Parameters of production and partial characterization of feline interleukin 2

Veterinary Immunology and Immunopathology, 19 (1988) 173-183 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

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P a r a m e t e r s of P r o d u c t i o n and P a r t i a l C h a r a c t e r i z a t i o n of F e l i n e I n t e r l e u k i n 2 RICHARD M. BAUER' and RICHARD G. OLSEN ''2'3

'Department of Veterinary Pathobiology, College of Veterinary Medicine, The Ohio State Univers ity, Columbus, OH 43210-1092 (U.S.A.) 2Comprehensive Cancer Center, The Ohio State University, 410 West 12th, Columbus, OH 43210 (U.S.A.) aTo whom correspondence should be addressed. (Accepted 7 March 1988)

ABSTRACT Bauer, R.M. and Olsen,R.G., 1988. Parameters of production and partial characterization of feline interleukin 2. Vet. Immunol. Immunopathol., 19: 173-183.

The conditions for the production of feline interleukin 2 (IL-2) from peripheral blood leukocytes (PBL) and splenocytes by concanavalin A (Con A) stimulation are described. Feline IL-2 was quantitated by measuring DNA synthesis in the murine IL-2-dependent cell line, CTLL-20. In addition, feline IL-2 was generated for the maintenance of long-term cultures of Con A-stimulated feline PBL and for biochemical characterization. Finally, IL-2 production was evaluated from the PBL of feline leukemia virus (FeLV)-infected cats. Con A at 9.6 ]~g/ml produced a plateau of peak IL-2 activity from 24 to 48 h following stimulation. The tumor promotor, phorbol myristic acetate, stimulated feline IL-2 production and enhanced Con A-stimulated feline IL°2 production. Fetal caff serum (FCS) was not required for IL2 production; however, FCS at 5% (v/v) allowed for maximal Con A-stimulated IL-2 production. Feline IL-2 generated from Con A-stimulated splenocytes migrated with an apparent molecular size of 13.7 to 23 kD by gel filtration chromatography and supported the proliferation of Con Aactivated feline PBL at a final concentration of 0.3 to 0.9 units/ml.

INTRODUCTION

Interleukin 2 (IL-2) is an immunoreguIatory lymphokine distinct from other factors based on biochemical characteristics and biologic activity (Aarden et al., 1979). Originally described as T cell growth factor (Morgan et al., 1976), IL-2 allows for proliferation and expansion of clonal T lymphocytes. That these T lymphocytes maintain both phenotype and immunologic function has been demonstrated (Baker et al., 1979; J.J. Farrar et al., 1980; Schreier et al.,, 1980; Gillis and Watson, 1981; Sredni and Schwartz, 1981; Lefrancois et al., 1984). 0165-2427/88/$03.50

© 1988 Elsevier Science Publishers B.V.

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In addition, IL-2 stimulates lymphokine-activated killer cells (Lotze et al., 1981; Cheever et al., 1982; Grimm et al., 1982; Rosenstein et al., 1984), enhances natural killer cell activity (Olabuenaga et al., 1983; Rook et al., 1983; Reddy et al., 1984), and induces the production of gamma interferon (W.L. Farrar et al., 1981). Furthermore, it is now recognized that IL-2 participates in the regulation of certain humoral immune responses (J.J. Farrar et al., 1982 ). Thus, lymphokine function, in particular IL-2, correlates with cell-mediated and certain humoral immune responses. In the cat, feline leukemia virus (FeLV) infection results in either a protective host response and clearance of viral infection or a progressive fatal wasting disease (reviewed in Rojko and Olsen, 1984; Olsen et al., 1986). In the progressive disease state, animals die of opportunistic secondary infection, attributed to FeLV-mediated immunosupression (Hardy et al., 1976; Hardy, 1980). Furthermore, it is predicted that a majority of cats recovering from FeLV infection remain immunocompromised and susceptible to secondary disease, including neoplasia (Essex et al., 1975). This may be a consequence of latent FeLV infection and reactivation; however, it is unclear what co-factors are involved in this process (Rojko et al., 1982a). A clinically similar scenario is described in human acquired immunodeficiency syndrome (AIDS) resulting from infection with the human immunodeficiency virus (HIV) (reviewed in Pinching and Weiss, 1986). In this case, opportunistic fatal infection is also attributed to retrovirus-mediated immunosuppression. Central to this immunosuppression, an IL-2 deficiency has been described (Cibannu et al., 1983; Murray et al., 1984). Whether this occurs in FeLV-infected cats is unknown; however, an IL-2 deficiency may account for disease progression and the failure of the animal to mount an effective immune response during FeLV infection. Rodent and human IL-2 have been purified (Smith, 1980; Smith et al., 1980; Watson and Mochiznki, 1980; Ruscetti and Gallo, 1981) and the conditions for IL-2 production have been described in chicken (Schnetzler et al., 1983), canine (Daeman et al., 1984), ovine (Ellis and DeMartini, 1985), porcine (Gasbarre et al., 1984), bovine (Baker and Knoblock, 1982; Namen and Magnuson, 1984; Brown and Grab, 1985), and non-human primate (Tatsumi and Yabe, 1986) systems. The induction of feline IL-2 has recently been described (Tompkins et al., 1987); however, feline IL-2 has not been quantitated, nor has the molecule been characterized. Furthermore, cell-mediated immunity is poorly defined in the cat, as cell surface markers and cloned cell lines for the study of feline immunology are unavailable. Therefore, it is anticipated that the findings of this study will provide the conditions necessary for the characterization of feline IL-2 and, as in the mouse and man, lead to a better understanding of feline immunology, especially as it pertains to the host response in infectious disease.

175 METHODS

Animals All cats used in these experiments were from The Ohio State University, Department of Veterinary Pathobiology specific-pathogen-freebreeding colony.

Tissue culture media and reagents RPMI 1640 (Gibco, Grand Island, NY) was supplemented with 1.0 mM Lglutamine (Difco, Detroit, MI ), 50 units/ml penicillin, 25 pg/ml streptomycin (Gibco), and 5 × 10 -~ 2-mercaptoethanol. Fetal calf serum (FCS; Sterile Systems, Logan, UT) was added to 5% (v/v) or as indicated. Concanavalin A (Con A; Sigma Chemical Co., St. Louis, MO ) was diluted to 32 ttg/ml in tissue culture media prior to use. Phorbol myristic acetate (PMA; Sigma ) was diluted to 100/zg/ml in tissue culture media and stored at - 70 ° C.

Preparation of peripheral blood leukocytes and splenocytes Peripheral blood leukocytes (PBL) and splenocytes were obtained and prepared as previously described (Cockerell et al., 1975; Rojko et al., 1982b). Briefly, PBL were separated by centrifugation using ficoll-hypaque (Pharmacia Fine Chemicals, Piscataway, NJ) and the interface mononuclear cells were washed in tissue culture media and resuspended to 1 × 107 viable cells/ ml. Splenocytes were cleared of red blood cells by 0.15 M NH4C1 treatment, washed in tissue culture media, and resuspended to 1 × 107 viable cells/ml.

Interleukin 2 production Feline IL-2 was produced at 37 °C in a humidified 5% CO2 incubator. Splenocyte and PBL cultures were of 5 or 10 ml volumes, in either 6-well tissue culture plates (Costar, Cambridge, MA) or in T-25 flasks (Corning, Corning, NY). Large-batch preparation of IL-2 was performed in 50 or 100 ml volumes in T-75 or T-150 flasks (Corning). Spent culture medium, the putative IL-2containing lymphocyte-conditioned medium (Ly-CM), was collected at various times after culture initiation, filtered (0.45 ttm), and stored at - 7 0 ° C until assayed for IL-2 activity. Feline PBL or splenocytes were stimulated with Con A at final concentrations of 1.6, 3.2, 6.4, or 9.6 ttg/ml. The initial cell density was from 0.5 to 3 × 106 viable cells/ml and FCS supplements were from 0 to 10% (v/v), as indicated. The effect of PMA on IL-2 production was tested in the presence and absence of Con A. PMA was added to a final concentration of 0.01 to 1.0/zg/ml, and the Ly-CM collected and stored as above.

176

Long-term culture of feline lymphocytes in IL-2-containing medium Feline PBL, which had been stimulated with Con A for 48 or 72 h, were maintained in culture media supplemented with 5% FCS and feline IL-2 (0.3 to 0.9 units/mt). In brief, following Con A-stimulation, non-adherent cells were washed in culture media and adjusted to a cell density of 1 X 106 cells/ml. Every 2 to 3 days, fresh medium containing IL-2 was added and the cell density readjusted to 1 X 10 ~ cells/ml. Control cultures contained no Ly-CM and cell proliferation was determined by measuring cell density.

Interleukin 2 microassay Preliminary experiments indicated tht feline Ly-CM stimulates the proliferation of the murine IL-2-dependent cell line, CTLL-20. Therefore, this line was utilized to quantitate IL-2 activity as described (Gillis et al., 1978) with minor modifications (Orosz et al., 1985). Briefly, CTLL-20 cells (100 zl at 5 X 105 cells/ml) were added to serially diluted Ly-Cm samples (100 ]ll in triplicate) and cultured at 37°C in a humidified, 5% CO2 incubator for 24 h. During the final 6 h of culture 0.5 ]lCi of tritiated thymidine (3H-TdR; New England Nuclear, Boston, MA) was added. The cultures were harvested onto glass filter strips with a semi-automatic sample harvester and 3H-TdR incorporation determined by liquid scintillation counting. Using a standard EL-4derived IL-2 preparation (Orosz et al., 1985), assigned a value of 100 units/ ml, units of IL-2 in each feline Ly-CM sample were calculated using the following formula (J.J. Farrar et al., 1980): sample dilution of cpm (50% max.) X 100 units/ml standard dilution of cpm (50% max.) - - u n i t s / m l in a sample.

Ammonium sulfate fractionation, gel filtration, and ion-exchange chromatography The Ly-CM from cultures of splenocytes stimulated with Con A was sequentially fractionated with ammonium sulfate. Briefly, solid ammonium sulfate was added to 50% saturation and stirred continuously for 1 h at 4 °C. Precipitates were collected by centrifugation at 10 0 0 0 x g for 20 min. The resulting supernatant was brought to 75% saturation with ammonium sulfate and the precipitate prepared as above. Each fraction was dialyzed exhaustively against 0.15 M phosphate-buffered saline (PBS), pH 7.1, tested for IL-2 activity, and then applied to a Superose 12, HR 10/20 gel filtration column (Pharmacia) equilibrated with PBS. For ion-exchange chromatography, IL-2-containing

177 fractions were applied to the Mono Q, HR 5/5 anion exchange column (Pharmacia) equilibriated with 0.05 M Tris-HC1, pH 8.0. Chromatography was controlled using the Pharmacia FPLC system with the LCC-500. Gel filtration was performed at 0.25 ml/min and a gradient from 0 to 100% NaC1 in 0.5 M Tris-HC1 was developed at 1.0 ml/min for ion-exchange chromatography. The effluents from the columns were filter sterilized and tested for IL-2 activity in the IL-2 microassay. RESULTS

Optimization of IL-2 production conditions Because cell density, Con A concentration, and FCS concentration are known to alter IL-2 production in other systems, these parameters were tested independently. First, an optimal Con A concentration was established. Feline PBL or splenocytes, at 1 × 106 cells/ml, were incubated with and without Con A at various concentrations in the presence of FCS at 5% (v/v) for 48 h. The LyCM from cultures without Con A contained no detectable IL-2 activity; however, IL-2 activity was detected in the Ly-CM of cultures stimulated with Con A, demonstrating that Con A stimulates IL-2 production and that 9.6/~g Con A/ml stimulates IL-2 production maximally. Next, feline splenocytes or PBL were stimulated with Con A at 9.6/lg/ml, and cell number or FCS concentration were varied. It was found that as the cell density was increased from 0.5 × 106 to 2 × 106 cell/ml, IL-2 activity measured in the Ly-CM increased. Changes in FCS concentration similarly affected the production of IL-2, with reduced levels of FCS resulting in diminished IL-2 production. However, IL-2 activity was detectable in the Ly-CM of cultures stimulated with Con A in the absence of FCS. Thus, IL-2 was maximally produced at a cell density of 2 × 106 cells/ml, in the presence of 5% (v/v) FCS, with 96/tg/ml Con A.

Kinetics of IL-2 production In order to further examine the conditions of feline IL-2 production, the generation of IL-2 over ~ime was determined by following the kinetics of the appearance of IL-2 in the Ly-CM. In this case, the kinetics of IL-2 production were determined by stimulating both feline PBL and splenocytes with Con A. The results of these experiments demonstrate that IL-2 activity was always present in the Ly-CM by 18 h after culture initiation (Fig. 1 ). However, when optimum Con A (9.6 pg/ml) concentrations were used, splenocytes produced IL-2 as early as 6 h after culture initiation. The IL-2 produced from PBL varied with time, with maximal activity detected from 18 to 48 h. In contrast, splenocyte cultures produced IL-2 with a plateau of peak activity from 24 to 48 h.

178

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Fig. 1. Con A-stimulated feline IL-2 production. PBL (open columns) and splenocytes (hatched columns) were cultured at an initial cell density of 1 × 106 cells/ml in RPMI-1640 containing 5% (v/v) FCS and Con A at 9.6 #g/ml. Ly-CM was collected and IL-2 activity determined in the IL2 microassay. TABLE 1 Effect of PMA on feline IL-2 production Con A~

PMA

(+ / - )

(#g/ml)

+ + + + -

--

1.0 0.1 0.01 0 1.0 0.1 0.01

Units/ml ILo2 activity +_S.D. b Spleen

PBL

21.5 +_6.64 19.72 4-7.2 18.9 +-7.51 8.23 _1.33 5.75 +_2.15 3.56 +_1.2 0.023 __+0.005

0.76 +0.72 0.645+_0.716 0.82 +-0.86 1.16 +-1.35 0.0 0.01 +_0.001 0.022 +_0.003

aPBL and splenocytes, at 1 X 106 cells/ml in media containing 5% (v/v) FCS, were treated with PMA in the presence ( + ) or absence ( - ) of Con A at 9.6/~g/ml. t~IL-2 activity in the Ly-CM of 48-h cultures was determined in the IL-2 microassay.

Effect of PMA on IL-2 production T h e e f f e c t o f P M A o n feline I L - 2 p r o d u c t i o n w a s m e a s u r e d f o l l o w i n g 36 h o f c u l t u r e , in t h e p r e s e n c e a n d a b s e n c e o f C o n A. A t a cell d e n s i t y o f 1 × 106 c e l l s / m l , w i t h 5% (v/v) F C S , P M A e n h a n c e d I L - 2 p r o d u c t i o n a t all c o n c e n t r a t i o n s t e s t e d , a n d p e a k I L - 2 p r o d u c t i o n w a s o b s e r v e d at a P M A c o n c e n t r a t i o n o f 1 . 0 / t g / m l ( T a b l e 1 ). I n a d d i t i o n , P M A s t i m u l a t e d I L - 2 p r o d u c t i o n in t h e a b s e n c e o f C o n A.

Continuous culture of feline lymphocytes C o n A - s t i m u l a t e d feline P B L w e r e m a i n t a i n e d in c u l t u r e f o r m o r e t h a n 60 d a y s ( > 20 p a s s a g e s ) b y I L - 2 s u p p l e m e n t a t i o n . F o l l o w i n g 48 o r 72 h o f C o n A

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Fig. 2. Growth profile of Con A-stimulated P B L m a i n t a i n e d in culture with feline IL-2. Con Astimulated P B L were cultured at an initial cell density of 1 X 106 cells/ml in feline IL-2 (0.3 to 0.9 u n i t s / m l ) - s u p p l e m e n t e d media. Cell density was determined daily a n d cells were recultured in fresh IL-2-containing media every t h i r d day at a n initial cell density of 1 X 106 cells/ml. Open triangles were control cultures, incubated without a n IL-2 source.

stimulation, non-adherent P B L were collected and recultured in IL-2-containing media (0.3 to 0.9 u n i t s / m l feline IL-2). The growth profile of these cells is demonstrated in Fig. 2. By adjusting the cell density every 2 to 3 days and adding fresh IL-2-supplemented media, proliferation and expansion of these cells was achieved. Failure to provide IL-2 in the media resulted in cell death by 48 to 72 h following IL-2 starvation.

Biochemical characterization of feline IL-2 Feline IL-2-containin~ supernatants from cultures of 1X 106 splenocytes/ ml stimulated with Con A at 6.4 or 9.6 ~g/ml were concentrated 100- to 200fold by sequential ammonium sulfate precipitation. The ammonium sulfateconcentrated IL-2 preparations were then applied to a molecular sizing column (Superose 12, HR 10/20; Pharmacia) and IL-2 activity was monitored in the effluent (Fig. 3). IL-2 activity eluted with an apparent molecular weight of 13.7 to 23 kD. During ion-exchange chromatography, feline IL-2 failed to bind to the Mono Q anion exchange column under the conditions used and eluted prior to the NaC1 gradient, suggesting that feline IL-2 has a net negative charge.

180

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Fig. 3. GelfiltrationchromatographyoffelineIL-2. Molecularweightstandardswerebovineserum albumin (67 kD), ovalbumin(43 kD ), chymotrypsinogen(23 kD), and ribonucleaseA ( 13.7kD). Open bars representfractionswhereIL-2 activitywas recovered. DISCUSSION In these studies, the conditions for the production of feline IL-2 were examined by optimizing lectin (Con A) concentration, cell concentration, and fetal calf serum requirements. Partial biochemical characterization and purification of feline IL-2 was performed by ammonium sulfate fractionation, gel filtration, and ion-exchange chromatography. In addition, the kinetics of feline IL-2 production and the effect of the phorbol ester PMA on IL-2 production were examined. For quantitation of IL-2 activity, it was found that feline IL-2 stimulated the proliferation of the murine IL-2-dependent cell line, CTLL-20, and therefore, the IL-2 microassay of Gilles et al. (1978) was used. Further, IL-2 activity was demonstrated by measuring the ability of the preparations to support the long-term proliferation of mitogen-stimulated feline PBLs. Peak IL-2 activity was obained from PBL and splenocytes stimulated at 2×108 cells/ml with 9.6 #g/ml Con A in the presence of 5% (v/v) FCS. In addition, the phorbol ester PMA could be used to stimulate IL-2 production as well as enhance Con A-stimulated IL-2 production. FCS was not required for IL-2 production, although optimum conditions for IL-2 production required FCS supplements. The kinetics of IL-2 production indicate that a plateau of maximal IL-2 activity can be obtained from 24 to 48 h after Con A stimulation, with splenocyte-derived IL-2 and PBL-derived IL-2 reaching maximum levels at 36 and 48 h, respectively. These results are consistent with the results of others for the production of IL-2 by rodent (Gillis et al., 1980), porcine (Gasbarre et al., 1984), bovine (Baker and Knoblock, 1982), and human (Alvarez et al., 1979 ) lymphocytes. That is, measurable IL-2 activity was found as early

181 as 6 h after Con A stimulation and IL-2 activity declined after 48 h of stimulation. It should be noted, however, t h a t the level of IL-2 activity produced by Con A-stimulated P B L was variable from animal to animal. This variability may be explained by differences in P B L composition and the nature of variability in out-bred species (Alvarez et al., 1979; B o n n a r d et al., 1980). Feline IL-2 activity was concentrated from Con A-stimulated splenocyte LyCM by a m m o n i u m sulfate precipitation and applied to a gel filtration column. IL-2 activity eluted with a molecular size in the range of 13.7 to 43 kD, consist e n t with the size of the IL-2 molecule of other animals and m a n (Gillis et al., 1980; J.J. Farrar et al., 1982; Daeman et al., 1984 Tatsumi and Yabe, 1986). The continuous culture of feline Con A-activated P B L s was achieved by IL2 supplementation and, while these IL-2 preparations allowed for profileration and expansion of IL-2-responsive PBL, these cells have not been characterized. However, the methodology for IL-2 production and the continuous culture of feline P B L is described, and this may be instrumental for understanding immunoregulatory functions and retroviral-induced disease in the feline model. ACKNOWLEDGEMENTS This work was supported in part by grants from N I H - N C I CA-31547, CA30338, and CA-1608.

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