Assessment of Myeloperoxidase Kidney

Activity in Whole Rat

L. M. HILLECASS, D. E. GRISWOLD, B. BRICKSON, AND C. ALBRIGHTSONWINSLOW

A method to quantitate myeloperoxidase (MPO) activity from rat whole kidney is described. Polymorphonuclear leukocyte (PMN) infiltration into tissue is a hallmark of acute inflammation. Historically, the degree of inflammation has been quantified by the identification and enumeration of PMNs histologically or by some other means. More recently, the enzyme activity of MPO, a marker enzyme for PMN, and freshly emigrated monocytes in many inflamed tissues has replaced these methods. The kidney, however, has been identified as a tissue from which MPO cannot be measured. Indeed, kidney homogenized by a standard extraction procedure was devoid of MPO activity. We modified the established methodology so that kidney was homogenized in 5 mM potassium phosphate buffer (PB) first and then centrifuged at 30,000gfor 30 min atCC prior to extraction. The resulting 30,000 g pellets expressed MPO activity after suspending them in 50 mM PB containing 0.5% hexadecyltrimethylammoniumbromide (HTAB). Interference in the assay was observed with supernatants from control and inflamed kidney, which appeared to be due to kidney-derived material forming a complex with HTAB. After washing the pellets twice, we noted that their extracts exhibited greater activity, and interference from supernatants was abolished. Using this method, we observed that acutely inflamed kidneys from rats treated with sheep nephrotoxic immunoglobulin G (IgG) had significantly elevated MPO activity over kidneys from control rats. Thus, the described technique allows for the routine assay of MPO in kidney tissue. Key Words: Nephrotoxic kocytes; Myeloperoxidase

IgG; Kidney inflammation;

Polymorphonuclear

leu-

INTRODUCTION The infiltration

of polymorphonuclear

leukocytes

(PMN)

in a tissue

is a charac-

teristic of acute inflammation and is now generally regarded to be indicative of the independent or collective action of chemotactic mediators (Snyderman and Uhing, 1988). The elucidation

of the chemotaxins

current scientific interest. Studies of inflammation

From

the Department

in many

of Pharmacology,

and their regulation

tissues

SmithKline

have estimated

Beecham

is the subject of much the degree

Pharmaceuticals,

plc,

of inflam-

King of Prussia,

Pennsylvania. Address maceuticals, Received

reprint

requests

plc, P.O. December

to:

L. M. Hillegass,

Box 1539, 1989;

Department

King of Prussia,

revised

and accepted

of Pharmacology,

SmithKline

Beecham

Phar-

PA 19406. April 1990. 285

Journalof Pharmacological Methods 24, 285-295 WFM) 0 1990 Elsevier Science Publishing

Co., Inc., 655 Avenue of the Americas, New York, NY lWl0

286

L. M. Hillegass et al. matory cell infiltrate by histologic observation, counting PMN after digestion, or by radioactive means (Katz and Strober, 1978; Griswold et al., 1987; Hellberg and KalIskog, 1989; Wahba et al., 1984). Recently, the myeloperoxidase (MPO) kinetic assay of Bradley et al. (1982) has become accepted as a reproducible and objective method to estimate reliably PMN infiltration into inflamed tissues. This colormetric method, which measures the activity of MPO found in the azurophilic granules present in PMN, correlates with other estimations of PMN infiltration in many tissues (Priebat et al., 1983; Allan et al., 1985; Chang et al., 1986; Smith et al., 1988). Kidney tissue, however, was found to be devoid of MPO activity even though PMN were observed histologically in a model of experimentally induced pyelonephritis (Ormrod et al., 1987). Indeed, these authors have found that the activity of MPO from exogenously added PMN in tissue homogenates from kidney, liver, and spleen could not be detected. In a model of myocardial infarction, Griswold et al. (1988) took 30,000 g pellets remaining from the extraction of creatine phosphokinase (CPK) in infarcted rat hearts and recovered MPO activity after performing a simple washing procedure with 5 mM phosphate buffer prior to extraction for MPO. The washing procedure removed 2-mercaptoethanol, which was present in the homogenization buffer for extraction of CPK, and which interfered with the MPO assay. This finding suggested that washing pellets from many tissues may permit the removal of substances that cause interference. Thus, it appeared that tissues deemed unsuitable for MPO analysis may express MPO activity after undergoing a similar washing step prior to the extraction of MPO. This paper describes a method to remove substances from kidney homogenates that interfere with the established MPO assay described by Bradley et al. (1982) and which may be applicable to other tissues. The results using this modification to measure MPO in kidneys from control rats and rats treated with nephrotoxic IgG to induce acute glomerulonephritis are presented.

METHODS Animals Male Sprague-Dawley rats (200-350 g) were obtained from Charles River Breeding Co., Wilmington, MA; male Munich-Wistar rats (180-220 g) were obtained from Harlan Sprague Dawley, Inc., Indianapolis, IN. Female Dorsett sheep (40-50 kg) were obtained from the Buckshire Corp., Perkasie, PA. Male Balb/c mice (18-25 g) were obtained from Charles River Breeding Laboratories, Kingston, NY. Isolation of Rat Clomeruli Glomeruli were isolated from pentobarbital-anesthetized by the method of Burlington and Cronkite (1973). Glomerular least 86% pure, determined by counting glomeruli and tubules Glomerular protein was determined by the method of Lowry

Sprague-Dawley rats preparations were at on a hemacytometer. et al. (1951).

Assessment of Myeloperoxidase Production

and Bioassay of Nephrotoxic

Activity

Serum

Sheep were initially injected subcutaneously with 6 mL saline or 6 mL freshly isolated rat glomeruli (23-39 mg glomerular protein/animal) emulsified in 6 mL complete Freund adjuvant (Difco Labs, Detroit, MI) to provide control serum and nephrotoxic serum (NTS). Complete Freund adjuvant was replaced with incomplete Freund adjuvant (Difco Labs) in subsequent bimonthly immunizations. Following each immunization, samples of sheep blood were taken. Sera from saline- (control serum) and rat glomeruli-injected sheep were heat-inactivated at 56°C for 1 hr, and bioassayed for induction of proteinuria in rats (see below). Immunization with rat glomeruli eventually induced nephrotoxicity in the sheep as determined by elevated blood urea nitrogen, whereas immunization with saline did not. At this time, the sheep were anesthetized and exsanguinated. Serum from the exsanguination was heat-inactivated and frozen for purification of immunoglobulin G (IgG). The nephrotoxicity of all sera was assessed by using a bioassay in which seruminduced proteinuria in Sprague-Dawley rats was measured. Twenty-four-hour urine samples were collected from the bioassay rats before and after intravenous (i.v.) injection of complement-inactivated control and glomeruli-injected sheep sera. A significant proteinuria in a 24-hr sample in bioassay rats was considered nephrotoxic. Injection of preimmune and control sheep sera (0.1-I .O mL) did not change baseline urinary protein excretion in the bioassay rats, whereas significant proteinuria (a ninefold increase) was achieved by nephrotoxic serum. Purification

of IgG From Sheep

Serum

Control sera and NTS from the sheep were extensively absorbed against rat red blood cells (RBCs) and filtered (0.45 km) prior to IgG purification by affinity column chromatography (GammaBind G, Genex Corp., Gaithersburg, MD). The purity of IgG was 99% as determined by gel electrophoresis. The concentration of the isolated IgG was determined spectrophotometrically at 280 nm. In Munich-Wistar rats, control sheep IgG (15 pg/g body weight [bwl) had no effect on urinary protein excretion, whereas nephrotoxic IgG (15 pg/g) produced a proteinuria similar to that induced by 0.1 mL of NTS, and this was used to induce nephrotoxic serum nephritis. Induction

of Nephritis

by Nephrotoxic

IgC

Male Munich-Wistar rats weighing 200-300 g were injected i.v. with 15 pg/g bw of control sheep IgC or sheep nephrotoxic IgG (inflamed kidney). After 3 hr, the animals

were

anesthetized

The abdominal

with

Ketamine

aorta was cannulated

phosphate-buffered

(60 mg/kg)

and pentobarbital

(21 mg/kg).

and the kidneys were perfused in situ with

saline with 20 mM Hepes,

pH 7.2. During

the perfusion

of the

kidneys, the animal was exsanguinated, the kidneys were removed, frozen on dry ice, and stored at -20°C for up to 2 weeks before thawing for homogenization. Kidney

Preparation

The method of Bradley et al. (1982) was used to extract tissue as described. Initially, 22 kidneys (nonperfused) from a preliminary study were homogenized (1:5, w/v)

287

288

L. M. Hillegass et al.

in 0.5% hexadecyltrimethylammonium bromide solution (HTAB, Sigma Chemical Co., St. Louis, MO) prepared in 0.05 M potassium phosphate buffer (pH 6.0, room temperature) using a Tekmar Tissumizer (Takmar Co., Cincinnati, Ohio) for three on/off cycles at 5-set intervals. The homogenates were immediately frozen on dry ice. Three freeze and thaw cycles were performed with sonication for 10 set between cycles. After the last sonication, the samples were chilled in an ice bath for 20 min and then centrifuged at 12,500 g for 15 min (4°C).

initial Kidney Homogenates Aliquots A, B, and C

Centrifugation LT A

7l B and C

u Supernatants harvested Pellets harvested

h--J B a Supernatants harvested Pellets harvested

II Discard supernatants Rehomogenize pellets 0 Centrggation C II Discard supernatants Rehomogenize pellets II Centrifugation 5 Supernatants harvested Pellets harvested

FIGURE 1. Flow diagram for the preparation of kidney homogenates to evaluate the effect of washing upon the expression of MPO from pellets and of supernatants for interference/ inhibition.

Assessment of Myeloperoxidase Activity

In later studies, this was modified so that normal nonperfused and perfused control and inflamed kidneys were homogenized (1:20, w/v -21 mL) in 5 mM PB (pH 6.0,4”C) for three on/off cycles of 5-set intervals. To ascertain the effect of washing upon the expression of MPO activity and the removal of inhibitory substances, homogenates from perfused kidneys were split into three aliquots (A-C, -7 mL, Figure 1) and centrifuged at 30,000 g for 30 min at 4°C. The above homogenization in PB and centrifugation procedure was repeated once more for the pellets of sample B and twice more for sample C. The supernatants from each sample were frozen on dry ice and stored at -20°C. After all of the pellets were accumulated in an ice bath, they were extracted by the following procedure: each was suspended in 0.5% HTAB buffer (1:5, original tissue wet w/v); frozen, thawed, and sonicated three times; incubated at 4°C for 20 min; and centrifuged at 12,500 g for 15 min (4°C). Myeloperoxidase

Assay

The extracts were assayed for MPO activity kinetically as described by Bradley et al. (1982). The rate at which a colored product formed during the MPO-dependent reaction of o-dianisidine (0.167 mg/mL; Sigma Chemical Co.) and hydrogen peroxide (0.0005%; Sigma Chemical Co.) was measured at 460 nm in a Perkin-Elmer spectrophotometer (Model 552A UV/VlS). One unit of MPO activity is defined as that which degrades 1 ymol of peroxideimin at 25°C. Supernatants A-C from the washes of 30,000 g pellets derived from perfused control and inflamed rat kidneys obtained prior to extraction were evaluated for their ability to affect the rate of chromophore formation of an active MPO extract. Mouse ears taken 60 min after being treated with 2 mg arachidonic acid, a treatment known to cause acute inflammation correlated with MPO (Chang et al., 1986), were homogenized in HTAB solution (one ear per mL) to provide a source of active enzyme (AA-MPO). An aliquot of the AA-MPO (0.015 mL) containing 0.0026 unit MPO was added to a cuvette containing 0.95 mL of assay reagent. The enzymatic reaction was allowed to proceed for 1 min while the absorbance at 460 nm was being recorded. Supernatants A-C (0.05 mL) were added to the progressing reaction and the rate of change in absorbance was measured for an additional 2-3 min. Statistics

Statistical analysis was performed significant.

by Student’s t test and p < 0.05 was considered

RESULTS

Nonperfused rat kidneys (n = 22) were homogenized in HTAB solution to extract MPO in an initial study and all were found to be completely devoid of MPO activity. Because interference and/or inhibition in the assay could account for the absence of activity, an aliquot (0.05 mL) of extract from kidney homogenized in HTAB solution was added to a reaction mixture 1 min after combining reagents and 0.0125 unit of AA-MPO. The enzymatic rate provided by the AA-MPO in this sample abruptly plateaued immediately after the addition of the extract. Prior to discard, the sample

289

290

1. M. Hillegass et al.

was inspected and it was observed that an opacity had formed suggesting thaillnterfering and/or inhibitory substances are present in rat kidney prepared in 0.5% HTAB buffer to extract MPO directly as described by Bradley et al. (1982). * In an effort to demonstrate some level of MPO activity in kidney tissue, normal kidneys (nonperfused, n = 4) were homogenized in 5 mM PB first. The homogenates were centrifuged at 30,000 g and the resulting pellets were then extracted in HTAB solution. These samples provided MPO activity (0.030 +- 0.004 unit [mean ? SEMI per g [wet wt.]) suggesting that homogenization of rat kidney in a buffer prior to extraction is required to reveal the enzyme. Although MPO activity was obtained from pellets after first homogenizing kidneys in 5 mM PB, it was considered likely that some interfering or inhibitory material may remain which could cause an underestimation of the actual activity. To evaluate this possibility and to validate the above result, perfused kidneys from rats treated with sheep control IgG (control tissue) and sheep nephrotoxic IgG (inflamed tissue) were homogenized in 5 mM PB. The homogenates were aliquoted into thirds so that the effect of two sequential washes of the pellets and the resulting activity of the MPO extracted from the washed pellets could be determined (Figure 1, aliquots A-C). Supernatants from the initial homogenates and the washes were also saved so that the progress of washing out interfering substances from the pellets could be followed. Homogenization of rat kidney in 5 mM PB prior to HTAB extraction yielded 30,000 g pellets which expressed modest MPO activity (Table I), whereas kidney samples were devoid of activity if initially homogenized in HTAB solution. The level of MPO activity obtained from inflamed rat kidneys was significantly greater than that from the control tissue (Table 1). Sequential washing of tissue pellets from both control and inflamed kidneys enhanced the MPO activity, however, the increase obtained

TABLE 1 Rat Kidney Myeloperoxidase (MPO). Effect of Washing Tissue Pellets Prior to HTAB Extraction* UNITS MPO/KIDNEY n 6

Control

Inflammation ates were

prepared

the 30,000 g pellets PB (see methods).

by treatment

with

IgG per g of bw. Kidney in 5 mM

PB, aliquoted

The 30,000

5 SEM data.

from

g pellets control

15 kg

homogen(A-C),

from B and C were washed

tracted in HTAB solution. a Significantly different * Mean

to.055

20.054

was induced

nephrotoxic

0.18Sa

0.171”

to.042

sheep

-to.010

kO.009

0.133”

4

0.027

0.024

0.020 t 0.014

Inflamed

C

B

A

(A-C)

and

in 5 mM were

at p < 0.05.

ex-

Assessment of Myeloperoxidase

Activity

from pellets B and C was not significantly different from that of the first pellets. Portions of the active extracts were also boiled for 3 min, which also inactivated them. This suggested that the enzymatic activity from the kidney extracts was not artifactual (data not shown). Supernatants A-C from control tissue were added to ongoing MPO assays so that the adequacy of washing to remove interfering/inhibitory substances from pellets could be determined. This study (Table 2) found that the enzymatic rate was decreased 13% by supernatant B. Supernatant C further reduced the rate to where it approximated that which was obtained by samples containing buffer to control for the effects of dilution (21% versus 34%, respectively). Supernatant A, however, increased the rate of absorbance twofold over that provided by exogenous AA-MPO. From an inspection of the cuvettes containing supernatant A, it was observed that an opacity had formed, which was unaccompanied by the continued development of chromophore. This was not observed in the assay cuvettes containing supernatants B and C. It was also found that none of the supernatants had MPO activity when assayed in the absence of exogenous MPO (data not shown). The means by which supernatant A caused interference/inhibition was studied by combining assay reagents and materials selectively. Opacity occurred when supernatant A and 0.05 mL HTAB solution were combined in a mixture lacking assay reagents and AA-MPO. When 0.1 mg bovine serum albumin (BSA) (Sigma Chemical Co., St. Louis, MO) was added, the development of the opacity was enhanced (Table 3). Wash supernatants B and C were unable to form opacity under these conditions. Also, a reaction mixture containing assay reagents, HTAB solution, and BSA remained clear and colorless.

TABLE 2 Effect of Supernatants from Rat Kidney Homogenates and Pellet Washes upon an Actively Progessing MPO Reaction* % CHANGE OF APPARENTENZYME RATE

SUPERNATANT

n

A(l-6)

6

+I08

8(-l-6)

6

-13

+ 4

C(l-6)

6

-21

2 4

5mMPB

3

-34

4 1

The reaction AA-MPO

was initiated

to a cuvette

This achieved unit so that

rapid

measured.’

supernatant

(A-C,

PB was added unadjusted * Mean

0.95

(+)

per or

increases

kidneys;

analysis. decrease

for dilution. 2 SEM data.

minute The

(-)

later,

see methods) sign indicates

from

Unit

at 460 nm.

of 0.25 absorbance

over this rate could

One

control

of 0.0026

mL assay reagents.

rate of 0.030imin

was set to a full deflection

accurately

increase

by the addition

containing

an absorbance

The recorder

2 15

the

be more 0.05

mL

or 5 mM either

standard

an rate

291

292

1. M. Hillegasset al. TABLE3 Effectof Supernatantsfrom Rat Kidneys in Combination with BSA and HTAB in the Absence of Reagent CHANGE IN ABSORBANCE~

BSA

0.000

BSA + HTAB

0.202

Each analysis consisted

preparations

C

0.000

0.000

0.003

+ 0.024’

k 0.001

0.000

of 0.85 mL 50 mM PB, 0.01 mL BSA (IO mg/

ml), 0.1 ml_ supernatant, HTAB solution.

B

A

SAMPLECOMEIINATION

and either

0.05 mL 50 mM

The data are the mean

in 5 mM PB, aliquoted

f

PB or 0.05 mL

SEM of four control

for A-C

supernatants

kidney

per group

(see methods). a OD

units per min at 460 nm.

b Chromophore

(orange)

was not produced.

DISCUSSION Myeloperoxidase

activity

from

inflamed

rat kidneys

could

not be demonstrated

in a model of pyelonephritis even though the presence of large numbers of neutrophils was confirmed histologically (Ormrod et al., 1987). These authors concluded that the enzyme was inactivated by renal tissue in HTAB extraction solution because the activity of exogenously added MPO was abolished after a 30-set incubation. However, it appears that extracellular MPO remains active in renal tissue, because Johnson et al. (1987a) were able to induce glomerular injury in the rat by infusing polymorphonuclear oxide and halide glomerular

leukocyte MPO and nontoxic concentrations of hydrogen perthrough the renal artery. They also found that MPO bound to

anionic

structures

(Johnson

system is activated in a PMN-mediated phritis in rats (Johnson et al., 1987b). developed, morphologically (Johnson et al., 1988). Thus, flamed

kidney

simply

quantify

from PMN

models

et al., 1987a) and that the MPO-Hz02-halide immune complex The MPO-mediated

resembled changes finding active MPO of renal disease

model of glomeruloneglomerular disease that

found in renal disease in man and measuring its activity in in-

may have greater

significance

than to

infiltration.

In preliminary studies, we found that extracts from kidney (nonperfused) suspended in HTAB solution (1:5, w/v) to extract MPO were enzymatically unresponsive suggesting MPO was inactive. When an aliquot (0.05 mL) from one of the extracts was added to an MPO reaction already in progress, it was found that the kinetically developing chromophore product of the MPO reaction was halted and that the reaction was unable to continue. Upon inspection, however, served to have formed an intense opacity which completely

this sample was obinterfered with the

assay. These results appear to support the conclusion of Ormrod et al. (1987) that HTAB kidney extracts inactivate MPO. The finding that an extract caused an interfering opacity to form suggested soluble kidney material may be responsible for

Assessment of Myeloperoxidase Activity the apparent

lack of MPO

activity

of opacity may be avoided We sought to overcome ence by modifying

in the extracts

and, therefore,

that the formation

by treating the tissue in some other way. the apparent enzyme inactivation and to avoid interfer-

the conditions

used for tissue homogenization

and enzyme

ex-

traction from the established method of Bradley et al. (1982). Active enzyme was found in whole rat kidney only after effecting a separation of the MPO containing structures (azurophilic granules from a PMN infiltrate and possibly tissue-bound MPO) from other kidney material prior to extraction. This was achieved by the centrifugation of kidney homogenate prepared in 5 mM phosphate buffer in a manner similar to that described by Griswold et al. (1988). Griswold et al. were able to extract

MPO

phosphate which

by HTAB

buffer

had been

perfused

normal,

after

solution

from

reclaiming

previously perfused

a tissue

the pellet

extracted control,

pellet from

for creatine

and inflamed)

which

was washed

myocardial

phosphokinase. homogenized

phate buffer, provided 30,000 g pellets from which extraction with HTAB solution. Pellets from control

in 5 mM

infarcted

rat hearts

Kidneys

(non-

first in 5 mM phos-

MPO activity was found after and inflamed kidneys, which

were washed further (two more times) prior to extraction, provided greater activity. This elevation did not differ significantly from the MPO activity in the first pellets, however. present inflamed

Nevertheless, animals, kidneys

increased

MPO models

may have been achieved

activity

rats treated over

control

material

it. Had the study enlisted

of tissue preparation,

from

utility of using MPO

suggests some interfering

removed

significance

Using this method perfused

in animal

this trend

and that washing

with

was originally

a larger

we were able to demonstrate sheep

animals.

activity as a quantitative

nephrotoxic This

of

result

that whole

IgG had significantly illustrates

assay of the evolution

as well as to serve as an indicator

number

in this group.

of PMN infiltration.

the potential

of renal disease Measurement

of MPO activity in inflamed kidney from rats treated with nephrotoxic serum has been used previously as a marker for PMN infiltration. Lianos (1988) isolated glomeruli via a sieving technique, which appears to have separated PMN and bound MPO from the rest of the tissue. These results suggest that the MPO interference and/or inactivation is derived from material unassociated with glomeruli and that the sieving technique achieved a washing procedure. A study was performed interference the pellets) expected

to elucidate

the nature of the development

of the observed

by adding supernatants from the initial homogenates (and after washing to an ongoing MPO reaction (AA-MPO). The initial supernatant (A),

to cause significant

interference,

caused

a rapid elevation

of absorbance

to develop kinetically which was suggestive of exaggerated MPO activity. parent activity was artifactual, however, because an inspection of these

The apsamples

revealed a whitish opacity had formed which was not accompanied by chromophore development. Washing pellets further provided wash supernatants that were unable to induce opacity. This suggests that soluble tissue-derived material and possibly buoyant organelles may be responsible for the observed interference. The contribution to opacity formation by other assay ingredients was also evaluated using a pseudo reaction mixture lacking reagents and MPO. The assay reagents (o-dianisidine and hydrogen peroxide) and MPO were not required for the

293

294

1. M. Hillegass et al.

formation of the opacity but supernatants and HTAB were. This suggests the development of opacity may have been due to kidney-derived material from ‘A’ supernatants forming a complex with the HTAB in the extraction solution. Because divalent cations often participate in complex formation, chelating agents may be able to block this development. We did not pursue this possibility, however. Instead, we were able to avoid this phenomenon by washing the tissue pellet twice prior to MPO extraction. We used this method because it is simple to perform and reproducible. The modification of the standard methodology of Bradley et al. (1982) just described, provides a convenient method for the estimation of inflammatory cell infiltration into whole kidney tissue without the need to isolate a particular portion, such as the glomeruli. The initial 5 mM phosphate buffer homogenization removed tissue-derived interfering material and allowed the demonstration and quantification of MPO activity from whole kidney. The washing procedure established by this study may possibly be applied to other inflamed tissues from which active MPO has not been found. Presumably, these tissues will also exhibit MPO activity upon using this modification and this should permit a wider application of MPO activity as a study parameter in inflammation research. The authors wish to thank Mrs. Audrey Tressler for her excellent secretarial assitance with this manuscript.

REFERENCES Allan C, Bhattacherjee P, Brook CD, Read NG, Parke A] (1985) Myeloperoxidase activity as a quantitative marker of polymorphonuclear leukocyte accumulation into an experimental myocardial infarct: The effect of ibuprofen on infarct size and polymorphonuclear leukocyte accumulation. 1 Cardiovasc Pharmacol 7:1154-1160. Bradley PP, Priebat DA, Christensen RD, Rothstein C (1982) Measurement of cutaneous inflammation: Estimation of neutrophil content with an enzyme marker. / invest Dermato/78:206-209. Burlington H, Cronkite EP (1973) Characteristics of cell cultures from renal glomeruli. Proc Sot Exp Biol Med 142:143-149. Chang j, Carlson RP, O’Neill-David L, Lamb B, Sharma RN, Lewis AJ (1986) Correlation between mouse skin inflammation induced by arachidonic acid and eicosanoid synthesis. Inflammation 10:205-214.

Griswold DE, Webb E, Schwartz L, Hanna N (1987) Arachidonic acid-induced inflammation: Inhibition by dual inhibitor of arachidonic acid metabolism, SK&F 86002. inflammation 11:189-199.

Griswold DE, Hillegass LM, Hill DE, Egan JW, Smith EF III (1988) Method for quantification of myocardial infarction and inflammatory cell infiltration in rat cardiac tissue. 1 Pharmacol Methods 20:225-235.

Hellberg POA, Kallskog TOK (1989) Neutrophil-mediated post-ischemic leakage in the rat kidney. Kidney

Int 36:555-561.

Johnson RJ, Couser WC, Chi EY, Adler S, Klebanoff SJ (1987a) New mechanism for glomerular injury: Myeloperoxidase-hydrogen peroxide-halide system. / C/in Invest 79:1379-1387. Johnson RJ, Klebanoff SJ, Ochi RF, Adler S, Baker P, Sparks L, Couser WG (1987b) Participation of the myeloperoxidase-H202-halide system in immune complex nephritis. Kidney Int 32:342-349. Johnson RJ, Guggenheim SJ, Klebanoff SJ, Ochi RF, Wass A, Baker P, Schulze M, Couser WG (1988) Morphologic correlates of glomerular oxidant injury induced by the myeloperoxidase-hydrogen peroxide-halide system of the neutrophil. Lab Invest 5:294-301. Katz SI, Strober W (1978) The pathogenesis of der-

Assessment of Myeloperoxidase Activity matitis herpetiformis. 75.

/ invest Dermatol

70:63-

Lianos EA (1988) Synthesis of hydroxyeicosatetraenoic acids and leukotrienes in rat nephrotoxic serum glomerulonephritis: Role of anti-glomerular basement membrane antibody dose, complement, and neutrophils. / C/in invest 82:427435. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folinphenol reagent. / Biol Chem 193:265-275. Ormrod DJ, Harrison CL, Miller TE (1987) Inhibition of neutrophil myeloperoxidase activity by selected tissues. / Pharmacol Methods 18:137-142. Priebat DA, Bradley PP, Christensen RD, Rothstein G (1983) The neutrophil response to polyvinyl

sponge

implantation.

Proc

Sot

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172:39-45.

Smith EW III, Egan JW, Bugelski PJ, Hillegass LM, Hill DE, Griswold DE (1988) Temporal relation between neutrophil accumulation and myocardial reperfusion injury. Am / Physiol 255:H1060H1068.

Snyderman R, Uhing RJ (1988) Phagocytic cells: Egress from marrow and diapedesis. In Inflammation:

Basic Principles

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Correlates.

Eds. Jl Gullin, IM Goldstein, and R Snyderman. New York: Raven Press, Ltd., pp. 309-323. Wahba AV, Barnes B, Lazarus GS (1984) Labeling of peripheral blood polymorphonuclear leukocytes with Indium-111: A new method for the quantitation of in-vivo accumulation of PMNLs in rabbit skin. / Invest Dermato/82:126-131.

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Assessment of myeloperoxidase activity in whole rat kidney.

A method to quantitate myeloperoxidase (MPO) activity from rat whole kidney is described. Polymorphonuclear leukocyte (PMN) infiltration into tissue i...
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