Veterinary hnmunology and Irnmunopathology, 29 ( 1991 ) 41-56
41
Elsevier Science Publishers B.V., Amsterdam
The effect of Pasteurella haemo!ytica A ! leukotoxic culture supernate on the in vitro proliferative response of bovine lymphocytes A.L. Majury ~ and P.E. Shewen 2 Department of Veterinary Microbiology and Immunology, University of Guelph, Guelph, N I G 2 W l, Canada (Accepted 7 August 1990)
ABSTRACT Majury, A.L. and Shewen, P.E., 1991. The effect of Pasteureila haemolytica A I leukotoxic culture supernate on the in vitro proliferative response of bovine lymphocytes. Vet. hmnunol, hnmunopathol., 29:41-56. The effect of sublethal concentrations of the Pasteurella haemolytica leukotoxic culture supernate on bovine lymphocyte blastogenesis was investigated. Blastogenesis in cultures stimulated with either concanavalin A (Con A) or pokeweed mitogen (PWM) was inhibited in the presence of the supernate, as was the response to purified protein derivative in lymphocytes from BCG-vaccinated cattle. Partially purified leukotoxin had a similar effect. Pre-incubation of the ieukotoxic supernate with a polyclonal rabbit antiserum raised to the immunogenic molecule of recombinant leukotoxin (r LktA ) abrogated this effect, implicating leukotoxin as the factor responsible for the inhibition. B cell enriched cultures tended to be more sensitive to leukotoxic effects than did T cell enriched cultures. Although only ruminant cells are susceptible to the lethal effects of P. haemoiytica ieukotoxin, the toxin did inhibit both Con A- and PWM-induced proliferation of human and dog lymphocytes. As well, at high leukotoxin doses, Con A-stimulated pig lymphocyte proliferation was reduced. Rabbit lymphocytes were not affected by leukotoxin in either Con A- or PWM-stimulated cultures. ABBREVIATIONS BCG, Bacillus Calnette-guerin; BHIB, brain heat infusion broth; Con A, concanavalin A; cpm, counts per minute; FCS, fetal calf serum; OD, optical density; PBL, peripheral blood iymphocytes; PPD, purified protein derivative; PWM, pokeweed mitogen.
INTRODUCTION .__ ,,.,u~ls, also known as shipping fever, is an important Pneumonic n~l~to,,r~ll'~"'cause of economic loss in the feedlot cattle industry (Jericho, 1979). . . . . .
~Present address: Department of Microbiology and Immunology, Queens's University, Kingston, Ontario, K7L 2N2, (Canada) 2Author to whom correspondence should be addressed.
0165-2427/91/$03.50
© 1991 - - Elsevier Science Publishers B.V.
42
A.U MAJURY AND P.E. SHEWEN
Pasteurella haemolytica serotype 1 (AI) is the most frequently isolated organism associated with this disease (Wessman and Hilker, 1968; Wray and Thompson, 1971; Frank and Smith, 1983). Actively growing P. haemolytica produces a soluble, heat-labile ruminant leukocyte-specific cytotoxin (leukotoxin ) (Kaehler et al., 1980; Berggren et al., 1981; Shewen and Wilkie, 1982 ). This leukotoxin is thought to contribute to the pathogenesis of pneumonic pasteurellosis by impairing primary lung defenses and subsequent immune response and/or by the induction of severe inflammation as the result of leukocyte lysis. Cytotoxic changes and decreased phagocytic ability have been described for bovine alveolar macrophages incubated with live P. haemolytica A 1 for 30 or 45 rain (Benson et al., 1978). Markham and Wilkie (1980) showed impaired bacterial uptake by bovine alveolar macrophages following incubation with P. haemolytica culture supernate. More recently, studies involving the incubation of alveolar macrophages with sublethal doses of crude leukotoxin resulted in decreased production of chemotactic factors (Markham et al., 1982). Furthermore, cell-mediated immune response, as measured using the leukocyte migration inhibition test, was inhibited in five calves experimentally infected by intratracheal inoculation of P. haemolytica (Borroughs et al., 1987). Although the pathogenic effects of cell lysis by the P. haemolytica leukotoxin may account for many of the pulmonary lesions characteristic of pneumonic pasteurellosis, the influence of effects on leukocyte function cannot be overlooked. These may be particularly important during the initial stages of infection when impaired phagocytic cell function might permit colonization, ..,~.h^^..~ . . . . . .~.n.d. rl,~r,~©e.-ri v-,,~,.-, I,J,-v-~,,.~,~ activation coma alter induction of an antigen-specific immune response. These concerns led to an investigation of the effects of sublytic levels of the P. haemolytica leukotoxin on lymphocyte function, as determined in vitro using the lymphocyte blastogenesis assay. , -"
MATERIALS AND METHODS
Preparation of the leukotoxin The method used was that described by Shewen and Wilkie (1982). Briefly, several colonies from an 18-h blood agar culture of P. haemolytica A 1 were inoculated into 500 ml of brain heart infusion broth (BHIB) in a 1000 mi Erlenmeyer flask and grown at 37°C on a rocking platform for 4.5 h. Bacteria were pelleted by centrifugation for 10 rain at 4000Xg then resuspended in 1000 ml of RPMI 1640 medium (GIBCO Laboratories, Grand Island, NY) containing 7% fetal calf serum (FCS) and incubated on a rocking platform for l h at 37°C. After centrifugation, at 6000×g for 15 rain, the supernate was filtered through a 0.22/lm millipore filter (MiUipore Corp., Bedford, MA), dialyzed against two changes of distilled water and lyophilized. Before use, the lyophilized material was reconstituted to the desired concentration
43
PROLIFERATIVE RESPONSE OF BOVINE LYMPHOCYTES
in the appropriate medium, filtered through a 0.22/lm filter, and cultured to confirm sterility. This preparation was endotoxin free when tested using the Limulus Amoebocyte Lysate assay (Whittaker M.A. Bioproducts Inc., Walkerville, MD).
Partial purification of the leukotoxin from other components of the culture supernate Neutral protease and neuraminadase are known to be present in P. haemolytica culture supernate (Otulakowski et al., 1983). These activities were separated by precipitation with acetic acid (pH4) to yield an acid precipitate containing all the enzyme activity but no cytotoxic activity and an acid supernate having no enzyme activity but capable of kiUing 100% of cells, as measured using the neutral red assay (Udoh, 1986). Briefly, crude lyophilized culture supernate was resuspended at 25 mg/ml in distilled water. Acetate buffer (0.01 M, pH 4) was added to this solution so that the original volume was tripled. The solution was refrigerated at 4 °C for 30 min before centrifugation at 10 000 g, for l 0 rain at 4 °C, such that a 100 -
\\
gO-
\\ \\ \\ \\ \\
7060-
4030-
2010-
0
-
|
|
O.~
0 Units
[77]
Heat-Wnactivatea
1
2
4
of Toxin ACtive
Toxin
Fig. 1. The effect of leukotoxic culture supernate on the proliferative response of bovine PBL stimulated with the mitogen Con A (2.5/~g/ml). Heat inactivation is the pretreatment of leukotoxic supernate by incubation at 56°C for 60 min. Sublethality occurred at 0.5 units oftoxin or less. A is significantly different from B (Student's t-test, P < 0 . 0 5 ) . Mean cpm for unstimulated cultures is 2298, that for stimulated (Con A) cultures is 158 560.
44
A.L. MAJURY AND P.E. SHEWEN
precipitate could form. The acid supernate was then dialysed against distilled water, filtered (0.22/zm ) and lyophilized before use in the blastogenesis assay.
Neutral red assayfor cytotoxicity The method used for measuring lcukotoxicity was the microplate cytotoxicity assay described by Greer and Shewen (1985 ) using the bovine leukemia-derived B lymphocyte cell line (BL3, originally obtained for G. Theilen, University of California, Davis) as the target cell. Cell viability was determined by the uptake of the vital dye neutral red and was measured by reading the optical density (OD) of each well at 540 nm with an automated spectrophotometer (Titretek Multiskan, Flow Laboratories, Mississauga, Ontario). Lyophilized P. haemolytica culture supernate reconstituted at 2 mg/ml and serially diluted two-fold was incubated with a 200/ll aliquot of BL3 cells at 1 × 106 cells/ml ( 1 h, 37 °C). The cells were then washed in saline and subsequently 100/zl of neutral red working solution (OD, 540 nm, 1.9; CI 825, Allied Chemical and Dye Corp., NY) were added to each well. The plates were incubated for a further 60 rain at 37°C. After a final wash in saline, the 100 90BO70-
\
c o
60-
\
~3
50
¢-
C
z~
40 30
\\ \\ \\ \\ \\
20 10
0
O. 125
O. 25 Units
[ ~
Heat
InactivateO
O. ~
1
2
4
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Active
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Fig. 2. The effect of leukotoxic culture supernate on the proliferative response of bovine PBL stimulated with the niitogen PWM (10/zg/ml). Heat inactivation is the pretreatment of leukotoxic supernate by incubation at 56°C for 60 min. Sublethality occurred at 0.5 units of toxin or less. A is significantly different from B (Student's t-test, P
41
Elsevier Science Publishers B.V., Amsterdam
The effect of Pasteurella haemo!ytica A ! leukotoxic culture supernate on the in vitro proliferative response of bovine lymphocytes A.L. Majury ~ and P.E. Shewen 2 Department of Veterinary Microbiology and Immunology, University of Guelph, Guelph, N I G 2 W l, Canada (Accepted 7 August 1990)
ABSTRACT Majury, A.L. and Shewen, P.E., 1991. The effect of Pasteureila haemolytica A I leukotoxic culture supernate on the in vitro proliferative response of bovine lymphocytes. Vet. hmnunol, hnmunopathol., 29:41-56. The effect of sublethal concentrations of the Pasteurella haemolytica leukotoxic culture supernate on bovine lymphocyte blastogenesis was investigated. Blastogenesis in cultures stimulated with either concanavalin A (Con A) or pokeweed mitogen (PWM) was inhibited in the presence of the supernate, as was the response to purified protein derivative in lymphocytes from BCG-vaccinated cattle. Partially purified leukotoxin had a similar effect. Pre-incubation of the ieukotoxic supernate with a polyclonal rabbit antiserum raised to the immunogenic molecule of recombinant leukotoxin (r LktA ) abrogated this effect, implicating leukotoxin as the factor responsible for the inhibition. B cell enriched cultures tended to be more sensitive to leukotoxic effects than did T cell enriched cultures. Although only ruminant cells are susceptible to the lethal effects of P. haemoiytica ieukotoxin, the toxin did inhibit both Con A- and PWM-induced proliferation of human and dog lymphocytes. As well, at high leukotoxin doses, Con A-stimulated pig lymphocyte proliferation was reduced. Rabbit lymphocytes were not affected by leukotoxin in either Con A- or PWM-stimulated cultures. ABBREVIATIONS BCG, Bacillus Calnette-guerin; BHIB, brain heat infusion broth; Con A, concanavalin A; cpm, counts per minute; FCS, fetal calf serum; OD, optical density; PBL, peripheral blood iymphocytes; PPD, purified protein derivative; PWM, pokeweed mitogen.
INTRODUCTION .__ ,,.,u~ls, also known as shipping fever, is an important Pneumonic n~l~to,,r~ll'~"'cause of economic loss in the feedlot cattle industry (Jericho, 1979). . . . . .
~Present address: Department of Microbiology and Immunology, Queens's University, Kingston, Ontario, K7L 2N2, (Canada) 2Author to whom correspondence should be addressed.
0165-2427/91/$03.50
© 1991 - - Elsevier Science Publishers B.V.
42
A.U MAJURY AND P.E. SHEWEN
Pasteurella haemolytica serotype 1 (AI) is the most frequently isolated organism associated with this disease (Wessman and Hilker, 1968; Wray and Thompson, 1971; Frank and Smith, 1983). Actively growing P. haemolytica produces a soluble, heat-labile ruminant leukocyte-specific cytotoxin (leukotoxin ) (Kaehler et al., 1980; Berggren et al., 1981; Shewen and Wilkie, 1982 ). This leukotoxin is thought to contribute to the pathogenesis of pneumonic pasteurellosis by impairing primary lung defenses and subsequent immune response and/or by the induction of severe inflammation as the result of leukocyte lysis. Cytotoxic changes and decreased phagocytic ability have been described for bovine alveolar macrophages incubated with live P. haemolytica A 1 for 30 or 45 rain (Benson et al., 1978). Markham and Wilkie (1980) showed impaired bacterial uptake by bovine alveolar macrophages following incubation with P. haemolytica culture supernate. More recently, studies involving the incubation of alveolar macrophages with sublethal doses of crude leukotoxin resulted in decreased production of chemotactic factors (Markham et al., 1982). Furthermore, cell-mediated immune response, as measured using the leukocyte migration inhibition test, was inhibited in five calves experimentally infected by intratracheal inoculation of P. haemolytica (Borroughs et al., 1987). Although the pathogenic effects of cell lysis by the P. haemolytica leukotoxin may account for many of the pulmonary lesions characteristic of pneumonic pasteurellosis, the influence of effects on leukocyte function cannot be overlooked. These may be particularly important during the initial stages of infection when impaired phagocytic cell function might permit colonization, ..,~.h^^..~ . . . . . .~.n.d. rl,~r,~©e.-ri v-,,~,.-, I,J,-v-~,,.~,~ activation coma alter induction of an antigen-specific immune response. These concerns led to an investigation of the effects of sublytic levels of the P. haemolytica leukotoxin on lymphocyte function, as determined in vitro using the lymphocyte blastogenesis assay. , -"
MATERIALS AND METHODS
Preparation of the leukotoxin The method used was that described by Shewen and Wilkie (1982). Briefly, several colonies from an 18-h blood agar culture of P. haemolytica A 1 were inoculated into 500 ml of brain heart infusion broth (BHIB) in a 1000 mi Erlenmeyer flask and grown at 37°C on a rocking platform for 4.5 h. Bacteria were pelleted by centrifugation for 10 rain at 4000Xg then resuspended in 1000 ml of RPMI 1640 medium (GIBCO Laboratories, Grand Island, NY) containing 7% fetal calf serum (FCS) and incubated on a rocking platform for l h at 37°C. After centrifugation, at 6000×g for 15 rain, the supernate was filtered through a 0.22/lm millipore filter (MiUipore Corp., Bedford, MA), dialyzed against two changes of distilled water and lyophilized. Before use, the lyophilized material was reconstituted to the desired concentration
43
PROLIFERATIVE RESPONSE OF BOVINE LYMPHOCYTES
in the appropriate medium, filtered through a 0.22/lm filter, and cultured to confirm sterility. This preparation was endotoxin free when tested using the Limulus Amoebocyte Lysate assay (Whittaker M.A. Bioproducts Inc., Walkerville, MD).
Partial purification of the leukotoxin from other components of the culture supernate Neutral protease and neuraminadase are known to be present in P. haemolytica culture supernate (Otulakowski et al., 1983). These activities were separated by precipitation with acetic acid (pH4) to yield an acid precipitate containing all the enzyme activity but no cytotoxic activity and an acid supernate having no enzyme activity but capable of kiUing 100% of cells, as measured using the neutral red assay (Udoh, 1986). Briefly, crude lyophilized culture supernate was resuspended at 25 mg/ml in distilled water. Acetate buffer (0.01 M, pH 4) was added to this solution so that the original volume was tripled. The solution was refrigerated at 4 °C for 30 min before centrifugation at 10 000 g, for l 0 rain at 4 °C, such that a 100 -
\\
gO-
\\ \\ \\ \\ \\
7060-
4030-
2010-
0
-
|
|
O.~
0 Units
[77]
Heat-Wnactivatea
1
2
4
of Toxin ACtive
Toxin
Fig. 1. The effect of leukotoxic culture supernate on the proliferative response of bovine PBL stimulated with the mitogen Con A (2.5/~g/ml). Heat inactivation is the pretreatment of leukotoxic supernate by incubation at 56°C for 60 min. Sublethality occurred at 0.5 units oftoxin or less. A is significantly different from B (Student's t-test, P < 0 . 0 5 ) . Mean cpm for unstimulated cultures is 2298, that for stimulated (Con A) cultures is 158 560.
44
A.L. MAJURY AND P.E. SHEWEN
precipitate could form. The acid supernate was then dialysed against distilled water, filtered (0.22/zm ) and lyophilized before use in the blastogenesis assay.
Neutral red assayfor cytotoxicity The method used for measuring lcukotoxicity was the microplate cytotoxicity assay described by Greer and Shewen (1985 ) using the bovine leukemia-derived B lymphocyte cell line (BL3, originally obtained for G. Theilen, University of California, Davis) as the target cell. Cell viability was determined by the uptake of the vital dye neutral red and was measured by reading the optical density (OD) of each well at 540 nm with an automated spectrophotometer (Titretek Multiskan, Flow Laboratories, Mississauga, Ontario). Lyophilized P. haemolytica culture supernate reconstituted at 2 mg/ml and serially diluted two-fold was incubated with a 200/ll aliquot of BL3 cells at 1 × 106 cells/ml ( 1 h, 37 °C). The cells were then washed in saline and subsequently 100/zl of neutral red working solution (OD, 540 nm, 1.9; CI 825, Allied Chemical and Dye Corp., NY) were added to each well. The plates were incubated for a further 60 rain at 37°C. After a final wash in saline, the 100 90BO70-
\
c o
60-
\
~3
50
¢-
C
z~
40 30
\\ \\ \\ \\ \\
20 10
0
O. 125
O. 25 Units
[ ~
Heat
InactivateO
O. ~
1
2
4
of Toxin [ ~
Active
Toxin
Fig. 2. The effect of leukotoxic culture supernate on the proliferative response of bovine PBL stimulated with the niitogen PWM (10/zg/ml). Heat inactivation is the pretreatment of leukotoxic supernate by incubation at 56°C for 60 min. Sublethality occurred at 0.5 units of toxin or less. A is significantly different from B (Student's t-test, P
