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Bovine interleukin 2 (IL-2) production and activity on bovine and murine cell lines
Veterinary Immunology and Immunopathology , 7 (1984) 119--130 Elsevier Science Publishers B.V., A m s t e r d a m -- Printed in The Netherlands
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BOVINE INTERLEUKIN 2 (IL-2) PRODUCTIONAND ACTIVITY ON BOVINE AND MURINE CELL LINES MICHELE MILLER-EDGE and GARY A. SPLITTER Department of Veterinary Science, U n i v e r s i t y of Wisconsin, Madison, Wisconsin 53706 (U.S.A.) (Accepted 27 February 1984) ABSTRACT Miller-Edge, M. and S p l i t t e r , G.A., 1984. Bovine i n t e r l e u k i n 2 (IL-2) production and a c t i v i t y on bovine and murine c e l l l i n e s . Vet. Immunol. Immunopathol., 7: 119-130.
Long-term growth of T c e l l cultures requires addition of I n t e r l e u k i n 2 (IL-2). In order to maintain bovine c u l t u r e s , optimal conditions for bovine IL-2 production were defined using peripheral blood mononuclear c e l l s (PBM). I r r a d i a t i o n and preculture enhanced IL-2 production possibly by reducing suppressor a c t i v i t y . IL-2 a c t i v i t y was also detected in Bovine Herpesvirus Type i - s t i m u l a t e d cultures. Unlike mitogen-stimulated cultures, a wide v a r i a t i o n in IL-2 a c t i v i t y was seen between supernatants produced by v i r u s stimulated c e l l s from d i f f e r e n t animals i n d i c a t i n g the clonal nature of antigen s p e c i f i c c e l l s from i n d i v i d u a l s . Bovine IL-~-dependent c e l l s used to q u a n t i t a t e IL-2 a c t i v i t y were characterized as: PNA , esterase negative, H4+ (anti l a - l i k e ) , B29+ (anti-pan T c e l l ) , and C5- (anti-monocyte). The observations that bovine IL-2 can maintain activated murine c e l l s , CTLL-20 and HT-2, could lead to the replacement of rat IL-2 with bovine IL-2 in long-term murine cultures. Conditions described here r e s u l t in large volumes of active medium.
INTRODUCTION The discovery of I n t e r l e u k i n 2 has allowed in v i t r o expansion of activated T lymphocytes for i n v e s t i g a t i o n of t h e i r role in immune responses (Gillis
et a l . , 1978; Watson, 1979; G i l l i s et a l . , 1980; Schreier et a l . ,
1980; Braciale et a l , 1981; F i t c h , 1981; Paul et a l . , 1981; Lamb et a l . , 1982).
Because IL-2 is an important requirement for these cultures, several
0165-2427/84/$03.00
© 1984 Elsevier Science Publishers B.V.
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methods of production and a number of assays for human and murine IL-2 have been described by others (Paetkau et a l . , 1976; G i l l i s et a l . , 1978; Coutinho et a l . , 1979; Watson et a l . , 1979; Inouye et a l . , 1980; Ruscetti et a l . , 1980; Granelli-Piperno et a l . , 1981; Stull and G i l l i s , However, l i t t l e
1981; Wu et a l . , 1982).
work has been done in other species.
Only recently has
IL-2-1ike a c t i v i t y been described in domestic animals such as the pig (Gasbarre et a l . , 1982), horse (Magnuson et a l . , 1983) and cow (Baker and Knoblock, 1982a,b; Miller-Edge and S p l i t t e r , 1983). In studying the role of cell-mediated immunity in infectious diseases of c a t t l e , addition of culture supernatants containing growth factor (IL-2) is required to maintain bovine T lymphocytes for in v i t r o functional studies. This work examines the effects of modifying culture conditions to enhance bovine IL-2 production and the a b i l i t y of bovine IL-2 to maintain long-range bovine and murine T cell cultures. MATERIALS AND METHODS Medi urn Serum-containing medium (SCM) consisted of RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with L-glutamine (2mM), p e n i c i l l i n (I00 u n i t s / m l ) , streptomycin (I00 ~g/ml), HEPES buffer (25mM), 2-mercaptoethanol (5xlO-5M), and 10% fetal c a l f serum (K.C. Biologicals, Inc., Lenexa, KS).
Serum-free
medium (SFM) contained RPMI 1640 supplemented with L-glutamine (2mM), p e n i c i l l i n (I00 u n i t s / m l ) , streptomycin (100 ~g/ml), HEPES buffer (25mM), 1% (v/v) MEM vitamin solution (GIBCO), 1% (v/v) nonessential amino acids (GIBCO), 1% ( v / v ) MEM sodium pyruvate solution (GIBCO), human t r a n s f e r r i n (I0 ~g/ml) (Sigma Chemical Co., St. Louis, MO), 0.125% (w/v) bovine serum albumin (Miles Laboratories, Elkhart, IN), and sodium selenite (2xlO-7M) (Sigma Chemical Co.). Preparation of IL-2 containing medium Peripheral blood mononuclear c e l l s (PBM) f o r IL-2 production were isolated from Guernsey cows by Ficoll-Hypaque (1.077 g/ml) separation. Supernatants assayed for IL-2 a c t i v i t y were prepared by culturing PBM with mitogen or antigen in 5 ml p l a s t i c tubes (Falcon No. 2054, Oxnard, CA).
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Tubes were incubated at 37°C in a humidified atmosphere of 5% CO2 and air. Supernatants were harvested by centrifugation, sterilized by passage through 0.22 ~m f i l t e r s (Millipore, Bedford, MA) and stored at -20°C until assayed. IL-2 production by bovine cells was compared by varying the following parameters: 1) cell concentration (i, 3, and 5x106 cells/ml); 2) media conditions (SFM, SCM); 3) mitogens (Phytohemagglutinin-M (PHA-M), (GIBCO) and Concanavalin A (Con A) (Miles Laboratories)), or antigen (UV-inactivated Bovine Herpesvirus Type I); 4) mitogen doses (PHA-M: 0.25, 0.5, I, 2% (v/v); Con A: I, 5, 10, 20 ~g/ml); 5) length of incubation period (24, 48, 72, 96 hours with mitogen; 3-8 days with antigen); and 6) cell pretreatment (irradiation at 1000 rads with a 137Cs source or preincubation for 2 days before lectin stimulation). Rat IL-2 was prepared by stimulating 3xi06 spleen cells/ml with 2.5 ~g/ml Con A in SCM for 24 hours. Biological assays f o r IL-2 a c t i v i t y Bovine IL-2-dependent c e l l s Long-term cultures of bovine IL-2-dependent T c e l l s were derived from a mixed lymphocyte culture (MLC) (Bonnard et a l . , 1980; Baker and Knoblock, 1982b).
Cells used in the assay had been maintained in 25% (v/v) bovine
l e c t i n - f r e e IL-2 f o r 3-6 weeks.
Cells were washed in PBS, resuspended in
fresh SCM, and ixlO 4 c e l l s / w e l l were added to 96-well round-bottom m i c r o t i t e r plates with a 1:2 d i l u t i o n of test supernatant.
Plates were incubated f o r
40 hours, pulsed with 1.0 ~Ci [3H] thymidine/well and harvested 6 hours l a t e r . Data are expressed as the mean of [3HI thymidine incorporation observed in t r i p l i c a t e cultures ± standard d e v i a t i o n . Murine IL-2-dependent c e l l s CTLL-20 and HT-2 cloned murine IL-2-dependent c e l l lin e s were the g i f t of Dr. Frank Fitch.
3xlO 4 CTLL-20 or IxlO 4 HT-2 c e l l s / w e l l were incubated with
75% test supernatant and I ~Ci/well [3H] thymidine f o r 24 hours. I d e n t i f i c a t i o n of cultured c e l l s Bovine c e l l s cultured with IL-2 were stained f o r T c e l l s with f l u o r e s cenated peanut a g g l u t i n i n (PNA-FITC) according to the method described by Usinger and S p l i t t e r (1981).
Cells were also cytocentrifuged and assessed f o r
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non-specific esterase as described by Yam et al. (1971).
In addition, they
were stained with monoclonal antibodies H4 (anti l a - l i k e ) , B29 (anti-pan T c e l l ) and C5 (anti-monocyte) by indirect immunofluorescence using FITC-rabbit anti-mouse IgG and IgM. Percentage of positively staining cells was determined by counting >200 cells and using the formula:
(number of
fluorescent c e l l s / t o t a l number of cells counted) x 100%.
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Fig. 1. Effect ~f cell treatment on IL-2 production. IL-2 was produced by stimulating lx10 cells/ml with Con A (2 ~g/ml) in SCM for 24, 48, or 72 hours after no pretreatment (~), incubation in RPMI 1640 only for 2 days (e), or i r r a d i a t i o n with 10qO rads ((~). Supernatants were tested on bovine IL-2dependent c e l l s . : [~H] Thymidine incorporation is expressed as counts per minute (cpm). [~H] thymidine incorporation in the presence of SCM, Con A, rat Con A or unstimulated culture supernatants did not exceed 2361±333 cpm.
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RESULTS
Preincubating cells for 2 days before stimulation with mitogen or irradiating ceils immediately prior to culture are alternative methods for enhancement of IL-2 production.
After preincubation or i r r a d i a t i o n , Ix106
PBM/ml were stimulated with 2 ug/ml Con A in SCM and supernatants tested on bovine IL-2 dependent cells.
Results are shown in Figure I .
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Fig. 2. Production of IL-2 in response to BHV-I. lx106 PBM/ml from each of four cows were stimulated with Ix10 PFU/ml UV-inactivated BHV-1 in SC~ for 3-8 days. Supernatants were tested on bovine IL-2-dependent cells. [ H] Thymidine incorporation is expressed as counts per minute (cpm). Unstimulated cultures did not produce detectable levels of active factor.
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IL-2 production occurred when PBM were preincubated in RPMI 1640 f o r 2 days and then stimulated with Con A.
I n t e r e s t i n g l y , the k i n e t i c s of IL-2
production shifted from a peak at 72 hours in untreated cultures to 48 hours in preincubated cultures.
I r r a d i a t i o n of c e l l s with I000 rads p r i o r to
stimulation enhanced IL-2 production in 48 hour supernatants but decreased a c t i v i t y by 72 hours. Several c u l t u r e parameters were tested to optimize IL-2 production by bovine PBM. Cell concentration, mitogen dose, incubation time, and medium were varied.
In general, our data supported those reported by Baker and
Knoblock (1982a).
That i s , 106 PBM were stimulated with I% PHA f o r 48 hours
or 2 ~g/ml Con A f o r 72 hours in SCM. Peak IL-2 production under serum-free
Dose-Dependent P r o l i f e r a t i o n of Bovine IL-2 D e p e n d e n t Cells i
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MLA 144(glbbon IL-2) Bovine
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Fig. 3. Dose-dependent proliferation of bovine IL-2-dependent cells using regression analysis. Serial dilutions of two bovine IL-2 samples (produced by stimulation Con A or BHV-1) and suBernatant from MLA 144 cultures were tested on bovine IL-2-dependent cells. [~H] Thymidine incorporation is expressed as counts per minute (cpm).
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conditions occurred at a lower dose of mitogen (I ~g/ml Con A or 0.5% PHA) than in serum-containing medium.
In addition, IL-2 present in serum-free
supernatants had greater a c t i v i t y than serum-containing samples. IL-2 is produced not only in response to stimulation by mitogen, but also antigen.
All the animals in t h i s study had been previously immunized with
Bovine Herpesvirus Type i (BHV-I) (Nasalgen, Jensen-Salsbery Laboratories, Kansas City, MO). Preliminary studies showed that the optimal p r o l i f e r a t i o n of l x i 0 6 PBM/ml with ix106 PFU/ml UV-inactivated BHV-1 occurred on days 5,6. responding and two low responding animals were chosen.
Two high
As can be seen in
Figure 2, there were also high and low IL-2-producing animals.
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correlated with their p r o l i f e r a t i v e response. Peak IL-2 a c t i v i t y occurred on day 3 as detected by IL-2-dependent c e l l s .
Cells from all of these animals
produced highly active supernatants when stimulated with Con A, comparable to levels of IL-2 produced by the high responder when stimulated with virus. To determine whether p r o l i f e r a t i o n of IL-2-dependent cells could be used as a quantitative measure of IL-2, we examined proliferation in the presence TABLE I C h a r a c t e r i z a t i o n of long-term bovine c e l l s by immunofluorescence
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Unlike bovine IL-2-dependent c e l l s , murine IL-2-dependent c e l l s were capable of detecting a c t i v i t y in rat Con A supernatants.
In a d d i t i o n , both
CTLL-20 and HT-2 c e l l s responded better to some bovine supernatants than to rat supernatants (Fig. 4).
Active supernatants were produced in response to
Con A or BHV-I. These r e s u l t s suggest that one active f a c t o r in bovine supernatants is IL-2. DISCUSSION IL-2 could be produced in response to Bovine Herpesvirus Type 1.
This
suggests that IL-2 is the product of antigen s p e c i f i c a c t i v a t i o n in the bovine and offers the potential to assess the function of c e r t a i n antigen responsive clones.
Our r e s u l t s indicate that bovine IL-2 can maintain murine IL-2
dependent c e l l l i n e s (Fig. 4).
These observations could lead to replacement
of rat IL-2 with bovine IL-2 in long-term murine cultures since large q u a n t i t i e s of active medium can be generated. One modification that enhanced production of IL-2 was preincubation of c e l l s f o r 2 days before Con A s t i m u l a t i o n (Fig. I ) .
Increased IL-2 a c t i v i t y
may r e s u l t from reduced suppressor f u n c t i o n which has been proposed previously by others (Northoff et a l . , 1980, Gullberg and Larsson, 1982; Pauly et a l . , 1982).
Other methods which reduce suppressor a c t i v i t y include i r r a d i a t i o n of
IL-2 producing cultures.
Bovine suppressor c e l l s have been generated in Con
A-stimulated cultures (Smith et a l . ,
1981) and were functional a f t e r 2000 rads
irradiation.
Therefore, i r r a d i a t i o n with i000 rads should not t o t a l l y abolish
suppression.
Our r e s u l t s showed that i r r a d i a t i o n enhanced IL-2 production at
48 hours (Fig. i ) . (Northoff et a l . ,
Several other i n v e s t i g a t o r s llave reported no e f f e c t 1980; Chouaib and F r a d e l i z i , 1982; Gullberg and Larsson,
1982) or enhancement (Inouye et a l . , 1980) at t h i s i r r a d i a t i o n dose. I r r a d i a t i o n e f f e c t s on IL-2 production have not been f u l l y resolved. Other i n v e s t i g a t o r s have reported producing IL-2 in other species in response to a s p e c i f i c antigen as well as mitogen (Andrus and L a f f e r t y , 1981; llonen and Salmi, 1982; Kaufmann et a l . , 1982). ability
Therefore, we examined the
of in vivo primed PBM to produce IL-2 upon r e s t i m u l a t i o n in v i t r o .
A c t i v i t y of supernatants from cultures stimulated with BHV-I, as detected on bovine IL-2-dependent c e l l s , corresponded with the p r o l i f e r a t i v e response. Since BHV-I has previously been shown to stimulate T c e l l s (Rouse and Babiuk, 1974; Rouse and Babiuk; 1975), and bovine IL-2-dependent c e l l s detected an
128
active f a c t o r , these results suggest that bovine PBM are also capable of producing IL-2 in response to an antigen. Long-term cultured bovine c e l l s were used to test f o r IL-2 a c t i v i t y . Observations that long-term cultured c e l l s were IL-2 responsive T c e l l s are as follows:
1) responder c e l l s are PNA p o s i t i v e , non-specific esterase negative;
la p o s i t i v e (using monoclonal antibody H4), C5- (anti-monocyte), and B2g+ (anti-pan T c e l l ) ; 2) long-term cultured c e l l s are unresponsive to mitogen, medium alone, or conditioned medium from unstimulated cultures; 3) supernatants from a known IL-2-producing cell l i n e (MLA 144) supported p r o l i f e r a t i o n (Rabin et a l . , 1981); 4) s t r i c t requirement of IL-2 f or survival and p r o l i f e r a t i o n of these c e l l s . >95% were dead w i t h i n 72 hours.
I f these c e l l s were not provided with IL-2, Results using monoclonal antibody H4 suggest
these c e l l s are activated T c e l l s ( l a +) as reported f o r other T c e l l s growing in continuous c u l tu r e (Metzgar et a l . , 1979). IL-2-dependent c e l l s require only IL-2 as a signal f o r p r o l i f e r a t i o n because they have already seen antigen and have been expanded f or long periods with IL-2.
Since these c e l l s are mitogen unresponsive, they can be used to
determine a dose-dependent curve fo r growth f a c t o r - c o n t a i n i n g samples (Fig. 3).
Regression analysis indicates that the a c t i v i t y measured by
p r o l i f e r a t i o n of these c e l l s is a l i n e a r function of the concentration.
Under
these conditions, a c t i v i t y can be converted to units as reported by G i l l i s et al.
(1978).
sample.
This allows comparison between assays using a standard reference
Therefore, long-term bovine T c e l l s are defined as being IL-2-
dependent.
This dependence was also confirmed by t h e i r a b i l i t y to p r o l i f e r a t e
in response to supernatants from a known IL-2-producing cell l i n e , MLA 144 (Rabin et a l . , 1980) (Fig. 3).
These c e l l s can most l i k e l y be generated in
any species in which IL-2-dependent c e l l l i n e s have not been established. This opens up i n v e s t i g a t i o n s into economically important species, such as the bovine.
Conditions f o r IL-2 production and assay are a f i r s t
s p e c i f i c T cell populations in v i t r o .
step for growing
These c e l l s can then be used to
elucidate t h e i r role in vivo in disease problems s p e c i f i c to the species. In addition to maintaining bovine c e l l s , these supernatants were capable of sustaining the growth of murine c e l l s , CTLL-20 and HT-2 (Fig. 4). c e l l s have previously been shown to be IL-2 dependent.
These
Therefore, the large
volumes of supernatants that can be produced using bovine PBM may substitute f o r rat Con A supernatants in long-term murine cultures.
129
ACKNOWLEDGMENTS This work was supported in part by USDA-SEA Grant Nos. 901-15-145, 12-14-3001-758, 59-2552-1-2-038-0, USDA Dairy Forage Laboratory and by the College of A g r i c u l t u r a l and L i f e Sciences.
The authors wish to thank
Dr. Arden Hardie for providing c a t t l e used in t h i s study.
MLA 144 c e l l s were
the kind g i f t of Dr. Gary Fathman and monoclonal antibody B29 the g i f t William C. Davis.
of
We also wish to acknowledge Dr. Frank Fitch for supplying
us with the murine IL-2 dependent c e l l s CTLL-20 and HT-2. REFERENCES Andrus, L. and Lafferty, K.J., 1981. Interleukin 2 production by alloantigen (H-2) activated T cells. AJEBAK, 59 : 413-426. Baker, P.E. and Knoblock, K.F., 1982a. Bovine costimulator. I. Production k i n e t i c s , p a r t i a l p u r i f i c a t i o n , and q u a n t i f i c a t i o n in serumifree Iscove's medium. Vet. Immunol. Immunopath., 3 : 365-380. Baker, P.E. and Knoblock, K.F., 1982b. Bovine costimulator. I I . Generation and maintenance of a bovine costimulator-dependent bovine lymphoblastoid cell l i n e . Vet. Immunol. Immunopath., 3 : 381-398. Bonnard, G.D., Yasaka, K. and Maca, R.D., 1980. Continued growth of functional human T lymphocytes: Production of human T-cell growth factor. Cell. Immunol., 51 : 390-401. Braciale, T . J . , Andrew, M.E. and Braciale, V.L., 1981. Heterogeneity and s p e c i f i c i t y of cloned l i n e s of i n f l u e n z a - v i r u s - s p e c i f i c c y t o t o x i c T lymphocytes. J. Exp. Med., 153 : 910-923. Chouaib, S. and F r a d e l i z i , D., 1982. The mechanism of i n h i b i t i o n of human IL-2 production. J. Immunol., 129 : 2463-2468. Coutinho, A., Larsson, E.L., Gr~nvik, K . I . and Andersson, J . , 1979. Studies on T lymphocyte a c t i v a t i o n . II. The target c e l l s for concanavalin A-induced growth factors. Eur. J. Immunol., 9 : 587-592. F i t c h , F.W., 1981. T lymphocyte clones having defined immunological functions. Transplantation, 32 : 171-176. Gasbarre, L.C., Urban, J r . , J.F. and Romanowski, R.D., 1982. Production and c h a r a c t e r i z a t i o n of a porcine T c e l l growth f a c t o r . Fed. Proc., 41 : 317 (abstr. No. 288). G i l l i s , S., Ferm, M.W., Ou, W. and Smith, K.A., 1978. T c e l l growth f a c t o r : parameters of production and a q u a n t i t a t i v e microassay for a c t i v i t y . J. l J ~ u n o l . , 120 : 2027-2032. G i l l i s , S., Smith, K.A. and Watson, J . , 1980. Biochemical c h a r a c t e r i z a t i o n of lymphocyte regulatory molecules. I I . P u r i f i c a t i o n of a class of rat and human lymphokines. J. Immunol., 124 : 1954-1962. G r a n e l l i - P i p e r n o , A., V a s s a l l i , J. and Reich, E., 1981. P u r i f i c a t i o n of murine T c e l l growth f a c t o r . A lymphocyte mitogen with helper activity. J. Exp. Med., 154 : 422-431. Gullberg, M, and Larsson, E.L., 1982. Studies on induction and e f f e c t o r functions of concanavalin A-induced suppressor c e l l s that l i m i t TCGF production. J. Immunol., 128 : 746-750. llonen, J. and Salmi, A., 1982. Detection of a n t i g e n - s p e c i f i c c e l l u l a r immune response by the in vivo production of T-cell growth factor. Scand. J. Immunol., 15 : 521-524. Inouye, H., Hank, J . A . , A l t e r , B.J. and Bach, F.H., 1980. TCGF production f o r cloning and growth of f u n c t i o n a l human T lymphocytes. Scand. J. Immunol., 12 : 149-154. Kaufmann, S.H.E., Hahn, H., Simon, M.M., R611inghoff, M. and Wagner, H., 1982. I n t e r l e u k i n 2 induction in Lyt 1+23- T c e l l s from L i s t e r i a monocytogeoes-immune mice. I n f e c t . Immun., 37 : 1292-1294.
180 Lamb, J.R., Eckels, D.D., Lake, P., Johnson, A.H., Hartzman, R.J. and Woody, J.N., 1982. A n t i g e n - s p e c i f i c human T lymphocyte clones: induction antigen s p e c i f i c i t y , and MHC r e s t r i c t i o n of Influenza virus-immune clones. J. Immunol., 128 : 233-238. Magnuson, N.S., Namen, A.E., Perryman, L.E. and Magnuson, J . A . , 1983. I n t e r l e u k i n 2 from horses. Fed. Proc., 42 : 446 (abstr. No. 905). Metzgar, R.S., B e r t o g l i o , J . , Anderson, J.K., Bonnard, G.D. and Ruscetti, F.W., 1979. Detection of HLA-DRw ( l a - l i k e ) antigens on human T lymphocytes grown in tissue c u l t u r e . J. Immunol., 122 : 949-953. Miller-Edge, M. and S p l i t t e r , G.A., 1983. I n t e r l e u k i n - 2 (IL-2) assays compared by measuring bovine IL-2. Fed. Proc., 42 : 447 (abstr. No. 911). Northoff, H., Carter, C. and Oppenheim, J . J . , 1980. I n h i b i t i o n of concanavalin A-induced human lymphocyte mitogenic f a c t o r ( I n t e r l e u k i n - 2 ) production by suppressor T lymphocytes. J. Immunol., 125 : 1823-1828. Paetkau, V., M i l l s , G., Gerhart, S. and ~ n t i c o n e , V., 1976. P r o l i f e r a t i o n of murine thymic lymphocytes in vivo is mediated by the concanavalin A-induced release of a lymphokine (costimulator). J. Immunol., 117 : 1320-1324. Paul, W.E., Sredni, B. and Schwartz, R.H., 1981. Long-term growth and cloning of non-transformed lymphocytes. Nature, 294 : 697-699. Pauly, J . L . , Russell, C.W., Planinsek, J.A. and Minowada, J . , 1982. Studies of cultured human T lymphocytes. I. Production of the T cell growthpromoting lymphokine I n t e r l e u k i n 2. J. Immunol. Methods, 50 : 173-186. Rabin, H., Hopkins I I I , R.F., Ruscetti, F.W., Neubauer, R.H., Brown, R.L. and Kawakami, T.G., 1980. Spontaneous release of a factor with properties of T cell growth f a c t o r fr~n a continuous l i n e of primate tumor T c e l l s . J. Immunol., 127 : 1852-1856. Rouse, B.T. and Babiuk, L.A., 1974. Host responses to Infectious Bovine R h i n o t r a c h e i t i s Virus. I I I . I s o l a t i o n and immunologic a c t i v i t i e s of bovine T lymphocytes. J. Immunol., 113 : 1391-1398. Rouse, 8.T. and Babiuk, L.A., 1975. Host defense mechanisms against Infectious Bovine R h i n o t r a c h e i t i s Virus. I I . I n h i b i t i o n of v i r a l plaque formation by immune peripheral blood lymphocytes. Cell. Immunol., 17 : 43-56. Ruscetti, F.W., Mier, J.W. and Gallo, R.C., 1980. Human T-cell growth factor: parameters for production. J. Supramol. S t r u c t . , 13 : 229-241. Schreier, M.H., Iscove, N.N., Tees, R., Aarden, L. and Von 8oehmer, H., 1980. Clones of k i l l e r and helper T c e l l s : growth requirements, s p e c i f i c i t y and retention of function in long-term c u l t u r e . Immunol. Rev., 51 : 315-336. Smith, W.G., Usinger, W.R. and S p l i t t e r , G.A., 1981. Bovine con A-induced suppressor c e l l s : generation, macrophage requirements and possible mechanisms of regulatory action, l~nunol., 43 : 91-100. S t u l l , D. and G i l l i s , S., 1981. C o n s t i t u t i v e production of I n t e r l e u k i n 2 by a T cell hybridoma. J. Immunol., 126 : 1680-1683. Usinger, W.R. and S p l i t t e r , G.A., 1981. Two molecularly independent surface receptors i d e n t i f y bovine T lymphocytes. J. Immunol. Methods, 45 : 209219. Watson, J . , 1979. Continuous p r o l i f e r a t i o n of murine a n t i g e n - s p e c i f i c helper T lymphocytes in c u l t u r e . J. Exp. Med., 150 : 1510-1519. Watson, J . , G i l l i s , S., Marbrook, J . , Mochizuki, D. and Smith, K.A., 1979. Biochemical and b i o l o g i c a l c h a r a c t e r i z a t i o n of lymphocyte regulatory molecules. I. P u r i f i c a t i o n of a class of murine lymphokines. J. Exp. Med., 150 : 849-861. Wu, Y., Ernberg, I . , Masucci, M.G., Johnson, D., K l e i n , E. and Klein, G., 1982. Human T cell growth f a c t o r (TCGF) produced by repeated s t i m u l a t i o n of non-adherent human lymphocytes. J. Immunol. Methods, 51 : 35-44. Yam, L.T., Liand, C.Y., Crosby, W.H., 1971. Cytochemical i d e n t i f i c a t i o n of monocytes and granulocytes. A.S.C.P., 55 : 283-290.
119
BOVINE INTERLEUKIN 2 (IL-2) PRODUCTIONAND ACTIVITY ON BOVINE AND MURINE CELL LINES MICHELE MILLER-EDGE and GARY A. SPLITTER Department of Veterinary Science, U n i v e r s i t y of Wisconsin, Madison, Wisconsin 53706 (U.S.A.) (Accepted 27 February 1984) ABSTRACT Miller-Edge, M. and S p l i t t e r , G.A., 1984. Bovine i n t e r l e u k i n 2 (IL-2) production and a c t i v i t y on bovine and murine c e l l l i n e s . Vet. Immunol. Immunopathol., 7: 119-130.
Long-term growth of T c e l l cultures requires addition of I n t e r l e u k i n 2 (IL-2). In order to maintain bovine c u l t u r e s , optimal conditions for bovine IL-2 production were defined using peripheral blood mononuclear c e l l s (PBM). I r r a d i a t i o n and preculture enhanced IL-2 production possibly by reducing suppressor a c t i v i t y . IL-2 a c t i v i t y was also detected in Bovine Herpesvirus Type i - s t i m u l a t e d cultures. Unlike mitogen-stimulated cultures, a wide v a r i a t i o n in IL-2 a c t i v i t y was seen between supernatants produced by v i r u s stimulated c e l l s from d i f f e r e n t animals i n d i c a t i n g the clonal nature of antigen s p e c i f i c c e l l s from i n d i v i d u a l s . Bovine IL-~-dependent c e l l s used to q u a n t i t a t e IL-2 a c t i v i t y were characterized as: PNA , esterase negative, H4+ (anti l a - l i k e ) , B29+ (anti-pan T c e l l ) , and C5- (anti-monocyte). The observations that bovine IL-2 can maintain activated murine c e l l s , CTLL-20 and HT-2, could lead to the replacement of rat IL-2 with bovine IL-2 in long-term murine cultures. Conditions described here r e s u l t in large volumes of active medium.
INTRODUCTION The discovery of I n t e r l e u k i n 2 has allowed in v i t r o expansion of activated T lymphocytes for i n v e s t i g a t i o n of t h e i r role in immune responses (Gillis
et a l . , 1978; Watson, 1979; G i l l i s et a l . , 1980; Schreier et a l . ,
1980; Braciale et a l , 1981; F i t c h , 1981; Paul et a l . , 1981; Lamb et a l . , 1982).
Because IL-2 is an important requirement for these cultures, several
0165-2427/84/$03.00
© 1984 Elsevier Science Publishers B.V.
120
methods of production and a number of assays for human and murine IL-2 have been described by others (Paetkau et a l . , 1976; G i l l i s et a l . , 1978; Coutinho et a l . , 1979; Watson et a l . , 1979; Inouye et a l . , 1980; Ruscetti et a l . , 1980; Granelli-Piperno et a l . , 1981; Stull and G i l l i s , However, l i t t l e
1981; Wu et a l . , 1982).
work has been done in other species.
Only recently has
IL-2-1ike a c t i v i t y been described in domestic animals such as the pig (Gasbarre et a l . , 1982), horse (Magnuson et a l . , 1983) and cow (Baker and Knoblock, 1982a,b; Miller-Edge and S p l i t t e r , 1983). In studying the role of cell-mediated immunity in infectious diseases of c a t t l e , addition of culture supernatants containing growth factor (IL-2) is required to maintain bovine T lymphocytes for in v i t r o functional studies. This work examines the effects of modifying culture conditions to enhance bovine IL-2 production and the a b i l i t y of bovine IL-2 to maintain long-range bovine and murine T cell cultures. MATERIALS AND METHODS Medi urn Serum-containing medium (SCM) consisted of RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with L-glutamine (2mM), p e n i c i l l i n (I00 u n i t s / m l ) , streptomycin (I00 ~g/ml), HEPES buffer (25mM), 2-mercaptoethanol (5xlO-5M), and 10% fetal c a l f serum (K.C. Biologicals, Inc., Lenexa, KS).
Serum-free
medium (SFM) contained RPMI 1640 supplemented with L-glutamine (2mM), p e n i c i l l i n (I00 u n i t s / m l ) , streptomycin (100 ~g/ml), HEPES buffer (25mM), 1% (v/v) MEM vitamin solution (GIBCO), 1% (v/v) nonessential amino acids (GIBCO), 1% ( v / v ) MEM sodium pyruvate solution (GIBCO), human t r a n s f e r r i n (I0 ~g/ml) (Sigma Chemical Co., St. Louis, MO), 0.125% (w/v) bovine serum albumin (Miles Laboratories, Elkhart, IN), and sodium selenite (2xlO-7M) (Sigma Chemical Co.). Preparation of IL-2 containing medium Peripheral blood mononuclear c e l l s (PBM) f o r IL-2 production were isolated from Guernsey cows by Ficoll-Hypaque (1.077 g/ml) separation. Supernatants assayed for IL-2 a c t i v i t y were prepared by culturing PBM with mitogen or antigen in 5 ml p l a s t i c tubes (Falcon No. 2054, Oxnard, CA).
121
Tubes were incubated at 37°C in a humidified atmosphere of 5% CO2 and air. Supernatants were harvested by centrifugation, sterilized by passage through 0.22 ~m f i l t e r s (Millipore, Bedford, MA) and stored at -20°C until assayed. IL-2 production by bovine cells was compared by varying the following parameters: 1) cell concentration (i, 3, and 5x106 cells/ml); 2) media conditions (SFM, SCM); 3) mitogens (Phytohemagglutinin-M (PHA-M), (GIBCO) and Concanavalin A (Con A) (Miles Laboratories)), or antigen (UV-inactivated Bovine Herpesvirus Type I); 4) mitogen doses (PHA-M: 0.25, 0.5, I, 2% (v/v); Con A: I, 5, 10, 20 ~g/ml); 5) length of incubation period (24, 48, 72, 96 hours with mitogen; 3-8 days with antigen); and 6) cell pretreatment (irradiation at 1000 rads with a 137Cs source or preincubation for 2 days before lectin stimulation). Rat IL-2 was prepared by stimulating 3xi06 spleen cells/ml with 2.5 ~g/ml Con A in SCM for 24 hours. Biological assays f o r IL-2 a c t i v i t y Bovine IL-2-dependent c e l l s Long-term cultures of bovine IL-2-dependent T c e l l s were derived from a mixed lymphocyte culture (MLC) (Bonnard et a l . , 1980; Baker and Knoblock, 1982b).
Cells used in the assay had been maintained in 25% (v/v) bovine
l e c t i n - f r e e IL-2 f o r 3-6 weeks.
Cells were washed in PBS, resuspended in
fresh SCM, and ixlO 4 c e l l s / w e l l were added to 96-well round-bottom m i c r o t i t e r plates with a 1:2 d i l u t i o n of test supernatant.
Plates were incubated f o r
40 hours, pulsed with 1.0 ~Ci [3H] thymidine/well and harvested 6 hours l a t e r . Data are expressed as the mean of [3HI thymidine incorporation observed in t r i p l i c a t e cultures ± standard d e v i a t i o n . Murine IL-2-dependent c e l l s CTLL-20 and HT-2 cloned murine IL-2-dependent c e l l lin e s were the g i f t of Dr. Frank Fitch.
3xlO 4 CTLL-20 or IxlO 4 HT-2 c e l l s / w e l l were incubated with
75% test supernatant and I ~Ci/well [3H] thymidine f o r 24 hours. I d e n t i f i c a t i o n of cultured c e l l s Bovine c e l l s cultured with IL-2 were stained f o r T c e l l s with f l u o r e s cenated peanut a g g l u t i n i n (PNA-FITC) according to the method described by Usinger and S p l i t t e r (1981).
Cells were also cytocentrifuged and assessed f o r
122
non-specific esterase as described by Yam et al. (1971).
In addition, they
were stained with monoclonal antibodies H4 (anti l a - l i k e ) , B29 (anti-pan T c e l l ) and C5 (anti-monocyte) by indirect immunofluorescence using FITC-rabbit anti-mouse IgG and IgM. Percentage of positively staining cells was determined by counting >200 cells and using the formula:
(number of
fluorescent c e l l s / t o t a l number of cells counted) x 100%.
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Fig. 1. Effect ~f cell treatment on IL-2 production. IL-2 was produced by stimulating lx10 cells/ml with Con A (2 ~g/ml) in SCM for 24, 48, or 72 hours after no pretreatment (~), incubation in RPMI 1640 only for 2 days (e), or i r r a d i a t i o n with 10qO rads ((~). Supernatants were tested on bovine IL-2dependent c e l l s . : [~H] Thymidine incorporation is expressed as counts per minute (cpm). [~H] thymidine incorporation in the presence of SCM, Con A, rat Con A or unstimulated culture supernatants did not exceed 2361±333 cpm.
123
RESULTS
Preincubating cells for 2 days before stimulation with mitogen or irradiating ceils immediately prior to culture are alternative methods for enhancement of IL-2 production.
After preincubation or i r r a d i a t i o n , Ix106
PBM/ml were stimulated with 2 ug/ml Con A in SCM and supernatants tested on bovine IL-2 dependent cells.
Results are shown in Figure I .
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Fig. 2. Production of IL-2 in response to BHV-I. lx106 PBM/ml from each of four cows were stimulated with Ix10 PFU/ml UV-inactivated BHV-1 in SC~ for 3-8 days. Supernatants were tested on bovine IL-2-dependent cells. [ H] Thymidine incorporation is expressed as counts per minute (cpm). Unstimulated cultures did not produce detectable levels of active factor.
124
IL-2 production occurred when PBM were preincubated in RPMI 1640 f o r 2 days and then stimulated with Con A.
I n t e r e s t i n g l y , the k i n e t i c s of IL-2
production shifted from a peak at 72 hours in untreated cultures to 48 hours in preincubated cultures.
I r r a d i a t i o n of c e l l s with I000 rads p r i o r to
stimulation enhanced IL-2 production in 48 hour supernatants but decreased a c t i v i t y by 72 hours. Several c u l t u r e parameters were tested to optimize IL-2 production by bovine PBM. Cell concentration, mitogen dose, incubation time, and medium were varied.
In general, our data supported those reported by Baker and
Knoblock (1982a).
That i s , 106 PBM were stimulated with I% PHA f o r 48 hours
or 2 ~g/ml Con A f o r 72 hours in SCM. Peak IL-2 production under serum-free
Dose-Dependent P r o l i f e r a t i o n of Bovine IL-2 D e p e n d e n t Cells i
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Fig. 3. Dose-dependent proliferation of bovine IL-2-dependent cells using regression analysis. Serial dilutions of two bovine IL-2 samples (produced by stimulation Con A or BHV-1) and suBernatant from MLA 144 cultures were tested on bovine IL-2-dependent cells. [~H] Thymidine incorporation is expressed as counts per minute (cpm).
125
conditions occurred at a lower dose of mitogen (I ~g/ml Con A or 0.5% PHA) than in serum-containing medium.
In addition, IL-2 present in serum-free
supernatants had greater a c t i v i t y than serum-containing samples. IL-2 is produced not only in response to stimulation by mitogen, but also antigen.
All the animals in t h i s study had been previously immunized with
Bovine Herpesvirus Type i (BHV-I) (Nasalgen, Jensen-Salsbery Laboratories, Kansas City, MO). Preliminary studies showed that the optimal p r o l i f e r a t i o n of l x i 0 6 PBM/ml with ix106 PFU/ml UV-inactivated BHV-1 occurred on days 5,6. responding and two low responding animals were chosen.
Two high
As can be seen in
Figure 2, there were also high and low IL-2-producing animals.
This
correlated with their p r o l i f e r a t i v e response. Peak IL-2 a c t i v i t y occurred on day 3 as detected by IL-2-dependent c e l l s .
Cells from all of these animals
produced highly active supernatants when stimulated with Con A, comparable to levels of IL-2 produced by the high responder when stimulated with virus. To determine whether p r o l i f e r a t i o n of IL-2-dependent cells could be used as a quantitative measure of IL-2, we examined proliferation in the presence TABLE I C h a r a c t e r i z a t i o n of long-term bovine c e l l s by immunofluorescence
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Unlike bovine IL-2-dependent c e l l s , murine IL-2-dependent c e l l s were capable of detecting a c t i v i t y in rat Con A supernatants.
In a d d i t i o n , both
CTLL-20 and HT-2 c e l l s responded better to some bovine supernatants than to rat supernatants (Fig. 4).
Active supernatants were produced in response to
Con A or BHV-I. These r e s u l t s suggest that one active f a c t o r in bovine supernatants is IL-2. DISCUSSION IL-2 could be produced in response to Bovine Herpesvirus Type 1.
This
suggests that IL-2 is the product of antigen s p e c i f i c a c t i v a t i o n in the bovine and offers the potential to assess the function of c e r t a i n antigen responsive clones.
Our r e s u l t s indicate that bovine IL-2 can maintain murine IL-2
dependent c e l l l i n e s (Fig. 4).
These observations could lead to replacement
of rat IL-2 with bovine IL-2 in long-term murine cultures since large q u a n t i t i e s of active medium can be generated. One modification that enhanced production of IL-2 was preincubation of c e l l s f o r 2 days before Con A s t i m u l a t i o n (Fig. I ) .
Increased IL-2 a c t i v i t y
may r e s u l t from reduced suppressor f u n c t i o n which has been proposed previously by others (Northoff et a l . , 1980, Gullberg and Larsson, 1982; Pauly et a l . , 1982).
Other methods which reduce suppressor a c t i v i t y include i r r a d i a t i o n of
IL-2 producing cultures.
Bovine suppressor c e l l s have been generated in Con
A-stimulated cultures (Smith et a l . ,
1981) and were functional a f t e r 2000 rads
irradiation.
Therefore, i r r a d i a t i o n with i000 rads should not t o t a l l y abolish
suppression.
Our r e s u l t s showed that i r r a d i a t i o n enhanced IL-2 production at
48 hours (Fig. i ) . (Northoff et a l . ,
Several other i n v e s t i g a t o r s llave reported no e f f e c t 1980; Chouaib and F r a d e l i z i , 1982; Gullberg and Larsson,
1982) or enhancement (Inouye et a l . , 1980) at t h i s i r r a d i a t i o n dose. I r r a d i a t i o n e f f e c t s on IL-2 production have not been f u l l y resolved. Other i n v e s t i g a t o r s have reported producing IL-2 in other species in response to a s p e c i f i c antigen as well as mitogen (Andrus and L a f f e r t y , 1981; llonen and Salmi, 1982; Kaufmann et a l . , 1982). ability
Therefore, we examined the
of in vivo primed PBM to produce IL-2 upon r e s t i m u l a t i o n in v i t r o .
A c t i v i t y of supernatants from cultures stimulated with BHV-I, as detected on bovine IL-2-dependent c e l l s , corresponded with the p r o l i f e r a t i v e response. Since BHV-I has previously been shown to stimulate T c e l l s (Rouse and Babiuk, 1974; Rouse and Babiuk; 1975), and bovine IL-2-dependent c e l l s detected an
128
active f a c t o r , these results suggest that bovine PBM are also capable of producing IL-2 in response to an antigen. Long-term cultured bovine c e l l s were used to test f o r IL-2 a c t i v i t y . Observations that long-term cultured c e l l s were IL-2 responsive T c e l l s are as follows:
1) responder c e l l s are PNA p o s i t i v e , non-specific esterase negative;
la p o s i t i v e (using monoclonal antibody H4), C5- (anti-monocyte), and B2g+ (anti-pan T c e l l ) ; 2) long-term cultured c e l l s are unresponsive to mitogen, medium alone, or conditioned medium from unstimulated cultures; 3) supernatants from a known IL-2-producing cell l i n e (MLA 144) supported p r o l i f e r a t i o n (Rabin et a l . , 1981); 4) s t r i c t requirement of IL-2 f or survival and p r o l i f e r a t i o n of these c e l l s . >95% were dead w i t h i n 72 hours.
I f these c e l l s were not provided with IL-2, Results using monoclonal antibody H4 suggest
these c e l l s are activated T c e l l s ( l a +) as reported f o r other T c e l l s growing in continuous c u l tu r e (Metzgar et a l . , 1979). IL-2-dependent c e l l s require only IL-2 as a signal f o r p r o l i f e r a t i o n because they have already seen antigen and have been expanded f or long periods with IL-2.
Since these c e l l s are mitogen unresponsive, they can be used to
determine a dose-dependent curve fo r growth f a c t o r - c o n t a i n i n g samples (Fig. 3).
Regression analysis indicates that the a c t i v i t y measured by
p r o l i f e r a t i o n of these c e l l s is a l i n e a r function of the concentration.
Under
these conditions, a c t i v i t y can be converted to units as reported by G i l l i s et al.
(1978).
sample.
This allows comparison between assays using a standard reference
Therefore, long-term bovine T c e l l s are defined as being IL-2-
dependent.
This dependence was also confirmed by t h e i r a b i l i t y to p r o l i f e r a t e
in response to supernatants from a known IL-2-producing cell l i n e , MLA 144 (Rabin et a l . , 1980) (Fig. 3).
These c e l l s can most l i k e l y be generated in
any species in which IL-2-dependent c e l l l i n e s have not been established. This opens up i n v e s t i g a t i o n s into economically important species, such as the bovine.
Conditions f o r IL-2 production and assay are a f i r s t
s p e c i f i c T cell populations in v i t r o .
step for growing
These c e l l s can then be used to
elucidate t h e i r role in vivo in disease problems s p e c i f i c to the species. In addition to maintaining bovine c e l l s , these supernatants were capable of sustaining the growth of murine c e l l s , CTLL-20 and HT-2 (Fig. 4). c e l l s have previously been shown to be IL-2 dependent.
These
Therefore, the large
volumes of supernatants that can be produced using bovine PBM may substitute f o r rat Con A supernatants in long-term murine cultures.
129
ACKNOWLEDGMENTS This work was supported in part by USDA-SEA Grant Nos. 901-15-145, 12-14-3001-758, 59-2552-1-2-038-0, USDA Dairy Forage Laboratory and by the College of A g r i c u l t u r a l and L i f e Sciences.
The authors wish to thank
Dr. Arden Hardie for providing c a t t l e used in t h i s study.
MLA 144 c e l l s were
the kind g i f t of Dr. Gary Fathman and monoclonal antibody B29 the g i f t William C. Davis.
of
We also wish to acknowledge Dr. Frank Fitch for supplying
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