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Comparative effects of bovine cytokines on cattle and bison peripheral blood mononuclear cell proliferation
~
Pergamon
Comp. hnmun. Microbiol. infi,ct. Dis. Vol. 20, No. 2, pp. 155 162. 1997 Published by Elsevier Science Ltd Printed in Great Britain PII: S0147-9571(96)00037-9 0147-9571/97 $17.0o + 0.o0
COMPARATIVE EFFECTS OF BOVINE CYTOKINES CATTLE AND BISON PERIPHERAL BLOOD MONONUCLEAR CELL PROLIFERATION MARK
G. S T E V E N S * , S T E V E N C. O L S E N a n d N O R M A N
ON
F, C H E V 1 L L E
National Animal Disease Center, USDA, Agriculture Research Service, 2300 Dayton Avenue, Ames, Iowa, 50010, U.S.A. (ReceivedJbr publication 28 October 1996)
Abstract--Proliferation of peripheral blood mononuclear cells (PBMC) from cattle and bison was measured following stimulation of PBMC with bovine cytokines. Bovine interleukin l fl (BOIL-1/1), interleukin 2 (BOIL-2) or granulocyte-macrophage colony-stimulating factor (BoGM-CSF) at 0.1-100 U/ml were incubated for 48 h with PBMC alone or with PBMC and various mitogens. These included concanavalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM) or Escherickia coli 055:B5 lipopolysaccharide (LPS) at 10 0.1 ~Lg/ml. BOIL-2 alone, but not BoIL-l/J and BoGM-CSF alone, induced proliferation of cattle and bison PBMC in the absence of mitogens. In addition, BoIL-lfl and BOIL-2, but not BoGM-CSF, enhanced proliferation of cattle and bison PBMC induced by mitogens. These results indicate that BolL-1/~' and BOIL-2 stimulate cattle and bison PBMC proliferation in a similar manner, whereas BoGM-CSF does not appear capable of stimulating either cattle or bison PBMC proliferation. Published by Elsevier Science Ltd Key words: Bovine cytokines, interleukin, bison
R 6 s u m ~ N o u s avons mesur6 la prolif6ration de cellules mononucl6aires sanguines p6riphdriques (PBMC) de bovins et de bisons apr6s stimulation par des cytokines d'origine bovine. Ces cellules mononucl6aires seules ou en pr~:sence de mitog6nes, ont 6t6 incub6es pendant 48 hr avec 10(~0.1 U/ml d'interleukine 1/3 (BoIL-lfl), d'interleukine 2 (BolL-2), ou de facteur de stimulation de colonie granulocytaire-macrophagique (BoGM-CSF) d'origine bovine. Concavalin A (Con A), phytohemagglutinin (PHA), mitogbne pokeweed (PWM), ou lipopolysaccharides (LPS) d'Esckerichia coli 055:B5 ont ~t6 utilis~s comme mitog6nes 5. une concentration de 1~0.1 ~g/ml. En l'abscence des mitog6nes, BOIL-2 fut la seule/t induire la prolifdration des PBMC de bovins et de bisons. En pr6sence des mitog6nes, BOIL-1 et BOIL-2 furent les seules 5. induire la prolif6ration des PBMC de bovins et de bisons. Ces rdsultats indiquent que BoIL-lfl et BOIL-2, contrairement 5. BoGM-CSF, stimulent la prolifdration des PBMC de bovins et de bisons. Published by Elsevier Science Ltd Mots-chff[~': Bovine cytokines, interleukine, bison
INTRODUCTION
Bovine (Bo) cytokines such as interleukin lfl (BoIL-lfl), interleukin 2 (BOIL-2) and granulocyte-macrophage colony-stimulating factor (BoGM-CSF) have been shown to have immunostimulatory activity in cattle [1 5]. The activity of BoGM-CSF is believed to be species specific [6]; however, BoIL-I// and BOIL-2 exhibit cross-reactivity on immune cells from other important food-producing animals such as sheep [7] and pigs [8]. Cattle, sheep and bison are related ruminants, belonging to the family Bovidae. Although bison *Author for correspondence. 155
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barely escaped extinction, they are currently raised in small numbers for meat production in the United States and large free-ranging herds are being preserved in Yellowstone National Park in the United States and in other parks and sanctuaries in Canada [9, 10]. Very little is known about the bison immune system and it has not been determined if bovine cytokines exhibit activity on bison immune cells, as has been demonstrated to occur in sheep and pigs. The objective of this study was to determine if bovine cytokines have similar activity on bison and bovine immune cells. This objective was accomplished by measuring the capacity of BoiL-l/J, BOIL-2 and B o G M - C S F to stimulate proliferation of normal or mitogen-activated bison and cattle peripheral blood mononuclear cells (PBMC). MATERIALS AND METHODS Animals and isolation of P B M C Six Polled Hereford heifer calves and six bison heifer calves were obtained from two separate ranches in Iowa (U.S.A.) and were used in the current study at 18 months of age. Blood was obtained by jugular venipuncture and PBMC were isolated by density gradient centrifugation using Histopaque 1083 (Sigma Chemical Co., St Louis, MO, U.S.A.), as described previously [5]. The PBMC were placed in R P M I 1640 medium (G1BCO, Grand Island, NY, U.S.A.) containing 25 m M Hepes, 2 m M L-glutamine, 10% fetal bovine serum (Hyclone Laboratories, Logan, UT, U.S.A.), 5 x 10 5 M 2-mercaptoethanol, 100 U/ml of penicillin and 100/zg/ml of streptomycin. hwubation oJ P B M C with cytokines and mitogens Based on our previous work with bovine PBMC [11, 12], mitogen concentrations were selected that would induce optimum to suboptimurn P B M C proliferation. These concentrations were 10, 1 and 0.1 llg/ml for concanavalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM) and Escherichia coil 055:B5 lipopolysaccharide (LPS) (Sigma). Concentrations of BoIL-I/J, BOIL-2 and B o G M - C S F were chosen based on our previous work with these cytokines [5, 12] and on their reported in vitro activity as indicated by the manufacturer (American Cyanamid, Princeton, N J, U.S.A.). The tested concentrations of BoiL-l/J, BOIL-2 and B o G M - C S F were 100, 10, 1 and 0.1 U/ml. Cattle and bison P B M C were adjusted to 4 × 10~' cells/ml and a 50/xl aliquot was added to each flat-bottom well of a 96-well microtiter plate. Duplicate PBMC cultures from each animal were incubated with each concentration of cytokine or mitogen alone or with a combination of each concentration of cytokine and mitogen. Cultures contained in 150 ~/l were incubated for 48 h at 3 7 C in 5% CO2 and then pulsed for 18 h with 1.0 #Ci of 3H-thymidine per well. Cells were then harvested and counted for radioactivity in a liquid scintillation counter as counts per minute (cpm). Cell proliferation results were expressed as mean cpm and standard error of the mean (SEM). Statistical analysis Statistical differences in the capacity of the various cytokines to stimulate proliferation of normal or mitogen-activated bison and bovine PBMC was determined by analysis of variance and Fisher's protected least significance difference. The P B M C samples obtained from each of the six cattle or bison were used as blocking factors in the statistical analysis, in that differences were determined among PBMC obtained from the same animal that had been incubated with or without the cytokines.
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RESULTS
Effect of bovine cytokines alone on cattle and bison PBMC proli/bration Incubation of cattle or bison PBMC with BOIL-2 in the absence of mitogens resulted in a dose-dependent and linear increase in proliferative responses (Fig. I(B)). Significantly (P _< 0.05) increased responses resulted when cattle or bison PBMC were incubated with relatively high levels of BOIL-2 (100 or 10 U/ml). However, BoIL-lfl or BoGM-CSF at 0.1-100 U/ml did not increase cattle and bison PBMC proliferative responses when compared with PBMC incubated alone (Fig. I(A) and (C)). Effect of bovine cytokines on mitogen-stimulated cattle and bison PBMC proliferation Significantly ( P < 0 . 0 5 or 0.01) increased proliferative responses resulted from incubating cattle or bison PBMC with BoIL-lfl plus either Con A (10/tg/ml), PHA (10/~g/ml) or LPS (1 pg/ml) when compared with PBMC incubated with each mitogen alone (Fig. 2(A)-(C)). In addition, PBMC from cattle and bison exhibited a similar dose-dependent proliferative response to BoIL-lfi (0.1-100 U/ml) when the PBMC were co-stimulated with 10/~g/ml of either Con A or PHA (Fig. 2(A) and (B)). Incubation of cattle or bison PBMC with BoIL-lfl (0.1-100 U/ml) plus each of the other tested concentrations of mitogens including Con A (1 and 0.1 pg/ml), PHA (1 and 0.1 #g/ml), LPS (10 and 0.1 /~g/ml) or PWM (10, 1 and 0.1 pg/ml) did not increase PBMC proliferation above that induced by incubating PBMC with each mitogen alone (data not shown). Proliferation of cattle and bison PBMC, which resulted from incubation with Con A (0.1 gg/ml), PHA (10/~g/ml) or LPS (l /~g/ml), was significantly ( P < 0.05 or 0.01) increased by the additional incubation of the PBMC with BolL-2 (Fig. 3(A)-(C)). In addition, similar dose-dependent proliferative responses to BOIL-2 occurred when cattle or bison PBMC were incubated with 0.1-100 U/ml of BOIL-2 combined with Con A (0.1 ~g/ml), PHA (10 l~g/ml) or LPS (1 #g/ml) (Fig. 3(A)-(C)). However, incubation of cattle or bison PBMC with BOIL-2 (0.1-100 U/ml) plus each of the other tested concentrations of mitogens including Con A (10 and 1.0/~g/ml), PHA (1 and 0.1 pg/ml), LPS (10 and 0.1 /~g/ml) or PWM (10, 1 and 0.1 #g/ml) did not change PBMC proliferation compared with PBMC incubated with each mitogen alone (data not shown). Proliferation induced by incubating cattle or bison PBMC with Con A, PHA, LPS or PWM at 10, 1 or 0.1 /~g/ml was not altered when the mitogen-stimulated PBMC were incubated with B o G M - C S F at 0.1-100 U/ml (data not shown). DISCUSSION Both BoIL-lfl and BOIL-2 had activity on bison PBMC, as assessed by their capacity to augment PBMC proliferation induced by stimulation with Con A, PHA or LPS. The resulting cross-reactivity of BoIL-lfl and BOIL-2 on bison immune cells parallels results from previous studies demonstrating that these cytokines exhibit activity on sheep and pig immune cells [7, 8]; however, PBMC from cattle and bison did not respond to either B o G M - C S F alone or B o G M - C S F when combined with three different concentrations of each of the four different mitogens. Few studies have been conducted with BoGM-CSF, although it has been shown that B o G M - C S F stimulates colony formation of bovine bone marrow progenitor cells[6] and appears to stimulate bovine macrophages and neutrophils [4, 13]. Murine G M - C S F has similar stimulatory activity on murine cells [14]
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Fig. 2. Effect o f B o l L - l f l o n m i t o g e n - s t i m u l a t e d cattle a n d b i s o n P B M C p r o l i f e r a t i o n . C a t t l e o r b i s o n P B M C w e r e i n c u b a t e d f o r 48 h w i t h BoiL-1/1 in c o m b i n a t i o n w i t h either (A) C o n A, (B) P H A o r (C) E. coli 055:B5 L P S . Cells w e r e t h e n p u l s e d f o r 18 h w i t h ~ H - t h y m i d i n e . Results a r e e x p r e s s e d as m e a n _+ S E M (n = 6). P B M C i n c u b a t e d with B o I L - l f l p l u s a m i t o g e n ( d e n o t e d *) a r e s i g n i f i c a n t l y different f r o m P B M C i n c u b a t e d w i t h the m i t o g e n a n d n o B o l L - l f l . (A) P _< 0.05, (B) P_< 0.01 a n d (C) P < 0.05. 159
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Treatments Fig. 3. Effect of BOIL-2 on mitogen-stimulated cattle and bison PBMC proliferation. Cattle or bison PBMC were incubated for 48 h with BOIL-2 in combination with either (A) Con A, (B) PHA or (C) E. colt 055:B5 LPS. Cells were then pulsed for 18 h with 3H-thyrnidine. Results are expressed as mean _+ SEM (n = 6). PBMC incubated with BOIL-2 plus a mitogen (denoted *) are significantly different from PBMC incubated with the mitogen and no BOIL-2, (A) P < 0.05, (B) P ~< 0.01 and ((') P ~< 0.05.
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but it also stimulates murine T lymphocyte proliferation[15, 16]. It remains to be determined if B o G M - C S F resembles murine G M - C S F by also stimulating lymphocytes even though B o - G M - C S F did not exhibit this activity in the current study. In a previous study, we demonstrated that activation of bovine P B M C to a cytokine-responsive state in vitro usually requires stimulation of P B M C with a mitogen and that both the type and concentration of mitogen determines if P B M C are able to respond to a particular cytokine[ll]. Induction of the cytokine-responsive state following stimulation of bovine P B M C with mitogens presumably results from mitogen-induced cytokine receptor expression, as has been shown to occur with mitogen°stimulated cells in other species[17]. On the basis of these results, different mitogens and mitogen concentrations were tested as bison and cattle P B M C activation signals in the current study in order to facilitate the capacity of P B M C to respond to the tested cytokines. All mitogens except PWM were effective as P B M C activation signals, which enabled cells to become responsive to bovine cytokines in the present study. Similar results were obtained in our previous study, which assessed the activity of nine different human interleukins on bovine P B M C in that co-stimulation of P B M C with Con A, P H A or LPS, but not with PWM, effectively activated P B M C to a cytokine-responsive state [11]. In the current study, bison P B M C consistently exhibited higher proliferation in response to stimulation with Con A, P H A or LPS than cattle PBMC. The significance of these findings is not clear, however, because the higher response of bison P B M C to mitogens did not always result in higher responses of these cells to cytokines. F o r example, bison P B M C when co-stimulated with Con A or P H A exhibited significantly increased proliferation in response to BOIL-lfl at a 10-100-fold lower concentration of this cytokine than cattle P B M C that were co-stimulated with the same mitogens. However, PHA-stimulated bison P B M C were less responsive to BOIL-2 than were PHA-stimulated cattle P B M C and LPS-stimulated bison P B M C were less responsive to BoIL-lfl and BOIL-2 than cattle P B M C which were co-stimulated with the same mitogens. Results from the current study indicated that only BOIL-2 at relatively high concentrations (10 100 U/ml) induced proliferation of cattle and bison P B M C which had not been co-stimulated with mitogens. These results are in agreement with previous studies which have noted that IL-2 receptors are constitutively expressed on certain blood lymphocytes in cattle [18] and that BOIL-2 induces m a m m a r y gland mononuclear cell and P B M C proliferation in cattle without a needed co-stimulus [19]. A similar type of IL-2 receptor expression may also occur on bison lymphocytes and this may explain the capacity of PBMC from these animals to respond to BOIL-2 without additional activation stimuli. Previous studies have indicated that BoIL-lfl, BOIL-2 and B o G M - C S F augments immunity to certain diseases in cattle [1-4]. As indicated here, BoIL-lfl and BOIL-2 had activity on bison immune cells, which suggests that these cytokines may also be immunostimulating therapeutic agents in bison. However, additional studies will be required to determine if B o G M - C S F has activity on bison immune cells and perhaps bison neutrophils or macrophages may be a more effective target for this cytokine than PBMC. REFERENCES 1. Reddy P, G., Blecha F., Minocha H. C., Anderson G. A., Morrill J. L., Fedorka-Cray P. J. and Baker P. E. (1989) Bovine recombinant interleukin-2 augments immunity and resistance to bovine herpesvirus infection. Vet. Immunol. Immunopathol. 23, 61 74.
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2. Reddy P. G., Reddy D. N., Pruiett S. E., Daley M. J., Shirley J. E , Chengappa M. M. and Blecha F. (1992) lnterleukin 2 treatment of Staphylococcus aureus mastitis. Cvtokine 4, 227 231. 3. Daley M. J., Coyle P. A., Williams T. J., Furda G.. Dougherty R. and Hayes P. W. (1991) Staphylococcus aureus mastitis: pathogenesis and treatment with bovine interleukin-lh' and interleukin-2. J. Dairy Sci. 74, 4413~4424. 4. Daley M. J., Williams T., Coyle P.. Furda G., Dougherty R. and Hayes P. (1993) Prevention and treatment of Staphylococcus aureus infections with recombinant cytokines. Qvtokine 5, 276 284. 5. Stevens M. G., Wilson L. K. and Gershwin L. J. (1992) Isolation and characterization of an IL-l-dependent IL-4-producing bovine C D 4 T cell clone. Cell. hmnunol. 140, 453 467. 6. Leong S. R., Flaggs G. M., Lawman M. J. and Gray P. W. (1989) Cloning and expression of the c D N A for bovine granulocyte-rnacrophage colony-stimulating factor. 12't. lmmunol, hnmunopathol. 21, 261 278. 7. Ellis J. A., Russell H. 1. and Du C. W. (1994) Effect of selected cytokines on the replication of Coo'nehacterium pseudotuberculosis and ovine lentiviruses in pulmonary macrophages. ~2~t. h~mmnol. hnmunopathol. 40, 31 47. 8. Shi J., G o o d b a n d R. D., Chengappa M. M.. Nelssen J. L., Tokach M. D., McVey D. S. and Blecha F. (1994) Influence of interleukin-I on neutrophil function and resistance to Str~7ytococcus suis in neonatal pigs. J. Leuk. Biol. 56, 88 92. 9. Connelly R. G. Northern Diseased Bi.~on. Report ~?/ the Ent'ironmental Assessment Pallel, pp. I 35. Federal Environmental Assesment Review Office, Hull, Quebec (1990). 10. Berger J. and C u n n i n g h a m C. Mating and conservation in small populations. In Bison. Berger J. and C u n n i n g h a m C. (Eds.). pp. 25 40. Columbia University Press, New York (1994). I I. Olsen S. C. and Stevens M. G. (1993) Effects of recombinant h u m a n cytokines on mitogen-induced bovine peripheral blood mononuclear cell proliferation. Cvtokhw 5, 498 505. 12. Stevens M, G. and Olsen S. C, (1994) In vilro efl'ecis of live and killed Brucella aborlu~s on bovine cytokinc and prostaglandin E~ production. Vet. lmmunol, hnnumopathol. 40, 149 161. 13. Sordillo L. M., Afseth G., Davies G. M. and Babiuk L. A. (1992) Effects of recombinant granulocyte-macrophage colony-stimulating factor on bovine peripheral blood and ..~tmmary gland neutrophil function in vitro. Can. J. ~'et. Res. 56, 16 21. 14. Metcalf D. (1985) The granulocyte-tnacrophagc colony-stimulating factors. Science 229, 16 22. 15. Kupper T., Flood P., Coleman D. and Horowitz M. (1987) Growth of an interleukin 2/interleukin 4-dependent T cell line induced by granulocyte-macrophage colony-stimulating facl ,r ( G M - C S F ) . . / . hnmunol. 138, 4288 4292. 16. W o o d s A., West J., Rasmussen R. and Bottomly K. (1987) Granulocyte-macrophage ,-olony stimulating factor produced by cloned L 3 T 4 a . class ll-restricted T cells induces HT-2 cells to proliferate. J. hmmmol. 138, 4293-4297. 17. Miyajima A.. Kitamura T., t-larada N., Yokota T. and Kenichi A. (1992) Cytokinc receptors and signal transduction. An,u. Ret:. hnmumd. 10, 295 331. 18. Franklin S. T., Young J. W. and Nonnecke B. J. (1994) Proliferation and phenotype of bovine mononuclear leukocytes in cultures stimulated with pokeweed mitogen. J. Dairy Sci. 77, 3592 3600. 19. Torte P. M., K o n u r P. K. and Oliver S. P. (1992) Proliferative response of m a m m a r y gland mononuclear cells to recombinant bovine interleukin-2. Ve/. b,mtmol, lmmum~pathol. 32, 351 358.
Pergamon
Comp. hnmun. Microbiol. infi,ct. Dis. Vol. 20, No. 2, pp. 155 162. 1997 Published by Elsevier Science Ltd Printed in Great Britain PII: S0147-9571(96)00037-9 0147-9571/97 $17.0o + 0.o0
COMPARATIVE EFFECTS OF BOVINE CYTOKINES CATTLE AND BISON PERIPHERAL BLOOD MONONUCLEAR CELL PROLIFERATION MARK
G. S T E V E N S * , S T E V E N C. O L S E N a n d N O R M A N
ON
F, C H E V 1 L L E
National Animal Disease Center, USDA, Agriculture Research Service, 2300 Dayton Avenue, Ames, Iowa, 50010, U.S.A. (ReceivedJbr publication 28 October 1996)
Abstract--Proliferation of peripheral blood mononuclear cells (PBMC) from cattle and bison was measured following stimulation of PBMC with bovine cytokines. Bovine interleukin l fl (BOIL-1/1), interleukin 2 (BOIL-2) or granulocyte-macrophage colony-stimulating factor (BoGM-CSF) at 0.1-100 U/ml were incubated for 48 h with PBMC alone or with PBMC and various mitogens. These included concanavalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM) or Escherickia coli 055:B5 lipopolysaccharide (LPS) at 10 0.1 ~Lg/ml. BOIL-2 alone, but not BoIL-l/J and BoGM-CSF alone, induced proliferation of cattle and bison PBMC in the absence of mitogens. In addition, BoIL-lfl and BOIL-2, but not BoGM-CSF, enhanced proliferation of cattle and bison PBMC induced by mitogens. These results indicate that BolL-1/~' and BOIL-2 stimulate cattle and bison PBMC proliferation in a similar manner, whereas BoGM-CSF does not appear capable of stimulating either cattle or bison PBMC proliferation. Published by Elsevier Science Ltd Key words: Bovine cytokines, interleukin, bison
R 6 s u m ~ N o u s avons mesur6 la prolif6ration de cellules mononucl6aires sanguines p6riphdriques (PBMC) de bovins et de bisons apr6s stimulation par des cytokines d'origine bovine. Ces cellules mononucl6aires seules ou en pr~:sence de mitog6nes, ont 6t6 incub6es pendant 48 hr avec 10(~0.1 U/ml d'interleukine 1/3 (BoIL-lfl), d'interleukine 2 (BolL-2), ou de facteur de stimulation de colonie granulocytaire-macrophagique (BoGM-CSF) d'origine bovine. Concavalin A (Con A), phytohemagglutinin (PHA), mitogbne pokeweed (PWM), ou lipopolysaccharides (LPS) d'Esckerichia coli 055:B5 ont ~t6 utilis~s comme mitog6nes 5. une concentration de 1~0.1 ~g/ml. En l'abscence des mitog6nes, BOIL-2 fut la seule/t induire la prolifdration des PBMC de bovins et de bisons. En pr6sence des mitog6nes, BOIL-1 et BOIL-2 furent les seules 5. induire la prolif6ration des PBMC de bovins et de bisons. Ces rdsultats indiquent que BoIL-lfl et BOIL-2, contrairement 5. BoGM-CSF, stimulent la prolifdration des PBMC de bovins et de bisons. Published by Elsevier Science Ltd Mots-chff[~': Bovine cytokines, interleukine, bison
INTRODUCTION
Bovine (Bo) cytokines such as interleukin lfl (BoIL-lfl), interleukin 2 (BOIL-2) and granulocyte-macrophage colony-stimulating factor (BoGM-CSF) have been shown to have immunostimulatory activity in cattle [1 5]. The activity of BoGM-CSF is believed to be species specific [6]; however, BoIL-I// and BOIL-2 exhibit cross-reactivity on immune cells from other important food-producing animals such as sheep [7] and pigs [8]. Cattle, sheep and bison are related ruminants, belonging to the family Bovidae. Although bison *Author for correspondence. 155
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barely escaped extinction, they are currently raised in small numbers for meat production in the United States and large free-ranging herds are being preserved in Yellowstone National Park in the United States and in other parks and sanctuaries in Canada [9, 10]. Very little is known about the bison immune system and it has not been determined if bovine cytokines exhibit activity on bison immune cells, as has been demonstrated to occur in sheep and pigs. The objective of this study was to determine if bovine cytokines have similar activity on bison and bovine immune cells. This objective was accomplished by measuring the capacity of BoiL-l/J, BOIL-2 and B o G M - C S F to stimulate proliferation of normal or mitogen-activated bison and cattle peripheral blood mononuclear cells (PBMC). MATERIALS AND METHODS Animals and isolation of P B M C Six Polled Hereford heifer calves and six bison heifer calves were obtained from two separate ranches in Iowa (U.S.A.) and were used in the current study at 18 months of age. Blood was obtained by jugular venipuncture and PBMC were isolated by density gradient centrifugation using Histopaque 1083 (Sigma Chemical Co., St Louis, MO, U.S.A.), as described previously [5]. The PBMC were placed in R P M I 1640 medium (G1BCO, Grand Island, NY, U.S.A.) containing 25 m M Hepes, 2 m M L-glutamine, 10% fetal bovine serum (Hyclone Laboratories, Logan, UT, U.S.A.), 5 x 10 5 M 2-mercaptoethanol, 100 U/ml of penicillin and 100/zg/ml of streptomycin. hwubation oJ P B M C with cytokines and mitogens Based on our previous work with bovine PBMC [11, 12], mitogen concentrations were selected that would induce optimum to suboptimurn P B M C proliferation. These concentrations were 10, 1 and 0.1 llg/ml for concanavalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM) and Escherichia coil 055:B5 lipopolysaccharide (LPS) (Sigma). Concentrations of BoIL-I/J, BOIL-2 and B o G M - C S F were chosen based on our previous work with these cytokines [5, 12] and on their reported in vitro activity as indicated by the manufacturer (American Cyanamid, Princeton, N J, U.S.A.). The tested concentrations of BoiL-l/J, BOIL-2 and B o G M - C S F were 100, 10, 1 and 0.1 U/ml. Cattle and bison P B M C were adjusted to 4 × 10~' cells/ml and a 50/xl aliquot was added to each flat-bottom well of a 96-well microtiter plate. Duplicate PBMC cultures from each animal were incubated with each concentration of cytokine or mitogen alone or with a combination of each concentration of cytokine and mitogen. Cultures contained in 150 ~/l were incubated for 48 h at 3 7 C in 5% CO2 and then pulsed for 18 h with 1.0 #Ci of 3H-thymidine per well. Cells were then harvested and counted for radioactivity in a liquid scintillation counter as counts per minute (cpm). Cell proliferation results were expressed as mean cpm and standard error of the mean (SEM). Statistical analysis Statistical differences in the capacity of the various cytokines to stimulate proliferation of normal or mitogen-activated bison and bovine PBMC was determined by analysis of variance and Fisher's protected least significance difference. The P B M C samples obtained from each of the six cattle or bison were used as blocking factors in the statistical analysis, in that differences were determined among PBMC obtained from the same animal that had been incubated with or without the cytokines.
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RESULTS
Effect of bovine cytokines alone on cattle and bison PBMC proli/bration Incubation of cattle or bison PBMC with BOIL-2 in the absence of mitogens resulted in a dose-dependent and linear increase in proliferative responses (Fig. I(B)). Significantly (P _< 0.05) increased responses resulted when cattle or bison PBMC were incubated with relatively high levels of BOIL-2 (100 or 10 U/ml). However, BoIL-lfl or BoGM-CSF at 0.1-100 U/ml did not increase cattle and bison PBMC proliferative responses when compared with PBMC incubated alone (Fig. I(A) and (C)). Effect of bovine cytokines on mitogen-stimulated cattle and bison PBMC proliferation Significantly ( P < 0 . 0 5 or 0.01) increased proliferative responses resulted from incubating cattle or bison PBMC with BoIL-lfl plus either Con A (10/tg/ml), PHA (10/~g/ml) or LPS (1 pg/ml) when compared with PBMC incubated with each mitogen alone (Fig. 2(A)-(C)). In addition, PBMC from cattle and bison exhibited a similar dose-dependent proliferative response to BoIL-lfi (0.1-100 U/ml) when the PBMC were co-stimulated with 10/~g/ml of either Con A or PHA (Fig. 2(A) and (B)). Incubation of cattle or bison PBMC with BoIL-lfl (0.1-100 U/ml) plus each of the other tested concentrations of mitogens including Con A (1 and 0.1 pg/ml), PHA (1 and 0.1 #g/ml), LPS (10 and 0.1 /~g/ml) or PWM (10, 1 and 0.1 pg/ml) did not increase PBMC proliferation above that induced by incubating PBMC with each mitogen alone (data not shown). Proliferation of cattle and bison PBMC, which resulted from incubation with Con A (0.1 gg/ml), PHA (10/~g/ml) or LPS (l /~g/ml), was significantly ( P < 0.05 or 0.01) increased by the additional incubation of the PBMC with BolL-2 (Fig. 3(A)-(C)). In addition, similar dose-dependent proliferative responses to BOIL-2 occurred when cattle or bison PBMC were incubated with 0.1-100 U/ml of BOIL-2 combined with Con A (0.1 ~g/ml), PHA (10 l~g/ml) or LPS (1 #g/ml) (Fig. 3(A)-(C)). However, incubation of cattle or bison PBMC with BOIL-2 (0.1-100 U/ml) plus each of the other tested concentrations of mitogens including Con A (10 and 1.0/~g/ml), PHA (1 and 0.1 pg/ml), LPS (10 and 0.1 /~g/ml) or PWM (10, 1 and 0.1 #g/ml) did not change PBMC proliferation compared with PBMC incubated with each mitogen alone (data not shown). Proliferation induced by incubating cattle or bison PBMC with Con A, PHA, LPS or PWM at 10, 1 or 0.1 /~g/ml was not altered when the mitogen-stimulated PBMC were incubated with B o G M - C S F at 0.1-100 U/ml (data not shown). DISCUSSION Both BoIL-lfl and BOIL-2 had activity on bison PBMC, as assessed by their capacity to augment PBMC proliferation induced by stimulation with Con A, PHA or LPS. The resulting cross-reactivity of BoIL-lfl and BOIL-2 on bison immune cells parallels results from previous studies demonstrating that these cytokines exhibit activity on sheep and pig immune cells [7, 8]; however, PBMC from cattle and bison did not respond to either B o G M - C S F alone or B o G M - C S F when combined with three different concentrations of each of the four different mitogens. Few studies have been conducted with BoGM-CSF, although it has been shown that B o G M - C S F stimulates colony formation of bovine bone marrow progenitor cells[6] and appears to stimulate bovine macrophages and neutrophils [4, 13]. Murine G M - C S F has similar stimulatory activity on murine cells [14]
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Fig. 2. Effect o f B o l L - l f l o n m i t o g e n - s t i m u l a t e d cattle a n d b i s o n P B M C p r o l i f e r a t i o n . C a t t l e o r b i s o n P B M C w e r e i n c u b a t e d f o r 48 h w i t h BoiL-1/1 in c o m b i n a t i o n w i t h either (A) C o n A, (B) P H A o r (C) E. coli 055:B5 L P S . Cells w e r e t h e n p u l s e d f o r 18 h w i t h ~ H - t h y m i d i n e . Results a r e e x p r e s s e d as m e a n _+ S E M (n = 6). P B M C i n c u b a t e d with B o I L - l f l p l u s a m i t o g e n ( d e n o t e d *) a r e s i g n i f i c a n t l y different f r o m P B M C i n c u b a t e d w i t h the m i t o g e n a n d n o B o l L - l f l . (A) P _< 0.05, (B) P_< 0.01 a n d (C) P < 0.05. 159
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Treatments Fig. 3. Effect of BOIL-2 on mitogen-stimulated cattle and bison PBMC proliferation. Cattle or bison PBMC were incubated for 48 h with BOIL-2 in combination with either (A) Con A, (B) PHA or (C) E. colt 055:B5 LPS. Cells were then pulsed for 18 h with 3H-thyrnidine. Results are expressed as mean _+ SEM (n = 6). PBMC incubated with BOIL-2 plus a mitogen (denoted *) are significantly different from PBMC incubated with the mitogen and no BOIL-2, (A) P < 0.05, (B) P ~< 0.01 and ((') P ~< 0.05.
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but it also stimulates murine T lymphocyte proliferation[15, 16]. It remains to be determined if B o G M - C S F resembles murine G M - C S F by also stimulating lymphocytes even though B o - G M - C S F did not exhibit this activity in the current study. In a previous study, we demonstrated that activation of bovine P B M C to a cytokine-responsive state in vitro usually requires stimulation of P B M C with a mitogen and that both the type and concentration of mitogen determines if P B M C are able to respond to a particular cytokine[ll]. Induction of the cytokine-responsive state following stimulation of bovine P B M C with mitogens presumably results from mitogen-induced cytokine receptor expression, as has been shown to occur with mitogen°stimulated cells in other species[17]. On the basis of these results, different mitogens and mitogen concentrations were tested as bison and cattle P B M C activation signals in the current study in order to facilitate the capacity of P B M C to respond to the tested cytokines. All mitogens except PWM were effective as P B M C activation signals, which enabled cells to become responsive to bovine cytokines in the present study. Similar results were obtained in our previous study, which assessed the activity of nine different human interleukins on bovine P B M C in that co-stimulation of P B M C with Con A, P H A or LPS, but not with PWM, effectively activated P B M C to a cytokine-responsive state [11]. In the current study, bison P B M C consistently exhibited higher proliferation in response to stimulation with Con A, P H A or LPS than cattle PBMC. The significance of these findings is not clear, however, because the higher response of bison P B M C to mitogens did not always result in higher responses of these cells to cytokines. F o r example, bison P B M C when co-stimulated with Con A or P H A exhibited significantly increased proliferation in response to BOIL-lfl at a 10-100-fold lower concentration of this cytokine than cattle P B M C that were co-stimulated with the same mitogens. However, PHA-stimulated bison P B M C were less responsive to BOIL-2 than were PHA-stimulated cattle P B M C and LPS-stimulated bison P B M C were less responsive to BoIL-lfl and BOIL-2 than cattle P B M C which were co-stimulated with the same mitogens. Results from the current study indicated that only BOIL-2 at relatively high concentrations (10 100 U/ml) induced proliferation of cattle and bison P B M C which had not been co-stimulated with mitogens. These results are in agreement with previous studies which have noted that IL-2 receptors are constitutively expressed on certain blood lymphocytes in cattle [18] and that BOIL-2 induces m a m m a r y gland mononuclear cell and P B M C proliferation in cattle without a needed co-stimulus [19]. A similar type of IL-2 receptor expression may also occur on bison lymphocytes and this may explain the capacity of PBMC from these animals to respond to BOIL-2 without additional activation stimuli. Previous studies have indicated that BoIL-lfl, BOIL-2 and B o G M - C S F augments immunity to certain diseases in cattle [1-4]. As indicated here, BoIL-lfl and BOIL-2 had activity on bison immune cells, which suggests that these cytokines may also be immunostimulating therapeutic agents in bison. However, additional studies will be required to determine if B o G M - C S F has activity on bison immune cells and perhaps bison neutrophils or macrophages may be a more effective target for this cytokine than PBMC. REFERENCES 1. Reddy P, G., Blecha F., Minocha H. C., Anderson G. A., Morrill J. L., Fedorka-Cray P. J. and Baker P. E. (1989) Bovine recombinant interleukin-2 augments immunity and resistance to bovine herpesvirus infection. Vet. Immunol. Immunopathol. 23, 61 74.
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2. Reddy P. G., Reddy D. N., Pruiett S. E., Daley M. J., Shirley J. E , Chengappa M. M. and Blecha F. (1992) lnterleukin 2 treatment of Staphylococcus aureus mastitis. Cvtokine 4, 227 231. 3. Daley M. J., Coyle P. A., Williams T. J., Furda G.. Dougherty R. and Hayes P. W. (1991) Staphylococcus aureus mastitis: pathogenesis and treatment with bovine interleukin-lh' and interleukin-2. J. Dairy Sci. 74, 4413~4424. 4. Daley M. J., Williams T., Coyle P.. Furda G., Dougherty R. and Hayes P. (1993) Prevention and treatment of Staphylococcus aureus infections with recombinant cytokines. Qvtokine 5, 276 284. 5. Stevens M. G., Wilson L. K. and Gershwin L. J. (1992) Isolation and characterization of an IL-l-dependent IL-4-producing bovine C D 4 T cell clone. Cell. hmnunol. 140, 453 467. 6. Leong S. R., Flaggs G. M., Lawman M. J. and Gray P. W. (1989) Cloning and expression of the c D N A for bovine granulocyte-rnacrophage colony-stimulating factor. 12't. lmmunol, hnmunopathol. 21, 261 278. 7. Ellis J. A., Russell H. 1. and Du C. W. (1994) Effect of selected cytokines on the replication of Coo'nehacterium pseudotuberculosis and ovine lentiviruses in pulmonary macrophages. ~2~t. h~mmnol. hnmunopathol. 40, 31 47. 8. Shi J., G o o d b a n d R. D., Chengappa M. M.. Nelssen J. L., Tokach M. D., McVey D. S. and Blecha F. (1994) Influence of interleukin-I on neutrophil function and resistance to Str~7ytococcus suis in neonatal pigs. J. Leuk. Biol. 56, 88 92. 9. Connelly R. G. Northern Diseased Bi.~on. Report ~?/ the Ent'ironmental Assessment Pallel, pp. I 35. Federal Environmental Assesment Review Office, Hull, Quebec (1990). 10. Berger J. and C u n n i n g h a m C. Mating and conservation in small populations. In Bison. Berger J. and C u n n i n g h a m C. (Eds.). pp. 25 40. Columbia University Press, New York (1994). I I. Olsen S. C. and Stevens M. G. (1993) Effects of recombinant h u m a n cytokines on mitogen-induced bovine peripheral blood mononuclear cell proliferation. Cvtokhw 5, 498 505. 12. Stevens M, G. and Olsen S. C, (1994) In vilro efl'ecis of live and killed Brucella aborlu~s on bovine cytokinc and prostaglandin E~ production. Vet. lmmunol, hnnumopathol. 40, 149 161. 13. Sordillo L. M., Afseth G., Davies G. M. and Babiuk L. A. (1992) Effects of recombinant granulocyte-macrophage colony-stimulating factor on bovine peripheral blood and ..~tmmary gland neutrophil function in vitro. Can. J. ~'et. Res. 56, 16 21. 14. Metcalf D. (1985) The granulocyte-tnacrophagc colony-stimulating factors. Science 229, 16 22. 15. Kupper T., Flood P., Coleman D. and Horowitz M. (1987) Growth of an interleukin 2/interleukin 4-dependent T cell line induced by granulocyte-macrophage colony-stimulating facl ,r ( G M - C S F ) . . / . hnmunol. 138, 4288 4292. 16. W o o d s A., West J., Rasmussen R. and Bottomly K. (1987) Granulocyte-macrophage ,-olony stimulating factor produced by cloned L 3 T 4 a . class ll-restricted T cells induces HT-2 cells to proliferate. J. hmmmol. 138, 4293-4297. 17. Miyajima A.. Kitamura T., t-larada N., Yokota T. and Kenichi A. (1992) Cytokinc receptors and signal transduction. An,u. Ret:. hnmumd. 10, 295 331. 18. Franklin S. T., Young J. W. and Nonnecke B. J. (1994) Proliferation and phenotype of bovine mononuclear leukocytes in cultures stimulated with pokeweed mitogen. J. Dairy Sci. 77, 3592 3600. 19. Torte P. M., K o n u r P. K. and Oliver S. P. (1992) Proliferative response of m a m m a r y gland mononuclear cells to recombinant bovine interleukin-2. Ve/. b,mtmol, lmmum~pathol. 32, 351 358.