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reperfusion
ejp European Journal of Pharmacology Environmental Toxicology and Pharmacology Section 292 (1994) 81-88
ELSEVIER
environmental toxicology and pharmacology
Assessment of myeloperoxidase activity in renal tissue after ischaemia/reperfusion D a v i d W . L a i g h t a,,, N a g i n L a d b, B r i a n W o o d w a r d
", J o h n F. W a t e r f a l l c
" Pharmacology Group, Bath UnirersiO', Clat,erton Down, Bath BA2 7AY, UK t, Roche Research Centre, 40 Broadwater Road, Welwyn Garden Ci~', Herts. A L7 3A Y, UK c Hoffman La-Roche Inc., 340 Kingsland Street, NJ 07110-1199, USA
Received 14 February 1994; revised MS received 6 April 1994; accepted 19 April 1994
Abstract
We have shown that a photometric assay of myeloperoxidase derived from rat blood polymorphonucleocytes employing 3,3',5,5'-tetramethylbenzidine as substrate is more sensitive than an established assay employing o-dianisidine. We went on to demonstrate that rat renal tissue is capable of inhibiting peroxidase activity. This activity approached 100% when the rat renal supernate was incubated at 60°C for 2 h and the assay was conducted in the presence of a 10-fold higher concentration of hydrogen peroxide (H202). Rat kidneys undergoing 45 min ischaemia and 1, 3 and 6 h reperfusion in vivo, exhibited significant increases in myeloperoxidase activity, indicating tissue polymorphonucleocyte accumulation. Monoclonal antibodies against rat intercellular adhesion molecule 1 (ICAM-1) and CD18 of/32-integrins administered both 5 min before a period of 45 min renal ischaemia (20/xg/kg i.v.) and at the commencement of 1 h reperfusion (20/~g/kg i.v.) reduced renal tissue polymorphonucleocyte accumulation. However, similar treatment with the parent murine antibody immunoglobulin G1 (IgG1) and an unrelated murine antibody, IgG2a, also significantly reduced renal tissue polymorphonucleocyte accumulation. In conclusion, we demonstrate that the rat renal suppression of peroxidase activity can be overcome by a combination of heat inactivation and the provision of excess assay H20 2. In addition, the available evidence suggests that murine monoclonai antibodies against rat adhesion molecules may exert non-specific actions in our model of renal ischaemia/reperfusion in vivo.
Keywords: Myeloperoxidase; Tetramethylbenzidine; Polymorphonucleocyte; Renal ischemia/reperfusion; Adhesion molecule
I. Introduction
Myeloperoxidase, a characteristic constituent of polymorphonucleocytes (Bradley et al., 1982a; Schultz and Kaminker, 1962), has been used as a biochemical marker for the tissue content of polymorphonucleocytes. The established photometric assay of myeloperoxidase employs o-dianisidine as the hydrogen donor for H 2 0 2 (Bradley et al., 1982b). However, tetramethylbenzidine may be used as a non-carcinogenic alternative hydrogen donor for the determination of peroxidase (Bos et al., 1981; Andrews and Krinsky, 1981; Holland et al., 1974) and can be shown to provide a more sensitive assay of human myeloperoxidase than o-dianisidine (Suzuki et ai., 1983). In an attempt to
* Corresponding author. Tel.: 44-0225-826826, ext. 5779; Fax: 440225-826114. 0926-6917/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 9 2 6 - 6 9 1 7 ( 9 4 ) 0 0 0 2 3 - 9
extend these findings, the sensitivities of tetramethylbenzidine and o-dianisidine assays were directly compared in an assay of myeloperoxidase derived from rat blood polymorphonucleocytes. Polymorphonucleocyte infiltration as assessed by tissue myeloperoxidase assay has been shown in numerous tissues including the inflamed rat dermis (Bradley et al., 1982b), the ischaemic/reperfused dog and rabbit myocardium (Mullane et al., 1985; Schierwagen et al., 1990) and the ischaemic/reperfused rat intestine (Grisham et al., 1986; Krawisz et al., 1984). A potential problem concerned with the assessment of myeloperoxidase in renal tissue is that peroxidase activity may be effectively inhibited or masked by renal tissue factors (Schierwagen et al., 1990; Hillegas et al., 1990; Ormrod et al., 1987). Schierwagen et al. (1990) have speculated that these factors could potentially include H 2 0 2 consuming systems. The initial aim of the present study was to employ measures that would overcome these
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peroxidase inhibitory factors. To this end, the recovery of exogenous horseradish peroxidase activity added to rat renal tissue was assessed as an index of inhibition. The validity of these measures was demonstrated by delineating a time course of polymorphonucleocyte accumulation after 45 min renal ischaemia in vivo. It was of further interest to attempt to modulate renal polymorphonucleocyte accumulation using recently available murine monoclonal antibodies against rat adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and CD18 of /32-integrins (Tamatani and Miyasaki, 1990).
2. Materials and methods
2.1. Isolation of rat blood polymorphonucleocytes Blood collected into heparin (10 U / m l ) from male CD Wistar rats anaesthetised with pentobarbital sodium (60 mg/kg i.p.) was carefully layered onto Ficoll-Paque lymphocyte separation medium and spun at 300 x g for 30 min. The lower reddish layer was mixed with an equal volume of 1.8% dextran in pH 7.4 phosphate buffered saline (PBS) and left to sediment for 40 min. The resultant upper layer was spun at 200 x g for 10 min and the pellet resuspended in first 5.8 ml 0.21% NaCI and then 0.5 ml 1 M KCI to lyse remaining erythrocytes. After washing in PBS, viable cells were enumerated in a Neubauer haemocytometer following dilution with 0.1% trypan blue. Differential cell counts indicated the yield of polymorphonucleocytes to be routinely > 90%. Samples were diluted in PBS to provide 106 polymorphonucleocytes/ml. 2.2. Extraction of myeloperoxidase from rat blood polymorphonucleocytes Aliquots of polymorphonucleocytes (lOS-lO ~) were transferred to vials and the volume subsequently made up to 5 ml with hexadecyltrimethylammonium bromide in 50 mM phosphate buffer pH 6.0 to give a final concentration of 0.5%. Polymorphonucleoeyte suspensions were then disrupted for 30 s using a Branson sonieator at 20% power and subsequently snap frozen in liquid nitrogen and thawed on three consecutive occasions before a final 30 s sonieation. Polymorphonueleoeyte suspensions were then incubated at 60"C for 2 h (vide infra) to verify the heat resistance of myeloperoxidase (see Sehierwagen et al., 1990). Incubated polymorphonueleoeyte suspensions were spun at 4000 × g for 12 rain and the myeloperoxidase-rieh renal supemate collected and stored at - 2 2 " C until assay.
2.3. Assay of myeloperoxidase from rat blood polymorphonucleocytes Myeloperoxidase was assayed photometrically in a 96-well plate incubated at 37°C using a Dynatech MR7000 plate reader controlled by a Toshiba 1000 microprocessor. The tetramethylbenzidine assay of myeloperoxidase was essentially as described by Suzuki et al. (1983). The assay mixture consisted of 20 /zl myeloperoxidase-rich supernate, 10/zl tetramethylbenzidine (final concentration 1.6 mM) dissolved in dimethylsulphoxide (DMSO) and 70 /zl H 2 0 2 (final concentration 0.3 mM) diluted in 80 mM phosphate buffer pH 5.4. The o-dianisidine assay of myeloperoxidase was essentially as described by Bradley et al. (1982b). The assay mixture consisted of 20 /zl myeloperoxidase-rich supernate and 80/xl H 2 0 2 (final concentration 0.147 mM) diluted in 50 mM phosphate buffer pH 6.0 containing o-dianisidine dihydrochloride (final concentration 0.526 mM). The reaction was initiated by the addition of H 2 0 2 and the increase in absorbance was determined over 3 min at 30 s intervals at either 630 nm (tetramethylbenzidine assay) or 460 nm (o-dianisidine assay).
2.4. Recovery of exogenous horseradish peroxidase activity from renal supernate As an index of the inhibition of peroxidase activity by renal tissue, the recovery of exogenous horseradish peroxidase activity was assessed from renal supernate derived from non-ischaemic rat kidneys in a photometric assay using tetramethylbenzidine. The activity of exogenous horseradish peroxidase in the presence of 0.5% hexadecyltrimethyiammonium bromide was defined as 100% recovery. In an attempt to enhance the recovery of exogenous horseradish peroxidase activity from renal supernate, the renal supernate was heat-inactivated by incubating at 60°C for 2 h in a water bath (Schierwagen et al., 1990) a n d / o r the assay H202 concentration was raised 10-fold. The assay mixture consisted of 10 p,l tetramethylbenzidine (final concentration 1.6 mM) dissolved in DMSO, 50/zl H202 (final concentration 0.3 or 3.0 raM) diluted in 80 mM phosphate buffer pH 5.4, 20 /zl heat-inactivated or untreated renal supernate or 20 /~l 0.5% hexadecyltrimethyiammonium bromide (as control) and 20/zl horseradish peroxidase (final concentration 8.7-43.3 p,g/1). Horseradish peroxidase activity was expressed as the initial rate of change in absorbance (SA/min) per unit concentration of enzyme (1 mg/l).
2.5. Rat model of renal ischaemia / reperfusion Male CD Wistar rats (300-400 g) were anaesthetised with pentobarbital sodium (60 m g / k g i.p.) and
D.W. Laight et al. / Eur. J. Pharmacol. Enciron. Toxicol. Pharmacol. Section 292 (1994)81-88
the body temperature maintained at 37°C by a warming pad. Both kidneys were exposed via flank incisions and kept moist with warmed .0.9% saline. After the administration of heparin (200 U / k g i.v.), the left renal pedicle was occluded by an atraumatic vascular clamp for 45 min. Reperfusion was elected for 0.5-6 h by removal of the clamp and visually confirmed before postischaemic and sham-operated kidneys were restored to the body cavity and the wounds sutured. Buprenorphine hydrochloride (0.5 mg/kg s.c.) was administered to provide post-operative analgesia. Renal reperfusion was terminated by cervical dislocation of the animal. Kidneys were then excised, blotted dry and weighed. Kidneys were subsequently immersed in 0.5% hexadecyltrimethylammonium bromide in 50 mM phosphate buffer pH 6.0 and stored at - 2 2 ° C until myeloperoxidase extraction and assay.
2.6. Renal myeloperoxidase extraction Frozen rat kidneys were thawed at room temperature and macerated with an Ultra-Turrax T25 blender for 30 s. Sample volumes were then made up to 10 ml with 0.5% hexadecyltrimethylammonium bromide before disruption and freeze-fractionation as described for the extraction of myeloperoxidase from rat blood polymorphonucleocytes. Samples were incubated at 60°C for 2 h in a water bath and then spun at 4000 × g for 12 min. The supernate was collected for myeloperoxidase assay.
2. 7. Renal myeloperoxidase assay Renal myeloperoxidase activity was assessed photometrically at 630 nm as described for the assessment of rat blood polymorphonucleocyte myeloperoxidase activity using tetramethylbenzidine as substrate. The assay mixture consisted of 20 ~l heat-inactivated renal supernate, 10 /~l tetramethylbenzidine (final concentration 1.6 mM) dissolved in DMSO and 70 p,l H202 (final concentration 3.0 mM) diluted in 80 mM phosphate buffer pH 5.4. Renal myeloperoxidase activity was expressed in units (U) where 1 U represents that amount of enzyme degrading 1/zmol H 2 0 2 / m i n under the stated conditions. Myeloperoxidase activity was standardised with respect to the wet weight of the sham-operated kidney (U/g).
2.8. Effect of monoclonal antibodies against polymorphonucleocyte adhesion on renal myeloperoxidase activity Murine anti-rat intercellular adhesion molecule-1 (ICAM-1) or murine anti-rat CD18 monoclonal antibody was administered to animals both 5 rain before 45 min renal isehaemia (20 ~ g / k g i.v.) and at the corn-
83
mencement of 1 h reperfusion (20/~g/kg i.v.) in vivo. The parent murine antibody, immunoglobulin G1 (IgG1) or an unrelated murine antibody, IgG2a, was also administered in an identical manner to serve as controls. Vehicle control animals received 0.9% saline (0.5 ml/kg i.v.) at the appropiate time points. Shamoperated contralateral kidneys provided control preparations for ischaemic/reperfused kidneys.
2.9. Effect of lgG1 on recovery of horseradish peroxidase activity from renal supernate In order to examine the possibility that murine antibodies might directly affect tissue peroxidase activity, the recovery of exogenous horseradish peroxidase activity was assessed from renal supernate derived from non-ischaemic kidneys exposed to murine IgG1 (40 t~g/kg i.v. total) in vivo. The activity of horseradish peroxidase in the presence of 0.5% hexadecyltrimethylammonium bromide was defined as 100% recovery.
2.10. Fluorescence-activated cell sorting (FACS) analysis Aliquots of rat blood polymorphonucleocytes (0.5 x 106 cells) were suspended in 50/zl PBS/0.2% sodium azide together with 4 tzg of anti-ICAM-1 or anti-CD18 monoclonal antibody or murine IgG1 or IgG2a antibody and incubated at - 4 ° C for 30 min. Cell suspensions were then washed twice in 1 ml PBS/0.2% sodium azide and resuspended in 50 /~1 PBS/0.2% sodium azide together with 0.5/zg fluorescently labelled bovine anti-murine IgG R-phycoerythrin conjugate F(ab') 2 fragment and incubated at - 4 ° C for 30 min. Cells were then washed twice and finally resuspended in PBS/0.2% sodium azide and stored at - 4 ° C until FACS analysis.
2.11. Materials Agents used were murine anti-rat ICAM-1 and murine anti-rat LFA-1/3 chain (CD18) monoclonal antibodies (British Biotechnology), murine IgG1, murine IgG2a and bovine anti-murine IgG R-phycoerythrin conjugate F(ab') 2 fragment (Sigma Immunochemieals), trypan blue, hydrogen peroxide and dextran (250 kDa) (British Drug House), sterile 0.9% saline for injection (Fresenius), buprenorphine hydroehloride (Reekitt and Colman), pentobarbital sodium (May and Baker), Ficoll-Paque (Pharmacia), 3,3',5,5'-tetramethylbenzidine, o-dianisidine dihydrochloride (3,3'-dimethoxybenzidine dihydrochloride), horseradish peroxidase (Type II), dimethylsulphoxide, sodium azide and hexadeeyllximethylammonium bromide (Sigma). 3,3',5,5'-tetramethylbenzidine was dissolved in absolute dimethylsulphoxide to give a concentration of 16 mM which was then diluted in the assay to the working concentration of 1.6
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mM. Dimethylsulphoxide was therefore present at a final concentration of 10% (v/v).
2.12. Statistics
6A/mln/mg/I Horserodish Peroxldose 40.0 35.0
The comparison of two group means was made using unpaired Student's t test while the comparison of a control group mean with each of a number of treatment group means was conducted using Dunnett's test (Wallenstein et al., 1980). The comparison of slopes of linear regression lines was conducted using Student's t test for parallelism (Tallarida and Murray, 1981). Values presented are means and standard error of the mean (S.E.M.) of n observations. Statistical significance was accepted at the 5% level.
30.0 25.0 20.0
15.0. 10.0.
~
~*
5.0. ¢t
0.0
"=" 0.3
3.0 Hydrogen Peroxide (raM)
3. Results
3.1. Assay of myeloperoxidase derived from rat blood polymorphonucleocytes Myeloperoxidase activity from rat blood polymorphonucleoeytes was linearly correlated with the number of polyrnorphonucleoeytes from which myeloperoxidase was extracted in either an assay using tetramethylbenzidine (r = 1.000, P < 0.01, n -- 5) or o-dianisidine (r -- 0.998, P < 0.01, n = 5) (Fig. 1). However, the tetramethylbenzidine assay of polymorphonucleocytederived myeloperoxidase was 3-fold more sensitive than the o-dianisidine assay (51.83 + 1.56 6 A / m i n per 106 polyrnorphonucleocytes compared to 18.08 + 1.07 8 A / r a i n per 106 polymorphonucleocytes, P < 0.01, n --5, Student's t test). The more sensitive tetramethyl-
dA/min i*
60.0"
40.0"
20.0.
I 0.0
1
3
10
Polyrnorphonucleocytes (x 105) Fig. 1. Myeloperoxidase activity derived from rat blood polymorphonucleocytes assessed as the initial rate of change of absorbance (SA/min) using 3,3',5,5'-tetramethylbenzidine (solid line) (n = 5) or o-dianisidine (dashed line) (n = 5) assays.Values are means (S.E.M.). *, P < 0.05 with respect to o-dianisidine myeloperoxidase assay.
Fig. 2. Horseradish peroxidase activity assessed as the initial rate of change of absorbance ( 6 A / m i n ) per unit concentration of enzyme in the presence of 0.5% hexadecyltrimcthylammonium b r o m i d e / 5 0 mM pH 6.0 phosphate buffer (control, open bars) (n = 4) or in the presence of renal supernate either incubated at 60°C for 2 h (crosshatched bars) or unincubated (closed bars) (n = 4): effects of 0.3 or 3.0 mM hydrogen peroxide in assay. Values are means (S.E.M.). '~, P < 0.01 with respect to appropriate control value.
benzidine assay was therefore adopted in subsequent studies.
3.2. Recovery of horseradish peroxidase activity from renal supernate The initial rate of increase of absorbance at 630 nm was linearly correlated with the concentration of horseradish peroxidase in the assay (8.7-43.3 ~g/1). The activity of exogenous horseradish peroxidase added to renal supernate derived from non-ischaemic rat kidneys was significantly depressed ( P < 0.01, n = 4, Student's t test) compared to the activity of exogenous horseradish peroxidase activity added to an equal volume of 0.5% hexadecyltrimethylammonium bromide (Fig. 2) and represented a recovery of only 9% (i.e., 91% inhibition). The activity of exogenous horseradish peroxidase added to renal supernate incubated at 60°C for 2 h was still significantly depressed ( P < 0.01, n = 4, Student's t test). Similarly, when H 2 0 2 was employed at a 10-fold higher concentration in the assay (3.0 mM), the activity of exogenous horseradish peroxidase added to untreated renal supernate remained significantly reduced ( P < 0.01, n = 4, Student's t test) compared to the appropriate control. However, when H 2 0 2 was present in the assay at 3.0 mM, the activity of exogenous horseradish peroxidase added to heat-inactivated renal supernate was not significantly different from the appropriate control activity. No myeloperoxidase activity could be detected in heat-inactivated non-ischaemic
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D. IV. Laight et aL / Eur. J. Pharmacol. Environ. Toxicol. Pharmacol. Section 292 (1994) 81-88
Renal wet weight (~)
Renal myeloperoxidase (U/g)
150.0-
3.5¢r
¢r
3.0¢r
2_
140.0-
2.5130.0-
2.0-
¢r
1.5-
120.0-
1.0110.0 0.5J 0.0
0.5
100.0 3
I
0.5
Reperfusion period (h)
1
1
3
Reperfusion period (h)
Fig. 3. Rat renal myeloperoxidase activity after 45 min ischaemia and various periods of reperfusion in vivo expressed per unit wet weight of the sham-operated kidney. Values are means (S.E.M.) (n = 6). *, P < 0.05 with respect to zero activity in sham-operated kidneys.
Fig. 4. Wet weight of rat kidneys after 45 rain ischaemia and various periods of reperfusion in vivo as a percentage of the wet weight of sham-operated kidneys. Values are means (S.E.M.) (n = 6). *, P < 0.05 with respect to 100%.
renal supernate per se in the absence of exogenous horseradish peroxidase.
addition, identical treatment with the parent murine IgG1 or unrelated murine IgG2a antibody produced a similar effect. In contrast, myeloperoxidase activity in kidneys undergoing 45 min ischaemia and 1 h reperfusion in animals receiving vehicle alone in vivo (0.5 ml/kg i.v. 0.9% saline) was not significantly affected. Myeloperoxidase activity in sham-operated kidneys was undetectable in these experiments. The recovery of exogenous horseradish peroxidase activity from renal supernate derived from non-ischaemic kidneys exposed
3.3. Renal myeloperoxidase activity after ischaemia / reperfusion When renal supernate was assayed under conditions where the recovery of exogenous horseradish peroxidase activity was near 100%, i.e., 2 h incubation at 60"C and 3.0 mM assay H202, kidneys undergoing 45 rain ischaemia and 1, 3 or 6 h reperfusion (but not 0.5 h reperfusion) in vivo showed significant myeloperoxidase activity, indicating polymorphonucleocyte accumulation (Fig. 3). Myeloperoxidase activity in sham-operated kidneys was undetectable. In preliminary experiments where renal supernate was not heat-inactivated and the assay concentration of H 2 0 2 was 0.3 mM, myeloperoxidase activity was undetectable in rat kidneys undergoing 45 min ischaemia and 1 or 3 h reperfusion. The wet weight of kidneys undergoing 45 rain ischaemia and 0.5-6 h reperfusion in vivo was significantly increased as a percentage of the wet weight of sham-operated kidneys (Fig. 4). Left kidney wet weight was 99.8 + 2.5% of the right kidney wet weight in a random sample of animals (n = 6).
3.4. Effect of antibodies on renal myeloperoxidase activity after ischaemia /reperfusion Treatment of animals with anti-ICAM-1 or antiCD18 monoelonal antibody ( 2 0 / z g / k g i.v. both 5 rain before isehaemia and at the commencement of reperfusion) was associated with significantly reduced myeloperoxidase activity in kidneys undergoing 45 rnin isehaemia and 1 h reperfusion in vivo (Fig. 5). In
Renal rnyeloperoxidose ( U / g )
2.0-
1.5-
1.0~r
o5
O.0
*
no
intervention
saline
~
*
-
CD18
~
antiICAM- I
IgGI
•
IgO2o
Fig. 5. Effects of monoclonal antibodies q a i n s t rat CD18 of/~2.integrins and rat intercellular adhesion molecule 1 (ICAM-1) or the parent murine antibody immunoglobulin G t (IgG1) and an unrelated murine IgG2a antibody or 0.9% saline vehicle, on myelopormddase activity in rat kidneys undergoing 45 min isehaemia and I h r e i ~ f u sion in vivo. Antibodies were administered both5 min before 46 isehaemia (20 ttg/k8 iv.) and at the commencement .of renerfusion (20/zg/k8 i.v.). 0.9% saline vehicle was administered ~ r ~ LV.) at identical time points. Values are means (S.E,M:) ( n = ~, *, P < 0.05 with respect to n o intervention.
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D.W. Laight et al./ Eur. J. Pharmacol. Environ. Toxicol. Pharmacol. Section 292 (1994) 81-88
to murine IgG1 (40 /zg/kg i.v. total) in vivo was not significantly different from 100%. Furthermore, FACS analysis showed that of the antibodies studied, only anti-CD18 monoclonal antibody was capable of binding to rat blood polymorphonucleocytes. In addition, antibody interventions (40/~g/kg i.v. total) did not significantly affect increases in the wet weight of rat kidneys undergoing 45 min ischaemia and 1 h reperfusion in vivo.
4. Discussion
The present findings agree with those of Suzuki et al. (1983) that a tetramethylbenzidine assay can provide a viable, safe alternative to the established o-dianisidine assay of myeloperoxidase. The difference in sensitivity between tetramethylbenzidine and o-dianisidine assays cannot be accredited to the higher concentration of H 2 0 2 in the case of the tetramethylbenzidine assay since when o-dianisidine was substituted for tetramethylbenzidine in an assay of horseradish peroxidase, the substituted o-dianisidine assay proved similarly less sensitive (personal observation). A corollary of the enhanced sensitivity of the described tetramethylbenzidine assay of myeloperoxidase is that the threshold of detection is lowered. This theoretically provides for the assessment of early polymorphonueleocyte accumulation during reperfusion after ischaemia which might be overlooked using a less sensitive o-dianisidine assay. The masking of exogenous horseradish peroxidase activity by rat renal supernate indicates the presence of rat renal tissue factors that interfere with the peroxidase assay. Hillegas et al. (1990) have suggested that such factors may include conjugates of kidney-derived protein and the detergent hexadecyltrimethylammonium bromide, used to solubilise myeloperoxidase. The level of inhibition (91%) exhibited by rat renal tissue assessed by the recovery of added horseradish peroxidase activity, was comparable to that reported by Sehierwagen et al. (1990) (88%) measuring the recovery of exogenous human myeloperoxidase activity in the rabbit kidney. However, in contrast to the findings of Sehierwagen et al. (1990) in the rabbit kidney, the rat renal inhibition of exogenous peroxidase activity was not abolished by heat inactivation alone. In fact, the level of rat renal inhibition remained significantly hi'gh at 49% after the rat renal supernate had been incubated at 60"C for 2 h. The discrepancy between studies suggests the existence of both heat labile and heat stable interfering factors in rat renal tissue. The 'observation that rat renal inhibition was effectively abolished by the provision of excess peroxide to heati n s t a t e d rat renal supernate implies that the proposed heat stable factors are peroxide consuming sys-
tems. A conceivable role for such systems was postulated by Schierwagen et al. (1990). It is unlikely that the systems are themselves peroxidase-like since no activity was detected in the renal supernate of nonischaemic rat kidneys per se. Hence, tissue catalase-like activity may be responsible. However, one prime candidate, the heat stable enzyme catalase, can probably be excluded since DMSO at 10% v/v, which was present in all tetramethylbenzidine assays, has been reported to abolish catalase activity in vitro (Rammler, 1967). Myeloperoxidase is an unusually heat-stable enzyme. Schierwagen et al. (1990) have reported that myeloperoxidase loses only 2% of activity after incubation at 60°C for 2 h. The present determination of myeioperoxidase from rat polymorphonucleocytes undergoing the same treatment similarly attests the heat stability of myeloperoxidase. This suggests that any tissue activity of myeloperoxidase would not be seriously compromised by this period of heat incubation. The applicability of procedures designed to optimise the recovery of exogenous peroxidase activity from renal supernate in vitro, was demonstrated by the delineation of a time course for renal polymorphonueleocyte accumulation using myeloperoxidase as a biochemical marker after ischaemia/reperfusion in vivo. As further verification, no myeloperoxidase activity could be detected in ischaemic/reperfused kidneys when the level of renal inhibition of added peroxidase activity was 91%. This emphasises the importance of assessing and overcoming the renal suppression of peroxidase activity before attempting ischaemia/reperfusion studies of renal polymorphonucleocyte accumulation using myeloperoxidase as a biochemical marker. This study does not discriminate between myeloperoxidase activity associated with infiltrated a n d / o r marginated polymorphonucleocytes and that associated with incidental polymorphonucleocytes within the renal vascular compartment. However, it is interesting to note that the histological studies of Hellberg et al. (1990) and Willinger et al. (1992) have documented polyrnorphonucleocyte margination in the rat kidney after 45 min ischaemia and periods of reperfusion predicted by the present study from myeloperoxidase measurements. It is also noteworthy that since renal blood flow to the isehaemic kidney is reduced during reperfusion (Cristol et al., 1993), a reduced flux of polymorphonucleocytes to the kidney would be expected. This would suggest that a rise in renal myeloperoxidase activity was due to a process of polymorphonucleocyte accretion by renal tissue after ischaemia/reperfusion. The efficacy of a standard dose of monoclonal antibodies against intercellular adhesion molecule-1 (ICAM-1) and CD18 of /3z-integrins to block renal polymorphonueleoeyte accumulation after ischaemia/ reperfusion, would suggest the involvement of these
D.W. Laight et al. / Fur. J. Pharrnacol. Environ. Toxicol. Pharmacol. Section 292 (1994) 81-88
adhesion molecules in vivo. This would agree with previous studies in vitro indicating the importance of ICAM-1 and CD18 to polymorphonucleocyte-endothelial cell adhesion (Argenbright et al., 1991; Pohlman et al., 1986). However, the present results must be qualified by the findings that the parent murine antibody, IgG1 in addition to an unrelated murine antibody, IgG2a, reproduced this blockade of renal polymorphonucleocyte accumulation. The reason(s) for this paradoxical result is unclear. One possibility worth investigating is that murine antibodies may reduce the circulating level of polymorphonucleocytes. There was no evidence that exogenous horseradish peroxidase activity was inhibited by renal supernate exposed to the parent murine antibody, IgG1. This indicates that murine antibodies do not directly affect myeloperoxidase activity in vitro. Taken overall, this suggests that murine monoclonal antibodies against rat adhesion molecules may be capable of depressing ischaemic/ reperfused renal polymorphonucleocyte accumulation at least in part by virtue of a non-specific action in vivo. FACS analysis which showed only the binding of antiCD18 monoclonal antibody to rat blood polymorphonucleocytes, implies that a common, non-specific action of murine antibodies at the level of the polymorphonucleocyte membrane can be excluded. The level of polymorphonucleocyte accumulation in ischaemic/ reperfused kidneys exposed to the vehicle (0.9% saline) alone was not significantly reduced, indicating that effects were related to antibody interventions. The gain in wet weight of ischaemic/reperfused kidneys suggests oedema formation. This occurred in the presence and absence of demonstrable ischaemic/ reperfused renal polyrnorphonucleocyte accumulation. This indicates "a largely polymorphonucleocyte-independent rise in renal microvascular permeability in this model of renal ischaemia/reperfusion (Wedmore and Williams, 1981; Williams et al., 1990). Oedema-promoring factors linked with coagulation (Gerdin and Saldeen, 1978) may be excluded since animals were heparinised. Roles for local autocoids a n d / o r vascular permeability factor (Collins et al., 1993) are conceivable. Alternatively, the integrity of the endothelial barrier may have been irreversibly damaged by ischaemia/ reperfusion. In summary, we have found that a tetramethylbenzidine assay provides a more sensitive photometric assessment of rat polymorphonucleocyte-derived myeloperoxidase than an established o-dianisidine assay. We have also determined that rat renal tissue factors which interfere with the assessment of rat renal peroxidase activity can be surmounted by a combination of heat inactivation and the provision of excess assay H 2 0 2. We have subsequently demonstrated renal polymorphonucleoeyte accumulation after i s e h a e m i a / reperfusion in vivo using myeloperoxidase as a bio-
87
chemical marker in a sensitive tetramethylbenzidine assay. Finally, our evidence suggests that murine monoclonal antibodies against rat adhesion molecules may be effective in reducing renal polyrnorphonucleocyte accumulation after ischaemia/reperfusion in vivo at least in part in a non-specific manner.
Acknowledgements David W. Laight is a CASE award Ph.D. student with Roche Research Centre.
References Andrews P.C. and N.I. Ka'insky, 1981, The reductive cleavage of myeloperoxidase in half produces enzymatically active hemimyeloperoxidase, J. Biol. Chem. 256, 4211. Argenbright, L.W., L.G. Letts and R. Rothlein, 1991, Monoclonal antibodies to the leukocyte membrane CD18 glycoprotein complex and to intercellular adhesion molecule-I inhibit leukocyteendothelial adhesion in rabbits, J'. Leuk. Biol. 49, 253. Bos, E.S., A.A. Van der Doelen, N. Van Roy and A.H.W.N. Schuurs, 1981, 3,3',5,5'-Tetramethylbenzidine as an Ames test negative chromogen for horse-radish peroxidase in enzyme-immunoassay, J. Immunoassay 2, 187. Bradley, P.P., R.D. Christensen and G. Rothstein, 1982a, Cellular and extracellular myeloperoxidase in pyogenic inflammation, Blood 60, 618. Bradley, P.P., D.A. Priebat, R.D. Christensen and O. Rothstein, 1982b, Measurement of cutaneous inflammation: estimation of neutrophll content with an enzyme marker, J. Invest. Dermatol. 78, 206. Collins, P.D., D.T. Connoily and T.J. Williams, 1993, Characterisation of the increase in vascular permeabifity induced by vascular permeability factor in vivo, Br. J. Pharmacol. 109, 195. Cristol, J.P., C. Thiemermann, J.A. Mitchell, C. Walder and J.R. Vane, 1993, Support of renal blood flow after ischaemic-reperfusion injury by endogenous formation of nitric oxide and of cyclooxygenase vasodilator metabolites, Br. J. Pharmacol. 109, 188. Gerdin, B. and T. Saldeen, 1978, Effects of fibrin degradation products on microvascular permeabifity, Thromb. Res. 13, 995. Orisham, M.B., L.A. Hernandez and D.N. Granger, 1986, Xanthine oxidase and neutrophil infiltration in intestinal ischaemia, Am. J. Physiol. 251, 0567. Hellberg, P.O.A., O.T. Kaellskog, (3. Oejteg and M. Wolgast, 1990, Peritubular capillary permeability and intravascular red blood cell aggregation after ischaemia: effects of neutrophils, Am. J. Physiol. 258, F1018. Hillegas, L.M., D.E. Griswold, B. Brickson and C. AlbrightsonWinslow, 1990, Assessment of myeloperoxidase activity in whole rat kidney, J. Pharmacol. Meth. 24, 285. Holland, V.R., B.C. Saunders, F.L Rose and A.L Walpole, 1974, A safer substitute for benzidine in the detection of blood, Tetrahedron 30, 3299. Krawisz, J.E., P. Sharon and W.F. Stenson, 1984, Quantitative assay for acute intestinal inflammation based on myeloperoxidas¢ activity. Assessment of inflammation in rat and hamster models, Gastroenterology 87, 1344. Mullane, K.M., R. Kraemer and B. Smith, 1985, Myeloporoxidase activity as a quantitative assessment of neutrophil infiltration ~into ischaemic myocardium, J. PharmacoL Meth. 14, 157.
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Ormrod, D.J., G.L. Harrison and T.E. Miller, 1987, Inhibition of neutrophil myeloperoxidase activity by selected tissues, J. Pharmacol. Meth. 18, 137. Pohlman, T.H., ICA. Stanness, P.G. Beatty, H.D. Ochs and J.M. Harlan, 1986, An endothelial cell surface factor(s) induced in vitro by lipopolysaccharide, interleukin 1, and t u m o u r necrosis factor-a increases neutrophil adherence by a C D w l 8 - d e p e n d e n t mechanism, J. Immunol. 136, 4548. Rammler, D.H., 1967, The effect of DMSO on several enzyme systems, Ann. NY Acad. Sci. 141, 291. Schierwagen, C., A.C. Bylund-Fellenius and C. Lundberg, 1990, Improved method for quantification of tissue PMN accumulation measured by myeloperoxidase activity, J. Pharmacol. Meth. 23, 179. Schultz, J. and K. Kaminker, 1962, Myeloperoxidase of the leukocyte of the normal human blood. I Content and Iocalisation, Arch. Biochem. Biophys. 96, 465. Suzuki, K., H. Ota, S. Sasagawa, T. Sakatani and T. Fujikura, 1983, Assay method for myeloperoxidase in human polymorphonuclear leukocytes, Anal. Biochem. 132, 345.
Tallarida, R.J. and R.B. Murray, 1981, Manual of Pharmacologic Calculations with Computer Programs (Springer-Verlag, New York) p. 11. Tamatani, T. and N. Miyasaki, 1990, Identification of monoclonal antibodies reactive with the rat homolog of ICAM-1, and evidence for a differential involvement of ICAM-I in the adherence of resting versus activated lyrnphocytes to high endothelial cells, Int. Immunol. 2, 165. Wallenstein, S., C.L. Zucker and J.L. Fleiss, 1980, Some statistical methods useful in circulation research, Circ. Res. 47, 1. Wedmore, C.V. and T.J. Williams, 1981, Control of vascular permeability by polymorphonuclear leukocytes in inflammation, Nature 289, 646. Williams, F.M., P.D. Collins, M. Tanniere-Zeller and T.J. Williams, 1990, The relationship between neutrophils and increased microvascular permeability in a model of myocardial ischaemia and reperfusion in the rabbit, Br. J. Pharmacol. 100, 729. Willinger, C.C., H. Schramek, K. Pfaller and W. Pfaller, 1992, Tissue distribution of neutrophils in postischaemic acute renal failure, Virchows Arch. B Cell Pathol. 62, 237.
ELSEVIER
environmental toxicology and pharmacology
Assessment of myeloperoxidase activity in renal tissue after ischaemia/reperfusion D a v i d W . L a i g h t a,,, N a g i n L a d b, B r i a n W o o d w a r d
", J o h n F. W a t e r f a l l c
" Pharmacology Group, Bath UnirersiO', Clat,erton Down, Bath BA2 7AY, UK t, Roche Research Centre, 40 Broadwater Road, Welwyn Garden Ci~', Herts. A L7 3A Y, UK c Hoffman La-Roche Inc., 340 Kingsland Street, NJ 07110-1199, USA
Received 14 February 1994; revised MS received 6 April 1994; accepted 19 April 1994
Abstract
We have shown that a photometric assay of myeloperoxidase derived from rat blood polymorphonucleocytes employing 3,3',5,5'-tetramethylbenzidine as substrate is more sensitive than an established assay employing o-dianisidine. We went on to demonstrate that rat renal tissue is capable of inhibiting peroxidase activity. This activity approached 100% when the rat renal supernate was incubated at 60°C for 2 h and the assay was conducted in the presence of a 10-fold higher concentration of hydrogen peroxide (H202). Rat kidneys undergoing 45 min ischaemia and 1, 3 and 6 h reperfusion in vivo, exhibited significant increases in myeloperoxidase activity, indicating tissue polymorphonucleocyte accumulation. Monoclonal antibodies against rat intercellular adhesion molecule 1 (ICAM-1) and CD18 of/32-integrins administered both 5 min before a period of 45 min renal ischaemia (20/xg/kg i.v.) and at the commencement of 1 h reperfusion (20/~g/kg i.v.) reduced renal tissue polymorphonucleocyte accumulation. However, similar treatment with the parent murine antibody immunoglobulin G1 (IgG1) and an unrelated murine antibody, IgG2a, also significantly reduced renal tissue polymorphonucleocyte accumulation. In conclusion, we demonstrate that the rat renal suppression of peroxidase activity can be overcome by a combination of heat inactivation and the provision of excess assay H20 2. In addition, the available evidence suggests that murine monoclonai antibodies against rat adhesion molecules may exert non-specific actions in our model of renal ischaemia/reperfusion in vivo.
Keywords: Myeloperoxidase; Tetramethylbenzidine; Polymorphonucleocyte; Renal ischemia/reperfusion; Adhesion molecule
I. Introduction
Myeloperoxidase, a characteristic constituent of polymorphonucleocytes (Bradley et al., 1982a; Schultz and Kaminker, 1962), has been used as a biochemical marker for the tissue content of polymorphonucleocytes. The established photometric assay of myeloperoxidase employs o-dianisidine as the hydrogen donor for H 2 0 2 (Bradley et al., 1982b). However, tetramethylbenzidine may be used as a non-carcinogenic alternative hydrogen donor for the determination of peroxidase (Bos et al., 1981; Andrews and Krinsky, 1981; Holland et al., 1974) and can be shown to provide a more sensitive assay of human myeloperoxidase than o-dianisidine (Suzuki et ai., 1983). In an attempt to
* Corresponding author. Tel.: 44-0225-826826, ext. 5779; Fax: 440225-826114. 0926-6917/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 9 2 6 - 6 9 1 7 ( 9 4 ) 0 0 0 2 3 - 9
extend these findings, the sensitivities of tetramethylbenzidine and o-dianisidine assays were directly compared in an assay of myeloperoxidase derived from rat blood polymorphonucleocytes. Polymorphonucleocyte infiltration as assessed by tissue myeloperoxidase assay has been shown in numerous tissues including the inflamed rat dermis (Bradley et al., 1982b), the ischaemic/reperfused dog and rabbit myocardium (Mullane et al., 1985; Schierwagen et al., 1990) and the ischaemic/reperfused rat intestine (Grisham et al., 1986; Krawisz et al., 1984). A potential problem concerned with the assessment of myeloperoxidase in renal tissue is that peroxidase activity may be effectively inhibited or masked by renal tissue factors (Schierwagen et al., 1990; Hillegas et al., 1990; Ormrod et al., 1987). Schierwagen et al. (1990) have speculated that these factors could potentially include H 2 0 2 consuming systems. The initial aim of the present study was to employ measures that would overcome these
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peroxidase inhibitory factors. To this end, the recovery of exogenous horseradish peroxidase activity added to rat renal tissue was assessed as an index of inhibition. The validity of these measures was demonstrated by delineating a time course of polymorphonucleocyte accumulation after 45 min renal ischaemia in vivo. It was of further interest to attempt to modulate renal polymorphonucleocyte accumulation using recently available murine monoclonal antibodies against rat adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and CD18 of /32-integrins (Tamatani and Miyasaki, 1990).
2. Materials and methods
2.1. Isolation of rat blood polymorphonucleocytes Blood collected into heparin (10 U / m l ) from male CD Wistar rats anaesthetised with pentobarbital sodium (60 mg/kg i.p.) was carefully layered onto Ficoll-Paque lymphocyte separation medium and spun at 300 x g for 30 min. The lower reddish layer was mixed with an equal volume of 1.8% dextran in pH 7.4 phosphate buffered saline (PBS) and left to sediment for 40 min. The resultant upper layer was spun at 200 x g for 10 min and the pellet resuspended in first 5.8 ml 0.21% NaCI and then 0.5 ml 1 M KCI to lyse remaining erythrocytes. After washing in PBS, viable cells were enumerated in a Neubauer haemocytometer following dilution with 0.1% trypan blue. Differential cell counts indicated the yield of polymorphonucleocytes to be routinely > 90%. Samples were diluted in PBS to provide 106 polymorphonucleocytes/ml. 2.2. Extraction of myeloperoxidase from rat blood polymorphonucleocytes Aliquots of polymorphonucleocytes (lOS-lO ~) were transferred to vials and the volume subsequently made up to 5 ml with hexadecyltrimethylammonium bromide in 50 mM phosphate buffer pH 6.0 to give a final concentration of 0.5%. Polymorphonucleoeyte suspensions were then disrupted for 30 s using a Branson sonieator at 20% power and subsequently snap frozen in liquid nitrogen and thawed on three consecutive occasions before a final 30 s sonieation. Polymorphonueleoeyte suspensions were then incubated at 60"C for 2 h (vide infra) to verify the heat resistance of myeloperoxidase (see Sehierwagen et al., 1990). Incubated polymorphonueleoeyte suspensions were spun at 4000 × g for 12 rain and the myeloperoxidase-rieh renal supemate collected and stored at - 2 2 " C until assay.
2.3. Assay of myeloperoxidase from rat blood polymorphonucleocytes Myeloperoxidase was assayed photometrically in a 96-well plate incubated at 37°C using a Dynatech MR7000 plate reader controlled by a Toshiba 1000 microprocessor. The tetramethylbenzidine assay of myeloperoxidase was essentially as described by Suzuki et al. (1983). The assay mixture consisted of 20 /zl myeloperoxidase-rich supernate, 10/zl tetramethylbenzidine (final concentration 1.6 mM) dissolved in dimethylsulphoxide (DMSO) and 70 /zl H 2 0 2 (final concentration 0.3 mM) diluted in 80 mM phosphate buffer pH 5.4. The o-dianisidine assay of myeloperoxidase was essentially as described by Bradley et al. (1982b). The assay mixture consisted of 20 /zl myeloperoxidase-rich supernate and 80/xl H 2 0 2 (final concentration 0.147 mM) diluted in 50 mM phosphate buffer pH 6.0 containing o-dianisidine dihydrochloride (final concentration 0.526 mM). The reaction was initiated by the addition of H 2 0 2 and the increase in absorbance was determined over 3 min at 30 s intervals at either 630 nm (tetramethylbenzidine assay) or 460 nm (o-dianisidine assay).
2.4. Recovery of exogenous horseradish peroxidase activity from renal supernate As an index of the inhibition of peroxidase activity by renal tissue, the recovery of exogenous horseradish peroxidase activity was assessed from renal supernate derived from non-ischaemic rat kidneys in a photometric assay using tetramethylbenzidine. The activity of exogenous horseradish peroxidase in the presence of 0.5% hexadecyltrimethyiammonium bromide was defined as 100% recovery. In an attempt to enhance the recovery of exogenous horseradish peroxidase activity from renal supernate, the renal supernate was heat-inactivated by incubating at 60°C for 2 h in a water bath (Schierwagen et al., 1990) a n d / o r the assay H202 concentration was raised 10-fold. The assay mixture consisted of 10 p,l tetramethylbenzidine (final concentration 1.6 mM) dissolved in DMSO, 50/zl H202 (final concentration 0.3 or 3.0 raM) diluted in 80 mM phosphate buffer pH 5.4, 20 /zl heat-inactivated or untreated renal supernate or 20 /~l 0.5% hexadecyltrimethyiammonium bromide (as control) and 20/zl horseradish peroxidase (final concentration 8.7-43.3 p,g/1). Horseradish peroxidase activity was expressed as the initial rate of change in absorbance (SA/min) per unit concentration of enzyme (1 mg/l).
2.5. Rat model of renal ischaemia / reperfusion Male CD Wistar rats (300-400 g) were anaesthetised with pentobarbital sodium (60 m g / k g i.p.) and
D.W. Laight et al. / Eur. J. Pharmacol. Enciron. Toxicol. Pharmacol. Section 292 (1994)81-88
the body temperature maintained at 37°C by a warming pad. Both kidneys were exposed via flank incisions and kept moist with warmed .0.9% saline. After the administration of heparin (200 U / k g i.v.), the left renal pedicle was occluded by an atraumatic vascular clamp for 45 min. Reperfusion was elected for 0.5-6 h by removal of the clamp and visually confirmed before postischaemic and sham-operated kidneys were restored to the body cavity and the wounds sutured. Buprenorphine hydrochloride (0.5 mg/kg s.c.) was administered to provide post-operative analgesia. Renal reperfusion was terminated by cervical dislocation of the animal. Kidneys were then excised, blotted dry and weighed. Kidneys were subsequently immersed in 0.5% hexadecyltrimethylammonium bromide in 50 mM phosphate buffer pH 6.0 and stored at - 2 2 ° C until myeloperoxidase extraction and assay.
2.6. Renal myeloperoxidase extraction Frozen rat kidneys were thawed at room temperature and macerated with an Ultra-Turrax T25 blender for 30 s. Sample volumes were then made up to 10 ml with 0.5% hexadecyltrimethylammonium bromide before disruption and freeze-fractionation as described for the extraction of myeloperoxidase from rat blood polymorphonucleocytes. Samples were incubated at 60°C for 2 h in a water bath and then spun at 4000 × g for 12 min. The supernate was collected for myeloperoxidase assay.
2. 7. Renal myeloperoxidase assay Renal myeloperoxidase activity was assessed photometrically at 630 nm as described for the assessment of rat blood polymorphonucleocyte myeloperoxidase activity using tetramethylbenzidine as substrate. The assay mixture consisted of 20 ~l heat-inactivated renal supernate, 10 /~l tetramethylbenzidine (final concentration 1.6 mM) dissolved in DMSO and 70 p,l H202 (final concentration 3.0 mM) diluted in 80 mM phosphate buffer pH 5.4. Renal myeloperoxidase activity was expressed in units (U) where 1 U represents that amount of enzyme degrading 1/zmol H 2 0 2 / m i n under the stated conditions. Myeloperoxidase activity was standardised with respect to the wet weight of the sham-operated kidney (U/g).
2.8. Effect of monoclonal antibodies against polymorphonucleocyte adhesion on renal myeloperoxidase activity Murine anti-rat intercellular adhesion molecule-1 (ICAM-1) or murine anti-rat CD18 monoclonal antibody was administered to animals both 5 rain before 45 min renal isehaemia (20 ~ g / k g i.v.) and at the corn-
83
mencement of 1 h reperfusion (20/~g/kg i.v.) in vivo. The parent murine antibody, immunoglobulin G1 (IgG1) or an unrelated murine antibody, IgG2a, was also administered in an identical manner to serve as controls. Vehicle control animals received 0.9% saline (0.5 ml/kg i.v.) at the appropiate time points. Shamoperated contralateral kidneys provided control preparations for ischaemic/reperfused kidneys.
2.9. Effect of lgG1 on recovery of horseradish peroxidase activity from renal supernate In order to examine the possibility that murine antibodies might directly affect tissue peroxidase activity, the recovery of exogenous horseradish peroxidase activity was assessed from renal supernate derived from non-ischaemic kidneys exposed to murine IgG1 (40 t~g/kg i.v. total) in vivo. The activity of horseradish peroxidase in the presence of 0.5% hexadecyltrimethylammonium bromide was defined as 100% recovery.
2.10. Fluorescence-activated cell sorting (FACS) analysis Aliquots of rat blood polymorphonucleocytes (0.5 x 106 cells) were suspended in 50/zl PBS/0.2% sodium azide together with 4 tzg of anti-ICAM-1 or anti-CD18 monoclonal antibody or murine IgG1 or IgG2a antibody and incubated at - 4 ° C for 30 min. Cell suspensions were then washed twice in 1 ml PBS/0.2% sodium azide and resuspended in 50 /~1 PBS/0.2% sodium azide together with 0.5/zg fluorescently labelled bovine anti-murine IgG R-phycoerythrin conjugate F(ab') 2 fragment and incubated at - 4 ° C for 30 min. Cells were then washed twice and finally resuspended in PBS/0.2% sodium azide and stored at - 4 ° C until FACS analysis.
2.11. Materials Agents used were murine anti-rat ICAM-1 and murine anti-rat LFA-1/3 chain (CD18) monoclonal antibodies (British Biotechnology), murine IgG1, murine IgG2a and bovine anti-murine IgG R-phycoerythrin conjugate F(ab') 2 fragment (Sigma Immunochemieals), trypan blue, hydrogen peroxide and dextran (250 kDa) (British Drug House), sterile 0.9% saline for injection (Fresenius), buprenorphine hydroehloride (Reekitt and Colman), pentobarbital sodium (May and Baker), Ficoll-Paque (Pharmacia), 3,3',5,5'-tetramethylbenzidine, o-dianisidine dihydrochloride (3,3'-dimethoxybenzidine dihydrochloride), horseradish peroxidase (Type II), dimethylsulphoxide, sodium azide and hexadeeyllximethylammonium bromide (Sigma). 3,3',5,5'-tetramethylbenzidine was dissolved in absolute dimethylsulphoxide to give a concentration of 16 mM which was then diluted in the assay to the working concentration of 1.6
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mM. Dimethylsulphoxide was therefore present at a final concentration of 10% (v/v).
2.12. Statistics
6A/mln/mg/I Horserodish Peroxldose 40.0 35.0
The comparison of two group means was made using unpaired Student's t test while the comparison of a control group mean with each of a number of treatment group means was conducted using Dunnett's test (Wallenstein et al., 1980). The comparison of slopes of linear regression lines was conducted using Student's t test for parallelism (Tallarida and Murray, 1981). Values presented are means and standard error of the mean (S.E.M.) of n observations. Statistical significance was accepted at the 5% level.
30.0 25.0 20.0
15.0. 10.0.
~
~*
5.0. ¢t
0.0
"=" 0.3
3.0 Hydrogen Peroxide (raM)
3. Results
3.1. Assay of myeloperoxidase derived from rat blood polymorphonucleocytes Myeloperoxidase activity from rat blood polymorphonucleoeytes was linearly correlated with the number of polyrnorphonucleoeytes from which myeloperoxidase was extracted in either an assay using tetramethylbenzidine (r = 1.000, P < 0.01, n -- 5) or o-dianisidine (r -- 0.998, P < 0.01, n = 5) (Fig. 1). However, the tetramethylbenzidine assay of polymorphonucleocytederived myeloperoxidase was 3-fold more sensitive than the o-dianisidine assay (51.83 + 1.56 6 A / m i n per 106 polyrnorphonucleocytes compared to 18.08 + 1.07 8 A / r a i n per 106 polymorphonucleocytes, P < 0.01, n --5, Student's t test). The more sensitive tetramethyl-
dA/min i*
60.0"
40.0"
20.0.
I 0.0
1
3
10
Polyrnorphonucleocytes (x 105) Fig. 1. Myeloperoxidase activity derived from rat blood polymorphonucleocytes assessed as the initial rate of change of absorbance (SA/min) using 3,3',5,5'-tetramethylbenzidine (solid line) (n = 5) or o-dianisidine (dashed line) (n = 5) assays.Values are means (S.E.M.). *, P < 0.05 with respect to o-dianisidine myeloperoxidase assay.
Fig. 2. Horseradish peroxidase activity assessed as the initial rate of change of absorbance ( 6 A / m i n ) per unit concentration of enzyme in the presence of 0.5% hexadecyltrimcthylammonium b r o m i d e / 5 0 mM pH 6.0 phosphate buffer (control, open bars) (n = 4) or in the presence of renal supernate either incubated at 60°C for 2 h (crosshatched bars) or unincubated (closed bars) (n = 4): effects of 0.3 or 3.0 mM hydrogen peroxide in assay. Values are means (S.E.M.). '~, P < 0.01 with respect to appropriate control value.
benzidine assay was therefore adopted in subsequent studies.
3.2. Recovery of horseradish peroxidase activity from renal supernate The initial rate of increase of absorbance at 630 nm was linearly correlated with the concentration of horseradish peroxidase in the assay (8.7-43.3 ~g/1). The activity of exogenous horseradish peroxidase added to renal supernate derived from non-ischaemic rat kidneys was significantly depressed ( P < 0.01, n = 4, Student's t test) compared to the activity of exogenous horseradish peroxidase activity added to an equal volume of 0.5% hexadecyltrimethylammonium bromide (Fig. 2) and represented a recovery of only 9% (i.e., 91% inhibition). The activity of exogenous horseradish peroxidase added to renal supernate incubated at 60°C for 2 h was still significantly depressed ( P < 0.01, n = 4, Student's t test). Similarly, when H 2 0 2 was employed at a 10-fold higher concentration in the assay (3.0 mM), the activity of exogenous horseradish peroxidase added to untreated renal supernate remained significantly reduced ( P < 0.01, n = 4, Student's t test) compared to the appropriate control. However, when H 2 0 2 was present in the assay at 3.0 mM, the activity of exogenous horseradish peroxidase added to heat-inactivated renal supernate was not significantly different from the appropriate control activity. No myeloperoxidase activity could be detected in heat-inactivated non-ischaemic
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Renal wet weight (~)
Renal myeloperoxidase (U/g)
150.0-
3.5¢r
¢r
3.0¢r
2_
140.0-
2.5130.0-
2.0-
¢r
1.5-
120.0-
1.0110.0 0.5J 0.0
0.5
100.0 3
I
0.5
Reperfusion period (h)
1
1
3
Reperfusion period (h)
Fig. 3. Rat renal myeloperoxidase activity after 45 min ischaemia and various periods of reperfusion in vivo expressed per unit wet weight of the sham-operated kidney. Values are means (S.E.M.) (n = 6). *, P < 0.05 with respect to zero activity in sham-operated kidneys.
Fig. 4. Wet weight of rat kidneys after 45 rain ischaemia and various periods of reperfusion in vivo as a percentage of the wet weight of sham-operated kidneys. Values are means (S.E.M.) (n = 6). *, P < 0.05 with respect to 100%.
renal supernate per se in the absence of exogenous horseradish peroxidase.
addition, identical treatment with the parent murine IgG1 or unrelated murine IgG2a antibody produced a similar effect. In contrast, myeloperoxidase activity in kidneys undergoing 45 min ischaemia and 1 h reperfusion in animals receiving vehicle alone in vivo (0.5 ml/kg i.v. 0.9% saline) was not significantly affected. Myeloperoxidase activity in sham-operated kidneys was undetectable in these experiments. The recovery of exogenous horseradish peroxidase activity from renal supernate derived from non-ischaemic kidneys exposed
3.3. Renal myeloperoxidase activity after ischaemia / reperfusion When renal supernate was assayed under conditions where the recovery of exogenous horseradish peroxidase activity was near 100%, i.e., 2 h incubation at 60"C and 3.0 mM assay H202, kidneys undergoing 45 rain ischaemia and 1, 3 or 6 h reperfusion (but not 0.5 h reperfusion) in vivo showed significant myeloperoxidase activity, indicating polymorphonucleocyte accumulation (Fig. 3). Myeloperoxidase activity in sham-operated kidneys was undetectable. In preliminary experiments where renal supernate was not heat-inactivated and the assay concentration of H 2 0 2 was 0.3 mM, myeloperoxidase activity was undetectable in rat kidneys undergoing 45 min ischaemia and 1 or 3 h reperfusion. The wet weight of kidneys undergoing 45 rain ischaemia and 0.5-6 h reperfusion in vivo was significantly increased as a percentage of the wet weight of sham-operated kidneys (Fig. 4). Left kidney wet weight was 99.8 + 2.5% of the right kidney wet weight in a random sample of animals (n = 6).
3.4. Effect of antibodies on renal myeloperoxidase activity after ischaemia /reperfusion Treatment of animals with anti-ICAM-1 or antiCD18 monoelonal antibody ( 2 0 / z g / k g i.v. both 5 rain before isehaemia and at the commencement of reperfusion) was associated with significantly reduced myeloperoxidase activity in kidneys undergoing 45 rnin isehaemia and 1 h reperfusion in vivo (Fig. 5). In
Renal rnyeloperoxidose ( U / g )
2.0-
1.5-
1.0~r
o5
O.0
*
no
intervention
saline
~
*
-
CD18
~
antiICAM- I
IgGI
•
IgO2o
Fig. 5. Effects of monoclonal antibodies q a i n s t rat CD18 of/~2.integrins and rat intercellular adhesion molecule 1 (ICAM-1) or the parent murine antibody immunoglobulin G t (IgG1) and an unrelated murine IgG2a antibody or 0.9% saline vehicle, on myelopormddase activity in rat kidneys undergoing 45 min isehaemia and I h r e i ~ f u sion in vivo. Antibodies were administered both5 min before 46 isehaemia (20 ttg/k8 iv.) and at the commencement .of renerfusion (20/zg/k8 i.v.). 0.9% saline vehicle was administered ~ r ~ LV.) at identical time points. Values are means (S.E,M:) ( n = ~, *, P < 0.05 with respect to n o intervention.
86
D.W. Laight et al./ Eur. J. Pharmacol. Environ. Toxicol. Pharmacol. Section 292 (1994) 81-88
to murine IgG1 (40 /zg/kg i.v. total) in vivo was not significantly different from 100%. Furthermore, FACS analysis showed that of the antibodies studied, only anti-CD18 monoclonal antibody was capable of binding to rat blood polymorphonucleocytes. In addition, antibody interventions (40/~g/kg i.v. total) did not significantly affect increases in the wet weight of rat kidneys undergoing 45 min ischaemia and 1 h reperfusion in vivo.
4. Discussion
The present findings agree with those of Suzuki et al. (1983) that a tetramethylbenzidine assay can provide a viable, safe alternative to the established o-dianisidine assay of myeloperoxidase. The difference in sensitivity between tetramethylbenzidine and o-dianisidine assays cannot be accredited to the higher concentration of H 2 0 2 in the case of the tetramethylbenzidine assay since when o-dianisidine was substituted for tetramethylbenzidine in an assay of horseradish peroxidase, the substituted o-dianisidine assay proved similarly less sensitive (personal observation). A corollary of the enhanced sensitivity of the described tetramethylbenzidine assay of myeloperoxidase is that the threshold of detection is lowered. This theoretically provides for the assessment of early polymorphonueleocyte accumulation during reperfusion after ischaemia which might be overlooked using a less sensitive o-dianisidine assay. The masking of exogenous horseradish peroxidase activity by rat renal supernate indicates the presence of rat renal tissue factors that interfere with the peroxidase assay. Hillegas et al. (1990) have suggested that such factors may include conjugates of kidney-derived protein and the detergent hexadecyltrimethylammonium bromide, used to solubilise myeloperoxidase. The level of inhibition (91%) exhibited by rat renal tissue assessed by the recovery of added horseradish peroxidase activity, was comparable to that reported by Sehierwagen et al. (1990) (88%) measuring the recovery of exogenous human myeloperoxidase activity in the rabbit kidney. However, in contrast to the findings of Sehierwagen et al. (1990) in the rabbit kidney, the rat renal inhibition of exogenous peroxidase activity was not abolished by heat inactivation alone. In fact, the level of rat renal inhibition remained significantly hi'gh at 49% after the rat renal supernate had been incubated at 60"C for 2 h. The discrepancy between studies suggests the existence of both heat labile and heat stable interfering factors in rat renal tissue. The 'observation that rat renal inhibition was effectively abolished by the provision of excess peroxide to heati n s t a t e d rat renal supernate implies that the proposed heat stable factors are peroxide consuming sys-
tems. A conceivable role for such systems was postulated by Schierwagen et al. (1990). It is unlikely that the systems are themselves peroxidase-like since no activity was detected in the renal supernate of nonischaemic rat kidneys per se. Hence, tissue catalase-like activity may be responsible. However, one prime candidate, the heat stable enzyme catalase, can probably be excluded since DMSO at 10% v/v, which was present in all tetramethylbenzidine assays, has been reported to abolish catalase activity in vitro (Rammler, 1967). Myeloperoxidase is an unusually heat-stable enzyme. Schierwagen et al. (1990) have reported that myeloperoxidase loses only 2% of activity after incubation at 60°C for 2 h. The present determination of myeioperoxidase from rat polymorphonucleocytes undergoing the same treatment similarly attests the heat stability of myeloperoxidase. This suggests that any tissue activity of myeloperoxidase would not be seriously compromised by this period of heat incubation. The applicability of procedures designed to optimise the recovery of exogenous peroxidase activity from renal supernate in vitro, was demonstrated by the delineation of a time course for renal polymorphonueleocyte accumulation using myeloperoxidase as a biochemical marker after ischaemia/reperfusion in vivo. As further verification, no myeloperoxidase activity could be detected in ischaemic/reperfused kidneys when the level of renal inhibition of added peroxidase activity was 91%. This emphasises the importance of assessing and overcoming the renal suppression of peroxidase activity before attempting ischaemia/reperfusion studies of renal polymorphonucleocyte accumulation using myeloperoxidase as a biochemical marker. This study does not discriminate between myeloperoxidase activity associated with infiltrated a n d / o r marginated polymorphonucleocytes and that associated with incidental polymorphonucleocytes within the renal vascular compartment. However, it is interesting to note that the histological studies of Hellberg et al. (1990) and Willinger et al. (1992) have documented polyrnorphonucleocyte margination in the rat kidney after 45 min ischaemia and periods of reperfusion predicted by the present study from myeloperoxidase measurements. It is also noteworthy that since renal blood flow to the isehaemic kidney is reduced during reperfusion (Cristol et al., 1993), a reduced flux of polymorphonucleocytes to the kidney would be expected. This would suggest that a rise in renal myeloperoxidase activity was due to a process of polymorphonucleocyte accretion by renal tissue after ischaemia/reperfusion. The efficacy of a standard dose of monoclonal antibodies against intercellular adhesion molecule-1 (ICAM-1) and CD18 of /3z-integrins to block renal polymorphonueleoeyte accumulation after ischaemia/ reperfusion, would suggest the involvement of these
D.W. Laight et al. / Fur. J. Pharrnacol. Environ. Toxicol. Pharmacol. Section 292 (1994) 81-88
adhesion molecules in vivo. This would agree with previous studies in vitro indicating the importance of ICAM-1 and CD18 to polymorphonucleocyte-endothelial cell adhesion (Argenbright et al., 1991; Pohlman et al., 1986). However, the present results must be qualified by the findings that the parent murine antibody, IgG1 in addition to an unrelated murine antibody, IgG2a, reproduced this blockade of renal polymorphonucleocyte accumulation. The reason(s) for this paradoxical result is unclear. One possibility worth investigating is that murine antibodies may reduce the circulating level of polymorphonucleocytes. There was no evidence that exogenous horseradish peroxidase activity was inhibited by renal supernate exposed to the parent murine antibody, IgG1. This indicates that murine antibodies do not directly affect myeloperoxidase activity in vitro. Taken overall, this suggests that murine monoclonal antibodies against rat adhesion molecules may be capable of depressing ischaemic/ reperfused renal polymorphonucleocyte accumulation at least in part by virtue of a non-specific action in vivo. FACS analysis which showed only the binding of antiCD18 monoclonal antibody to rat blood polymorphonucleocytes, implies that a common, non-specific action of murine antibodies at the level of the polymorphonucleocyte membrane can be excluded. The level of polymorphonucleocyte accumulation in ischaemic/ reperfused kidneys exposed to the vehicle (0.9% saline) alone was not significantly reduced, indicating that effects were related to antibody interventions. The gain in wet weight of ischaemic/reperfused kidneys suggests oedema formation. This occurred in the presence and absence of demonstrable ischaemic/ reperfused renal polyrnorphonucleocyte accumulation. This indicates "a largely polymorphonucleocyte-independent rise in renal microvascular permeability in this model of renal ischaemia/reperfusion (Wedmore and Williams, 1981; Williams et al., 1990). Oedema-promoring factors linked with coagulation (Gerdin and Saldeen, 1978) may be excluded since animals were heparinised. Roles for local autocoids a n d / o r vascular permeability factor (Collins et al., 1993) are conceivable. Alternatively, the integrity of the endothelial barrier may have been irreversibly damaged by ischaemia/ reperfusion. In summary, we have found that a tetramethylbenzidine assay provides a more sensitive photometric assessment of rat polymorphonucleocyte-derived myeloperoxidase than an established o-dianisidine assay. We have also determined that rat renal tissue factors which interfere with the assessment of rat renal peroxidase activity can be surmounted by a combination of heat inactivation and the provision of excess assay H 2 0 2. We have subsequently demonstrated renal polymorphonucleoeyte accumulation after i s e h a e m i a / reperfusion in vivo using myeloperoxidase as a bio-
87
chemical marker in a sensitive tetramethylbenzidine assay. Finally, our evidence suggests that murine monoclonal antibodies against rat adhesion molecules may be effective in reducing renal polyrnorphonucleocyte accumulation after ischaemia/reperfusion in vivo at least in part in a non-specific manner.
Acknowledgements David W. Laight is a CASE award Ph.D. student with Roche Research Centre.
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