Granulocyte Plasma Membrane Damage by Leukotoxic Supernatant from Pasteurella haemolytica Al and Protection by Immune Serum Barbara Styrt, Robert D. Walker, John C. White, Linda D. Dahl and John C. Baker

ABSTRACT

peuvent etre induites partiellement par une leucotoxine secretee par ce Bovine respiratory disease caused microorganisme. Nous avons examine by Pasteurella haemolytica may be l'effet de surnageants leucotoxiques de partially mediated by a leukotoxin Pasteurella sur la fuite de l'enzyme secreted by the microorganism. We cytosolique lactate deshydrogenase et examined the effect of leukotoxic de l'enzyme lysosomiale arylsulphaPasteurella supernatants on leakage of tase de granulocytes bovins. La perte the cytosol enzyme lactate dehydroge- du lactate deshydrogenase (94%) etait nase and the lysosomal enzyme beaucoup plus importante que celle de arylsulfatase from bovine granulo- l'arylsulphatase, durant une periode cytes. Lactate dehydrogenase release dincubation de 30 minutes. (94%) was much higher than arylsulfaLes surnageants de Pasteurella ont tase release (38%) over 30 minutes of inhibe la production de superoxide incubation. The Pasteurella super- par les granulocytes stimules a des natants inhibited superoxide produc- concentrations qui provoquaient aussi tion by stimulated granulocytes at la mort cellulaire telle que mesuree par concentrations which also caused l'incapacite d'exclure le bleu de substantial cell death as measured by trypan. Ces deux effets cytotoxiques failure to exclude trypan blue. Both ont ete empeches par du serum de toxic effects were prevented by serum veaux immunises par aerosol. from aerosol-immunized calves, and La comparaison de serums de protection appeared to be antibody- foetus bovins avec du serum absorbe

specific by comparison with fetal bovine serum or with serum absorbed against intact P. haemolytica. These findings suggest that the leukotoxin may selectively disrupt the granulocyte plasma membrane, and that antibody directed against a surface component of the microorganism is also capable of protecting against the leukotoxin effect.

contre P. haemolytica intact semble demontrer que la protection est due a des anticorps specifiques. Ces observations suggerent que la leucotoxine rupture la membrane plasmatique des granulocytes et que l'anticorps dirige contre un composant de surface du microorganisme est egalement capable de proteger contre l'effet leucotoxique.

RESUME

INTRODUCTION

Les maladies respiratoires bovines causees par Pasteurella haemolytica

Pasteurella haemolytica is a major cause of bovine respiratory disease in

feedlot-aged calves (1,2). This organism produces a variety of potential virulence factors including an endotoxin and a secreted leukotoxin (3-7). The leukotoxin has been characterized as a glycoprotein which has sequence homologies with Escherichia coli alpha-hemolysin (8,9). The P. haemolytica leukotoxin causes cell damage and suppression of oxidative metabolism in ruminant leukocytes, but has little effect against white cells from other species (10). This toxin may serve as a virulence factor both by permitting the bacteria to escape host defenses and by promoting release from damaged leukocytes of substances which can damage lung tissue. However, the mechanism of its toxicity is not well understood. The following studies were designed to examine the interaction of leukotoxic supernatant from P. haemolytica with isolated bovine granulocytes, addressing aspects of the mechanism of cell damage and potential approaches to

cytoprotection. MATERIALS AND METHODS

Cytochrome C, superoxide dismutase, Histopaque-1077@, and 4methylumbelliferyl sulfate were obtained from Sigma Chemical, St. Louis, Missouri. Sodium fluoride was from Mallinckrodt. Bovine blood was obtained by venipuncture from healthy adult dairy cattle and collected in a heparinized 50 mL syringe.

Department of Medicine (Styrt, White), Department of Large Animal Clinical Sciences (Baker) and Department of Microbiology and Public Health (Walker, Dahl), Michigan State University, East Lansing, Michigan 48824. This work was supported in part by Michigan State University and the United States Department of Agriculture, and presented in part at the 69th Annual Meeting of the Conference of Research Workers in Animal Diseases, Chicago, 1988 (Abstract #357). Submitted May 9, 1989.

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the serum was harvested by centrifugation and assayed for anti-P. haemoFor granulocyte isolation, blood was diluted with an equal volume of lytica antibodies. An additional hour phosphate-buffered saline (PBSS), at room temperature and an overnight layered over a Histopaque cushion, absorption at 40C were required to and centrifuged at room temperature reduce the anti-P. haemolytica antiand 1200 rpm (300 g) for 40 min. The body titer to 1:8 as determined by pellet containing erythrocytes and ELISA. granulocytes was reserved, and eryOF throcytes were removed with two or PREPARATION LEUKOTOXIC SUPERNATANT three cycles of hypotonic saline lysis. Pasteurella haemolytica A1, from a The isolated granulocytes were then suspended in Hanks' balanced salt 1% inoculum of an overnight culture, solution (HBSS) and counted in a was grown in RPMI-1640, supplemented with 7% fetal bovine serum, at hemocytometer. 370C for 5 h in an oscillating water ANTISERUM PREPARATION bath. Bacteria were removed by Antiserum was obtained from three centrifugation at 10,000 x g for 15 min beef-bred calves, 150-200 kg, immun- at 40C and the supernatant culture ized by an inhalation exposure to was filtered through 0.2,m filters. viable, log-phase P. haemolytica Al Immediately following filtration the (biotype A, serotype 1), (2.0 x 109 fluids were aliquoted and frozen at colony-forming units [CFU]/mL of -70° C to retain leukotoxic activity. 0.01 M PBSS; pH 7.2) as described (11). Ten days after the second CYTOSOL AND GRANULE MARKERS inhalation exposure serum was colLeakage of granulocyte cytosol lected from all animals and pooled. contents was measured as in previous Anti-P. haemolytica antibody titer studies (13), by determining appearwas determined by an enzyme-linked ance in the media of the cytosolic immunosorbent assay (ELISA) as enzyme lactate dehydrogenase described (12). (LDH), assayed by standard spectrophotometric methods. Plasma memABSORPTION OF ANTIbrane integrity was also assessed as the P. HAEMOLYTICA ANTIBODY ability of cells to exclude trypan blue FROM CALF SERA Because of the difficulty of finding dye. Lysosomal hydrolase release was calves which lack detectible antibody measured by assaying arylsulfatase, to P. haemolytica, an absorption a using standard fluorometric assay procedure was used to provide lowThis (14). enzyme was chosen as a antibody-containing serum as a lysosome marker because it is an control. Serum from normal calves, abundant in lysosomal hydrolase with anti-Pasteurella haemolytica titers of 1:32, was absorbed against bovine granulocytes (15), which killed, intact P. haemolytica grown on contain very little of enzymes such as brain heart infusion agar, supple- lysozyme and beta-glucuronidase, mented with 1% horse serum, 1% yeast commonly used as lysosome markers extract and 5% defibrinated sheep in human granulocytes. (16,17) blood at 370 C in a 5% CO2 environ- ASSAYS OF MARKER RELEASE ment, and suspended in PBSS conTo measure toxicity of the P. taining 1% thimersol. The bacterial haemolytica supernatant, serial dilususpension was centrifuged at 10,000 x g for 10 min at 40 C, and washed twice tions were incubated with granuloin thimersol free PBSS with the cytes in HBSS for 30 min at 370C with resultant pellet resuspended in normal agitation. An aliquot was centrifuged calf serum. The serum-bacterial at 40C and 1000 rpm (220g) for 10 min suspension was incubated overnight at and the supernatant reserved for 40C. The serum-bacterial suspension enzyme assays. After a drop was taken was clarified by centrifugation and the for trypan blue determination, the serum was decanted and mixed with a remaining cells were lysed with 0.1% second bacterial pellet. This suspen- Triton X-100 for measurement of total sion was incubated for 45 min at 370 C, cellular enzyme content. CELL ISOLATION

OXIDATIVE METABOLISM

Superoxide was measured as the superoxide-dismutase-inhibitable reduction of cytochrome C using spectrophotometric scanning as in our previous studies (18). Sodium fluoride (20 mM), a presumed activator of guanosine-triphosphate-binding proteins (19), was used as a standard stimulus. Granulocytes were incubated with Pasteurella supernatant for 5 min at 370 C with agitation, the stimulus was added, and incubation was continued for another 30 min; cell suspensions were centrifuged for 10 min at 1000 rpm (220 g) and 40 C, and the supernatants were scanned to determine optical density (OD) 550-OD540. STATISTICAL TREATMENT

Results are expressed as mean ± SEM of stated number of

experiments indicated.

unless

otherwise

RESULTS CELL PREPARATIONS

Granulocytes isolated by the methods described consist predominantly of neutrophils, together with eosinophilic granulocytes and a few contaminating mononuclear cells. In prior experience with Wright's stain differential counts of typical preparations, over 90% of cells have been classified as neutrophils. In these studies, cell viability was 96 ± 1% by trypan blue dye exclusion at the end of 17 experiments in cells not exposed to P. haemolytica supernatants. Cell concentrations in these experiments averaged 6.2 ± 1.2 x 106 per mL in the final experimental suspension, with 92 ± 1% neutrophils. EFFECTS ON CELLULAR MARKERS

The effect of P. haemolytica leukotoxic supernatant on cell viability and enzyme release is shown in Fig. 1. Serial dilutions of the supernatant produced a dose-dependent increase in plasma membrane permeability as measured by uptake of trypan blue and release of the cytosol enzyme LDH. No significant arylsulfatase release was seen at sublethal concentrations of supernatant; at increasing concentrations, arylsulfatase release also rose in a dose-dependent fashion 147

801

3

trypan uptake

W

LDH release

T

I

D arylsulfatase release 110

1100

i1

40

I 11000

I

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ji IIII~

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Fig. 1. Dose-response for effect of leukotoxic supernatants on isolated bovine granulocytes, expressed as percentage release of total lactate dehydrogenase and arylsulfatase and as percentage of cells which did not exclude trypan blue. Mean ± SEM of four experiments. Left hand bars (marked 0) represent control cells without toxin, and remaining bars represent cells treated with indicated dilutions of toxic supernatant.

TOXIN DILUTION

Fig. 2. Inhibition of superoxide production by leukotoxic supernatant: percent decrease compared with granulocytes not exposed to supernatant. Mean ± SEM of three experiments.

tions of antibody retained a normal capacity for superoxide production which was not suppressed by the addition of leukotoxin. However, this protective effect was lost with progressive dilution of the antiserum.

but was always lower than LDH release at the same dose. At the highest concentration of supernatant used (1:10 dilution), 94 ± 2% of total ANTISERUM EFFECT ON cellular LDH but only 38 ± 7% of TRYPAN BLUE UPTAKE total arylsulfatase appeared in the Incubation with antiserum also medium. prevented plasma membrane damage EFFECTS ON OXIDATIVE METABOLISM by the leukotoxin as measured by trypan blue dye uptake. As shown in Figure 2 shows the inhibition of Fig. 3, exclusion of trypan blue was superoxide production by stimulated closely related to preservation of the granulocytes which had been incu- respiratory burst. bated with varying concentrations of leukotoxic P. haemolytica supernat- EFFECT OF ANTISERUM ant before stimulation. A dose- ADDED AFTER TOXIN dependent suppression of the respiraWhen cells were incubated with toxin tory burst was noted. The highest concentration of leukotoxic supernat- for 5 min, then stimulated with sodium ant, which inhibited superoxide fluoride for 30 min, with antiserum production by 80 ± 10%, was chosen added 5 min after stimulation, the for cytoprotection assays, to facilitate results shown in Fig. 4 were obtained. The supernatant used in these experithe detection of protective effects. ments had somewhat decreased leukoANTISERUM EFFECT ON toxic potency. Nevertheless, the highest RESPIRATORY BURST INHIBITION antiserum dilution offered no signifiFigure 3 shows the effect of a 5 min cant protection compared with no incubation of granulocytes with antiserum, in terms of either plasma antiserum before leukotoxin addition, membrane damage assessed by trypan followed by stimulation with fluoride. blue uptake or suppression of superoCells incubated with high concentra- xide production.

148

ANTISERUM

DILUTION

Fig. 3. Inhibition of superoxide production, and cell death measured as trypan blue uptake, in granulocytes exposed to leukotoxic supernatant after incubation with serial dilutions of serum from immunized calves. Mean ± SEM of five to eight values at each concentration. Left hand bars (marked 0) represent cells not exposed to antiserum, and remaining bars !dilutions of represent cells exposed to indicated antiserum. _1

!

-

v1

1

-

__A'_

EFFECT OF FETAL BOVINE SERUM

To determine whether the protection seen with antiserum preincubation was actually due to its antibody content, fetal bovine serum was used as a control. As shown in Fig. 5, fetal bovine serum offered no protection against the toxin. However, this serum itself showed some inhibition of superoxide production. Therefore, an additional control was thought to be

advisable. EFFECT OF ABSORBED ANTISERUM

As an additional control for antibody specificity, serum was absorbed against intact P. haemolytica as described under Methods above. As shown in Fig. 6, the protective effect of absorbed normal serum was at least tenfold less than that of unabsorbed specific antiserum.

DISCUSSION The neutrophil has been implicated in the pathogenesis of bovine pneumonic pasteurellosis, in that the

FBS EFFECT

ANTISERUM AFTER TOXIN 0/

x trypan

°

80'

inhibition

T+AAS

T

('4 -0

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*

40'

ANTISERUM

S T

80

JABSORBED

*F

*

*

*

*

* * *

O 40

'O 40L T+AS I

0

1:1000 1:100

1:10

Fig. 4. Experiments similar to Fig. 3 but with immune serum added five minutes after cell stimulation. Mean of two experiments.

course of infection is reportedly attenuated or altered in neutrophildepleted animals (20,21). The contribution of the neutrophil may be partially due to the release of an extracellular leukotoxin by P. haemo-

lytica. This leukotoxin thus could serve as a virulence factor in several ways: by protecting the bacterium from host defenses and by causing release of tissue-damaging neutrophil products such as lysosomal enzymes and oxygen metabolites. While generation of oxygen metabolites requires stimulation of the cell, release of lysosomal enzymes could result either from active degranulation or from cell death and disintegration. We examined the effect of leukotoxincontaining supernatants from P. haemolytica cultures on several of these granulocyte functions. Superoxide production was used as a measure of granulocyte generation of oxygen metabolites. This was not promoted but, indeed, was profoundly depressed by Pasteurella supernatant. This would be predicted from the literature on suppression of chemiluminescence by Pasteurella (10,22); however, it is noteworthy that we saw substantial cell death, as measured by LDH release and inability to exclude trypan blue, at the same concentrations of toxin which inhibited superoxide production, while previous

Fig. 5. Lack of cytoprotection by fetal bovine serum. T = cells incubated with toxic supernatant but no serum before stimulation, T + F = toxic supernatant plus fetal bovine serum, F = fetal bovine serum alone. Mean of two experiments.

studies have indicated depression of chemiluminescence at much lower toxin concentrations than those producing cell death (10). Further studies are needed to determine whether these discrepancies arise from differences in the specific oxygen metabolite measured, differences in incubation time, or use of different stimuli. Whichever of these is the case. the current experiments and published reports all show a suppression of oxidative metabolism, indicating that phagocyte-derived oxygen metabolites are not likely to be involved in Pasteurella-toxin-derived lung damage. Release of granule contents, on the other hand, could be either active or passive. If the toxin caused active release of granulocyte granule contents, this could contribute to secondary killing of both granulocytes and other host cells. Active secretion is not suggested by our results, which showed no significant arylsulfatase release at sublethal toxin concentrations; nor did granule disruption appear to be an immediate effect of cell death, since little arylsulfatase was released at toxin concentrations producing substantial LDH leakage. In fact, it appeared from the discordance between arylsulfatase and LDH release that lysosome membranes might be relatively protected from the toxin effect as compared with the

.10 1 100 110 11(W) Fig. 6. Comparison of protection from toxic supernatant (T) by immune (AS) and absorbed (AAS) serum. Mean of two experiments.

plasma membranes. This would suggest that release of granule contents might be only a late consequence of cell death and gradual disintegration. On the other hand, differences between arylsulfatase and LDH release might also reflect differing toxin susceptibility of different granule types within the cell, as well as the different contributions of eosinophils and neutrophils to total granulocyte enzyme content and enzyme discharge. Experiments are in progress to examine these various possibilities. A primary toxin effect mediated through plasma membrane permeabilization would be consistent with the ion fluxes demonstrated by Clinkenbeard et al (23) in bovine lymphoma cells, and would suggest that action of this toxin resembles that of bacterial toxins such as streptolysin O which lyses neutrophils by binding to membrane cholesterol, and differs from that of other bacterial leukotoxins such as staphylococcal leukocidin and streptolysin S which have cytotoxic activity that is accompanied by massive degranulation (24). However, further study is needed to confirm or refute such a mechanism of action. All of these studies were performed with crude Pasteurella supernatants. Since procedures to purify the leukotoxin have often resulted in an inactive product (4), most functional studies currently use culture supernatant preparations, although structural studies can now benefit from the cloning of the leukotoxin gene (8). Thus we cannot rule out the possibility 149

that supernatant constituents other REFERENCES than the leukotoxin, for example one of various Pasteurella exoenzymes 1. BLANCHARD-CHANNELL MT, ASHFAQ MK, KADEL WL. Efficacy of a (25) or endotoxin, may have been streptomycin-dependent, live Pasteurella responsible for some of the results we haemolytica vaccine against challenge saw. Any of these substances could be exposure to Pasteurella haemolytica in present in our crude preparation, and cattle. Am J Vet Res 1987; 48: 637-642. we are embarking on studies with 2. WIKSE SE. Feedlot cattle pneumonias. Vet Clin North Am (Food) 1985; 1: 289-3 10. partially purified toxin to clarify this 3. CHANG YF, RENSHAW HW. Pasteurpoint. We suggest that endotoxin is ella haemolytica leukotoxin: Comparison unlikely to be a valid alternative of 5tchromium-release, trypan blue dye explanation since studies of other exclusion, and luminol-dependent chemiluminescence-inhibition assays for sensitivendotoxin effects on neutrophils have ity in detecting leukotoxin activity. Am J generally shown stimulation or Res 1986; 47: 134-138. enhancement of responsiveness (26); 4. Vet GENTRY MJ, CONFER AW, WEINhowever, this issue will be resolvable BERG ED, HOMER JT. Cytotoxin (leukotoxin) production by Pasteurella only when sufficient quantities of haemolytica: Requirement for an ironleukotoxin which is both pure and containing compound. Am J Vet Res 1986; active can be prepared. 47: 1919-1923. The protection of granulocytes by 5. GENTRY MJ, CONFER AW, CRAVEN serum from immunized calves is RC. Effect of repeated in vitro transfer of Pasteurella haemolytica Al on encapsulaconsistent with results reported by tion, leukotoxin production, and virulence. Moore et al (12) but was unexpected Clin Microbiol 1987; 25: 142-145. given the reported lack of correlation 6. JKAEHLER KL, MARKHAM RJF, MUSbetween serum antibody titers and COPLAT CC, JOHNSON DW. Evidence of cytocidal effects of Pasteurella haemolypneumonia in some studies (1). tica on bovine peripheral blood mononuCytoprotection in our assays may clear leukocytes. Am J Vet Res 1980; 41: have been due to antibody reacting 1690-1693. with bacterial surface antigens, 7. O'BRIEN JK, DUFFUS WPH. Pasteurella judging from the lack of protection haemolytica cytotoxin: Relative susceptibility of bovine leucocytes. Vet Microbiol with fetal bovine serum and the 1987; 13: 321-334. reduced protection with absorbed 8. LO RYC, STRATHDEE CA, SHEWEN serum. However, the high concentraPE. Nucleotide sequence of the leukotoxin tions of serum required for protection genes of Pasteurella haemolytica Al. Infect Immun 1987; 55: 1987-1996. suggest that only a small proportion of the serum antibody raised by aerosol 9. STRATHDEE CA, LO RYC. Extensive homology between the leukotoxin of immunization is capable of neutralizPasteurella haemolytica Al and the alphaing the leukotoxin. Failure of serum hemolysin of Escherichia coli. Infect antibodies to protect in vivo could Immun 1987; 55: 3233-3236. result from inability to mobilize 10. CHANG YF, RENSHAW HW, MARTENS RJ, LIVINGSTON CW Jr. Pasteuenough antibody from the circulation rella haemolytica leukotoxin: Chemilumiinto the respiratory tract. Alternanescent responses of peripheral blood tively, serum antibody may be largely leukocytes from several different mammalian species to leukotoxin- and opsoninirrelevant to pulmonary disease but treated living and killed Pasteurella may prevent bovine pneumonic haemolytica and Staphylococcus aureus. pasteurellosis from entering a dissemiAm J Vet Res 47: 67-74. nated or bacteremic stage. Studies in 1 1. WALKER RD,1986; HOPKINS FM, SCHULTZ progress on passive immunization TW, McCRACKEN MD, MOORE RN. Changes in leukocyte populations in may help to answer these questions.

ACKNOWLEDGMENTS We thank Diane Bannerman and Tammy Burley for manuscript preparation.

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