Effects of Pasteurella haemolytica Leukotoxic Culture Supernatant on Bovine Neutrophil Aggregation Peter Conlon, Michele Gervais, Savita Chaudhari and Jennifer Conlon

ABSTRACT

Pasteurella haemolytica Al leukotoxic culture supernatant was evaluated for its ability to cause aggregation of bovine peripheral neutrophils. Neutrophils were isolated by a hypotonic lysis method and incubated with zymosanactivated plasma (ZAP), leukotoxic culture supernatant, antileukotoxin serum, calcium and magnesium-free media, p-bromophenacyl bromide and protein kinase C inhibitors. Aggregation was evaluated by changes in infrared light transmittance. Leukotoxic culture supernatant caused neutrophils to aggregate, and this effect was significantly removed by preincubation with antileukotoxin serum. Aggregation to ZAP and leukotoxin was dependent on the presence of extracellular calcium. Activation of protein kinase C by phorbol myristate acetate induced aggregation which was reduced by staurosporine; however, aggregation to leukotoxin did not involve protein kinase C activation. Phospholipase A2 inhibition did not alter the aggregation response to ZAP or to leukotoxin. The in vitro measurement of neutrophil aggregation induced by the leukotoxin of P. haemolytica reflects cytoskeletal and other activation events that may contribute to the intense inflammatory process which this organism induces in the lungs of cattle.

neutrophiles peripheriques bovins. Les neutrophiles, isoles par une methode de lyse hypotonique, ont ete incubes en presence de plasma active au zymosan (PAZ), d'un surnageant de culture ou d'un antiserum dirige contre la leucotoxine, dans un milieu sans calcium ou magnesium, ou en presence du bromure de p-bromophenacyle ou d'inhibiteurs de la proteine kinease c. L'agregation des neutrophiles a ete evaluee par turbidimetrie en lumiere infrarouge. Le surnageant de culture a entraine l'agregation de neutrophiles; un effet qui etait significativement bloque par une preincubation avec l'antiserum contre la leucotoxine. L'agregation des neutrophiles par le PAZ et la leucotoxine etait dependante de la presence de calcium extra-cellulaire. L'activation de la proteine kinease c par l'acetate de phorbol myristate induisait une agregation qui pouvait etre reduite par la staurosporine; par contre, I'agregation causee par la leucotoxine n'impliquait pas l'activation de la proteine kinase c. L'inhibition par la phospholipase A2 n'a pas modifie l'agregation causee par le PAZ ou la leucotoxine. La mesure in vitro de l'agregation des neutrophiles induite par la leucotoxine de P. haemolytica reflete des processus d'activation, cytosquelettique ou autres, qui peuvent contribuer a la reponse inflammatoire intense que ce microorganisme provoque au niveau pulmonaire chez les bovins. (Traduit par Dr Mario Jacques)

RESUME

INTRODUCTION Nous avons evalue la capacite d'un surnageant d'une culture de Pasteurella haemolytica Al, contenant une leucotoxine, a causer l'agregation de

Bovine respiratory disease is a serious economic and animal welfare problem. One of the most important

organisms in the

pathogenesis of the disease is Pasteurella haemolytica, which produces a leukotoxin shown to be lethal to bovine leukocytes, including neutrophils (1). The leukotoxin is composed of 105,000 molecular weight subunits of aggregates with an apparent molecular weight of >400,000 (2). During P. haemolytica infection in cattle, neutrophils invade the lung in great numbers. Through their production of inflammatory products, these cells may increase the degree of lung damage characteristic of the disease. Chemically-induced depletion of peripheral neutrophils in cattle has been reported to be both protective (3), and not protective (4), against the deleterious effects of P. haemolytica infection. Generally, as an early event in inflammation, neutrophils exhibit increased adherence to the endothelium of blood vessels and to each other. This may be an early response to chemotaxins which increases the number of neutrophils near the site of microbial colonization, thus amplifying the inflammatory process. Peripherally, neutropenia may be seen (5), and at the site of inflammation, tissue damage may occur due to release of oxygen radicals and enzymes (6). Since it is assumed that neutrophil-neutrophil aggregation is relevant to the inflammatory process, and that in vitro aggregation responses correlate with neutrophil adherence in vivo, measuring in vitro aggregation can assess these cells' responses to various mediators of inflammation and to pharmacological agents (7). Increased understanding of the cellular events involved in the activation of bovine neutrophils by P. hae-

Department of Biomedical Sciences (P. Conlon, Gervais, Chaudhari) and Department of Veterinary Microbiology and Ontario Veterinary College, University of Guelph, Guelph, Ontario NIG 2W1. This study was funded by the Natural Sciences and Engineering Research Council of Canada. Submitted February 3, 1992.

Can J Vet Res 1992; 56: 199-203

Immunology (J. Conlon),

199

molytica, and their place in pulmonary inflammation, is vital to the understanding of the pathogenesis of the disease caused by this organism. The purpose of the present study, was to determine if P. haemolytica leukotoxin can cause aggregation of bovine neutrophils and to examine some cellular mechanisms potentially involved in the interaction of leukotoxin and cells during this process. MATERIALS AND METHODS NEUTROPHIL ISOLATION

Using acid-citrate dextrose (ACD) as the anticoagulant, 20 mL blood samples were obtained by venipuncture from healthy, adult Holstein cows which were housed and handled following guidelines in the Guide to the Care and Use of Experimental Animals published by the Canadian Council on Animal Care. The blood was centrifuged for 20 min at 1000 x g and the serum, buffy coat and top two-thirds of the red cells removed and discarded. Neutrophils were then purified using a hypotonic lysis method. Two mL of hypotonic phosphate-buffered saline (PBS; pH = 7.2) was added to each vacutainer and this was followed, forty seconds later, by 1 mL of hypertonic PBS. The tubes were then centrifuged for 3 min and the supernatant discarded. This lysing process was performed three times. Neutrophil pellets were washed three times in 3 mL isotonic PBS and resuspended in 2 mL Hanks' balanced salt solution (HBSS; pH = 7.4). Wright-stained smears were made to verify the purity of the isolate. Cell counts were performed using a Coulter Counter, and the cells diluted to a final live neutrophil concentration of 1 x 106 cells/mL. Cell viability was assessed using trypan blue stain. Neutrophils were kept on ice throughout the experiments. Neutrophil viability was routinely above 90%7o, and was not significantly reduced by any in vitro treatment when compared to incubation with buffer. P. HAEMOLYTICA Al LEUKOTOXIC

CULTURE SUPERNATANT (PHLCS) AND CULTURE MEDIUM (CM)

The PHLCS was produced from 18 h blood agar cultures of the organism. Colonies were inoculated 200

into brain-heart infusion broth and incubated for 4.5 h at 370C. The broth was centrifuged and the bacterial pellet resuspended in twice its volume of RPMI 1640 containing 7%7o fetal bovine serum, and incubated for 1 h at 370C. The bacteria were then removed by centrifugation and filtration, and the supernatant dialyzed against water in 8000 molecular weight cut off dialysis tubing (Fisher Scientific, Toronto, Ontario) for 48 h and lyophilized. As determined by Limulus amoebocyte assay (Whittaker Bioproducts Inc., Walkersville, Maryland), no endotoxin was detectable in the PHLCS. The sensitivity of this assay is 0.10 endotoxin units (EU)/mL. Sixty mg (by total weight) of the lyophilized PHLCS was reconstituted in 2 mL HBSS, serial dilutions performed with HBSS, and the PHLCS kept on ice throughout the experiment. All concentrations reported are those in the cuvette at the time of aggregation measurement. In a water bath, culture supernatant was heat-treated (HT-PHLCS) at 56°C for 1 h. Toxin inactivation was confirmed by a neutral red assay on bovine BL3 lymphoma cells. Culture medium (CM), was processed similarly to culture supernatant, except for the absence of bacterial inoculation into the brainheart infusion broth. SERUM PREPARATION

Six-to-eight week old New Zealand White rabbits were bled prior to vaccination. Pasteurella haemolytica recombinant leukotoxin, prepared by the method of Strathdee and Lo (8), (LKTA; cut from an SDS-page gel and emulsified and dialyzed to remove SDS) at 300 mg protein/dose was then administered intramuscularly, twice at a two week interval, with 100 jg of the adjuvant Quil A (Cedarlane Inc., Hornby, Ontario). Five days after the second vaccination, a blood sample was taken and tested for antileukotoxin activity using the neutral red cytotoxicity assay (9). If this response was inadequate, a third vaccination one week after the second vaccination was administered. One week after the last vaccination (second or third boost) the rabbits were anesthetized with xylazine (Rompun®, Haver, Etobicoke, Ontario) and ketamine (Ketaset®, Austin Laboratories, Joliette, Quebec) and

exsanguinated. The blood was then centrifuged and the serum was collected, evaluated for leukotoxic activity as above and stored at - 20°C until used. At this time, equal volumes of leukotoxic culture supernatant and normal rabbit serum, anti-LKTA, or HBSS as a control were incubated together at 37°C for 30 min. ZYMOSAN-ACTIVATED PLASMA AND DRUGS

To produce zymosan-activated plasma (ZAP), bovine plasma was incubated with 2 mg/mL zymosan at 37°C for 45 min, the zymosan removed by centrifugation and the plasma diluted 1:3 in HBSS and kept at 37°C. p-Bromophenacyl bromide was dissolved in ethyl alcohol, quercetin in ethanol, stearylamine in dimethyl sulfoxide and staurosporine in dimethyl sulfoxide. All drugs were obtained from Sigma Chemical Co., St. Louis, Missouri and were diluted to their final concentrations in HBSS, except for EDTA which was dissolved in Ca- and Mg-free HBSS. AGGREGATION ASSAY

Neutrophils (4.0 x 105 cells in 400 yL HBSS with or without Ca and Mg) were combined in a siliconized glass cuvette with a further 50 AL of HBSS or the drug or serum being studied, and 50 jiL of ZAP or PHLCS. The PHLCS and serum (or HBSS as a control) were preincubated for 30 min at 37°C before being added to the cell suspension. After the cuvette was placed in the aggregometer (ChronoLog Corp., Haverstown, Pennsylvania) its temperature was kept at 37°C and continuous stirring was provided by a stir bar. Aggregation was measured by changes in optical density as determined by infrared transmittance. Maximum aggregation amplitude was calculated automatically by the instrument, and time of maximum aggregation was measured on the chart tracings. DATA ANALYSIS

By means of a statistical software program (SAS Institute, Inc., Cary, North Carolina), within animal differences among treatment responses were compared using paired Student t-tests. The data were also analyzed by analysis of variance (ANOVA). Where the ANOVA indicated differences

among treatments, multiple range tests (least significant differences) were performed to detect the presence of specific differences between treatments. A 95'70 confidence level (p < 0.05) was used in all tests.

RESULTS EFFECTS OF ZAP, PHLCS, HT-PHLCS, CM AND HT-CM

In order to determine if PHLCS would cause neutrophil aggregation, we incubated the cells with it, or with ZAP (Table I). Neutrophils consistently aggregated in response to PHLCS at all but the lowest concentration examined, and the responses were significantly greater than that produced by HBSS as a control stimulus, although less than that produced by ZAP. The aggregation profile generated by PHLCS differed from that resulting from challenge with ZAP. With the latter agent, there was a long lag phase which preceded a sharp rise in the rate of aggregation to a plateau, while with PHLCS aggregation began almost immediately and proceeded to increase until it plateaued. The greatest aggregation response due to PHLCS occurred at a concentration of 1.5 mg/mL. The response at this concentration was significantly greater than that produced by 6.0, 0.03 and 0.003 mg/mL, but did not differ from that produced by 3.0 or 0.3 mg/mL. The degree of aggregation caused by PHLCS at 0.003 mg/mL was not significantly different from that produced by HBSS. As compared to the effect of ZAP, time of maximum aggregation was significantly less for PHLCS at all concentrations. To ensure that components of the culture medium did not significantly contribute to the aggregation response, neutrophils were challenged with CM at 3.0 mg/mL. Aggregation due to CM, both before and after heattreatment, did not significantly differ from that produced by HBSS, and was significantly less than that resulting from challenge with PHLCS. The contribution of heat-labile components in the PHLCS to the aggregation response was determined by heat-treating the culture supernatant. This HT-PHLCS did not induce any more aggregation than did HBSS or CM, and produced

TABLE I. The effects of zymosan-activated plasma (ZAP), Hanks' balanced salt solution (HBSS) and P. haemolydca leukotoxic culture supernatant (PHLCS) on the aggregation response of bovine

peripheral neutrophilsa

Maximum

aggregation

Maximum

response

aggregation

Stimulus time (min)b (units)b ZAP 40.9 ± 10.4 15.7 ± 4.8 HBSS 6.4 ± 2.0A 4.6 ± 3.5 PHLCS (6.0 mg/mL) 24.7 ± 4.3BC 8.6 ± 1.9AB PHLCS (3.0 mg/mL) 28.1 ± 5.9BDE 10.7 ± 1.7ACDEF PHLCS (1.5 mg/mL) 31.1 ± 7.2DF 12.6 ± 1.2CGH PHLCS (0.3 mg/mL) 28.2 ± 4A4CEF 12.9 ± 2.1IDG PHLCS (0.03 mg/mL) 13.5 ± 3.9 12.6 ± 1.7EHI PHLCS (0.003 mg/mL) 6.3 ± 2.2A 8.3 ± 3.*BF aMean ± standard deviation of two experiments on each of five cows bWithin a column, means with the same superscript do not significantly differ from each other (p < 0.05 by analysis of variance and least significant differences)

TABLE II. The effects of zymosan-activated plasma (ZAP), Hanks' balanced salt solution (HBSS), P. haemolytca leukotoxic culture supernatant (PHLCS), heat-treated PHLCS (HT-PHLCS), culture medium (CM) and heat-treated culture medium (HT-CM) on the aggregation response of bovine peripheral neutrophilss Maximum

aggregation

Maximum response aggregation Stimulus (units)b time (min)b ZAP 35.9 ± 4.6 12.3 ± 7.4 HBSS 7.7 ± 3.5ACD 6.5 ± 5.3 PHLCS (3.0 mg/mL) 21.1 ± 4.4 10.0 ± 3.5 HT-PHLCS (3.0 mg/mL) 11.4 ± 3.3ABD 18.3 ± 7.4 CM (3.0 mg/mL) 10.1 ± 3.4A 14.8 ± 6.6 HT-CM (3.0 mg/mL) 11.5 ± 2ABC 25.8 ± 3.8 a Mean ± standard deviation of five experiments bWithin a column, means with the same superscript do not significantly differ from each other (p < 0.05 by analysis of variance and least significant differences) TABLE III. The effect of preincubation with normal (NRS) or heat-treated rabbit serum (HT-NRS), or with normal (anti-LKTA) or heat-treated (HT-anti-LKTA) antibody raised against recombinant P. haemolytica leukotoxin, on the aggregation of bovine neutrophils induced by zymosan-activated plasma (ZAP) and P. haemolytica leukotoxic culture supematant (PHLCS; 3.0 mg/mL)a Maximum aggregation response (units)b 27.3 ± 6.1C

Maximum aggregation time (min)b 12.4 ± 2.3

Stimulus PHLCS alone Anti-LKTA + PHLCS 7.9 ± 3.1 D 12.1 ± 5.5 HT-anti-LKTA + PHLCS 9.4 ± 4.9D 8.5 ± 4.7 NRS + PHLCS 30.0 ± 6.1C 12.3 ± 3.2 HT-NRS + PHLCS 32.8 ± 9.IABC 9.4 ± 3.4 ZAP 38.6 ± 6.OAB 9.8 ± 2.4 Anti-LKTA + ZAP 32.4 ± 34BC 12.6 ± 6.4 aMean ± standard deviation of four experiments bWithin a column, means with the same superscript do not significantly differ from each other (p < 0.05 by analysis of variance and least significant differences)

significantly less than did PHLCS (Table II). EFFECT OF ABSENCE OF Ca AND Mg

Aggregation responses to ZAP in the absence of Ca and Mg were significantly reduced to 12.90%o of the result

with calcium and magnesium. However, with PHLCS used as the challenge, the corresponding value was 122.4%. When EDTA (2 mM/mL) was added to the PHLCS, it was evident that the culture supernatant was the source of the needed ions since 201

TABLE IV. The effect of p-bromophenacylbromide (PBPB) on the aggregation response of bovine peripheral neutrophils to zymosan-activated plasma (ZAP) and P. haemolytica leukotoxic culture supernatant (PHLCS)9 Maximum aggregation response (units) PHLCS ZAP (3.0 mg/mL)

Stimulus PBPB concentration 0 38.3 ± 1 x 10-6 M 28.5 ± 1 X 10-7 M 36.0 ± 1 x 10-8 M 33.0 ± aMean ± standard deviation

8.5 28.3 ± 11.6 27.7 ± 5.5 26.0 ± 5.1 31.3 ± of four experiments

4.0 5.0 4.7 6.8

Maximum aggregation time (min) PHLCS ZAP (3.0 mg/mL) 14.4 12.2 13.1 13.2

± ± ± ±

3.1 2.4 3.1 2.6

12.7 13.8 10.9 11.9

+ ± + ±

1.2 1.4 0.4 2.2

TABLE V. The effect of stearylamine and quercetin on the aggregation response of bovine neutrophils to P. haemolytica leukotoxic culture supernatant (PHLCS; 3.0 mg/mL)p

Maximum aggregation response (units) Quercetin Stearylmine

Maximum aggregation time (min) Stearylamine Quercetin

Drug concentration 0 35.8 ± 8.1 35.8 ± 8.1 13.08 ± 1 X 10-4 M 15.8 ± 13.6b 23.0 ± 8.0b 8.4 ± 1 X 10-5 M 31.8 ± 11.6 34.3 ± 8.5 13.5 ± 1 x 10-6 M 25.0 ± 3.7b 30.8 ± 4.6 12.6 ± 1 X 10-7 M 31.3 ± 8.8 34.5 ± 11.2 13.4 ± a Mean ± standard deviation of four experiments bWithin a column, p < 0.05 versus no drug present by paired t-test

1.5

3.5b 0.5 1.1 0.8

13.08 10.9 13.4 13.0 13.1

± ± ± ± ±

1.5 2.8 2.3 1.1 1.1

TABLE VI. The effects of zymosan-activated plasma (ZAP), phorbol myristate acetate (PMA) and P. haemolytica leukotoxic culture supernatant (PHLCS), in the absence and presence of staurosporine, on the aggregation response of bovine peripheral neutrophilsa

Maximum aggregation response (units) No stauro-

sporine

I

Staurosporine x 10-7 M

Maximum aggregation time (min) No staurosporine

Staurosporine 1 x 10-7 M

16.7 ± 10.9

12.6 ± 2.2

11.6 ± 2.1 7.9 ± 4.6

11.9 ± 1.4 12.4 + 2.2

Stimulus ZAP 42.7 ± 8.5 14.3 ± 6.2b PHLCS (3.0 mg/mL) 32.3 ± 9.0 28.8 ± 9.8 PMA (1 x 10 -7 M) 34.3 ± 10.1 18.3 ± 10.7b aMean ± standard deviation of six experiments bp < 0.05 versus no staurosporine present by paired t-test

EFFECTS OF DRUGS, (TABLES IV, V AND VI)

The phospholipase inhibitor p-bromophenacyl bromide (PBPB) at concentrations from 1 x 10-6 to x 10-8 M had no significant effect on neutrophil aggregation responses to ZAP or to 3.0 mg/mL PHLCS. To determine what importance protein kinase C activity might have in the aggregation of bovine neutrophils, we incubated the cells with the positively charged enzyme inhibitor stearylamine or the uncharged, membrane stabilizing inhibitor quercetin and then challenged with PHLCS. Both drugs inhibited aggregation responses at 1 x 10-4 M. Stearylamine also inhibited aggregation at 1 x 10-6 M. Time of maximum aggregation was significantly shortened by stearylamine at 1 X 10-4 M, but there was no effect of quercetin at any concentration. Since the effect of stearylamine was inconsistent and such high concentrations of the drugs may have produced nonspecific effects, and also in order to more specifically study protein kinase C activity and neutrophil aggregation, the protein kinase C-stimulating drug phorbol myristate acetate (PMA) was used as a stimulus and the enzyme inhibitor staurosporine used as an antagonist. The degree of aggregation induced by PMA at 1 x 10-8 M was less than that of ZAP, but not significantly so; however, it produced the most rapid aggregation response. Time of maximum aggregation was significantly shorter with PMA than with ZAP. Staurosporine at 1 x 10-7 M significantly reduced the proaggregatory effect of both ZAP and PMA, but had no effect on the response to PHLCS. Time of maximum aggregation was not altered by staurosporine, regardless of the stimulus used. DISCUSSION

aggregation was significantly reduced maximum aggregation was not signifto 52.9% of the result without EDTA. icantly different among any of the treatments. The anti-LKTA antibody EFFECTS OF ANTIBODY did not exert any inhibitory effects on PREINCUBATION (TABLE III) ZAP-induced aggregation, nor did Preincubation of PHLCS with both normal rabbit serum produce any sigheat-treated and normal anti-LKTA nificant inhibition of PHLCS-induced significantly reduced maximum aggre- aggregation. Heat-treating did not gation response induced by the leuko- alter the inhibitory effects of antitoxic culture supernatant. The time of LKTA antiserum. 202

To our knowledge, this is the first report of the ability of P. haemolytica leukotoxic culture supernatant to cause aggregation of bovine neutrophils. The PHLCS caused aggregation to begin virtually immediately after its contact with the cells and continued to a peak from 8-13 min later. Maximum aggregation occurred at a concentration of 1.5 mg/mL, with the effect being lost

between 0.03 and 0.003 mg/mL. this latter drug inhibited protein Czuprinski et al (10) have demon- kinase C. We found that aggregation strated that dilute concentrations of induced by PHLCS is not protein partially purified leukotoxin stimulate kinase C dependent since staurosporine cytoskeletal alterations in bovine did not alter PHLCS' effect. neutrophils, as evidenced by shape Bomalaski et al (15) reported that a changes (which were also produced by phospholipase A2-activating protein ZAP). These changes may be early produced aggregation of human evidence of the cells' tendency to neutrophils. From our results, it is eviaggregate in response to the leukotoxin. dent that activation of phospholipase Although the culture supernatant A2 by ZAP or P. haemolytica leukoused in these experiments is known to toxin does not contribute to bovine contain other proteins, the loss of neutrophil aggregation since there was aggregating activity after heat treat- no significant change in aggregation ment, and the failure of the culture responses in the presence of p-bromomedium to produce any significant phenacyl bromide. The results of this study indicate that aggregation, strongly suggest that aggregation is caused by the leuko- P. haemolytica culture supernatant toxin, which is heat-labile (11). It has initiates one of the earliest events in the recently been reported, however, that process of neutrophil activation and a neutral metalloprotease isolated from that this activation is probably due to culture supernatants of P. haemolytica the leukotoxin produced by the bacteAl is also heat-labile (12). In our rium. It adds to the body of evidence study, the highly significant reduction which implicates the neutrophil as a in the aggregating ability of the leuko- key cell type involved in the pathotoxic culture supernatant resulting genesis of "shipping fever" pneumonia. from preincubation with anti-LKTA antiserum would argue against the proACKNOWLEDGMENTS tease being a major contributor to the results seen. The aggregation "bluntThe authors thank John Burger for ing" effect was specific to this antibody since similar effects were not seen his technical assistance. when normal rabbit serum was used. Our results also indicate that aggregaREFERENCES tion is dependent on extracellular calcium, does not involve the activation of phospholipase A2 and does 1. BALUYUT CS, SIMONSON RR, BEMRICK WJ, MAHESWARAN SK. Interaction of not depend on stimulation of protein Pasteurella haemolytica with bovine neutrokinase C by the toxin. phils: Identification and partial characterizaPhorbol esters, such as PMA, which tion of a cytotoxin. Am J Vet Res 1981; 42: 1920-1926. directly activate protein kinase C and CHANG YF, YOUNG R, POST D, by-pass receptor stimulation by agonists 2. STRUCK DK. Identification and characterisuch as C5a (13), cause adhesion of zation of the Pasteurella haemolytica human neutrophils without causing leukotoxin. Infect Immun 1987; 55: 2348-2354. changes in intracellular calcium concentrations (14). In our study, PMA- 3. SLOCOMBE RF, MALARK J, INGERSOLL R, DERKSEN FJ, ROBINSON NE. Imporinduced aggregation of bovine neutrotance of neutrophils in the pathogenesis of phils was significantly reduced by acute pneumonic pasteurellosis in calves. Am staurosporine, thus confirming that J Vet Res 1985; 46: 2253-2258.

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