson et al., 1983; Simpson et al., 1988), but they have the disadvantage of being time-consuming, and quantification of the accumulation is difficult with these methods. In other studies, PMNs have been labeled with a radioactive isotope, but this has another major limitation-the method requires the cells to be taken out and labeled in vitro and then returned to the circulation, a procedure that could cause activation of the cells (Lundberg and Arfors, 1983; Romson et al., 1982). A frequently employed method is to use myeloperoxidase (MPO) as a marker enzyme for measuring PMN accumulation (Bradley et al., 1982; Crisham et al., 1986; Krawisz et al., 1984; Lundberg and Arfors, 1983; Mullane et al., 1985). This enzyme is essential for the oxygen-dependent bactericidal system of PMNs (Klebanoff, 1971). In man, the MPO content per neutrophil is greater than 5% of the dry weight (Schultz and Kaminker, 1962). Mononuclear leukocytes are known to have a low content of MPO, and this enzyme is therefore suitable as a marker for PMNs, as proposed by Bradley et al. (1982). However, MPO recovery from various tissues is known to be poor, and in many studies in which MPO has been used as a marker enzyme, the PMN accumulation has thus probably been underestimated (Ormrod et al., 1987). In this study, we have introduced a novel approach to improve the recovery of MPO in several tissues, for example the myocardium, thereby circumventing the problem with enzyme inhibition by the tissue. The improved method was applied for determination of MPO activity in rabbit hearts subjected to ischemia and reperfusion and treated with superoxide dismutase or with IB4, a monoclonal antibody directed toward the leukocyte adhesion complex, Mac-l. MATERIALS
AND METHODS
MPO Assay MPO activity was assayed by measuring the HZOz-dependent oxidation of 3,3’,5,5’-tetramethylbenzidine (TMB) (Suzuki et al., 1983). In its oxidized form, TMB has a blue color, which was measured spectrophotometrically at 650 nm. The reaction mixture for analysis consisted of 25 PL tissue sample, 25 FL TMB (final concentration 0.16 mM; Sigma, St. Louis, MO, USA) dissolved in dimethylsulfoxide (DMSO) and 200 FL H202 (final concentration 0.24 mM; Merck, Darmstadt, West Germany) diluted in 0.08 M phosphate buffer pH 5.4. The reaction was performed in a 96-well microtiter plate. The mixture was incubated for 5 min at 37°C and stopped with 25 FL bovine catalase (final concentration 13.6 pg/mL; Boehringer Mannheim GmbH, West Germany). To ensure linearity of the reaction during this time period, MPO standards (human leukocyte, myeloperoxidase, 0.004-0.5 U/mL; the Green Corporation, Osaka, Japan) were included in each assay. One unit of MPO activity was defined as the amount of enzyme reducing 1 pmol peroxide/min.
Improved Method for Measuring PMN Accumulation
MPO Recovery Rabbit tissue samples, 500 mg each, from the kidney, liver, myocardium, skeletal muscle (iliopsoas), and skin were excised and frozen (-20°C). The samples were thawed at room temperature and homogenized in 0.05 M potassium phosphate buffer, pH 6.0, containing 0.5% hexadecyltrimethylammonium bromide (HTAB; Fluka Chemic AC, Buchs, Switzerland) at 0°C for 15 sec. After centrifugation (1700 x g, 30 min, 4”C), 1 mL of the supernatant was removed and heated to temperatures ranging between 4 and 60°C for periods of 0 to 24 hr on a thermostated waterbath. The supernatant was again centrifuged (10,000 x g, 5 min, 4°C) and thereafter mixed with a known amount of MPO (=“exogenous”), and assayed for enzyme activity as described above.
INDUCTION
OF MYOCARDIAL
ISCHEMIA/REPERFUSION
IN RABBITS
The rabbit model for coronary artery occlusion followed by reperfusion described in detail by Downey et al. (1987) was used. Briefly, rabbits were anesthetized with pentobarbital (20 mg/kg), a tracheostomy was performed, and the animals were ventilated with a respirator. The blood pressure in the carotid artery was recorded. The heart was exposed by a left thoracotomy, and a diagonal branch of the left coronary artery was occluded for 45 min. The ischemic tissue was then reperfused by release of the ligature. Treatment was given by IV injection into the ear vein 10 min before reperfusion. After 3 hr of reperfusion, the heart was excised for MPO determination. One biopsy specimen was taken within the occluded area of the left ventricle affected (=occluded area), and another was obtained from the right ventricle (= nonoccluded area). The animals were divided into three groups and treated as follows: 1. Saline only (n = 6) 2. Recombinant human superoxide dismutase (r-h SOD), 15 mg/kg (Pharmacia AB, Uppsala, Sweden) (n = 6) 3. IB4, 2 mg/kg (from Dr. Samuel Wright, Rockefeller University, USA) (n = 6) In addition, a fourth group of normal, nonoperated, untreated animals (n = 6) was included (4). PMN accumulation in rabbit myocardium from the above four groups was determined by a modification of the method of Grisham et al. (1986), using MPO as a marker enzyme. Myocardial tissue (500 mg) was homogenized in 10 mL of 0.02 M potassium phosphate buffer, pH 7.4. One milliliter of the homogenate was centrifuged at 10,000 x g for 5 min at 4°C. The first supernatant was discarded. The pellet was then resuspended in 1 mL of 0.05 M potassium phosphate buffer (pH 6.0) containing 0.5% HTAB. The suspension was frozen and thawed once, sonicated in a Branson sonifier cell disruptor, incubated at 60°C for 2 hr in a water-bath, and then centrifuged once again at 10,000 x g for 5 min. The supernatant was assayed for MPO activity as described above.
181
182
C. Schietwagen et al. Statistics
Data are presented as mean values + SD. For calculation of statistical significance, Student’s unpaired t test was used. In the incubation experiments using different temperatures (Figure I), statistical differences were evaluated using one-way analysis of variance (ANOVA) with a multiple range testing according to the method of least significant differences. A p value of ~0.05 was considered significant and is denoted in the figures with an asterisk. RESULTS MPO Recovery The recovery of a known amount of exogenously added MPO from myocardial homogenate after incubation of the homogenate at different temperatures and time periods was studied. As shown in Figure 1, the MPO recovery was only 65% when incubated at 4°C for 2 hr. However, after incubation of the homogenates at 60°C for 2 hr almost 100% of the exogenously added MPO was recovered. Incubation of the myocardial homogenates at 60°C for 2-24 hr resulted in complete MPO recovery. Pure MPO enzyme incubated at the same temperature and for the same length of time as the homogenates showed a slight decrease in activity with time, and had lost 2% of its activity at 2 hr and 17% at 24 hr, From these results, it was concluded that after incubation of tissue homogenates for 2 hr at 6O”C, the recovery was optimal, and this procedure was used in all subsequent experiments.
100
0 0
20
60
40 Temperature
(C “)
FIGURE 1. Recovery of exogenously added MPO from myocardial tissue incubated for 2 hr at temperatures ranging between 4 and 60°C. Mean + SD, n = 6 (at 6O”C, n = 12). Asterisks indicate a significant difference (p < 0.05) as compared to the 4°C value.
Improved Method for Measuring PMN Accumulation
Figure 2 shows the recovery of exogenously added MPO from homogenates of kidney, liver, myocardial, skeletal muscle tissue, and skin tissue. Only 5% of the exogenously added MPO was recovered from kidney homogenates at 4°C. However, after 2 hr of heating at 6O”C, the recovery was close to 100%. The MPO recovery from liver and skeletal muscle after a 2-hr incubation at 4°C was also low, but it increased to 100% after 2-hr of heating at 60°C. The MPO recovery from skin tissue was already 100% prior to heat incubation. MPO Activity in lschemic/Reperfused
Myocardium
MPO determination after incubation at 60°C for 2 hr was performed to quantify the PMN accumulation in ischemic-reperfused rabbit myocardium. After ligation of the left coronary artery for 45 min and reperfusion for 3 hr, the MPO activity was measured in a sample from the occluded area (the left ventricle) and the nonoceluded area in the right ventricle. For comparison, corresponding measurements were made in normal nonoperated animals. The results are presented in Figure 3. Myocardial tissue within the occluded area showed a fivefold increase in MPO activity as compared with intact myocardium from normal, nonoperated and untreated animals (p < 0.05). The effects of treatment with IB4, a monoclonal antibody recognizing the leukocyte adhesion molecule Mac-l, and r-hSOD (recombinant human superoxide dismutase), a superoxide radical scavenger administred IV prior to reperfusion of the ischemic myocardium, were also studied. Pretreatment with IB4 significantly decreased (p < 0.05) the myocardial MPO activity by 80%, but r-hSOD had no significant effect (Figure 3).
*
0 degr. H Kidney
Liver
Myocardium
Iliopsoas
60 degr.
Skin
FIGURE 2. Recovery of exogenously added MPO from kidney (n = 5), liver (n = 5), myocardium (n = 12), iliopsoas (n = 5), and skin (n = 5). The left and right bars represent MPO recovery before and after heat incubation (6O”C, 2 hr), respectively. Mean + SD. A significant increase (p < 6.05) in MPO recovery after heat incubation is indicated in the figure by an asterisk.
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non-occluded
q occluded
Control
r-hSOD
IB4
area
area
Intact Myocardium
FIGURE 3. Myocardial MPO activity in the nonischemic area (left bar) and ischemic area (right bar) in the four groups of animals: control (II = 6), r-hSOD-treated (n = 6) and IB4treated (n = 6) animals, and normal nonoperated untreated animals (intact myocardium) (n = 6). A significant increase (p < 0.05) in MPO activity was observed in the ischemic area of the control animals as compared with the same area of intact myocardium from nonoperated animals. IB4 treated animals showed a significant (p < 0.05) reduction in MPO activity in the ischemic area as compared with control treated animals (see asterisk). Mean 2 SD.
DISCUSSION
We have here demonstrated that the recovery of MPO activity in various tissues can be increased to 100% by the procedure of incubating the tissue homogenates at 60°C for 2 hr. The applicability of the method was illustrated by a fivefold increase in the MPO activity in infarcted myocardium, which could be prevented by pretreatment of the animals with the monoclonal antibody IB4. MPO is an enzyme that is located exclusively in PMNs and is therefore frequently used as a marker for quantifying PMN accumulation (Bradley et al., 1982; Grisham et al., 1986; Krawisz et al., 1984; Lundberg and Arfors, 1983; Mullane et al., 1985). For one of the substrates, TMB, an optimal final concentration of 0.16 mM was found to give an adequate color reaction without precipitation (data not shown). The optimal final concentration for the other substrate, H202, was found to be 0.24 mM and the pH-optimum was around 5.0 (data not shown). These results are in accordance with data published previously by Suzuki et al. (1983). The inhibition of exogenously added MPO activity by various tissues, and the large differences in MPO recovery between different organs found in this study, confirm previously reported findings (Ormrod et al., 1987). Only 5% of the MPO activity added to a kidney homogenate that had not been heat-inactivated could be recovered. In the liver, myocardium, and skeletal muscle the corresponding figures
Improved Method for Measuring PMN Accumulation
were 50,70, and 80% respectively. In the skin, on the other hand, 100% of the added MPO activity was recovered. MPO is an unusually heat-stable enzyme, and here we have utilized this specific feature of the enzyme as a tool to eliminate the activity of other factors that interfere with the MPO activity assay. The MPO recovery was 100% in all the tissues studied after incubation of their homogenates at 60°C for 224 hr, demonstrating that the factors interfering with the MPO enzyme were effectively eliminated by this procedure. Possible deleterious effects of this treatment on the MPO enzyme itself were also studied. A 17% decrease in the MPO activity was observed after 24 hr of incubation at 60°C. After incubation for periods of up to 6 hr, however, there was no appreciable loss of activity. An incubation time of 2 hr at 60°C can therefore be recommended for the tissues studied here. Measurement of MPO activity in tissue samples is complicated not only by the presence of factors inhibiting or masking the enzyme activity as discussed above, but also by the presence of other peroxidases, e.g., hemoglobin peroxidase, that can interfere with the assay by consuming H202 (Marklund, 1979). This problem can be circumvented by a two-step homogenization procedure as described by Grisham et al. (1986). In principle, the tissue is homogenized in a hypotonic buffer to achieve complete lysis of the cells, with the MPO-containing granule remaining intact. After centrifugation to sediment the granule, the supernatant, containing less than 5% of the total MPO activity, is discarded (Grisham et al., 1986). A detergent HTAB is then added to obtain lysis of the PMN granule and release of the MPO enzyme. The technique described earlier was used for measuring PMN accumulation in infarcted myocardium with use of MPO as a marker enzyme, followed by heat incubation of the final supernatant. A fivefold increase in MPO activity was found in the occluded area of the rabbit heart subjected to 45 min of ischemia and 3 hr of reperfusion, as compared with intact myocardial tissue. This is in accordance with the findings by Mullane et al. (1985) in the dog after 90 min of ischemia and 3 hr of reperfusion. In our study, pretreatment with r-hSOD had no significant effect on the MPO activity in the ischemic-reperfused myocardium. In intestinal ischemia in cats, however, treatment with SOD, a radical scavenger, decreased the mucosal MPO activity (Grisham et al., 1986). Animals treated with the monoclonal antibody IB4 showed significantly lower MPO activity in the occluded area than untreated animals. Earlier studies have shown that mabs recognizing Mac-l on the PMN cell surface membrane effectively reduce PMN accumulation in various in vivo inflammatory models (Arfors et al., 1987; Hernandez et al., 1987; Price et al., 1987; Simpson et al., 1988). In conclusion, we have demonstrated that heat incubation of tissue samples eliminates the problem of incomplete MPO recovery in myocardial, kidney, liver, and skeletal muscle tissue. Using this technique, PMN accumulation-e.g., in myocardial infarction-can be accurately measured with the use of MPO as a marker enzyme. This improved technique should provide a useful tool in future studies of acute inflammation. The authors are grateful to Ms. lnger Asberg for performing Catharina Wollert for preparation of the manuscript.
the myocardial infarction and to Mrs.
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REFERENCES Arfors KE, Lundberg C, Lindbom L, Lundberg K, Beatty PC, Harlan JM (1987) A monoclonal antibody to the membrane glycoprotein complex CD18 inhibits polymorphonuclear leukocyte accumulation and plasma leakage in vivo. Blood 69:338-340. Bradley PP, Priebat DA, Christensen RD, Rothstein G (1982) Measurement of cutaneous inflammation: Estimation of neutrophil content with an enzyme marker. / invest Dermato/78:206-209. Downey JM, Miura T, Eddy LJ, Chambers DE, Mellert T, Hearse DJ, Yellon DM (1987) Xanthine oxidase is not a source of free radicals in the ischemic rabbit heart. / MO/ Cell Cardio/19:10531060. Engler RL, Dahlgren MD, Peterson MA, Dobbs A, Schmid-Schonbein CW (1986) Accumulation of polymorphonuclear leukocytes during 3-h experimental myocardial ischemia. Am I fhysiol 251:93-100. Engler RL, Schmid-Schonbein GW, Pavelec RS (1983) Leukocyte capillary plugging in myocardial ischemia and reperfusion in the dog. Am 1 Pathol 111:98-111. Grisham MB, Hernandez LA, Cranger DN (1986) Xanthine oxidase and neutrophil infiltration in intestinal ischemia. Am / fhysiol251:567-574. Hernandez LA, Grisham MB, Twohig B, Arfors KE, Harlan JM, Granger DN (1987) Role of neutrophils in ischemia/reperfusion-induced microvascular injury. Am / Physio/253:699-703. Klebanoff SJ (1971) Intraleukocytic microbicidal defects. Ann Rev Med 22:39-62. Krawisz JE, Sharon P, Stenson WF (1984) Quantitative assay for acute intestinal inflammation based on myeloperoxidase activity Assessment of inflammation in rat and hamster models. Castroenterology 87:1344-1350. Lundberg C, Arfors KE (1983) Polymorphonuclear leukocyte accumulation in inflammatory dermal sites as measured by 5’Cr-labeled cells and myeloperoxidase. Inflammation 7~247-255. Marklund S (1979) A simple specific method for the determination of the hemoglobin content of tissue homogenates. C/in Chim Acta 92:229-234.
Mullane KM, Kraemer R, Smith B (1985) Myeloperoxidase activity as a quantitative assessment of neutrophil infiltration into ischemic myocardium. / Pharmacol Methods 14:157-167. Mullane KM, Read N, Salmon JA, Moncada S (1984) Role of leukocytes in acute myocardial infarction in anaesthetized dogs: relationships to myocardial salvage by antiinflammatory drugs. 1 Pharmacol Exp Ther 288:510-522. Nourshargh S, Hellewell PC, Rampart M, Jose PI, Harlan JM, Williams TJ (1987) Accumulation of polymorphonuclear leukocytes in allergic inflammation and inhibition by MOAB 60.3. Br I Pharmaco/90:56. Ormrod DJ, Harrison CL, MillerTE (1987) Inhibition of neutrophil myeloperoxidase activity by selected tissues. / Pharmacol Methods 18:137-142. Price TH, Beatty PG, Corpuz SR (1987) In vivo inhibition of neutrophil function in the rabbit using monoclonal antibody to CD18. J lmmunol 139:4174-4177. Romson JL, Hook BG, Kunkel SL, Abrams CD, Schork MA, Lucchesi BR (1983) Reduction of the extent of ischemic myocardial injury by neutrophil depletion in the dog. Circulation 67:10161023. Romson JL, Hook BG, Rigot VH, Schork MA, Swanson DP, Lucchesi BR (1982) The effect of ibuprofen on accumulation of indium-Ill-labelled platelets and leukocytes in experimental myocardial infarctions. Circulation 66:1002-1011. Schultz J, Kaminker K (1962) Myeloperoxidase of the leukocyte of normal human blood. I Content and localization. Arch Biochem Biophys 96:465467. Simpson PJ, Todd Ill RF, Fantone JC, Mickelson JK, Griffin JD, Lucchesi BR (1988) Reduction of experimental canine myocardial reperfusion injury by a monoclonal antibody (anti-MO 1, antiCDllb) that inhibits leukocyte adhesion. / C/in invest 81:624-629. Suzuki K, Ota H, Sasagawa S, Sakatani T, Fujikura T (1983) Assay method for myeloperoxidase in human polymorphonuclear leukocytes. Anal Biochem 132:345-352.
