Veterinary Immunology and lmmunopathology, 29 ( 1991 ) 57-68
57
Elsevier Science Publishers B.V., Amsterdam
Preliminary investigation of the mechanism of inhibition of bovine lymphocyte proliferation by Pasteurella haemolytica A 1 leukotoxin A.L. Majur3,~ and P.E. Shewen 2 Department of VeterinaryMicrobiology and Immunology, Universityof Guelph, Guelph, Ontario, N1G 2WI, Canada (Accepted 7 August 1990)
ABSTRACT Majury, A.L. and Shewen, P.E., 1991. Preliminary investigation of the mechanism of inhibition of bovine lymphocyte proliferation by Pasteurella haemolytica Al leukotoxin. Vet. Immunol. Immunopathol., 29: 57-68.
Pasteurella haemolytica Al leukotoxic culture supernate has been shown to inhibit bovine lymphocyte blastogenesis induced by concanavalin A (Con A), pokeweed mitogen (PWM) and purified protein derivative (PPD). The various mechanisms by which this inhibition could be overcome were investigated in an effort to determine at which stage of cell activation the leukotoxin exerted its inhibitory effect. For both Con A and PWM stimulated cultures, the addition of partially purified bovine interleukin l reduced the leukotoxin-induced inhibition. Recombinant interleukin 2 had a similar effect. Addition of the glycolipid, monosialoganglioside was also able partially to overcome the inhibition. ABBREVIATIONS Con A, concanavalin A; cpm, counts per minute; GM l, monosialoganglioside; IL-l, interleukin l; IL-2, interleukin 2; LPS, lipopolysaccharide; PPD, purified protein derivative; PWM, pokeweed mitogen.
INTRODUCTION
Pasteurella haemolytica AI has long been implicated as the most important etiologic agent associated with bovine pneumonic pasteurellosis (Scott and Farley, 1932; Carter, 1954; Collier, 1968; RehmtuUa and Thomson, 1981 ), a major cause of economic loss in feedlot cattle (Martin et al., 1980). Actively growing P. haemolytica produce a soluble, heat-labile ruminant rePresent address: Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, K7L 2N2, Canada. 2Author to whom correspondence should be addressed.
58
A.L. MAJURY AND P.E. SHEWEN
leukocyte-specific cytotoxin (Kaehler et al., 1980; Berggren et al., 198 l; Shewen and Wilkie, 1982). The genes coding for this leukotoxin have been cloned (Lo et al., 1985), and nucleotide sequence analysis revealed genes coding for two structural molecules: a l 01.9 kDa protein which is antigenic and probably incorporates the binding portion of the molecule and a nonantigenic, smaller 19.8 kDa protein necessary for biologic activity (Lo et al., 1987). The leukotoxin genes, lktA and lktC, respectively, share extensive homology with genes coding for the HlyA and HlyC alpha-haemolysin proteins of Escherichia coli (Strathdee and Lo, 1987 ). These two similar toxins are calcium-dependent, pore-forming cytolysins (Clinkenbeard et al., 1989a) and their homologous regions reveal a distinct pattern oftandemly repeated amino acid domains (Strathdee and Lo, 1987) which may prove important to cell receptor binding and/or host cell specificity. The importance of the cytolytic effects of leukotoxin to the pathogenesis of pneumonic pasteurellosis are well recognized, but its effects on cell function with relevance to disease cannot be overlooked. At sublethal doses, P. haemolytica leukotoxic culture supernate has been shown to impair bacterial uptake by bovine alveolar macrophages (Markham and Wilkie, 1979) and to inhibit the luminol-dependent chemiluminescent response of neutrophils (Chang et el., 1985 ). Pasteurella haemolytica leukotoxic culture supernate also inhibits concanavalin A (Con A), pokeweed mitogen (PWM) and purified protein derivative (PPD) induced bovine lymphocyte proliferation (Majury and Shewen, 1991 ). In an attempt to understand these inhibitory effects we investigated mechanisms by which leukotoxin-induced inhibition of in vitro bovine Ivmnhaevta prt~llforatiOn m l a h t h ~ c~var~nma
MATERIALS AND METHODS
Preparation of the leukotoxin The method of production was adapted from that used by Shewen and Wilkie (1982) and is described in detail in Majury and Shewen ( 1991 ).
Blastogenesis assay This method was based upon that described by Maluish and Strong (1986) and is also described in Majury and Shewen ( 1991 ). The effect of the leukotoxin was determined as the percent inhibition (or enhancement) of the proliferative response using the following formula: Percent inhibition-
cell control-cell test × 100 cell control
This calculation was applied only when the mean counts per minute (cpm) for the cells with mitogen but no toxin was significantly different from the cpm of the cells with mitogen and toxin (Student's t-test, P_< 0.05 ).
59
INHIBITION OF BOVINE LYMPHOCYTE PROLIFERATION
Sub!ethality occurred at a l / 8 dilution of the 2 mg/ml leukotoxin preparation; i.e. 0.5 units ofleukotoxin, where 1 unit was defined as a 1/4 dilution of the 2 mg/ml leukotoxic preparation. lnterleukin 1 Partially purified bovine interleukin 1 (IL- 1 ) was a gift from J.A. Lederer, Madison, WI. It was prepared from culture supernatants of bovine alveolar macrophages stimulated for 18 h with lipopolysaccharide (LPS) ( 10 pg/ml) and purified using size exclusion high pressure liquid chromatography 80 70 t CONCANAVALIN A BLASTOGENESIS
C
~0 t-
._o ,~_ 50 c-
_c
40
o
o
30 1
B T
20 10 |
O.'00 9O
0 '. -2 5 - ~
0.13
' 0.5
!
80J
PWN BLASTOGENESIS
70
o:
6O
50 -o
40
...... e
30
20. 100 I
i
0.125 II
0.5 Units of Toxin with IL-1 ITET~ NO I L - 1 0.25
1
Fig. 1. The effect of the addition of IL-I (partially purified bovine, 16 units/well on leukotoxininduced inhibition of bovine peripheral blood lymphocytes stimulated with Con A (2.5/zg/ml or PWM ( 10 pg/ml). Sublethality occurred at 0.5 units of toxin or less. A is significantly different from B, but not significantly different from C (Student's t-test, P_< 0.05). Mean cpm for unstimulated cultures is 744, that for stimulated (Con A) cultures is 118 683, that for stimulated (PWM) cultures is 87 340.
60
A.L. MAJURY AND P.E. SHEWEN
(Lederer and Czuprynski, 1989). Each millilitre contained 1280 units as measured by the murine thymocyte assay (Gery et al., 1972) and each well received 16 units. Interleukir. 2 Recombinant human intcdeukin 2 (IL-2) was purchased from Genzyme, Mississauga, Ontario. Recombinant human IL-2 has been shown to stimulate bovine lymphocytes (Fong and r~,,,,~,~L,,,j.,.,! 986). Each well received 50 units, where one unit was defined as that amount of IL-2 which caused half maximal
IOO CONCANAVALIN A BLASTOGENESIS 8O
6O 5O 40 30
t
2O 10 O O 100 90~
0.125
0.25
0.5
1
PWM BLASTOGENESIS "11"
0
0.125
0.25
0.5
1
Units of Toxin IwithlL-2 ~ NO I L - 2
Fig. 2. The effect of the addition of IL-2 (recombinant human, 50 units/well) on leukotoxininduced inhibition of bovine peripheral blood lymphocytes stimulated with Con A (2.5/zg/ml) or PWM ( l0/~g/ml). Sublethality occurred at 0.5 units of toxin or less. A is significantly different from B, but not significantly different from C (Student's t-test, P
57
Elsevier Science Publishers B.V., Amsterdam
Preliminary investigation of the mechanism of inhibition of bovine lymphocyte proliferation by Pasteurella haemolytica A 1 leukotoxin A.L. Majur3,~ and P.E. Shewen 2 Department of VeterinaryMicrobiology and Immunology, Universityof Guelph, Guelph, Ontario, N1G 2WI, Canada (Accepted 7 August 1990)
ABSTRACT Majury, A.L. and Shewen, P.E., 1991. Preliminary investigation of the mechanism of inhibition of bovine lymphocyte proliferation by Pasteurella haemolytica Al leukotoxin. Vet. Immunol. Immunopathol., 29: 57-68.
Pasteurella haemolytica Al leukotoxic culture supernate has been shown to inhibit bovine lymphocyte blastogenesis induced by concanavalin A (Con A), pokeweed mitogen (PWM) and purified protein derivative (PPD). The various mechanisms by which this inhibition could be overcome were investigated in an effort to determine at which stage of cell activation the leukotoxin exerted its inhibitory effect. For both Con A and PWM stimulated cultures, the addition of partially purified bovine interleukin l reduced the leukotoxin-induced inhibition. Recombinant interleukin 2 had a similar effect. Addition of the glycolipid, monosialoganglioside was also able partially to overcome the inhibition. ABBREVIATIONS Con A, concanavalin A; cpm, counts per minute; GM l, monosialoganglioside; IL-l, interleukin l; IL-2, interleukin 2; LPS, lipopolysaccharide; PPD, purified protein derivative; PWM, pokeweed mitogen.
INTRODUCTION
Pasteurella haemolytica AI has long been implicated as the most important etiologic agent associated with bovine pneumonic pasteurellosis (Scott and Farley, 1932; Carter, 1954; Collier, 1968; RehmtuUa and Thomson, 1981 ), a major cause of economic loss in feedlot cattle (Martin et al., 1980). Actively growing P. haemolytica produce a soluble, heat-labile ruminant rePresent address: Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, K7L 2N2, Canada. 2Author to whom correspondence should be addressed.
58
A.L. MAJURY AND P.E. SHEWEN
leukocyte-specific cytotoxin (Kaehler et al., 1980; Berggren et al., 198 l; Shewen and Wilkie, 1982). The genes coding for this leukotoxin have been cloned (Lo et al., 1985), and nucleotide sequence analysis revealed genes coding for two structural molecules: a l 01.9 kDa protein which is antigenic and probably incorporates the binding portion of the molecule and a nonantigenic, smaller 19.8 kDa protein necessary for biologic activity (Lo et al., 1987). The leukotoxin genes, lktA and lktC, respectively, share extensive homology with genes coding for the HlyA and HlyC alpha-haemolysin proteins of Escherichia coli (Strathdee and Lo, 1987 ). These two similar toxins are calcium-dependent, pore-forming cytolysins (Clinkenbeard et al., 1989a) and their homologous regions reveal a distinct pattern oftandemly repeated amino acid domains (Strathdee and Lo, 1987) which may prove important to cell receptor binding and/or host cell specificity. The importance of the cytolytic effects of leukotoxin to the pathogenesis of pneumonic pasteurellosis are well recognized, but its effects on cell function with relevance to disease cannot be overlooked. At sublethal doses, P. haemolytica leukotoxic culture supernate has been shown to impair bacterial uptake by bovine alveolar macrophages (Markham and Wilkie, 1979) and to inhibit the luminol-dependent chemiluminescent response of neutrophils (Chang et el., 1985 ). Pasteurella haemolytica leukotoxic culture supernate also inhibits concanavalin A (Con A), pokeweed mitogen (PWM) and purified protein derivative (PPD) induced bovine lymphocyte proliferation (Majury and Shewen, 1991 ). In an attempt to understand these inhibitory effects we investigated mechanisms by which leukotoxin-induced inhibition of in vitro bovine Ivmnhaevta prt~llforatiOn m l a h t h ~ c~var~nma
MATERIALS AND METHODS
Preparation of the leukotoxin The method of production was adapted from that used by Shewen and Wilkie (1982) and is described in detail in Majury and Shewen ( 1991 ).
Blastogenesis assay This method was based upon that described by Maluish and Strong (1986) and is also described in Majury and Shewen ( 1991 ). The effect of the leukotoxin was determined as the percent inhibition (or enhancement) of the proliferative response using the following formula: Percent inhibition-
cell control-cell test × 100 cell control
This calculation was applied only when the mean counts per minute (cpm) for the cells with mitogen but no toxin was significantly different from the cpm of the cells with mitogen and toxin (Student's t-test, P_< 0.05 ).
59
INHIBITION OF BOVINE LYMPHOCYTE PROLIFERATION
Sub!ethality occurred at a l / 8 dilution of the 2 mg/ml leukotoxin preparation; i.e. 0.5 units ofleukotoxin, where 1 unit was defined as a 1/4 dilution of the 2 mg/ml leukotoxic preparation. lnterleukin 1 Partially purified bovine interleukin 1 (IL- 1 ) was a gift from J.A. Lederer, Madison, WI. It was prepared from culture supernatants of bovine alveolar macrophages stimulated for 18 h with lipopolysaccharide (LPS) ( 10 pg/ml) and purified using size exclusion high pressure liquid chromatography 80 70 t CONCANAVALIN A BLASTOGENESIS
C
~0 t-
._o ,~_ 50 c-
_c
40
o
o
30 1
B T
20 10 |
O.'00 9O
0 '. -2 5 - ~
0.13
' 0.5
!
80J
PWN BLASTOGENESIS
70
o:
6O
50 -o
40
...... e
30
20. 100 I
i
0.125 II
0.5 Units of Toxin with IL-1 ITET~ NO I L - 1 0.25
1
Fig. 1. The effect of the addition of IL-I (partially purified bovine, 16 units/well on leukotoxininduced inhibition of bovine peripheral blood lymphocytes stimulated with Con A (2.5/zg/ml or PWM ( 10 pg/ml). Sublethality occurred at 0.5 units of toxin or less. A is significantly different from B, but not significantly different from C (Student's t-test, P_< 0.05). Mean cpm for unstimulated cultures is 744, that for stimulated (Con A) cultures is 118 683, that for stimulated (PWM) cultures is 87 340.
60
A.L. MAJURY AND P.E. SHEWEN
(Lederer and Czuprynski, 1989). Each millilitre contained 1280 units as measured by the murine thymocyte assay (Gery et al., 1972) and each well received 16 units. Interleukir. 2 Recombinant human intcdeukin 2 (IL-2) was purchased from Genzyme, Mississauga, Ontario. Recombinant human IL-2 has been shown to stimulate bovine lymphocytes (Fong and r~,,,,~,~L,,,j.,.,! 986). Each well received 50 units, where one unit was defined as that amount of IL-2 which caused half maximal
IOO CONCANAVALIN A BLASTOGENESIS 8O
6O 5O 40 30
t
2O 10 O O 100 90~
0.125
0.25
0.5
1
PWM BLASTOGENESIS "11"
0
0.125
0.25
0.5
1
Units of Toxin IwithlL-2 ~ NO I L - 2
Fig. 2. The effect of the addition of IL-2 (recombinant human, 50 units/well) on leukotoxininduced inhibition of bovine peripheral blood lymphocytes stimulated with Con A (2.5/zg/ml) or PWM ( l0/~g/ml). Sublethality occurred at 0.5 units of toxin or less. A is significantly different from B, but not significantly different from C (Student's t-test, P
