Quantitation of eosinophil and neutrophil infiltration into rat lung by specific assays for eosinophil peroxidase and myeloperoxidase Application in a Brown Norway rat model of allergic pulmonary inflammation

JOURNAL Of ii!EY’cAL

ELSEVIER

Journal of Immunological

Methods 198 ( 1996) 1- 14

Quantitation of eosinophil and neutrophil infiltration into rat lung by specific assays for eosinophil peroxidase and myeloperoxidase Application in a Brown Norway rat model of allergic pulmonary inflammation Thorsten Schneider, Andrew C. Issekutz

*

Department of Pediatrics and Microbiology-immunology. Dalhousie Unicersity Halifax, Canada Received 8 March 1996; revised 20 May 1996; accepted

12 June 1996

Abstract Conditions for measuring selectively eosinophil peroxidase (EPOJ and the neutrophil myeloperoxidase (MPO) in inflamed rat lung were determined. EPO could be specifically measured with o-phenylene diamine as chromogen at pH 8.0 in the presence of 3 mM bromide and MPO with tetramethylbenzidine as chromogen at pH 5.0 in the absence of bromide but with the EPO inhibitor, resorcinol. Aeroallergen challenge of sensitized Brown Norway rats with ovalbumin, but not with saline, resulted in a pronounced eosinophilic lung inflammation with some focal hemorrhages and an increase in lung wet weights. Quantitation of the eosinophil and neutrophil accumulation required lyophilization of lung samples, a hypotonic wash to remove contaminating hemoglobin, which interfered with the MPO assay, followed by extraction with the detergent cetyltrimethylammonium chloride. Based on lung EPO and MPO activities and standardization of enzyme activity with purified eosinophils and neutrophils, the total number of eosinophils and neutrophils in the lungs was calculated at 24 h (n = 191, 48 h (n = 9) and 72 h (n = 4) after challenge, as 56 + 6.4 X 10 6, 119 f 28 X lo6 and 108 f 33 X lo6 for eosinophils, respectively, and 94 + 6.8 X 106, 49 f 5.0 X lo6 and 32 f 5.5 X lo6 for neutrophils, respectively. We conclude that, with the assay conditions outlined here, EPO and MPO can be used to quantitate the tissue infiltration of eosinophils and neutrophils in the rat even in mixed inflammatory reactions. Keywords: Asthma; Hypersensitivity;

Airway;

Leukocyte

recruitment;

Animal model: Allergy

1. Introduction Abbreviations: BAL(F). bronchoalveolar lavage (fluid); BN, Brown Norway; CTAB, cetyltrimethylammonium bromide: CTAC, cetyltrimethylammonium chloride; EPO. eosinophil peroxidase; MPO. myeloperoxidase; OPD, o-phenylene diamine; PBS, phosphate buffered saline: RBC, red blood cells; TMB. 3’.5,5’-tetramethylbenzidine dihydrochloride. * Corresponding author. At: Department of Pediatrics, Izaak Walton Killam Hospital for Children, 5850 University Avenue, Halifax, Nova Scotia B3J 3G9. Canada. Tel.: (902) 428-8491: Fax: (902) 428-3217. 0022.1759/96/$15.00 Copyright PII SOO22- 1759(96)00143-3

Airway inflammation is a characteristic feature of allergic asthma and is considered to play an important role in asthma pathogenesis (Busse, 1993; Lukacs et al., 1995). Following contact with allergen, inflammatory and immune cells such as eosinophils, neutrophils, lymphocytes and monocytes are recruited

to the

specific

role of the different

0 1996 Elsevier Science B.V. All rights reserved.

lungs

of

the

allergic

subject.

inflammatory

The

cells is

not completely understood. Eosinophils. which are histologically the predominant cell type. are believed to be important effector cells, capable of mediating tissue damage and contributing to the development of airway hyperresponsiveness (Leff. 1994: Reed, 1994; Seminario and Gleich. 1994). The role of neutrophils is controversial. Infiltration of the lungs by neutrophils in response to allergen has been shown by many investigators. although it is not a consistent finding in human asthma. Neutrophils can produce potent inflammatory mediators and toxic oxygen radicals and proteases. suggesting that they also might be important (Busse. 1993; Lukacs et al.. 1995). A variety of animal models. with at least some features of human asthma. exist (Piechuta et al.. 1979; Murphy et al.. 1986: Abraham et al.. 1988; Hutson et al.. 1988; Gundel et al., 1992; Kung et al.. 1994: Woolley et al.. 1995). A model in the Brown Norway (BN) rat mimics human asthma in several aspects. This strain is a high IgE responder to allergic sensitization (Pauwels et al.. 1979: Waserman et al.. 1992). and following allergen challenge of sensitized animals, early and late asthmatic reactions occur (Renzi et al., 1993b), associated with pulmonary intlammation and bronchial hyperresponsiveness (Elwood et al.. 1991). For investigation of the regulation of leukocyte migration to the lung in animal models, quantitating the tissue content of these cells is essential. Different techniques have been employed such as: (a) ex vivo radiolabelling of leukocytes and quantitation of the lung content by gamma-counting (Hultkvist-Bengtsson et al., 19911, (b) histology of perfused and fixed lungs (Kung et al.. 1994). (cl enzymatic dispersion by tissue mincing and digestion with collagenase followed by cell counting (Renzi et al.. 199313). Quantitation of bronchoalveolar lavage (BALI cells is sometimes used alone or in combination with one of the outlined techniques. However, these techniques have limitations. Enzymatic dispersion and cell counting or histological quantitation of fixed lung are labor intensive and may be subject to non-specific cell loss or variation due to heterogenous involvement of lung tissue, respectively. BAL cell analysis alone might not fully reflect the inflammatory response in the tissue. Studies with radiolabelled eosinophils have been hampered by difficulties in purifying rat or guinea pig blood eosinophils.

Exudate eosinophils have been used for migration studies in rat (Sanz et al., 199.5) and guinea pig (Faccioli et al., 1991). but they may be activated, or at least primed and may differ from blood eosinophils in the expression of adhesion molecules (Walker et al.. 1993). Furthermore, in vitro handling of cells may also cause cell activation (Berends et al.. 1994; Youssef et al., 1995). An alternative method for quantitating the tissue content of granulocytes is the detection of peroxidase in tissue homogenates or extracts. since eosinophils and neutrophils both contain substantial amounts of peroxidase. However, discriminating between eosinophils and neutrophils in mixed inflammatory reactions. as occurs in diseases like asthma and atopic dermatitis. is important for understanding the specific mechanisms involved in their accumulation in the tissue and the role of these two cell types in the response. It has been shown that in human (Bolscher et al.. 1984). mouse (Strath et al.. 1985) and guinea pig (Tagari et al.. 1993). the peroxidases contained in eosinophils (eosinophil peroxidase, EPO) and in neutrophils (myeloperoxidase. MPO) differ in structure and biochemical activity. For example, human (Bozeman et al.. 1990) and guinea pig (Tagari et al.. 1993) EPO has a preference for Br- oxidation. which can be utilized to discriminate between these two enzymes. Rat eosinophils have been reported to contain peroxidase (Archer et al., 1965) but conditions for detection of and discrimination between EPO and MPO in tissues in the rat have. to our knowledge not previously been described. Accordingly. we have investigated conditions, which allow specific detection of rat EPO and MPO in the lung of allergen-challenged. sensitized BN rats as a means of quantitating the eosinophil and neutrophil components of the resulting pulmonary inflammation.

2. Materials and methods

Male, inbred Brown Norway (BN/SSN) rats. weighing 125 149 g were purchased from HarlanSprague Dawley (Indianapolis. IN). The animal experiments were in accordance with protocols ap-

T. Schneider. A.C. Issekutz / Journal of Inmunological Methods 198 (1996) l-14

proved by our University Animals.

Committee

2.2. Isolation of rat peritoneal

on Laboratory

neutrophils

Since the peritoneal cavity of naive BN rats contained a considerable number of resident eosinophils (2-3 X 106), closed peritoneal lavage was performed first to remove resident cells by injection of 50 ml of sterile saline i.p. under ketamine (Warner-Lambert Canada, Scarborough, ON) and Innovar (Janssen Pharmaceutical, Mississauga, Ont.) anesthesia (0.15 ml of a 4: 1 mixture of ketamine (50 mg/ml) and Innovar (50 pg/ml fentanyl citrate and 2.5 mg/ml droperidol) s.c./lOO g body weight), followed by gravity drainage through a 21 gauge needle. Lipopolysaccharide (E. coli, 0.55:B5, List Biologicals, Campbell, CA) (5 pug in 2 ml saline) was injected i.p. and the peritoneal lavage (50 ml) was repeated 3-16 h later and collected into 2 ml acid citrate dextrose (Fenwal-Travenol, Malton, ON, Canada) and 250 U of heparin. After centrifugation (200 x g for 10 min at 4”C), red blood cells (RBC) in the pellet were lysed with cold incubation (10 min at 4°C) in 5 ml of lysis buffer (0.155 M NH,Cl, 10 mM KHCO,, 0.1 mM EDTA, pH 7.4). After washing with phosphate buffered saline (PBS), 20-30 X lo6 neutrophils were obtained with > 95% viability and > 90% purity, assessed by Trypan Blue dye exclusion and staining of cytocentrifuge preparations with Diff-Quik (Baxter Healthcare, Miami. FL). Contaminating cells were a few percent macrophages and lymphocytes but < 1% eosinophils. 2.3. Isolation of rat peritoneal

eosinophils

To elicit an eosinophil rich exudate, 1 ml horse serum (Hyclone Laboratories, Logan, UT) was injected i.p. three times at 2-day intervals and a peritoneal lavage was performed 24 h after the last injection. After centrifugation (200 X g for 10 min at 4”C), cells were resuspended in 3 ml of Ca2+ and Mg’+-free Tyrode’s solution, pH 7.4, containing 10% rat plasma and 0.01% EDTA and were layered on a discontinuous Percoll (Pharmacia Fine Chemicals, Dorval, Quebec, Canada) gradient, consisting of 45%, S5%, 60%, 65% Percoll (based on 100% = isotonic Percoll, made up with 10 X concentrated

3

Tyrode’s), each layer containing 10% rat plasma and 0.01% EDTA. The gradient was centrifuged at 400 X g for 30 min at 4°C. The purest eosinophil fraction was recovered at the 60-65% Percoll interphase yielding 3-6 X lo6 eosinophils with > 80% purity by phloxine staining (Randolph, 1944) and cytocentrifuge smears stained with Diff-Quik. Contaminating cells were mainly macrophages with < 1% neutrophils. Viability of the cells by Trypan Blue exclusion was > 95%. 2.4. Enzyme extraction from eosinophils and neutrophils

peritoneal

exudate

Purified neutrophils and eosinophils were resuspended at 5 X lo6 or 1 X lO’/ml in 0.3 M sucrose, containing 0.22% cetyltrimethylammonium chloride (CTAC) (Aldrich Chemical Company, Inc. Milwaukee, WI) or cetyltrimethylammonium bromide (CTAB) (Sigma Chemical Co., St. Louis, MO). After vortexing for 1 min, the suspensions were freezethawed ( - 70°C) once, spun at 10 000 X g for 10 min at room temperature and the supematant (called hereafter neutrophil or eosinophil extract> was stored at -70°C until analyzed. 2.5. Assays of EPO and MPO activity toneal cell extracts

with peri-

The peroxidase substrates o-dianisidine, o-phenylene diamine (OPD) and 3’,5,5’-tetramethylbenzidine dihydrochloride (TMB) (Sigma Chemical Co.) were used. Serial dilutions of eosinophil and neutrophil extract were prepared in 10 mM citrate, pH 5.0, 0.22% CTAC with or without 6 mM KBr. 75 ~1 of these dilutions was added to wells of a 96-well flat-bottomed tissue culture plate (Nunc, GIBCOBRL, Mississauga, Ont.). The substrate solutions consisted of 3 mM o-dianisidine or OPD in 10 mM chloride-free citrate buffer, pH 5.0 and 8.8 mM H,O,, freshly prepared from a 30% stock H,O, (Fisher Scientific Co., Montreal, Que.). TMB (3 mM) was dissolved in a 5 mM citrate buffer, pH 5.0, because of precipitation at higher buffer concentrations. Buffer alone was pipetted into separate wells. The reaction was started by adding 75 PI substrate solution and after 15 min at 22°C protected. from light, the reaction was stopped by addition of 150 ~1

cold 4 N H,SO,. Absorbance was read at 490 nm (OPD and o-dianisidine) or 450 nm (TMB) on a Thermomax microplate reader (Molecular Devices, Menlo Park, CA). Blank values from buffer only wells were subtracted. To enhance the specificity of the MPO assay, the EPO inhibitor resorcinol (Fisher Scientific Company) was used. Resorcinol and eosinophil and neutrophil extracts were diluted in 10 mM citrate, pH 5.0. containing 0.22% CTAC. Extracts and resorcinol dilutions were mixed I : I. An equivalent to 40 000 extract eosinophil eosinophils/well and neutrophil extract equivalent to 20000 neutrophils/well was used. The substrate solution consisted of 3 mM TMB in distilled water, 8.8 mM H,O,. The reaction was stopped after 15 min with 4 N HsSOA. 2.6. Sensitization

and allergen challeqe

of BN ruts

Ovalbumin (OA, grade V, Sigma) was prepared at 2 mg/ml in 0.9% sterile, pyrogen-free NaCl and precipitated at I : 1 ratio with AI( (45 mg/ml, Imject Alum, Pierce, Rockford, IL) following the instructions of the manufacturer. Animals were sensitized by injection of a total of 1 mg OA (1 ml suspension) subcutaneously at two OA/AI(OH), different sites on the back of the neck. At the same time, 10” heat-killed Bordetella pertussis bacilli (gift from S. Halperin. Halifax, NS) in 0.5 ml saline were given i.p. as an adjuvant. 14 days later. the rats were placed in a 2 I- 1 sealed plexiglass chamber and challenged with an aerosol of 0.5% OA in saline. delivered at 3 liters/min by an ultrasonic nebulizer (Monaghan 670, Monaghan. Littleton, CO) at setting 7. Rats were challenged for I h and the challenge was repeated 5 h later. as described by Kung et al. (1994) in a murine model of allergic pulmonary eosinophilia. A control group of sensitized rats was challenged with saline. 2.7. Dissection

and lung pet$isiorz

At various time points after allergen challenge. the rats were anesthetized by i.p. administration of ketamine (50 mg/kg) and xylazine (IO mg/kg, Rompun, Chemagro, Etobicoke, Ont.. Canada). The abdominal cavity was opened. a 25 gauge butterfly needle was inserted into the inferior vena cava and

100 U of heparin in I ml saline was injected. The abdominal aorta was severed and at the same time, Tyrode’s (pH 7.4. 37°C) solution (with 1.8 mM CaCl: and 2 mM MgC12) was infused into the inferior vena cava. After 25 ml of Tyrode’s, the chest was opened and another 25 ml of Tyrode’s solution was infused via the vena cava above the diaphragm. This was followed by IO ml of chloridefree 0.3 M sucrose, 5 mM phosphate, pH 7.4, containing 0.2% EDTA. This consistently cleared the lung vasculature free of blood cells. 2.8. Bronchoalwolar nutiori

1aLxzge und BAL cell detenni-

The trachea was cannulated and 7 ml of cold (4°C). Ca2+ and Mg’+-free. pyrogen-free PBS with 0.1%~ EDTA. was instilled, withdrawn and this was repeated twice, followed by a last lavage with the sucrose buffer. Cells in the bronchoalveolar lavage fluid (BALF) were sedimented by centrifugation (10 min at 200 X g, 4°C) and resuspended in PBS. Total leukocyte counts were determined by crystal violet stain (Sigma Chemical Co.). Eosinophils were counted using 0.05% phloxine B (Sigma Chemical Co.) in 50% propylene glycol (Sigma Chemical Co.) in water. Cytocentrifuge preparations were stained with Diff-Quik and at least 200 cells were counted. Total BAL eosinophils are reported based on the phloxine B stain, the BAL neutrophil. lymphocyte and therophage counts were calculated from the differential on the stained cytocentrifuge preparations and the total leukocyte count. Eosinophil counts by phloxine stain and by Diff-Quik-stained slides were in good agreement. 2.9. Eqvrne

extruction from lungs

After BAL, the lungs were removed and weighed. Samples of parenchyma from each lobe, approximating 350 mg (lo-208 of the total lung wet weight), were pooled and stored at -70°C until being freeze-dried. For enzyme extraction, lyophilized samples were weighed, homogenized in 50 mM Hepes. pH 8.0 at 0.5% dry w/v with a pestle homogenizer (Talboys Engineering Corp., Emerson, NJ) in glass tubes for 1 min at setting 30-40 with IO- 15 passages. spun at 10 000 X g for 30 min at

i? Schneider,

A.C. Issekutz/

Journal

4°C and the supernatant was discarded. Less than 5% (3.5 f l.l%, IZ= 11) of EPO activity was found in this supematant. The pellet was resuspended in 0.5% CTAC in distilled water to the original volume, rehomogenized and spun again as before. This resulted in a pellet with a clear supematant and a thin lipid layer on the top. An aliquot of the clear supernatant, referred to as lung extract, was taken for analysis of EPO and MPO activity. Extracts were generally analyzed on the day of extraction but could be stored at -20°C without detectable loss in EPO or MPO activity (over several months) even after repeated freeze-thawing. 2.10. EPO and MPO detection in lung extracts Lung extracts were diluted I/ 10 in 50 mM Hepes, pH 8.0 (EPO dilution buffer) or 10 mM citrate buffer pH 5.0 (MPO dilution buffer). Aliquots of 75 ~1 of each sample were pipetted into four wells of a 96-well tissue culture plate. Cold stop solution (4 N HZ SO,, containing also 2 mM resorcinol for the EPO assay) was added to two wells (150 pi/well) to stop the reaction at t = 0 s (background OD). The EPO-substrate solution consisted of 50 mM Hepes, pH 8.0, 6 mM KBr, 3 mM OPD, 8.8 mM H,O, and the MPO-substrate solution was 3 mM TMB, 120 /.LM resorcinol and 2.2 mM H,O, in distilled water. Substrate solution (75 ~1) was added to each well, the reaction was stopped after 30 s (EPO) or 2 min (MPO) with 150 ~1 of cold stop solution, and the OD,,, nm (EPO) or OD,,, nm (MPO) was determined. As an additional control, 75 ~1 dilution buffer was placed into four wells, 75 ~1 substrate buffer was added followed by 150 ~1 stop solution after 30 s or 2 min. No color reaction was observed in these controls. All reagents were used at room temperature and the reaction was carried out at 22°C. The enzyme activities of the lung samples were calculated by subtracting the mean background OD and are expressed as change of OD/min. 2.1 I. Calculation of the total number of eosinophils and neutrophils in the lungs and MPO activity correction formula To develop a standard curve for calculating the number of eosinophils in the lungs based on the

of Immunological

Methods

I98 (1996) I-14

5

enzyme activities, BAL eosinophils were harvested at 48 h post challenge from sensitized BN rats and the BALF from rats with high eosinophil counts were pooled. Of this preparation (86% eosinophils, 3% neutrophils, 11% macrophages and lymphocytes), 10 X lo6 BAL eosinophils (in a volume of 50 ~1 PBS) were injected, without further purification, into a piece of non-inflamed control lung (200 mg wet weight), using a 0.5 ml insulin syringe with a 28 gauge needle. This tissue was then treated as described under Section 2.9. The EPO activity of the resulting extract was tested at different dilutions and the activity plotted against the theoretical eosinophil equivalents in the dilution of lung extract. This standard curve was used to estimate the eosinophil number in test lung extracts and the lungs. On this extract from the eosinophil injected control lung, the MPO activity was also measured and correlated with the EPO activity. Endogenous activities of the control lung tissue alone were subtracted. The MPO activity attributable to the 3% neutrophils in the eosinophil preparation, was below the detection limit for the MPO assay, based on the absolute number of neutrophils present. The resulting linear regression was therefore used as a correction of EPO spillover to the MPO assay (activity) in lung extracts. For calculating the neutrophil content of the lungs, a similar approach was used by injecting 53 X 10h peritoneal neutrophils (85% pure, 3.75% eosinophils) into control lung tissue (200 mg wet weight), which was then lyophilized and extracted. Different dilutions of this extract were analyzed for MPO activity and this standard curve was used for estimation of the neutrophil content of the lungs. Compared to a direct CTAC extraction of the used eosinophil and neutrophil preparations, 68% and 70%, respectively, of the injected activity was recovered in the lung extracts.

2.12. Data analysis All data are reported as arithmetic means. Error bars represent 1 SEM. The difference between duplicate wells was I 3% of the mean value. Differences between groups of rats were analyzed by Student’s unpaired t test using the StatView data analysis system version 4.5 (Abacus Concepts, Inc.. Berke-

ley. CA). A 11 value significant.

< 0.05 was considered

to be

Table

I

Effect of bromide ions on MPO and EPO activity with different chromogen\

3. Results

A,,,, 3

14

I

18

I

OPD

Enrqme

cell equivalents/well “1

cell equivalents/well

/----+

E3 0

m2

z 01 0

d Fig.

I.

5x103 1x104 cell equivalents/well

Peroxidasr activity of eohinophil and neutrophil extracts

%,ith variou’r chromogens and effect of bromide ions. Extracts 01 purified peritoneal eosinophilh (0 trophils (m TMB

+KBr.

+KBr.

0

no KBr) and neu-

0 no KBr) were essayed with r,-diankidine.

and OPD chromogens in the presence or absence of 3 mM

final KBr.

Extracts (in 0.22%

citrate. pH 5.0. 0.23%

CTAC

CTAC)

were diluted in IO mM

with or without 6 mM

these, substrate holution was added in 10 mM (TMB citrate buffer pH S.O. X.8 mM H,O,. 15 min with 4 N H2S0,.

KBr. To in S mM)

Reaction was stopped after

Values are means of duplicate wells

from one representative experiment of three.

(fold increase)

6

TMB

To find conditions for specific detection of rat EPO. the activity of eosinophil and neutrophil extracts with three different chromogens was investigated, as shown in Fig. 1. For all chromogens. reactions were cell number dependent and linear. o-Dianisidine was the least sensitive of the chromogens used for either detection of rat EPO or MPO, even at optimal wavelength of absorption (525 nm). In the absence of bromide ions, the eosinophil extract showed a higher activity than the neutrophil

A EP,j (fold increase)

Chromogen +Dianisidintt

2Y5

activity of extracts from purified peritoneal eosinophils

and neutrophils was investigated in the presence or absence of 3 mM

KBr.

Shown

is the increase in enzyme

activity

(A)

by

bromide ions compared to the activity without KBr as determined hy the \lope of the regression line5 for the linear part of the reaction curves in Fig.

I

extract with OPD as chromogen, whereas the opposite was the case for o-dianisidine and TMB. Bozeman et al. (1990) have demonstrated that bromide (optimally 3 mM) increases the activity of human EPO and to a lesser extent MPO, when added in the form of the detergent CTAB or as KBr. Accordingly. we investigated the effect of 3 mM final KBr on the activity of the extracts. Bromide stimulated EPO and MPO catalyzed oxidation of all three chromogens, but the effect on EPO activity was more pronounced (Fig. I ). To determine the effects of bromide and chromogen on the EPO and MPO activities, linear regressions for the linear part of the curves in Fig. I were performed. and the fold increase in the slope for each line is shown in Table I. The greatest effect of bromide was seen in the increase in EPO activity (295.fold) with TMB as chromogen. whereas the effect on MPO was most pronounced (1 l-fold) with OPD. Despite this, OPD allowed the most specific detection of EPO with minimal interference by MPO (Fig. I) and the difference between EPO and MPO activities with OPD as chromogen was further increased if bromide was present (Table 2). KBr could be substituted by CTAB in this assay, which gave a very similar result (data not shown). To further optimize conditions for specific detection of rat EPO. we next investigated EPO and MPO activity with OPD in the presence of bromide at pH 7-10. MPO activity declined with higher pH, whereas EPO activity was relatively constant in this pH range (data not shown). The pH optimum of human EPO is reported to be higher than that of MPO (Bos et al.. 1981) and to be pH 8.0 for EPO-catalyzed oxidation of OPD in the mouse (Strath et al., 1985) and between 6 and 8

T. Schneider, A.C. Issekutz/Joumal Table 2 Effect of chromogen MPO activity

and bromide

ions on the ratio of EPO to

Chromogen

+KBr(Aa,o/A,,ol

-KBr(An,o/A,,o)

o-Dianisidine OPD TMB

1.8 21.3 4.2

0.4 12.9 0.1

Enzyme activity of extracts from purified peritoneal eosinophils and neutrophils was investigated in the presence or absence of 3 mM KBr and with three different chromogens. Shown is the ratio of EPO activity/MPO activity I A,,, / AMpo) for the combinations of chromogen and presence or absence of bromide ions as determined by the slope of the regression lines for the linear part of the curves in Fig. 1.

in the rat (Archer et al., 1965). We therefore decided to use pH 8.0 for detection of rat EPO. Fig. 2 shows an evaluation of the effect of control lung extract on measurement of EPO or MPO activity in the granulocyte extracts (Fig. 2). At pH 8.0 and with 3 mM final bromide, the EPO activity was 2.5fold higher than the MPO activity per cell, based on the slope of the linear portion of the curves (Fig. 2). With 2 1.5 mM OPD, the reaction remained linear with increasing cell number up to an OD of

7

of Immunological Methods 198 (1996) l-14

more than 3. It is noteworthy that, after H,SO, was added to stop the reaction, oxidation of the chromogens slowly continued when bromide was present. This could be prevented by addition of 2 mM resorcinol to the H 2SO,.

of a specific rat MPO assay

3.2. Development

We observed that the neutrophil extract, in the absence of bromide ions, catalyzed a greater oxidation of TMB than the eosinophil extract (Fig. 1). Therefore, based on this difference, the possibility of developing an assay, specific for rat MPO with minimal interference by EPO, was examined. The activity of rat MPO in the absence of bromide ions was found to be optimal at pH 5 (data not shown). A variety of EPO inhibitors have been reported for other species (Cramer et al., 1980; Strath et al., 1985; Bozeman et al., 1990). Of the inhibitors tested (azide, aminotriazol, resorcinol), only resorcinol consistently inhibited rat EPO more than MPO, under the conditions used. In a representative experiment of inhibition by resorcinol, shown in Fig. 3, the 50% inhibitory concentration was 10 PM for EPO and 1oo+

t

eosinophils

-N-

neutrophils

_

eosinophils in control lung extract

MPO

1 0.1

0

1x104 2x104 cell equivalents/well

Fig. 2. Optimal conditions for measuring EPO activity. Eosinophil and neutrophil extracts were diluted in 10 mM Hepes, pH 8.0. containing 0.22% CTAB (6 mM BrY 1 or in control lung extract also in 0.22% CTAB and diluted l/IO in 10 mM Hepes, pH 8.0, 0.22% CTAB. The substrate solution was 3 mM OPD and 8.8 mM HzOz in 10 mM Hepes, pH 8.0. Intrinsic activity of control lung extract was 0.099 and was therefore subtracted from all appropriate test wells. Reaction was stopped after 15 min. The values for symbols 0 and W overlap at all points on the figure and therefore cannot be discriminated. Shown are means of duplicate wells from one of three similar experiments.

1

10

100

1000

10000

resorcinol [PM] Fig. 3. Inhibition of peroxidase activity by resorcinol. Resorcinol and eosinophil and neutrophil extracts (in 0.22% CTAC) were diluted in 10 mM citrate, pH 5.0, containing 0.22% CTAC. Eosinophil extract equivalent to 40000 eosinophils/well and neutrophil extract equivalent to 20000 neutrophils/well was used. An equal volume (75 ~1) of substrate solution (3 mM TMB in distilled water with 8.8 mM H,Ozl was added. Reaction was stopped after 15 min with 4 N H,SO,. Inset: Discrimination between rat MPO and EPO, as determined by the ratios of their activities. was optimal at about 60 FM final resorcinol. Shown are means of duplicate wells from one representative experiment of three.

3 +

neutrophils

-o-

eosinophils /

1 -w-

MPO

-o-

EPO

-t

MPOIEPO

cell equivalents/well

$0, WI Fig. -I. Effect of H20z concentration on MPO/EPO discrimination. Extract from purified peritoneal eosinophils and neutrophils (in 0.22% CTAC) were diluted in IO mM citrate buffer, pH 5.0 to a final concentration equivalent to 25X IO” cells/well. H,O, wu diluted in the substrate solution (3 mM TMB. I20 PM resorcinol in distilled water). and an equal volume of this mixture (75 ,ul) was added to the wells to give the indicated final concentration. Reaction wah stopped after 2 min with 4 N H,SO,. Shown are means of duplicate!. from one representative experiment of four.

300 FM for MPO. The difference between EPO and MPO activity, as determined by the ratios at OD,,,, ,,,,, (Fig. 3, inset) was greatest at about 60 FM resorcinol. Subsequently. 60 PM resorcinol final concentration was incorporated into the MPO assay. The effect of H,O, on EPO and MPO activity was also investigated. as shown in Fig. 4. The MPO activity was maximal with about 0.5 mM H,O,. whereas the best discrimination between MPO aid EPO activities was seen at 2 I mM. However. higher concentrations were inhibitory for both enzymes. Subsequently. I. 1 mM H,O, was used in the MPO assay. Finally, Fig. 5 shows optimized conditions for specific detection of rat MPO. Based on the slope of the lines, the MPO activity was 7.6-fold higher than the EPO activity on a per cell basis, and this was in the range of S- 1l-fold in five independent experiments with different extracts. 3.3. Allergen challetlgr of’sensiti~ed ruts crud yuuntitatim of lutlg rosinophils utd rleutrophils Challenge of sensitized BN rats with 0.5% OA aerosol resulted in accumulation of eosinophils and

Fig. i. Optimal conditiona for measuring MPO activity. Eosinophil and neutrophil extracts (5X lob/ml in 0.22% CTAC) were diluted in IO mM citrate. pH 5.0, containing 0.22% CTAC. To these. substrate solution. consisting of 3 mM TMB in distilled water. I20 PM reaorcinol (60 PM final concentration in well). 1.1 mM H1O,. was added. Reaction was stopped after 10 min at room temperature with 4 N H,SO,. Shown are means of duplicates from one of three similar experiments,

neutrophils and to a lesser extent of mononuclear cells in the BALF. as shown in Fig. 6. There was no intlammatory reaction in sensitized, saline-exposed

14m 12-

I

eos.

neutr.

lymph.

macroph.

Fig. 6. Leukocyte accumulation in BALF after OA challenge of sensitized BN rats. Numbers of BALF cells at 24 h (n = 19). 38 h (II = 9) and 72 h (n = 4) after challenge are shown. Total cell numbers in saline-challenged control rats (n = 9) at 24 h were: 1.8*2.8x IO’ eosinophils, 3.1 + 1.6X IO3 neutrophils, 5.6+1.1 X IO’ lymphocytes and 9.7+ 1.7X IO’ macrophages. Cell numbers were significantly increased (p < 0.01 for eosinophils and neutrophils and pi 0.05 for macrophages and lymphocytes) in the OA-challenged group. Values are means i SEM.

T. Schneider. A.C. Issekutz/Joumal

rats investigated at 24 h and the inflammatory cell influx in the OA-challenged group was significantly increased for all cell types ((p < 0.01 for eosinophils and neutrophils, and p < 0.05 for macrophages and lymphocytes) at this time point. Neutrophils peaked at 24 h (2.7 f 0.35 X 106) and declined rapidly thereafter. In contrast, eosinophils continued to accumulate, reaching a mean of 10.1 + 2.4 X IO6 72 h after the challenge, comprising up to 86% of BALF cells at this time. There appeared to be a slow but ongoing increase in lymphocyte and macrophage counts during this time. We observed a 50% increase of lung wet weights after lung perfusion and BAL in allergen-challenged rats compared to the saline-challenged control group (( p < 0.0001, data not shown). Therefore, lung tissue was lyophilized to standardize the homogenization and extraction based on the dry weight to compensate for this. Final extractions were done with CTAC in water to allow addition of KBr as a bromide source for the EPO assay and to allow ready adjustment of the pH by dilution of the extract in the appropriate buffer. The kinetics of OPD and TMB oxidation was studied with lung extracts from control rats and from sensitized rats 24 h after allergenchallenge (data not shown). It was found that the EPO assay was linear for 30-60 s if AOD/min was I 3. The MPO assay was linear for about 2 min for AOD/min I 1.5. Outside of this range, the time curves developed hyperboloid shapes. Since only the initial, linear rate of oxidation can be assumed to be proportional to the peroxidase concentration (Bozeman et al., 19901, in this case the MPO or EPO content of the lung, the reaction was stopped after 30 s for the EPO assay or after 2 min for the MPO assay, and the results are reported as change of OD/min. Testing extracts at different dilutions (l/320l/2.5 in the appropriate dilution buffer) showed that the EPO assay was linear at dilutions 2 l/5 whereas the MPO assay was linear at dilutions 2 l/IO (data not shown). Thus, extracts were tested at l/IO dilutions in both assays. Despite extensive perfusion of the pulmonary vasculature and BAL, some red blood cells (RBC) remained in the lungs due to RBC extravasation during the inflammatory reaction in sensitized, allergen-challenged animals. TMB reacts also with hemoglobin (Liem et al., 1979). and we found that

9

qflmmunological Methods 198 (1996) I-1-1

this interfered with the MPO detection. Using a spectrophotometric measure of hemoglobin content, we estimated that about 3 ~1 of blood was present in specimens (ca. 1/IO of the entire lung tissue) of inflamed lung. Therefore, a hypotonic washing step was added to the extraction protocol as described under Section 2. To investigate the efficiency of the washing procedure, 15 ~1 normal rat blood (five times the highest estimate of RBC contamination) was injected into a blood-free control lung specimen, and the MPO activity (AOD 450 nm/min) of the supernatant after the washing step (wash-SN) and of the final CTAC extract was measured. The endogenous MPO activity of the blood-free control lung piece was 0.09, of blood-injected lung after a hypotonic washing step was 0.04 and of the wash-SN was 0.62 ( AOD/min). Hence, even a massive RBC contamination could be effectively removed with this technique. The results in Fig. 7 show that 24 h after allergen-challenge, the EPO activity in lung extracts of OA-challenged rats was 67-fold and MPO activity was 5-fold increased compared to saline-challenged control animals (( p < 0.0001). MPO activities

0 24 i

EPO

MPO

;z

h after challenge

MPO (corrected)

Fig. 7. Time course of lung EPO and MPO activities after allergen challenge. Lung EPO and MPO activities were determined as described under Section 2. MPO activities were corrected for EPO contamination as described in Section 3. Peroxidase activities in saline-challenged control rats at 24 h were: 0.01 +0.003 @PO). 0.05 ~0.005 (MPO), 0.048+0.005 (corrected MPO). EPO and MPO activities were significantly increased in the OA-challenged group (p < 0.0001). Number of animals (n) were as in Fig. 6.

160,

peaked at 24 h and subsequently declined steadily whereas EPO activities peaked at 48 h after challenge.

Correlating the EPO activity of the eosinophil-injetted lung extract at different dilutions (see Section 2) with its MPO activity revealed a linear relationship between these two parameters (not shown). Linear regression analysis (R’ = 0.996) showed. that 5.6% of the measured EPO activity had to be subtracted from the measured MPO activity to compensate for EPO detection by the MPO assay. This is how the corrected MPO values shown in Fig. 7 were derived. On the other hand. the EPO assay did not

I 24 o 48 h after . 72 challenge

120 ZI p .5 801 I =v) 8 40

0’

-_L

eosinophils

h

neutrophils

Fig. Y. Total eosinophils and neutrophils in the lungs of OA-chnllenged. sensitized BN rdts at various times after challenge. Values were calculated based on the standard curves of EPO and MPO acnvity in control lung injected with known numbers of eosinophil\ and neutrophils

as in Fig.

8.

Means&SEM

with

number

of

animals 01) as in Fig. 6.

MPO in the extract of the neutrophil-injected lung tissue (not shown). In order to estimate the actual number of eosinophils and neutrophils in the lungs of OA-challenged, sensitized rats, standard curves were generated from EPO and MPO activity in extracts of control lung pieces injected with known numbers of eosinophils and neutrophils. These results are shown in Fig. 8. Based on these curves, we estimated the total number of eosinophils and neutrophils in the lungs at various times after challenge, as shown in Fig. 9. We considered the possibility. that the immune reaction in the lung may have induced degranulation of eosinophils. resulting in an unreliable estimate of the number of eosinophils in the tissue. Therefore. the EPO activities of purified peritoneal eosinophils, elicited by i.p. injection of horse serum, and BAL eosinophils at 48 h and 72 h post challenge were compared and the BAL eosinophils were found to possesa similar (about 10% higher) EPO activities to the peritoneal eosinophils (data not shown). detect

eosinophils/ml extract x lo5

neutrophils/ml extract x 1 O5 Fig. 8. Standard curves for calculation of total eosinophilh (ii) nrutrophils

(B)

in the lungs. Control (non-inflamed)

injected with purified eosinophils ( A) or neutrophils extracted as in Section 1. The EPO and MPO

and

lung wah

(~7)and

activitieh were

correlated with the theoretical eosinophil or neutrophil equivalents in the dilution of lung extract (linear regression: R‘ = O.YY6 ( A 1 and R’ = 0.996 CL?)).

4. Discussion

then

We have developed assays for detection 01 eosinophils and neutrophils in the lung tissue of BN rats based on peroxidase measurements. utilizing

T. Schneider, A.C. Issekut: / Journal

of Immunological

biochemical differences between the eosinophil peroxidase and the neutrophil myeloperoxidase. Our results indicate that the activity of rat EPO and MPO is stimulated by bromide ions as has been shown for the human (Bozeman et al., 1990) and guinea pig peroxidases (Tagari et al., 1993). As in human and guinea pig, rat EPO activity is stimulated to a greater extent by bromide than MPO activity and this phenomenon can be exploited to distinguish between the two peroxidases. However, our results also show that the degree of Br- stimulation depends on the chromogen and that enzyme activity is a function of both the chromogen and presence or absence of bromide. The stimulatory effect of bromide ions on EPO activity can vary as much as 1Cfold (for o-dianisidine) and 295fold (for TMB) increase in activity, as shown in Table 1. Halide ions are the physiological substrates for MPO and EPO, which use H,O, to oxidize them and to produce antimicrobial agents such as HOBr or HOCl. Human EPO has been shown to be much less active in Cl- oxidation than in Br- oxidation (Bozeman et al., 1990) and the same appears to be true for rat EPO. Since oxidized halide ions themselves are highly oxidizing, they can non-enzymatically oxidize chromogens commonly employed in detecting peroxidase enzymes as has been shown for HOC1 and HOBr-driven oxidation of TMB (Andrews and Krinsky, 1982; Bozeman et al., 1990). Therefore, the observed rate of chromogen oxidation depends on both direct peroxidase-catalyzed oxidation and indirect non-enzymatic oxidation. The rate of direct oxidation of TMB by EPO appears to be much slower than the EPO-catalyzed oxidation of bromide, but TMB is readily and quickly oxidized by HOBr, which likely explains the dramatic increase in TMB oxidation rate when bromide was present (Table 1, Fig. 1). Rat EPO has a preference in OPD oxidation compared to MPO, as has been noted previously for the mouse enzymes (Strath et al., 1985). This preference is further increased by bromide (Table 2, Fig. l), making the combination of OPD with bromide ions especially suitable for detection of rat EPO. On the other hand, TMB and o-dianisidine were more readily oxidized by rat MPO compared to EPO, when bromide was absent (Fig. 1, Table 2). Although the reaction buffer used was essentially Cl-free, chloride was introduced by TMB (because the

Methods 198 (1996) I- I4

11

dihydrochloride-derivative had to be used) and by the detergent CTAC. Therefore, we cannot determine, whether a direct or Cl--mediated reaction accounts for this effect. TMB was more sensitive than o-dianisidine for detecting MPO and TMB was also more specific (Fig. 1, Table 2). Hence, TMB was employed in the development of a selective MPO assay. In order to enhance the selectivity of MPO detection in the presence of significant amounts of EPO, it was found that an EPO inhibitor was required. Of the three EPO inhibitors tested, resorcinol in our hands was the only one, which consistently inhibited rat EPO to a greater degree than MPO. It is not clear, why the other inhibitors (azide, aminotriazol), which have been reported to selectively inhibit human EPO (Cramer et al., 1984; Bozeman et al., 1990). failed to selectively inhibit rat EPO compared to MPO. Species differences as well as differences in the assay condition used could account for this. The precise mechanism of inhibition is not known for any of the inhibitors, and they might act at different stages. We found e.g. that 60 /_LMresorcinol inhibited EPO and MPO completely, when the MPO assay was performed at pH 8.0. Each of the assay parameters (pH, halide ions, chromogen, hydrogen peroxide concentration, and even the buffer system used (Suzuki et al., 1983)) could potentially influence enzyme activity and the behavior of an inhibitor. Bozeman et al. (1990) used assay conditions similar to ours and found selective inhibition of human EPO with azide, aminotriazol and dapsone. It appears therefore likely, that there are species differences in the biochemical properties of EPO and MPO, at least between human and rat. Tagari et al. (1993) investigated some biochemical properties of guinea pig MPO and EPO. In contrast to our studies, they found that purified peritoneal guinea pig eosinophils had a higher activity than neutrophils on a per cell basis with TMB as chromogen, even in the absence of bromide ions. Although their assay system was quite different from ours, there might also be biochemical differences between the guinea pig and the rat enzymes, or the peroxidase content of guinea pig eosinophils might be higher or the MPO content of neutrophils lower compared to the rat. Structural and functional differences between rat EPO and MPO were observed as early as 1965

(Archer et al., 1965) but no attempts for measuring both enzymes selectively in solid tissues have to our knowledge previously been undertaken. To investigate the applicability of the in vitro assay conditions to measuring the inflammatory response in a solid tissue, we induced eosinophil and neutrophil infiltration into the lungs of OA-sensitized BN rats by aerosol challenge. The immunization and challenge protocol used by us resulted in a massive infiltration of neutrophils and eosinophils into the alveolar space as assessed by BAL and into the lung parenchyma as evaluated by lung peroxidase measurements and confirmed by histology (not shown). In accordance with other studies in this model (Renzi et al., 1993a; Tominaga et al.. 1995: Yu et al.. 1995). there was an early but transient influx of neutrophils and a delayed but longer lasting influx of eosinophils (Fig. 6, 9). Accurate quantitation of the eosinophil and neutrophil accumulation in the lung tissue required the development of a specialized protocol for enzyme extraction and detection. Freeze drying was needed to compensate for the increase in the wet weight of the lung tissue. which appeared to be dependent on the degree of inflammation. There was very little release of enzyme into the supernatant during the first wash homogenization without detergent, although all of the contaminating hemoglobin was released, suggesting that even after lyophilization and homogenization most of the granules stay intact or the peroxidases remain membrane-bound and can be sedimented. Our experiments with injection ol exudate eosinophils and neutrophils into control lung tissue followed by the lyophilization and enzyme extraction protocol (Fig. 8). validate the specificity of the assays for measuring the response in the lungs. This is further supported by the time course of EPO and MPO activities. which shows increase of EPO activity from 24 to 48 h. but at the same time a drop of MPO activity (Fig. 7). In a careful comparison of the MPO and EPO assays, there was virtually no spillover of MPO activity into the EPO assay, but some EPO activity was detected by the MPO assay (data not shown). However, a correction formula could easily be derived, so that with a known EPO activity, reliable MPO data can be obtained even when eosinophils predominate in the tissue analyzed (Fig. 7 and Fig. 9). Since both assays can be performed on the same

extract. specific information about the participation of eosinophils and neutrophils in the inflammatory reaction is readily obtained. Calculations of absolute cell numbers in the tissue based on peroxidase measurements may be affected. at least in theory. by variations in the peroxidase content between cells. depending on their degree of maturation or state of activation. which might be associated with partial degranulation. However, Strath et al. (1985) found no difference in the EPO activity per cell between different tissues of the mouse. This agrees with our finding that the EPO content per cell in BAL and peritoneal eosinophils is comparable. Thus, this may not be a major factor in practice. A major advantage of the method described here for the rat is that there is no need for leukocyte isolation requiring donor animals and in vitro handling of cells. which may induce functional alterations such as changes in adhesion molecule expression (Berends et al., 1994; Youssef et al.. 1995). Our results make clear that careful control over the assay conditions is required to guarantee selectivity of MPO and EPO assays. The detergent CTAB (HTAB) is often used for tissue peroxidase extractions because it is known to extract EPO and MPO very well (Bos et al.. 1981). However, since bromide ions are introduced with this detergent, it should be replaced by CTAC. when MPO measurements are to be done. Studies which disregard this could easily come to faulty MPO measurements, even with eosinophil contamination of only a few percent, because of the potent stimulation of EPO by Br-. Furthermore. assays developed for one species should be verified for EPO versus MPO specificity, when employed in a different species. due to possible differences in the behavior of EPO and MPO under the specific assay conditions as suggested by our findings here. Relatively little is known about the eosinophil and neutrophil numbers in the lung tissue itself and how this relates to their appearance in the BALF in the BN rat model. Earlier studies reported eosinophil counts between 6-16 X IOh. in the lung tissue at 32 h post challenge, using enzymatic dispersion of lung cells (Renzi et al.. 1993a; Laberge et al., 1995). This contrasts with our quantitation indicating 56 X lOh eosinophils by lung EPO assay at 24 h (Fig. 9). Thus. the tissue response observed by us appeared to be much greater than had been previously described

T. Schneider. A.C. Issekutz/

Journal of Immunological

in the BN rat. The prolonged (1 h) challenge protocol, administered twice within 6 h, which was adapted from Kung et al. (1994), who employed it in a murine model of allergic pulmonary inflammation, might be one reason for this. Additionally, nonspecific cell loss and incomplete release of cells from connective tissue, might complicate enzymatic lung dispersion, perhaps partially accounting for the differences. In conclusion, we have shown, that under the specific assay conditions outlined in this paper, rat EPO and MPO activity can be used to quantitate the infiltration of the lungs by eosinophils and neutrophils, even in a mixed eosinophilic-neutrophilic inflammation. This technique should be useful for investigating the regulation of granulocyte extravasation and should especially be helpful for defining the specific role of neutrophils and eosinophils in lung inflammation models in the rat.

Acknowledgements This work was supported by a grant from the Respiratory Health Network of Centres of Excellence (Inspiraplex). T. Schneider is a recipient of a scholarship of the German Academic Exchange Service (DAAD). The authors gratefully acknowledge the excellent technical assistance of Carol Jordan and Derek Rowter. We also thank Dr. Linda Ayer for helpful review of the manuscript and Annette Morris for production of Bordetella pertussis vaccine.

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