Veterinary Immunology and Immunopathology, 33 ( 1992 ) 51-68 Elsevier Science Publishers B.V., Amsterdam

51

Effects of Pasteurella haemolytica A 1 leukotoxin on bovine neutrophils: degranulation and generation of oxygen-derived free radicals S.K. M a h e s w a r a n a, D.J. Weiss a, M.S. K a n n a n b, E.L. T o w n s e n d a, K . R . R e d d y a, L.O. W h i t e l e y a a n d S. S r i k u m a r a n c ~Departments of Veterinary Pathobiology, and bVeterinary Biology, College of Veterinary Medicine, University of Minnesota, St. Paul, MN 55108, USA eDepartment of Veterinary Science, University of Nebraska, Lincoln, NE 68583-0905, USA (Accepted 8 August 1991 )

ABSTRACT Maheswaran, S.K., Weiss, D.J., Kannan, M.S., Townsend, E.L., Reddy, K.R., Whiteley, L.O. and Srikumaran, S., 1992. Effects ofPasteurella haemolytica A1 leukotoxin on bovine neutrophils: degranulation and generation of oxygen-derived free radicals. Vet. lmmunol. Immunopathol., 33:5168. To further define the role of Pasteurella haemolytica A 1 leukotoxin in the pathogenesis of bovine pneumonic pasteurellosis, its in vitro effects on bovine neutrophils were investigated. Leukotoxincontaining culture supernatant, from P. haemolytica, stimulated a neutrophil respiratory burst as measured by the generation of oxygen-derived free radicals O2- and H202. This effect was immediate because preincubation of neutrophils with the culture supernatant for 5 min or longer substantially suppressed this respiratory burst. This suppression was due to cytolysis of the neutrophils. Prolonged incubation of neutrophils with the same culture supernatant caused further cytolysis and degranulation. Heat-inactivated P. haemolytica culture supernatant that had lost its cytotoxic properties failed to stimulate respiratory burst by neutrophils. Furthermore, the respiratory burst, cytolysis and degranulation were abrogated only by leukotoxin-neutralizing monoclonal and polyclonal antibodies, but not by antibodies against the lipopolysaccharide. These studies show that the leukotoxin component in the culture supernatant was responsible for the generation of oxygen-derived free radicals and proteolytic enzymes from neutrophils which may participate in direct lung injury. ABBREVIATIONS HBSS, Hank's balanced salt solution; KDO, 2-keto-3-deoxyoctonate; LDH, lactate dehydrogenase; LKT, leukotoxin; LPS, lipopolysaccharide; MAb, monoclonal antibody; OD, optical density; PMA, phorbol myristate acetate. Correspondence to: S.K. Maheswaran, Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Minnesota, St. Paul, MN 55108, USA

52

S.K. MAHESWARAN ET AL.

INTRODUCTION

Bovine pneumonic pasteurellosis is the most important disease of cattle in North America and Britain both clinically and economically (Yates, 1982; Allan et al., 1985 ). Although it is a multifactorial disease involving interaction between infection by a variety of microorganisms and stress factors, Pasteurella haemolytica biotype A, serotype 1 (A1) has been established as the primary agent inducing the clinical disease and pulmonary tissue damage (Collier, 1968; Lillie, 1974; Dyer, 1982; Wikse, 1985). The disease has been reproduced experimentally in cattle by either intrathoracic or intratracheal administration of logarithmic growth phase P. haemolytica A1 organisms alone (Panciera and Corstvet, 1984; Ames et al., 1985 ). The predominant pulmonary lesion is lobar pneumonia characterized by neutrophil influx and fibrin deposition in the alveoli, capillary thrombosis, and the formation of foci of coagulation necrosis surrounded by a zone of bacteria intermixed with degenerate neutrophils (Rehmtulla and Thomson, 1981 ). These hallmark pulmonary lesions have been documented in both naturally occurring and experimentally induced pneumonic pasteurellosis (Rehmtulla and Thomson, 1981; Allan et al., 1985; Ames et al., 1985 ). Neutrophils are known to participate in lung injury associated with bovine pneumonic pasteurellosis (Slocombe et al., 1985; Breider et al., 1987). Of particular interest is that neutrophil depletion attenuated the lung injury and pathophysiologic alterations seen in the intact animal. This is similar to the adult respiratory distress syndrome in humans where there is extensive evidence which shows that neutrophils play an important role in mediating the acute lung injury (Tate and Repine, 1983; Weiland et al., 1986 ). Pasteurella haemolytica A 1 produces several potential virulence factors including endotoxin (Rimsay et al., 1981 ), an extracellular leukotoxin (LKT) (Benson et al., 1978; Berggren et al., 1981 ), neuraminidase (Frank and Tabatabai, 1981 ) and a neutral protease (Otulakowski et al., 1983). Of these factors, the LKT has received the most attention. Leukotoxin is a protein which at high concentrations is cytocidal to bovine neutrophils, macrophages, monocytes, and lymphocytes (Kaehler et al., 1980 ). This cytotoxicity is caused by the formation of pores in the cell membrane which allows the influx of calcium and results in a sequence of cell-damaging events (Clinkenbeard et al., 1989). The mechanisms by which neutrophils induce lung injury in pneumonic pasteurellosis is poorly understood. It has been shown in vitro that bovine neutrophils, exposed to logarithmic growth phase P. haemolytica A 1 cells or its culture supernatant, responded with an initial respiratory burst followed by a precipitous decrease as measured by luminol-dependent chemiluminescence (Chang et al., 1985). However, heat-killed bacteria caused a more gradual rise and sustained respiratory burst. These results suggest that a sol-

PASTEURELLA HAEMOLYTICA AI LEUKOTOXIN AND BOVINE NEUTROPHILS

53

uble heat-labile factor from P. haemolytica A 1, possibly the LKT, modulated the respiratory burst in bovine neutrophils. Other investigators (Czuprynski and Noel, 1990) have found that high concentration of crude LKT rapidly diminished the chemiluminescenceresponse of bovine neutrophils to opsonized zymosan. Furthermore, dilute LKT did not directly stimulate neutrophil chemiluminescence, nor did it alter the chemiluminescence response to opsonized zymosan. The data from Styrt et al. (1990) also suggested that leukotoxic culture supernatant from P. haemolytica A 1 suppressed the respiratory burst of bovine granulocytes induced by sodium fluoride. Since the data from these two laboratories showed that LKT did not directly stimulate the release of oxygen radicals but did suppress the respiratory burst induced by other known agonists, it was suggested that toxic oxygen radicals were not likely to be involved in lung damage in pneumonic pasteurellosis. In contrast to these findings, our results show that the LKT from P. haemolytica A1 itself stimulates the release of toxic oxygen radicals, namely superoxide anion (O~-) and hydrogen peroxide (H202), from bovine neutrophils. Our results also demonstrate that prolonged incubation of the neutrophils with LKT causes cytolysis and release of lysosomal enzymes such as myeloperoxidase and arylsulfatase. Preliminary results of these studies have been communicated earlier (Maheswaran et al., 1990). MATERIALSANDMETHODS

Leukotoxin preparation Leukotoxin was prepared by growing P. haemolytica A1 strain 12296 in RPMI-1640 tissue culture medium (Whittaker MA Bioproducts, Walkersville, MD) and collecting the logarithmic growth phase culture supernatant as described by Penaredondo et al. ( 1988 ). The culture supernatant was separated from the bacteria by centrifugation and was filter-sterilized (0.45/zm pore size). The supernatant was then concentrated five-fold by means of a spiral fiber apparatus (Model CH2S, Amicon, Danvers, MA) equipped with a spiral cartridge (Model S 1Y30) and dialyzed against pyrogen-free distilled water using the dialyzing mode of the same apparatus. The retentate was lyophilized and stored in a desiccator at 4 ° C. This culture supernatant, referred to as crude LKT, contained a mixture of LKT, lipopolysaccharide (LPS), capsular polysaccharide, and unknown proteins (Whiteley et al., 1990). The leukotoxic activity was quantified by a previously described colorimetric assay using the BL-3 bovine lymphoma cell line (obtained from R. Thielen, University of California, Davis, CA) and expressed in LKT units (Vega et al., 1987 ). The protein concentration in the crude LKT was determined with the Bio-Rad protein assay kit (Bio-Rad Laboratories, Richmond, CA) and the amount of LPS was estimated by determination of 2-keto-3-deoxyocton-

54

S.K. MAHESWARAN ET AL.

ate (KDO) content (Karkhanis et al., 1978). The crude LKT utilized in our assays ranged from 122 to 2000 LKT U m g - 1dry weight. This consisted of 550/zg protein m g - ~and 2-3 pg KDO mg- 1.

Preparation of LPS Lipopolysaccharide from P. haemolytica A 1, strain 12296 was extracted by the phenol-water method described by Rimsay et al. ( 1981 ) and lyophilized. The resulting LPS contained 68 #g KDO m g - ~dry weight.

Antibodies Monospecific antiserum against the LKT, referred to as polyclonal antibodies (anti-LKT), was produced by immunizing Pasteurella-free rabbits (Hazleton Research Products, Denver, PA) with purified LKT. The production, characterization, and specificity of LKT-neutralizing anti-LKT have been previously reported (Whiteley et al., 1990). Polyclonal antibodies against LPS (anti-LPS), were produced by the same method using purified LPS (Rimsay et al., 1981 ). The specificity of anti-LPS was determined in an indirect enzyme-linked immunosorbent assay using highly purified LPS derived from P. haemolytica A 1 (Penaredondo et al., 1988 ). The murine monoclonal LKT-neutralizing antibody (MAb-601 ) was produced by the hybridoma technology (Gentry and Srikumaran, 1991 ). Another murine monoclonal LKT-neutralizing antibody designated MAb-La was obtained from R.E. Corstvet (Louisiana State University, Baton Rouge, LA ). The heavy-chain isotype of MAb-601 was IgG~, while MAb-La was IgM. The neutralizing activity of both MAbs was confirmed with a colorimetric microtiter assay (Vega et al., 1987 ). The murine monoclonal antibody MAb-IH3 to the LPS ofP. haemolytica, wlSich was produced in our laboratory, was of the IgG3 isotype (Penaredondo et al., 1988). Ascites fluids of these three MAbs were used in these experiments. Control mouse ascites fluid (Sigma Chemicals, St. Louis, MO ) was produced in BALB/c mice implanted with the NS-1 myeloma. MAb-601 was depleted of LKT-neutralizing antibody by passage through protein G agarose (Schleicher & Schuell, Keene, NH ).

Reagents Phorbol myristate acetate (PMA), superoxide dismutase, (3360 U mg-~ ), ferricytochrome C, potassium ferricyanide, phenol red, horseradish peroxidase ( 100 U m g - 1), dimethyl sulfoxide and reagents for the lactate dehydrogenase, arylsulfatase and myeloperoxidase assays were obtained from Sigma Chemicals, St. Louis, MO. Hank's balanced salt solution (HBSS) and HBSS without Ca 2+ and Mg z+ were obtained from Gibco Laboratories, Grand Is-

PASTEURELLA HAEMOLYTICA A I LEUKOTOXIN AND BOVINE NEUTROPHILS

55

land, NY. A stock solution of PMA, containing 1 mg ml-~ in dimethyl sulfoxide, was stored frozen at - 7 0 ° C and diluted in HBSS at the time of use. All the reactants used in the superoxide anion and hydrogen peroxide assays were dissolved in HBSS.

Bovine neutrophils Bovine peripheral blood was obtained from normal Holstein cattle with a 5% solution of disodium ethylenediaminetetra-acetate as anticoagulant and centrifuged at 700 × g for 1 h over a discontinuous Percoll (Sigma) densitygradient technique as described previously (Weiss et al., 1989). The Percoll and water used for hypotonic lysis were autoclaved for 90 min to destroy LPS contaminants. The granulocytes were pelleted with the erythrocytes. The erythrocytes were hypotonically lysed. A second or third hypotonic lysis of residual erythrocytes was done as needed. The granulocyte cell suspensions were diluted in HBSS to the desired concentration. The purity of the neutrophils varied from 80 to 98% with eosinophils, but not mononuclear cells, as contaminants. Only cell suspensions containing more than 95% neutrophils were used in these studies. Viability, as determined by the trypan blue exclusion test, was more than 98%.

Assayfor superoxidegeneration Superoxide (O~-) generation was measured by the superoxide dismutaseinhibitable reduction of ferricytochrome C (Johnston et al., 1978 ) with modifications. The reaction mixture consisting of 200/tl neutrophils ( 1 X 107 cells ml -t ) and 50pl ferricytochrome C ( 18.7 mg ml -I in HBSS) in the presence and absence of 100/tl PMA ( 1/zg m l - ~in HBSS ), in the presence and absence of 20 #l superoxide dismutase ( 1 mg m l - t in HBSS, 3360 U m g - ~), was incubated at 37 °C for 5 rain after adding HBSS to bring the volume up to 800 /d. Different concentrations of LKT, diluted in HBSS, were pre-warmed for 5 min at 37°C and 200/zl was added to the reaction mixtures containing neutrophils. To determine the effects of antibodies against LKT or LPS on O~production, 400 #l of ascites fluid containing MAbs or antisera (anti-LKT or anti-LPS) was added to the reaction mixture containing neutrophils before the addition of an optimized concentration of LKT (4 U). The tubes containing the various reaction mixtures were incubated for an additional 25 min at 37 °C for color development and placed in an ice bath. The tubes were centrifuged and absorbance of the supernatant fluids was measured at 550 nm. Assays were performed in triplicate or quadruplicate. The absorbance values were converted to nmol O~- using a molar extinction coefficient of 2.1 × 104 c m - ~. The resu!ts were expressed as nmol O~- produced by 106 neutrophils.

56

S.K. MAHESWARAN ET AL.

Assayfor hydrogen peroxide production Hydrogen peroxide ( H202)-generation was determined by the peroxidasemediated oxidation of phenol red (Pick and Mizell, 1981 ) with modifications. Neutrophils (75/tl of 2.67 × 107 cells m1-1 ) were mixed with 210/zl of 0.1% phenol red and 60/tl horseradish peroxidase ( 100 U m l - 1) in the presence and absence of 45 #1 PMA ( 1 /lg m l - 1 in HBSS), in the presence and absence of 45 #1 mouse ascites fluid containing MAbs or antisera (anti-LKT or anti-LPS). HBSS was added to bring the volume up to 390/tl and the reaction mixtures were incubated at 37 °C for 5 rain. Different concentrations of LKT (75 #1 containing 4-64 U ) were pre-warmed at 37 °C for 5 min, added to the reaction mixture and incubation was continued for 30 min for color development. After the incubation, 75 #1 of 3 N NaOH were added to stop the reaction, and optical density (OD) was determined at 606 nmol. Assays were performed in triplicate or quadruplicate. The amount of H202 produced was calculated from a standard curve by linear regression. The results were expressed as nmoles of H202 generated per 2 × 106 neutrophils.

Enzyme assays Neutrophils were suspended at a concentration 4 × 10 6 cells m l - 1 in HBSS in the presence and absence of different concentrations of LKT (0.05-25 U m l - l ) or purified LPS ( 100 ng- 100/tg m l - 1) and incubated at 37 ° C for different time periods. All experiments were done in triplicate. The neutrophils were subsequently removed by centrifugation at 3 0 0 × g for 10 min and the supernatant frozen and saved for various enzyme assays. Neutrophil cytotoxicity was assessed by measurement of release of the cytosolic enzyme, lactate dehydrogenase ( L D H ) , utiliT.ing a colorimetric assay kit (Sigma, procedure no. 500). Neutrophil degranulation was measured by monitoring arylsulfatase B-(Fluharty and Edmond, 1978 ) and myeloperoxidase- (Henson et al., 1978 ) release. These two enzymes were chosen as lysosomal markers because they are present in bovine neutrophils in measurable concentrations (Gennaro et al., 1978). Enzyme release was reported in terms of percentage of maximal enzyme release ( 100% ), a value obtained from the enzyme activity in a lysate prepared by incubating an aliquot of neutrophils with 1% Triton X- 100. Spontaneous enzyme release was also determined in each experiment. To determine the effects of antibodies against leukotoxin or LPS on cytotoxicity and lysosomal enzyme release from neutrophils, 50/tl of mouse ascites fluid containing MAbs or antisera (anti-LKT or anti-LPS ) was added to the neutrophil suspension containing 5 × 106 cells m l - 1 and incubated for 15 min at 37°C. Fifty microliters of LKT (8.5 U ) , pre-warmed for 5 min was added to the neutrophil mixture and incubated for 40 min at 37 ° C on a rotary

57

PASTEURELLA HAEMOLYTICA A1 LEUKOTOXIN AND BOVINE NEUTROPHILS

shaker. The neutrophils were removed by centrifugation and the supernatant was frozen for various enzyme assays.

Statistical analysis Results shown in the figures were expressed as m e a n values + standard deviation (SD). Significance was determined using the unpaired Student's ttest, one- or two-way analysis o f variance, or the Bonferroni multiple comparison test. A P value o f less than 0.05 was considered statistically significant. Each o f the experiments presented in this paper was performed at least three times and all produced similar results or response patterns. RESULTS

Superoxide anion and hydrogen peroxide-generation by LKT-treated neutrophils To determine the effect o f LKT on activation of bovine neutrophils, 10 6 cells ml-~ were exposed to different concentrations o f crude LKT, and the generation o f O~- was measured. The concentration o f crude LKT required to elicit peak O~- generation was not the same in all experiments but the response pattern was the same. Factors, such as the source o f bovine neutrophils and day-to-day handling variables had an effect on this response pattern. However, results showed concentration-dependent O2-generation by bovine neutrophils after exposure to crude LKT (Fig. l (A) ). The generation of O~- was immediate because preincubation of neutrophils with crude LKT

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58

S.K. M A H E S W A R A N E T AL.

for a period of 5 min or more, before adding the other reactants, decreased the amount of measurable Oz by approximately 90% (based on four separate determinations). This decrease in OF generation was attributed to substantial cytolysis of neutrophils as determined by the release of the cytosol enzyme LDH and inability to exclude trypan blue. Since the crude LKT preparation contained LPS as a contaminant, it was necessary to exclude that the O~- production might be related to activation by LPS. Heat-inactivated crude LKT ( 121 °C for 30 min ), which resulted in loss of its cytocidal property but contained LPS, failed to stimulate O~- production (Fig. 2 ). LKT-neutralizing MAb-601 at 1:1000 and 1:20 000 dilutions abrogated O~- production (Fig. 3 (A)). MAb-La, which was also a LKT-neutralizing antibody but had a lower neutralizing titer than MAb-601, significantly decreased O~- at a 1:1000 dilution ( P < 0 . 0 5 ) but not at a 1:20 000 dilution. In contrast, MAb-IH3 that was directed against the LPS of P. haemolytica A 1 and control mouse ascites fluid, did not eliminate O~- production. Furthermore, MAb-601, depleted of LKT-neutralizing IgG~ antibody, did not suppress O~- production (based on three separate experiments ). Polyclonal antibodies against LKT (anti-LKT) dose-dependently suppressed the LKT-induced O~- production by neutrophils (Fig. 4). This suppressive effect was statistically significant ( P < 0.05 ). In contrast, anti-LPS or normal rabbit serum even at high concentrations did not suppress O~- production. These results indicate that LKT but not LPS was the active component in the P. haemolytica A 1 culture supernatant that was responsible for the generation of O~- from activated neutrophils. Bovine neutrophils exposed to crude LKT also generated H202 (Fig. 1 (B) ). This effect was dose-dependent and the same variable factors which affected O~- generation also had an effect on H202 production. Results from these 12, E oI_ ~01:: o

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Fig. 3. Inhibition of O~- production (A), or H202 production (B), in neutrophils by LKTneutralizing monoclonal antibody (MAb). Neutrophils were preincubated for 5 min at 37°C with 1:1000 (bar 2) or 1:20 000 dilution (bar 3) of LKT-neutralizing MAb-601, 1:1000 (bar 4) or 1:20 000 dilution (bar 5 ) of LKT-neutralizing MAb-La, 1:1000 (bar 6) or 1:20 000 dilution (bar 7) of anti-LPS MAb-IH3, and then exposed to P. haemolytica crude LKT containing 4 LKT units (A) or 16 LKT units (B) as described in the text. Data reflect the average +SD from three samples in a single experiment. Bar 1 represents neutrophils not incubated with MAb. The same experiment was carried out three times with similar results• 15

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Fig. 4. Inhibition of O~-production in neutrophils by LKT-neutralizing polyclonal antibodies (anti-LKT). Neutrophils were preincubated (5 min) with 1:100 (bar 2) or 1:1000 (bar 3) or 1:20 000 dilution (bar 4) ofanti-LKT, 1:100 (bar 5) or 1:20 000 dilution (bar 6) of anti-LPS polyclonal antibodies and then exposed to P. haemolytica crude LKT containing 4 LKT units. O~- production was measured as described in the text. Bar 1 represents neutrophils not incubated with polyclonal antibodies. The data represent the average + SD from three samples in an experiment that is representative of three. studies suggested t h a t h i g h e r c o n c e n t r a t i o n o f c r u d e L K T was r e q u i r e d for H 2 0 2 p r o d u c t i o n t h a n for O~- g e n e r a t i o n . T o e x c l u d e the possibility t h a t the H 2 0 2 g e n e r a t i o n was d e r i v e d f r o m the c o n t a m i n a t i n g L P S in the c r u d e L K T , t w o a d d i t i o n a l studies were d o n e . H e a t - i n a c t i v a t i o n o f c r u d e L K T a b r o g a t e d H202 production (data not shown). Additionally, LKT-neutralizing monoclonal a n t i b o d i e s (MAb-601 a n d M A b - L a ) suppressed H 2 0 2 p r o d u c t i o n (Fig.

60

S.K. M A H E S W A R A N E T AL.

3B). Both MAb-601 and MAb-La at a 1:1000 but not at 1:20 000 dilution, significantly suppressed H202 production. In contrast, MAb-601, depleted of LKT-neutralizing IgG, antibody, had no suppressive effect on H202 production (based on three separate experiments). MAb-IH3 which was directed against the LPS did not suppress H202 production at 1:1000 but did suppress slightly at 1:20 000 dilution. However, this suppressive effect was not statistically significant ( P = 0.669).

Effect of L K T on PMA activation of neutrophils To assess the effect of LKT on the response of neutrophils to PMA, cells were preincubated with an optimized concentration of LKT ( 16 U) for different time intervals (0-40 min ) and then stimulated with 100/tl PMA ( 1/~g m l - ] ) to trigger O~- or H202 release. A time-dependent suppression of O~and HzOz generation was noted (Figs. 5 (A) and 5 (B)). This suppression was related to cytotoxicity of the neutrophils as measured by uptake of trypan blue and release of LDH. MAb-601 prevented this LKT-induced suppression of neutrophil response to PMA (based on four separate determinations).

Effect of L K T on enzyme release by neutrophils To determine the effect of crude LKT on neutrophil cytolysis and lysosomal degranulation, 4 × 106 ceils m l - ~were exposed to various concentrations of LKT for 20, 40 and 60 min and the enzyme release assessed. There was a concentration- and time-dependent release of the cytosolic enzyme LDH 30A

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(based on six separate determinations). Release of the lysosomal granule constituent arylsulfatase B, increased in the same concentration- and timedependent fashion as LDH, although the magnitude was much lower than LDH at the same concentration (based on six separate determinations). Figure 6 shows the relationship between the release of lactate dehydrogenase, myeloperoxidase, and arylsulfatase B from neutrophils exposed for 60 min at various concentrations of LKT. A lethal LKT concentration of 25 U m l - 1 released less arylsulfatase B than 2.5 or 5 U LKT. This is a paradoxical finding, and could be due to inactivation of the enzyme by some substance present in the crude LKT. On the other hand, myeloperoxidase, which is also a

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Fig. 7. Inhibition of LKT-induced neutrophil cytotoxicity (A) and lysosomal enzyme release (B and C) by MAbs and polyclonal antibodies. Neutrophils were preincubated for 15 min at 37°C with 1:100 dilution of anti-LPS polyclonal antibodies (d), 1:100 dilution of anti-LKT polyclonal antibodies (e), 1:100 dilution of anti-LPS, MAb-IH3 (f), 1:100 dilution of LKTneutralizing MAb-La (g), 1: 100 dilution of LKT-neutralizing MAb-601 (h), and then incubated with 8.5 units ofP. haemolytica crude LKT for 40 min at 37°C. Results are expressed as in Fig. 6. Bar (a) is buffer control, (b) is neutrophils exposed to only LKT and (c) is neutrophils preincubated with a 1:100 dilution of normal serum collected from a pasteurella-free rabbit. Each value represents average + SD of 11-14 determinations. Statistical significance is identified on the figure.

lysosomal granule constituent, was released at sublethal concentration of LKT and peaked at a LKT concentration of 0.5 U m l - i, at which only _+38% of the total LDH was released. Leukotoxin-neutralizing monoclonal antibodies (MAb-601 and MAb-La) or polyclonal antibodies (anti-LKT) significantly ( P < 0.05 ) decreased the

PASTEURELLA HAEMOLYTICA AI LEUKOTOXIN AND BOVINE NEUTROPHILS

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cytolysis and degranulation of neutrophils induced by the crude LKT (Fig. 7 ). In contrast, normal rabbit serum, anti-LPS or MAb-IH3 did not have any effects on cytolysis or degranulation of neutrophils. Purified LPS from P. haemolytica A1 at a concentration of 100/~g ml -~ incubated with neutrophils for 120 min had no effect on cell viability as measured by LDH release. However, there was a substantial, statistically significant ( P < 0.0001 ) increase in myeloperoxidase release but not release of arylsulfatase B (based on six separate experiments). DISCUSSION

Studies from several laboratories have shown that the pulmonary injury, which occurs in the first 24 h of experimental pneumonic pasteurellosis in cattle, is associated with a marked influx of neutrophils into the lung (Ames et al., 1985; Slocombe et al., 1985; Walker et al., 1985; Breider et al., 1987; Whiteley et al., 1990). Perhaps the most compelling evidence for an obligate role of neutrophils in the genesis of the disease is the finding that prior neutrophil depletion from live calves either attenuated or abrogated the lung pathology observed in experimental pneumonic pasteurellosis (Slocombe et al., 1985 ). Studies with natural cases of the disease have also confirmed that a marked neutrophil influx into the alveoli preceded pneumonic lesions (Lay et al., 1986 ). The influx of neutrophils may be an important step in the lung injury since these cells release various inflammatory mediators including proteases and toxic oxygen species which are capable of degrading components of the lung (Sibille and Reynolds, 1990 ). Despite the presence of these potentially toxic neutrophils, the specific role, if any, of the neutrophils in the pathophysiology of bovine pneumonic pasteurellosis has yet to be thoroughly investigated. The LKT of P. haemolytica A 1 is believed to be involved in the pathogenesis of the disease because it allows the bacteria to survive by destroying the phagocytic cells in the lung (Whiteley et al., 1990). Indeed, we have shown recently, using immunohistochemical techniques, that LKT is present in the alveolar space and is bound to the membranes of degenerating inflammatory cells (Whiteley et al., 1990). The LKT gene has been cloned (Strathdee and Lo, 1987 ) and DNA sequence analysis indicates that it shares extensive homology with other pore-forming cytolysins from Morganella morganii, Proteus vulgaris, Proteus mirabilis, Actinobacillus actinomycetemcomitans and Escherichia coli (Welch, 1987; Seeger and Suttorp, 1987; Koronakis et al., 1987; Kolodrubetz et al., 1989: Lally et al., 1989). Since the cytolytic effect of P. haemolytica LKT is dependent on the presence of calcium (Clinkenbeard et al., 1989), LKT may play a role in augmenting the inflammatory response in the lung by several mechanisms (Whiteley et al., 1990 ), including activation of the respiratory burst and degranulation of bovine neutrophils. This study shows that the culture supernatant from P. haemolytica A 1, in

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addition to its cytocidal activity on bovine neutrophils, can stimulate a respiratory burst and cause release ofproteolytic enzymes from lysosomes. Stimulation of the respiratory burst was an immediate event, while cytolysis and release of proteolytic enzymes by degranulation occurred over a period of time. The factor in the culture supernatant responsible for the above activities was identified as LKT. Generation of oxygen-derived free radicals, such as O~and H202, was used as a measure of stimulation of respiratory burst. That the generation of OS and H202 was caused by LKT and not by contaminants, especially LPS, was confirmed by three findings. First, heat-inactivated crude LKT that had lost its cytocidal properties for neutrophils, failed to stimulate O2 and H202 production. Second, LKT-neutralizing MAbs (MAb-601 and MAb-La) dose-dependently suppressed O~- and H202 production. Third, LKT-neutralizing polyclonal antibodies also suppressed O~- in a dose-dependent fashion. Czuprynski and Noel (1990) recently reported that P. haemolytica A 1 LKT did not directly stimulate an oxidative burst by neutrophils as measured by chemiluminescence. Our data provide evidence to the contrary. This discrepancy may be ascribed to differences in the methods used for the production of crude LKT and the assay systems used in the two laboratories. However, the results of this study confirm previous observations (Czuprynski and Noel, 1990; Styrt et al., 1990) that preincubation of neutrophils with high concentrations of P. haemolytica A 1 LKT, suppressed the respiratory burst by neutrophils stimulated with known agonists. Our findings that this observed suppression of respiratory burst was indeed due to cytolysis of the neutrophils also confirms these earlier findings. Neutrophil myeloperoxidase and arylsulfatase B were released from cells exposed to crude LKT. It was noteworthy that arylsulfatase B release paralleled cytotoxicity as measured by LDH release (Fig. 6) which confirms earlier findings (Styrt et al., 1990). However, at LKT concentrations of 25 U ml-1 and higher, there was 92.21 _+6.78% of maximal LDH release but only 4.7 _+ 1.76% of maximal arylsulfatase B release. This may possibly be due to inactivation of the enzyme by a substance present in the crude LKT or failure of enzyme release from lysosomal granules despite cell lysis. Release of myeloperoxidase, another component of lysosomes, commenced at a sublethal LKT concentration and levelled off at higher LKT concentrations. Myeloperoxidase release may be by active secretion because it was released by sublethal concentrations of LKT. That cytotoxicity and the release of lysosomal enzymes, namely myeloperoxidase and arylsulfatase B, were indeed caused by the LKT was shown by two studies. First, only the LKT-neutralizing MAbs (MAb-601 and MAb-La ) prevented cytotoxicity and release of lysosomal enzymes. The MAb-IH3, directed against the P. haemolytica LPS which reacted serologically (Vega et al., 1987 ) but did not neutralize its biological activity, did not have any protective effect on neutrophils. Second, only the polyclonal LKT-neutralizing

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antibodies (anti-LKT) and not anti-LPS had a protective effect against cytotoxicity and release of lysosomal enzymes. The enzymes which are contained within the lysosomes of bovine neutrophils are not well-characterized. Bovine neutrophils contain three types of lysosomal granules, the larger granules contain cationic peptides (Gennaro et al., 198 3 ) while the other two granules contain most of the myeloperoxidase, hydrolytic enzymes, and lactoferrin (Grieve and Mattila, 1989 ). Results from our studies do not indicate which of the granules are degranulated by the P. haemolytica Al LKT. Our results found no evidence for linking purified LPS from P. haemolytica with the cytocidal effect on bovine neutrophils. However, they demonstrate that there was a substantial increase in myeloperoxidase release from the lysosomes when neutrophils were exposed to LPS. Studies are in progress to further elucidate this phenomenon. The contribution of the P. haemolytica A 1 LKT in the pathogenesis of bovine pneumonic pasteurellosis has been considered mainly in the context of its cytocidal properties against ruminant neutrophils, monocytes/macrophages, and lymphocytes (Benson et al., 1978; Kaehler et al., 1980; Berggren et al., 1981 ). The studies reported here demonstrate that the LKT is also endowed with a capacity to stimulate respiratory burst and cause degranulation of neutrophils. The toxic oxygen radicals, generated as a result of the respiratory burst, and the proteases released as a result of degranulation, can initiate harmful inflammatory reactions and lung damage and may be responsible for the tissue necrosis. It is possible that other biologically active cytokines may also be liberated simultaneously. This invites speculation as to whether these activities do occur in the bovine lung and are relevant in the pathogenesis of the disease. ACKNOWLEDGMENTS This work was supported by US Department of Agriculture special grant 88-CSRS-34116-4095. We thank Robert D. Nelson, Ph.D. from the Department of Surgery, University of Minnesota School of Medicine for helpful discussions. Published as paper no. 17986, Scientific Journal of the Minnesota Agricultural Experiment Station.

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