Preliminary investigation of the mechanism of inhibition of bovine lymphocyte proliferation by Pasteurella haemolytica A1 leukotoxin

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-

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_c

40

o

o

30 1

B T

20 10 |

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0 '. -2 5 - ~

0.13

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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

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t

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0.125

0.25

0.5

1

PWM BLASTOGENESIS "11"

0

0.125

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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<0.05, *P<0.10). Mean cpm for unstimulated cultures is 6739, that for stimulated (Con A) cultures is 197 420, that for stimulated (PWM) cultures is 187 868.

INHIBITION OF BOVINE LYMPHOCYTE PROLIFERATION

61

incorporation of 3H-thymidine in 4 × 103 CTLL (IL-2 dependent) cells in culture.

G!ycotipid The glycolipid, monosialoganglioside (GM 1 ), was purchased from Sigma Chemical Company, St. Louis, MO. Each well received 500 ng. For the assay in which both GM 1 and IL-2 were added to cell cultures, each well received 500 ng of GM 1, and 50 units of IL-2. 90. 80-

PREACTIVATION with CON A

70. 60.

40302010-

0

0.~25

0.25

0.5

1

70 PREACTIVATION with PWM

40

--

30

20 10

0.125

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0.25 0.5 1 Units of T o x i n Preoctivoted I~-~1 Not P r e o c t i v n t e d

Fig. 3. The effect of preactivating bovine lymphocyte cultures for 24 h with the mitogens Con A (2.5/zg/ml) or PWM ( 10/zg/ml ) prior to the addition of leukotoxin. 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, *P<0.10). Mean cpm for unstimulated cultures is 6972, that for stimulated (Con A, not preactivated) cultures is 125 729, that for stimulated (Con A, preactivated) cultures is 128 350, that for stimulated (PWM, not preactivated) cultures is 158 637, that for stimulated (PWM, preactivated) cultures is 171 464.

62

A.L. MAJURY AND P.E. SHEWEN

RESULTS

The effect of bovine IL-1 on leukotoxin-induced inhibition Serial two-fold dilutions of sublethal doses of leukotoxin imposed a titratable inhibitory effect upon peripheral blood lymphocytes stimulated with Con A or PWM. The exogenous monokine IL- 1 was added to the cell assay system in order to overcome a functional monoeyte defect, and inhibition of mitogen 90 BO

70 6O

--

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o

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Q00

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0.5

1.0

9°/~ BLASTOGENESIS

5 £3 o o

0.13

025

05

U n i t s of T o x i n

With GM 1

~

No GM 1

Fig. 4. The effect of the addition of GM 1 (500 ng/well) on leukotoxin-induced inhibition of Con A (2.5 pg/ml) or PWM ( l0 pg/ml) stimulated bovine peripheral blood lymphocyte cultures. 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. l 0). Mean cpm for unstimulated cultures is 8992, that for stimulated (Con A) cultures is 88 234, that for stimulated (PWM) cultures is 101 501.

63

INHIBITION OF BOVINE LYMPHOCYTE PROLIFERATION

indut:cd blastogenesis was ,.,,,,,,,,,,,,,,.,., ....,.,..,,~ . . . . . . - . . . . . . . . . . . . . purified bovine IL-1 to cell cultures (Fig. 1 ).

~....... j

The effect of recombinant human IL-2 on leukotoxin-induced inhibition The lymphokine IL-2 plays a vital and central role in the ir,duction of lymphocyte proliferation. As shown in Fig. 2, the addition of IL-2 (r IL-2, human, 50 units/well) partially overcame the inhibitory effects of the P. haemolytica leukotoxin on mitogen-induced blastogenesis. 7O CONCANAVALIN A BLASTOGEESIS

5O

8

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o

40-

30-

o

2010.

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| i

I

0.00

I

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40

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30. 20-

B --'---

10-

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0

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0.5 0~25 Units of Toxin NO GM 1 and I L - 2 G M 1 Qnd I L - 2 0.125

1

Fig. 5. The effect of the addition ofGM 1 (500 ng/weli ) and IL-2 (r human, 50 units/well ) on leukotoxin-induced inhibition of bovine peripheral blood iymphocytes cultures stimulated with the mitogens Con A (2.5 pg/ml) or PWM ( 10 #g/ml). Sublethality occurred at 0.5 units of toxin or less. A is significantly different from B (Student's t-test, P < 0.05 ). Mean cpm for unstimulated cultures is 65, that for stimulated (Con A) cultures is 120 693, that for stimulated (PWM) cultures is 66 730.

64

A.L. MAJURY AND P.E. SHEWEN

Preactivation of lymphocytes prior to the addition of leukotoxin Preactivation of lymphocytes with Con A or PWM for 24 h prior to the addition of leukotoxin reduced the amount of inhibition, although not always to a significant degree. This effect was more notable in Con A-stimulated ~han in PWM-stimulated lymphocyte cultures (Fig. 3 ).

The effects of the glycolipid, GM 1, on leukotoxin-induced inhibition The glycolipid, GM 1, was tested for its ability to overcome kukotoxininduced inhibition. GM 1 was able to diminish the effects of leukotoxin on mitogen-induced lymphocyte blastogenesis (Fig. 4). Simultaneous addition of GM 1 (500 ng) and r IL-2 (50 units) further reduced this inhibitory effect (Fig. 5 ). DISCUSSION

Pasteurella haemolytica produces a leukotoxin (Benson et al., 1978) known to be important in the pathogenesis of pneumonic pasteurellosis. Alveolar macrophages, blood monocytes, lymphocytes and neutrophils of ruminants are all susceptible to the cytolytic effects of leukotoxin (Kaehler et al., 1980; Berggren et al., 1981; Shewen and Wilkie, 1982). At sublethal doses leukotoxic culture supernatant will impair bacterial uptake by bovine alveolar macrophages (Markham and Wilkie, 1979) and inhibit the luminol-dependent chemiluminescent response of neutrophils (Chang et al., 1985 ). Recently, it was found that sublethal doses of leukotoxin inhibited bovine lymphocyte proliferation in a titratable fashion (Majury and Shewen, 1991 ). In the present study leukotoxin-induced inhibition of lymphocyte prolifer=u,,.= was ,~,~=vvu by Ul~ ~UUlUUn of the cytokines, iL-i ano I L - 2 . several mechanisms for such an effect are possible. For example, leukotoxin-induced inhibition of macrophage function may also inhibit IL-I release, therefnre making IL- 1 unavailable to induce lymphocyte activation or IL-2 production. Such a defect may be corrected through the addition of exogenous IL-1. Exogenous IL-2, on the other hand, could compensate for a primary lymphocyte defect. Miller-Edge and Splitter (1986) suggest that when responsiveness is restored through IL-2 supplementation, a failure of IL-2 production by the lymphocyte is probably responsible. Other possibilities include impaired IL2 binding, altered IL-2 receptor expression or a lower number of immunoreactive cells. In this study IL-2 abrogated the inhibition more dramatically in Con A than PWM stimulated cultures. This may be a direct consequence of Con A's mitogenic specificity for T rather than B lymphocytes, such that T lymphocyte activation would lead to endogenous IL-2 release resulting in increased IL-2 receptor expression on the cell surface, thereby further enhancing the action of exogenous IL-2. Preactivation of peripheral blood lymphocytes for 24 h before the addition

INHIBITION OF BOVINE LYMPHOCYTE PROLIFERATION

65

of leukotoxin also partially abrogated the inhibition. Preactivation may have allowed adequate IL-l or IL-2 production to occur prior to leukotoxin exposure. In cattle, IL-2 production begins as early as 3 h post-stimulation (Weinberg et al., 1988). The effect was again greater in Con A than PWM stimulated lymphocyte cultures. Pretreatment may have served the same function as the addition of exogenous IL-2, since it would have allowed endogenous IL-2 production prior to insult by the leukotoxin, thus inducing enhanced IL-2 receptor expression. The glycolipid, GM 1, partially overcame the leukotoxin-induced inhibition. The effects of this glycolipid were investigated because of the role of glycolipids as receptors for various bacterial toxins (Fishman and Brady, 1982 ). The inhibitory effects of glycolipids on lymphocyte proliferation are also well documented (Parker et al., 1984; Robb, 1986; Jackson et al., 1987; Marcus et al., 1987). This would suggest that GM 1 is not acting to overcome the inhibition induced by leukotoxin through non-specific lymphocyte stimulation. However, it is possible that GM 1 is interfering with the leukotoxincell association either via direct GM l-leukotoxin interaction or via alteration of the cell membrane or cell membrane receptor through an, as yet, unidentified mechanism. Further work is necessary to determine if such an interaction does exist. GM l is the natural receptor for cholera toxin (Holgrem et al., 1973), and this toxin's B subunit, upon cell binding, is known to increase intracellular calcium in peripheral blood lymphocytes and quiescent 3T3 cells in vitro (Fishman, 1986; Speigel and Fishman, 1987). This suggests an association of glycolipids with calcium channels, such that binding of the B subunit may, in turn, perturb these channels (Craig and Cuatrecasas, 1975 ). The P. haemolytica leukotoxin has been shown to induce calcium influx in the lymphocytes of a bovine leukemic cell line (BL3) (Clinkenbeard et al., 1 9 8 9 b ). It is therefore possible that leukotoxin, like cholera toxin, may affect C a 2 + f l u x via a glycolipid on the cell surface or via a second cell surface component associated with the glycolipid. Calcium ions play a central role in many cytolytic toxic mechanisms. One mechanism acts through alpha-actinin. Increased cytosolic C a 2 + concentration causes a dissociation ofactin microfilaments from alpha-actinin leading to the development of weakened sites in the cell membrane where the cytoskeleton has become dissociated (Orrenius et al., 1989 ). Since glycolipids, particularly GM 1, have been shown to interact with alphaactinin (Kellie et al., 1983), the possibility that a toxin-glycolipid-alpha-actinin-Ca 2+ interaction is important to toxin-induced cell deregulation is further reinforced. Addition of GM l together with human r IL-2 resulted in marked abrogation of the leukotoxin-induced inhibition. GM I is known to interact with IL2 and the IL-2 receptor (Robb, 1986), such that this interaction will inhibit proliferation in IL-2 dependent cell lines; hence, a decreased ability of exog-

66

A.L. MAJURY AND P.E. SHEWEN

enous IL-2 to overcome inhibition might have been expected. If GM 1 does interact with leukotoxin as previously speculated perhaps most, if not all, of the exogenous GM 1 became associated with leukotoxin leaving the IL-2 free to stimulate lymphocyte proliferation. This question could be directly addressed through future experimental studies. Whether or not leukotoxin acts to deregulate cells via glycolipid, interleukins, calcium and/or some alternative mechanism remains to be clarified. Regardless of the mechanism, it is of interest that IL-2 is able to overcome the leukotoxin-induced inhibition of lymphocyte proliferation. An efficacious vaccine which protects calves from pneumonic pasteurellosis has been developed from a bacteria-free P. haemolytica AI leukotoxic culture supernate (Shewen and Willde, 1988). Since local adminstration of IL-2 with antigen has been shown to stimulate humoral immunity (Kawamura et al., 1985), this formation, together with the knowledge that IL-2 overcomes leukotoxin-induced inhibition in the blastogenesis assay may warrant further investigation of a leukotoxin-IL-2 combination vaccine. Studies of immunodeficient athymic mice, of the n u \ nu genotype, infected with vaccinia virus or with a recombinant vaccinia-IL-2 preparation (Hexner et al., 1987; Ranshaw et al., 1987) revealed that mice infected with the recombinant virusIL-2 recovered rapidly whereas those infected with control virus developed progressive disease. An animal infected with pasteurellosis may be in an immunocomprimised state and therefore unlikely to mount an appropriate immune response if vaccinated. Combination leukotoxin-IL-2 vaccines may therefore be appropriate, especially for vaccination in the face of disease or in the stressed and immunocomprimised animal. ACKNOWLEDGEMENTS

This work was supported by the Natural Sciences and Engineering Research Council of Canada and the Ontario Ministry of Agriculture and Food. The assistance of Heather Edwards in blastogenesis assays is also gratefully acknowledged.

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Markham, R.J.F. and Wilkie, B.N., 1979. Interaction between Pasteurella haemolytica and bovine alveolar macrophages: Cytotoxic effect on macrophages and impaired phagocytosis. Am. J. Vet. Res., 41: 18-22. Martin, S.W., Meek, A.H., Davis, D.G., Thomson, R.G., Johnson, J.A., Lopez, A., Stephens, L., Curtis, R.A., Prescott, J.F., Rosendal, S., Savan, M., Zubaidy, A.J. and Bolton, M.R., 1980. Factors associated with mortality in feedlot cattle: The Bruce county beef project. Can. J. Comp. Med., 44: 1-10. Miller-Edge, M. and Splitter, G., 1986. Detection of impaired T cell-mediated immune responses to herpesvirus (BHV- 1 ) in cattle. Vet. Immunoi. Immunopathol., 13: 1-18. Orrenius, S., McConkey, D.J., Bellomo, G. and Nicotera, P., 1989. Role of Ca 2+ in toxic cell killing. T.I.P.S., 10: 281-285. Parker, J., Caldini, G., Krishnamarti, C., Ahrens, P.B. and Ankel, H., 1984. Binding ofinterleukin 2 to gangliosides. FEBS, Lett., 170:391-395. Ranshaw, I.A., Andrew, M.E., Phillips, S.M., Boyle, D.B. and Canpar, E.H., 1987. Recovery of immunodeficient mice from a vaccinia virus/IL2 recombinant infection. Nature, 329: 545546. Rehmtulla, A.J. and Thomson, R.G., 1981. A review of the lesions in shipping fever of cattle. Can. Vet. J., 22: 1-8. Robb, R.J., 1986. The suppressive effect ofgangliosides upon IL-2 dependent proliferation as a function of inhibition of IL-2 receptor association. J. Immunol., 136:971-976. Scott, J.P. and Farley, H., 1932. Preliminary bacteriological report on shipping fever. J. Am. Vet. Med. Assoc., 80:173-186. Shewen, P.E. and Wilkie, B.N., 1982. Cytotoxin of Pasteurella haemolytica acting on bovine leukocytes. Infect. Immun., 35:91-94. Shewen, P.E. and Wilkie, B.N., 1988. Vaccination of calves with leukotoxi,: culture supernatant from Pasteureila haemolytica. Can. J. Vet. Res., 52: 30-36. Spiegel, S. and Fishman, P.H., 1987. Gangliosides as bimodal regulators of cell growth. Proc. Natl. Acad. Sci. U.S.A., 84:141-145. Strathdee, C.A. and Lo, R.Y.C., 1987. Extensive homology between the leukotoxin of Pasteurella haemolytica Al and the alpha-haemolysin of Escherichia coil Infect. Immun., 55: 3233-3236. Weinberg, A.D., Magnuson, N.S., Reeves, R., Wyatt, C.R. and Magnuson, J.A., 1988. Evidence for two discrete phases of IL-2 production in bovine lymphocytes. J. Immunol., 141: 11741179.