Bovine interleukin 2: Production and characterization

Bovine interleukin 2: Production and characterization

Veterinary Immunology and Immunopathology, 18 (1988) 165-172 Elsevier Science Publishers B.V., A m s t e r d a m - - P r i n t e d in T h e N e t h e ...

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Veterinary Immunology and Immunopathology, 18 (1988) 165-172 Elsevier Science Publishers B.V., A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

165

B o v i n e I n t e r l e u k i n 2: P r o d u c t i o n and Characterization S U R Y A P R A K A S H R. S A M B H A R A a n d E. L E E B E L D E N

Department of Molecular Biology, College of Agriculture, University o[ Wyoming, Laramie, WY 82071 (U.S.A.) (Accepted 5 August 1987)

ABSTRACT Sambhara, S.R. a n d Belden, E.L., 1988. Bovine interleukin 2: production a n d characterization. Vet. Immunol. Immunopathol., 18: 165-172. T h e production of bovine IL 2 was studied and IL 2 was partially characterized. P M A at 5 n g / m l + Concanavalin A at 5/~g/ml t r e a t m e n t of peripheral blood mononuclear cells gave a greater yield of IL 2 activity in the s u p e r n a t a n t s t h a n Con A, P M A or sodium periodate t r e a t m e n t s alone. Macrophage depletion increased yields as did the addition of indomethacin, a prostaglandin E2 inhibitor. Bovine IL 2 was sensitive to trypsin, relatively stable at p H 2-9, 2-ME resistant and sensitive to increasing molar concentrations of urea. T h e activity of bovine IL 2 was reduced by over 45% at 70°C for 30 min and 95% at 90°C for 30 min. Bovine IL 2 was more stable at 4°C t h a n at room temperature a n d the stability at room temperature could be improved by inclusion of 1% B SA. Bovine IL 2 eluted from D E A E - S e p h a d e x as a broad peak with 0.1-0.2 M NaC1. Peak activity corresponded to a molecular weight of approximately 16 000 daltons on Sephadex G-100.

INTRODUCTION

Interleukin 2 (IL 2) it a lymphokine produced by stimulated T lymphocytes ( Smith et al., 1979 ). IL 2 participates in a variety of in vitro immune responses such as enhancing natural killer cells through induction of gamma interferon, induction of lymphotoxin and maintenance of activated T lymphocytes (Baker et al., 1978; Weigent et al., 1983; Reem and Yeh, 1984; Svedersky et al., 1985). Human and mouse IL 2 have been studied extensively but much less is known about IL 2 and its action in economically important large animal species. IL 2 from pigs, chickens, sheep, horses and cattle have recently been partially purified ( Gasbarre et al., 1982; Magnuson et al., 1983; Brown and Grab, 1985; Ellis and DeMartini, 1985; Vainio et al., 1986). We report conditions for the production of IL 2 from bovine peripheral lymphocytes, as well as partial purification and characterization. 0165-2427/88/$03.50

© 1988 Elsevier Science Publishers B.V.

166 MATERIALS AND METHODS

Culture medium Serum free medium similar to that of Kawamoto et al. (1983) was prepared by combining a 2:1:1 ratio of R P M I 1640 medium, Dulbecco's modified Eagle's medium and Nutrient mixture F-12 ( H a m ) . The medium was supplemented with the following: human transferrin 10/~g/ml, insulin 10 ~g/ml, human serum albumin 1 mg/ml, 2-mercaptoethano110 ~tM, sodium selenite 1 X 10 3 zM, oleic acid 4/~g/ml, 2-aminoethano110 #M, penicillin 100 units/ml, streptomycin 100 #g/ml and H E P E S buffer 10 mM. The p H was adjusted to 7.1.

Isolation of peripheral blood lymphocytes and IL 2 production Bovine peripheral blood mononuclear cells ( P B M ) were obtained as described by Belden et al. (1981). Briefly, whole heparinized (40 units/ml) bovine blood was diluted 1 : 2 with cold H a n k ' s balanced salt solution ( H B S S ) . The suspension ( 30 ml ) was layered onto 15 ml Ficoll/Hypaque ( density 1.075 ) and centrifuged for 30 min at 1200 g. The P B M rich band was washed three times in H B S S and viable cell counts were obtained by trypan blue staining. The P B M were adjusted to 1X 107 cells/ml in medium. Mitogen stimulation was with Con A at 5/~g/ml/1 X 107 P B M / m l for 48 h unless otherwise stated. The cells were removed from the conditioned medium ( C M ) by centrifugation and excess Con A was blocked by adding ~-methyl-D-mannoside at a concentration of 20 mg/ml of CM. Sheep, guinea pig and human IL 2 were prepared in the same manner.

Bovine IL 2 dependent cells and IL 2 assay Long-term cultures of bovine IL 2 dependent T cells were derived from mixed lymphocyte culture (Baker and Knoblock, 1982). The cells were maintained at least 3 weeks in 12.5% CM and were washed three times in HBSS. The cells were suspended at 1 X 106 cells/ml in medium and 1 X l0 s cells/well were added to 96 well round-bottomed microtiter plates with the IL 2 test supernatant. The plates were incubated for 48 h at 37°C, pulsed with 0.5/~Ci of tritiated thymidine in 50 ttl and incubated for an additional 18 h. The cells were harvested and thymidine incorporation was measured in a liquid scintillation counter. The results were expressed as counts per minute (cpm). Sheep and human IL 2 dependent cell lines were also obtained as described above.

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Effect of P M A and sodium periodate on IL 2 production The P B M were suspended to 1 X 107 cells/ml in medium. Cells were exposed to 5 ng/ml phorbol-12-myristate-13 acetate ( P M A ) or 5 ltg/ml Con A or both. A portion of P B M was treated with 10 m M sodium periodate in PBS for 20 min at 0 ° C, washed three times in H B S S and suspended in medium at 1 × 107 cells/ml. The cells were incubated, IL 2 test supernatants were collected at 12h intervals and treated with ~-methyl-D-mannoside.

Role of adherent cells in IL 2 production Adherent cells were removed by two 90-min cycles of incubation on plastic at 37 ° C. Con A was added to adherent cell depleted and undepleted cell suspensions and cells were incubated. IL 2 test supernatants were collected at 12h intervals for 6 days. Indomethacin, a PGE2 inhibitor, at 0.5 zg/ml, was added along with Con A to undepleted cells and incubated at 37 ° C. Supernatants were collected and assayed as above.

Characterization of IL 2 in conditioned medium IL 2 CM was heated to 70, 80 or 90 ° C for 10 or 30 min. After heat treatment, the samples were immediately cooled to 0 ° C. The stability of IL 2 in CM was also tested for 3 and 6 weeks at room temperature and at 4 ° C with or without the inclusion of 1% bovine serum albumin (BSA). To test pH stability, IL 2 CM was dialyzed in 50 volumes of appropriate pH buffer (pH 1-13) for 8 h under sterile conditions. Samples were then dialyzed against culture medium for 24 h. The IL 2 CM was treated with trypsin (40 z g / m l of IL 2 CM) and the mixture was incubated at 37°C for 8 h in 10 m M Tris-HC1 buffered saline (pH 8.2 ) containing I m M CaC12. After incubation the mixture was dialyzed against PBS for 48 h. IL 2 CM was incubated at room temperature in dialysis bags in 0.5, 1, 2, 4, 6 and 8 M urea for 8 h. Samples were then dialyzed against PBS. The effect of 2-mercaptoethanol (2-ME) on IL 2 activity was tested by dialyzing against 0.75 M 2-ME for 6 h at room temperature followed by dialysis against PBS.

Partial purification Precipitation was carried out after the method of Welte et al. (1982) using 80% a m m o n i u m sulfate saturation. After overnight stirring at 4 ° C, the precipitate was centrifuged (1000 g, 40 m i n ) , dissolved in 0.01 M Tris-HC1 (pH 7.8) and dialyzed against the same buffer for 48 h.

168 The protein solution obtained from the ammonium sulfate precipitation was applied to a column of D E A E Sephadex (Pharmacia) equilibrated in 0.01 M Tris-HC1 buffer. A linear salt gradient from 0-0.5 M NaC1 was applied and the fractions were dialyzed against P B S after collection. Fractions from the anion exchange column which showed IL 2 activity were pooled, concentrated by ultrafiltration and applied to a column of Sephadex G-100 (Pharmacia) equilibrated with PBS. Elution was carried out with the same buffer and fractions were dialyzed and tested for activity.

Species cross-reactivity of bovine IL 2 The ability of bovine IL 2 CM to support sheep and human IL 2 dependent cells for 1 month was tested. Similarly the ability of IL 2 CM from sheep, human and guinea pigs to support bovine IL 2 dependent cells was also tested. IL 2 dependent cell derivation, IL 2 production and assay conditions were as above. RESULTS AND DISCUSSION

Effect of P M A and sodium periodate on IL 2 production The kinetics of IL 2 production induced by Con A + PMA, P M A alone, Con A alone and sodium periodate t r e a t m e n t are shown in Fig. 1. Con A + P M A treatment gave a greater yield of activity than other treatments and the presence of IL 2 activity could be detected as early as 6 h post-treatment (data not shown). Hirano et al. (1984) observed that P M A enhanced phytohemagglutinin ( P H A ) induced IL 2 production by 10 fold compared with that induced by P H A alone. P M A induces macrophages to produce factors such as IL 1 which affect the activation of T cells for IL 2 production. Perhaps the difference was less in our studies because of the presence of macrophage replacing factor, 2-ME, in the culture medium. Sodium periodate induces extensive blastogenesis via oxidation of terminal sugars on cell surface glycoproteins ( Smith et al., 1985 ). We observed that IL 2 activity in the supernatants reached maximum levels 24 h post-treatment with sodium periodate; however, production was less than that obtained with Con A and Con A + P M A treatments.

Role of adherent cells in IL 2 production The adherent cell depleted cultures gave more IL 2 activity in the supernatants when stimulated with Con A than did undepleted cultures (Fig. 2). In humans (Chouaib et al., 1984) the adherent cells mediate negative signals by producing prostaglandin E2 in addition to positive signals through IL 1. Prostaglandins were shown to inhibit proliferative responses of lymphocytes by

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Fig. 1. The effect of P M A ( ~ - - - [ ~ ) , P M A + C o n A ( 0 - - - 0 ) , periodate ( + - - - + ) on IL 2 production.

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Fig. 3. The effectof indomethacin on IL 2 production. Indomethacin+ Con A ( + --- + ) and Con A alone ( [] --- [] ). preventing IL 2 production by T helper cells. In the present study we observed more IL 2 activity in the supernatants of cells treated with indomethacin, a PGE2 inhibitor, which suggests t h a t a similar regulation of IL 2 production is present in the bovine ( Fig. 3 ).

Effect of heat, pH, enzyme and chemical treatment on IL 2 Bovine IL 2 activity was reduced by 20% at 70°C for 10 min and by 45% at 70°C for 30 min. Exposure to 90°C for 10 and 30 min resulted in 90 and 95% reductions in activity, respectively. In examining storage stability it was found t h a t minimal activity was lost after 3 weeks or 6 weeks at 4 ° C. Six weeks at room temperature resulted in 60% reduced activity; however, when 1% BSA was included only 25% of the activity was lost after 6 weeks. W h e n stability at different pH was examined a graded response was observed with 84% reduction at pH 1, intermediate reductions from 30% to 10% through a pH of 6, no reduction at pH 7 and 6, 20 and 60% reductions at pH 8, 9 and 10. IL 2 activity was destroyed by exposure to trypsin. However, t r e a t m e n t with 2-ME had no effect on IL 2 activity. Activity was reduced by 25% by 0.5 M urea, 30% by 2 M urea and 90% by 8 M urea. These characteristics are similar to those of h u m a n and mouse IL 2 (Watson et al., 1982).

171 TABLE 1 Species c r o s s - r e a c t i v i t y o f b o v i n e I L 2 IL 2 source

Bovine Ovine Human G u i n e a pig

L y m p h o c y t e source Bovine

Ovine

Human

+ + + +

+ + + NT

+ NT

NT = Not tested.

Partial purification Fractions showing IL 2 activity were eluted from DEAE-Sephadex at 0.1-0.2 M NaC1 with a broad peak centering at 0.15 M. Namen and Magnuson (1984) reported that bovine IL 2 eluted as a broad peak at 0.075 M NaC1. Brown and Grab (1985) reported that bovine IL 2 eluted from DEAE-Sephacryl with between 0.135-0.165 M NaC1. Our results are more consistent with the latter. When the pooled active fractions from DEAE-Sephadex were placed on Sephadex G-100 the IL 2 activity eluted in a fraction with a peak corresponding to a molecular weight of 16 000 daltons. Namen and Magnuson (1984) reported a molecular weight of 25 000 daltons on Sephadex G-100 and heterogeneous molecular weights of 14 400, 16 800 and 20 200 daltons on SDS-PAGE. However, Brown and Grab (1985) reported that bovine IL 2 activity corresponded to molecular weights of 20 000 and 23 000 daltons. The differences observed may be due to variable glycosylation of bovine IL 2.

Species cross-reactivity of bovine IL 2 The ability of bovine IL 2 to support IL 2 dependent lymphocytes from cattle, sheep and humans as well as the ability ofIL 2 from other species to support bovine IL 2 dependent lymphocytes is presented in Table 1. Bovine IL 2 supported sheep IL 2 dependent cells and IL 2 from sheep, guinea pig and human lymphocytes supported bovine IL 2 dependent cells. Carter et al. (1986) reported that recombinant human IL 2 supported long-term growth of bovine cytotoxic lymphocytes. Cross-reactivity between sheep and cattle is understandable because of the evolutionary relationship. However, the lack of reciprocal cross-reactivity between human and bovine IL 2 is more difficult to explain. Sequencing studies of IL 2 from different species as well as studies of the structures of IL 2 receptors may clarify these cross-reactivities and species restricted activities.

172 ACKNOWLEDGEMENT

Published with the approval of the Director, Wyoming Agricultural Experiment Station, as Journal Article No. 1515. REFERENCES Baker, P.E. and Knoblock, K.F., 1982. Bovine costimulator. II. Generation and maintenance of a bovine eostimulator-dependent bovine lymphoblastoid cell line. Vet. Immunol. Immunopathol., 3:381 397. Baker, P.E., Gillis, S., Ferm, N.M. and Smith, K.A., 1978. The effect of T cell growth factor on the generation of cytolytic T cells. J. Immunol., 121: 2168-2173. Belden, E.L., McCroskey-Rothwell, M.K. and Strelkauskas, A.J., 1981. Subpopulations of bovine lymphocytes separated by rosetting techniques. Vet. Immunol. Immunopathol., 2: 467-474. Brown, W.C. and Grab, D.J., 1985. Biological and biochemical characterization of bovine interleukin 2. Studies with cloned bovine T cells. J. Immunol., 133: 3184-3190. Carter, J., Magnuson, N.S., Davis, W.C., Mason, P.H., Magnuson, J.A., Talmadge, J.E. and Barrs, P.J., 1986. Development and maintenance of bovine cytotoxic lymphocytes with recombinant human interleukin-2. Immunology, 57: 123-129. Chouaib, S., Chatenoud, L., Klatzmann, D. and Fradelizi, D., 1984. The mechanisms of inhibition of human IL 2 production. PGE 2 induction of suppressor T lymphocytes. J. Immunol., 132: 1851-1857. Ellis, J.A. and DeMartini, J.C., 1985. Ovine interleukin 2: partial purification and assay in normal sheep and sheep with ovine progressive pneumonia. Vet. Immunol. Immunopathol, 8:15-25. Gasbarre, L.C., Urban, J.F., Jr. and Ramanowski, R.D., 1982. Production and characterization of porcine T cell growth factor. Fed. Proc., 41: 317. Hirano, T., Fujimoto, K., Teranishi, T., Nishino, N., Onoue, K., Maeda, S. and Shimada, K., 1984. Phorbol ester increases the level of interleukin 2 mRNA in mitogen stimulated human lymphocytes. J. Immunol., 132: 2165-2167. Kawamoto, T., Sato, J.D., Lie, A., McClure, D.B. and Sato, G.H., 1983. Development of a serum free medium for growth of NS-1 hybridomas. Anal. Biochem., 130:445 453. Magnuson, N.S., Namen, A.E., Perryman, L.E. and Magnuson, J.A., 1983. Studies on interleukin 2 from horses. Fed. Proc., 42: 446. Namen, A.E. and Magnuson, J.A., 1984. Production and characterization of bovine interleukin 2. Immunology, 52: 469-475. Reem, G.H. and Yeh, N.H., 1984. Interleukin 2 regulates expression of its receptor and synthesis of gamma interferon by human T lymphocytes. Science, 225: 429-430. Smith, K.A., Gillis, S., Baker, P.E., McKenzie, D. and Ruscetti, F.W., 1979. T cell growth factor mediated T cell proliferation. Ann. N.Y. Acad. Sci., 332: 423-432. Smith, L.A., Cohen, D.A., Lachman, L.B. and Kaplan, A.M., 1985. Sodium periodate induced T cell mitogenesis: an analysis of the requirement for Ia and IL 1. J. Immunol., 135:1137 1144. Svedersky, L.P., Nedwin, G.E., Goeddel, D.V. and Palladino, M.A., Jr., 1985. Interferon gamma enhances induction of lymphotoxin in recombinant interleukin 2 stimulated peripheral blood mononuclear cells. J. Immunol., 134: 1604-1608. Vainio, O., Ratcliffe, M.J.H. and Leanderson, T., 1986. Chicken T-cell growth factor: use in the generation of a long term cultured T-cell line and biochemical characterization. Scand. J. Immunol., 23: 135-142. Watson, J., Frank, M.B., Mochizuki, D. and Gillis, S., 1982. The biochemistry and biology of interleukin 2. Lymphokines, 6: 95-135. Weigent, D.A., Stanton, G.J. and Johnson, H.M., 1983. Interleukin 2 enhances natural killer cell activity through induction of gamma interferon. Infect. Immun., 41:992 997. Welte, K.. Wang, C.Y., Mertelsmann, R., Venuta, S., Feldman, S.P. and Moore, M.A.S., 1982. Purification of human interleukin 2 to apparent homogeneity and its molecular heterogeneity. J. Exp. Med., 156:454 464.