- Email: [email protected]
[47] Procedures for studying binding of interferon to mouse cells
[47]
BINDING OF MOUSE INTERFERON
321
lated Hu-IFN-y was obtained with a specific activity of 11,000 Ci/mmol. ~ This is a specific activity at least 10- to 100-fold higher than reported for other ~2SI-labeled interferons. This specific activity corresponds to the incorporation of an average of about two molecules of phosphate per molecule of Hu-IFN-y. The high specific activity of 32p-labeled Hu-IFN-v and its convenient preparation make it a useful reagent for studying the receptor for human immune interferon. Acknowledgments We thank Dr. Jerome A. Langerfor carefulreviewof this text and SophieCuber for her assistance in the preparation of this manuscript. NOTE ADDED IN PROOF: The specific radioactivity reported here for 3-'P-labeled Hu-IFNy ( I 1,000 Ci/mmol) is based on the antiviral activity (as determined by the cytopathic effect inhibition assay measured with vesicular stomatitis virus and WISH cells) and the specific activity of Hu-IFN-y (2 x I117units/mg); all data in the text were based on this value. The specific radioactivity based on the total input protein was calculated to be 3800 Ci/mmol. This is the minimum value since the protein concentration in the phosphorylated sample was too low to be measured. Based on this minimum value, the parameters were calculated to be as follows: Kj = 4.3 x 10 ~ M, Rt 97 pM (5200 receptors/cell), and a total of about one molecule of phosphate incorporated per molecule of Hu-IFN-T. It is likely that the true wdues tbr these parameters are delimited by these values and those given in the texl.
[47] P r o c e d u r e s for S t u d y i n g B i n d i n g o f I n t e r f e r o n to M o u s e Cells
By
MICHEL AGUET
The aim of binding experiments is to reveal a specific ligand receptor interaction and thereby provide insight into the early events that initiate a ligand-specific biological response. Therefore, experimental conditions to determine binding site saturation and specific competitive binding inhibition I on entire cells have to be compatible with those required to elicit a relevant response. Further experimental restrictions arise from the requirements for interpreting binding data, notably with a view to applying the law of mass action. Isolated purified receptors would provide a more appropriate tool for accurate determination of association and dissociation constants than whole cells. Numerous functional aspects of receptor P. Cuatrecasas and M. D. Hollenberg, Adv. Protein Chem. 30, 251 (1976).
METHODS IN ENZYMOLOGY.VOL. 119
Copyright © 1986by Academic Press. Inc. All rightsof reproduction in any form reserved.
322
INTERFERON RECEPTORAND UPTAKEOF INTERFERON
[47]
binding, however, require analysis of binding properties on intact cells. The procedures described below are restricted to the use of living cells. Media and Reagents RPMI-1640 tissue culture medium (Gibco), containing 10 mM HEPES buffer, supplemented with 10% fetal calf serum (KC Biological) Dulbecco's modified Eagle's minimal essential medium (Gibco), containing 10 mM HEPES buffer, supplemented with 10% fetal calf serum (KC Biological) Phosphate buffered saline, pH 7.4 (PBS): 0.137 M NaCI, 10 mM Na2HPO4, 3 mM KH2PO4 NaJ251 (New England Nuclear), in pH 8-10 aqueous solution (reductant free), carrier free (about 17 Ci/mg), 100-200 mCi/ml Lactoperoxidase (Sigma), from bovine milk, lyophilized powder, 80100 units/mg protein H202, 30% (8.8 M) Sodium azide, 0.25 M Affi-Gel-10 (Bio-Rad) Sephadex G-25, prepacked disposable columns PD-10 (Pharmacia) Labeling of Mouse C243 Cell Interferon with 12sI Highly purified mouse C243 cell interferon induced by Newcastle disease virus comprises three major molecular weight speciesfl As indicated by NH2-terminal homologies and common antigenic properties with human interferons, the components with molecular weights of 24,000 and 30,000, which represent up to 90% of the total interferon, belong to the fl type, whereas the 19,000 molecular weight species behaves as IFN-a. 3 For convenience this heterogeneous material is referred to as interferon in this chapter and the antiviral activity is indicated in terms of reference units/ ml. According to our own experience lactoperoxidase catalyzed iodination4 of this interferon yields the best results with regard to both iodine incorporation and recovery of biological activity. Preparation of Lactoperoxidase Coupled to Sepharose Beads About 0.5 ml of wet Affigel-10 freshly washed with ice cold distilled water on a small fritted glass funnel is added to 0.5 ml of 0.1 M sodium 2 M. Aguet and B. Blanchard, Virology 115, 249 (1981). 3y. Kawade, M. Aguet, and M. G. Tovey, Antiviral Res. 2, 155 (1982). 4 G. S. David and R. A. Reisfeld,Biochemistry 13, 1014(1974).
[47]
BINDING
OF MOUSE INTERFERON
323
phosphate buffer (pH 7.0) containing 400/zg lactoperoxidase per ml as = 114). determined by optical density measurement at 412 nm (]tTl ",,~412m M nm The gel is kept in suspension overnight at 4° and subsequently washed with 20 ml of 1 M NaCI and excess PBS prior to resuspension in 10 ml PBS. Under these conditions lactoperoxidase is entirely coupled to the Sepharose to give a final concentration of 20 /zg/ml of gel suspension; unreacted coupling sites of the gel are inert after this incubation period. Provided that the described steps are carried out under sterile conditions this gel suspension is stable at 4° for at least a year. (Addition of sodium azide as preservative would result in inactivation of the enzyme.) Labeling Procedure Two millicuries of fleshly generated carrier-flee J25I (100 mCi/ml) is added to 100/xl of an interferon solution in PBS adjusted to 100 ~g protein per ml. Then 20/zl of Affigel-10-1actoperoxidase gel suspension is added prior to initiation of the iodination which is done by addition of 10/zl of diluted H2Oz to give a final concentration of 10-4 M. After 20 rain incubation at room temperature under continuous agitation the reaction is stopped by addition of 20/zl of 0.25 M sodium azide. Separation of free iodine is carried out on a prepacked Sephadex G-25 column equilibrated with PBS containing 1% fetal calf serum. Labeled interferon is eluted in a pool of 2.5 m! and subsequently diluted 8-fold in RPMI-1640 containing 10% fetal calf serum. Aliquots of 1 ml are kept frozen at -70 °. Under these conditions the material is stable for approximately 2 months; the loss of specific radioactivity follows the decay of J25I. This procedure yields iodinated interferon with a specific radioactivity of approximately 50 /.tCi//zg of protein while 25 to 50% of the biological activity is recovered. Procedure for Studying Binding to Cells Growing in Suspension The expression of mouse interferon receptors depends on a rather rapid turnover rate 2 and is markedly increased on exponentially growing cells as compared to cells resting at saturation (unpublished observation). To yield reproducible results binding experiments should be carried out on cells which have been cultured for at least 8 hr. Changes in cell culture conditions such as temporary temperature shifts or centrifugation of the cells result in decreased receptor expression with a latency of 1-2 hr upon reincubation at 370.2 Therefore, monitoring J25I-labeled interferon binding at 37° requires careful addition of labeled interferon to precultivated samples of cell suspensions while avoiding any perturbation of culture condi-
324
INTERFERON RECEPTOR AND UPTAKE OF INTERFERON
[47]
tions. In contrast receptor expression on cells kept at 4° in tissue culture medium remains virtually unchanged over at least 12 hr. Cell density is a critical variable in experiments designed to calculate constants defined by mass action law. It is only below a critical limit that the amount of ligand bound per cell at a given ligand concentration becomes independent of cell density. L2 This critical density or receptor concentration corresponds to the binding constant Kd and has to be verified experimentally. In the case of mouse L 1210 cells which express about 103 receptors per cell this critical threshold value corresponds to about 107 cells per ml. 2 In experiments carried out under culture conditions at 37° cells are incubated with uSI-labeled interferon in tissue culture flasks at cell densities compatible with exponential growth (i.e., 0.5-1.5 × 106 cells per ml for most lymphoid cells in nonagitated culture). Cell associated radioactivity can be determined simply by washing three times at 200 g for 5 min with ice cold culture medium containing 1% fetal calf serum and subsequently transferring the cells into counting vials. Dissociation of specifically bound ligand during this procedure is negligible. Multiple samples can be washed efficiently by transferring the cells into U-bottomed microtiter plates (Greiner) with 1-2 x l 0 6 cells per well. The plates are centrifuged at 200 g for 5 min in the cold and the supernatants discarded. Prior to addition of fresh medium the cell pellets are disrupted by stirring on a vortex. Before the last centrifugation the cells are transferred by means o f a multichannel pipette into U-bottomed minivials placed into the wells of a fiat bottomed microtiter plate (Greiner) that serves as a tray. Interferon incubation at 4° can be carried out directly in U-bottomed microtiter plates at a cell density of 0.5-1.0 x 107 cells per ml with 200/zl of cell suspension per well. To determine nonspecific binding, specific binding sites have to be saturated with unlabeled interferon. Therefore, an excess of interferon in a final concentration of 5 × 104 units/ml is added simultaneously with labeled interferon. Washing cells by repeated centrifugation to remove unbound ligand is certainly more time consuming than other commonly applied single step procedures such as centrifugation on sucrose gradients or oil. However, it has the advantage of maintaining the cells viable in a physiological medium and therefore of allowing recultivation or consecutive incubations with different ligands (e.g., to reveal specifically bound interferon by means of anti-interferon antibodiesS).
5 H. Arnheiter, M. Ohno, M. Smith, B. Gutte, and K. C. Zoon, Proc. Natl. Acad. Sci. U.S.A. 80, 2539 (1983).
[47]
BINDING OF MOUSE INTERFERON
325
Procedure for Studying Binding to Cells Growing as Monolayer Cultures Binding of ~25I-labeled interferon to monolayer cells is best carried out in Costar 3524 24-well plates on exponentially growing cells that reach confluency. 6 To improve cell adherence the plates are pretreated with a 1% solution of gelatine in PBS which is applied for a few minutes at room temperature and subsequently removed prior to drying the plates under sterile conditions. For most monolayer cells beginning confluency corresponds to approximately 105 cells per well. To avoid perturbation of culture conditions interferon incubation at 37° is carried out in a waterbath with prewarmed media and interferon samples. After incubation cells are washed three times by addition of 1.0 ml per well of ice cold culture medium which should remain in contact with the cells for about 1 rain prior to being removed. Subsequently, cell-associated radioactivity is determined by solubilization of the cells with 0.5 ml 1 N NaOH for 1 hr at 37 ° and transferring the lysates into counting vials. Nondisplaceable binding is determined as described in the previous section. Interpretation of Binding D a t a Binding data are most commonly analyzed by mass action law to estimate binding affinity and number of binding sites involved.W The limitations have been already emphasized elsewhere, 8,9 yet two aspects should be briefly recalled. As stated above cell density is a critical parameter for interpreting binding data by mass action law. To define a ligandreceptor interaction, mass action law requires that the reaction be reversible, bimolecular, and in an equilibrium state. Obviously these criteria are more convincingly fulfilled at low temperature, whereas at 37° dynamic processes such as receptor turnover, ligand internalization and degradation, or ligand induced modulation of receptor expression represent additional variables difficult to define by mass action law. Therefore, it is important to distinguish between experiments designed to determine or compare physical binding properties and experiments conceived to investigate dynamic events involved or induced by receptor binding under physiological conditions. To our knowledge a generally valid mathematical model to describe these dynamic processes has not yet been proposed. 6 M. Aguet, 1. Gresser, A. G. Hovanessian, M. T. Bandu, B. Blanchard, and D. Blangy, Virology 114, 585 (1981). 7 G. Scatchard, Ann. N . Y . Acad. Sci. 51, 660 (1949). 8 I. M. Klotz, Science 217, 1247 (1982). 9 M. Aguet and K. E. Mogensen, in " I n t e r f e r o n " (I. Gresser, ed.), Vol. 5, p. 1. Academic Press, New York, 1984.
BINDING OF MOUSE INTERFERON
321
lated Hu-IFN-y was obtained with a specific activity of 11,000 Ci/mmol. ~ This is a specific activity at least 10- to 100-fold higher than reported for other ~2SI-labeled interferons. This specific activity corresponds to the incorporation of an average of about two molecules of phosphate per molecule of Hu-IFN-y. The high specific activity of 32p-labeled Hu-IFN-v and its convenient preparation make it a useful reagent for studying the receptor for human immune interferon. Acknowledgments We thank Dr. Jerome A. Langerfor carefulreviewof this text and SophieCuber for her assistance in the preparation of this manuscript. NOTE ADDED IN PROOF: The specific radioactivity reported here for 3-'P-labeled Hu-IFNy ( I 1,000 Ci/mmol) is based on the antiviral activity (as determined by the cytopathic effect inhibition assay measured with vesicular stomatitis virus and WISH cells) and the specific activity of Hu-IFN-y (2 x I117units/mg); all data in the text were based on this value. The specific radioactivity based on the total input protein was calculated to be 3800 Ci/mmol. This is the minimum value since the protein concentration in the phosphorylated sample was too low to be measured. Based on this minimum value, the parameters were calculated to be as follows: Kj = 4.3 x 10 ~ M, Rt 97 pM (5200 receptors/cell), and a total of about one molecule of phosphate incorporated per molecule of Hu-IFN-T. It is likely that the true wdues tbr these parameters are delimited by these values and those given in the texl.
[47] P r o c e d u r e s for S t u d y i n g B i n d i n g o f I n t e r f e r o n to M o u s e Cells
By
MICHEL AGUET
The aim of binding experiments is to reveal a specific ligand receptor interaction and thereby provide insight into the early events that initiate a ligand-specific biological response. Therefore, experimental conditions to determine binding site saturation and specific competitive binding inhibition I on entire cells have to be compatible with those required to elicit a relevant response. Further experimental restrictions arise from the requirements for interpreting binding data, notably with a view to applying the law of mass action. Isolated purified receptors would provide a more appropriate tool for accurate determination of association and dissociation constants than whole cells. Numerous functional aspects of receptor P. Cuatrecasas and M. D. Hollenberg, Adv. Protein Chem. 30, 251 (1976).
METHODS IN ENZYMOLOGY.VOL. 119
Copyright © 1986by Academic Press. Inc. All rightsof reproduction in any form reserved.
322
INTERFERON RECEPTORAND UPTAKEOF INTERFERON
[47]
binding, however, require analysis of binding properties on intact cells. The procedures described below are restricted to the use of living cells. Media and Reagents RPMI-1640 tissue culture medium (Gibco), containing 10 mM HEPES buffer, supplemented with 10% fetal calf serum (KC Biological) Dulbecco's modified Eagle's minimal essential medium (Gibco), containing 10 mM HEPES buffer, supplemented with 10% fetal calf serum (KC Biological) Phosphate buffered saline, pH 7.4 (PBS): 0.137 M NaCI, 10 mM Na2HPO4, 3 mM KH2PO4 NaJ251 (New England Nuclear), in pH 8-10 aqueous solution (reductant free), carrier free (about 17 Ci/mg), 100-200 mCi/ml Lactoperoxidase (Sigma), from bovine milk, lyophilized powder, 80100 units/mg protein H202, 30% (8.8 M) Sodium azide, 0.25 M Affi-Gel-10 (Bio-Rad) Sephadex G-25, prepacked disposable columns PD-10 (Pharmacia) Labeling of Mouse C243 Cell Interferon with 12sI Highly purified mouse C243 cell interferon induced by Newcastle disease virus comprises three major molecular weight speciesfl As indicated by NH2-terminal homologies and common antigenic properties with human interferons, the components with molecular weights of 24,000 and 30,000, which represent up to 90% of the total interferon, belong to the fl type, whereas the 19,000 molecular weight species behaves as IFN-a. 3 For convenience this heterogeneous material is referred to as interferon in this chapter and the antiviral activity is indicated in terms of reference units/ ml. According to our own experience lactoperoxidase catalyzed iodination4 of this interferon yields the best results with regard to both iodine incorporation and recovery of biological activity. Preparation of Lactoperoxidase Coupled to Sepharose Beads About 0.5 ml of wet Affigel-10 freshly washed with ice cold distilled water on a small fritted glass funnel is added to 0.5 ml of 0.1 M sodium 2 M. Aguet and B. Blanchard, Virology 115, 249 (1981). 3y. Kawade, M. Aguet, and M. G. Tovey, Antiviral Res. 2, 155 (1982). 4 G. S. David and R. A. Reisfeld,Biochemistry 13, 1014(1974).
[47]
BINDING
OF MOUSE INTERFERON
323
phosphate buffer (pH 7.0) containing 400/zg lactoperoxidase per ml as = 114). determined by optical density measurement at 412 nm (]tTl ",,~412m M nm The gel is kept in suspension overnight at 4° and subsequently washed with 20 ml of 1 M NaCI and excess PBS prior to resuspension in 10 ml PBS. Under these conditions lactoperoxidase is entirely coupled to the Sepharose to give a final concentration of 20 /zg/ml of gel suspension; unreacted coupling sites of the gel are inert after this incubation period. Provided that the described steps are carried out under sterile conditions this gel suspension is stable at 4° for at least a year. (Addition of sodium azide as preservative would result in inactivation of the enzyme.) Labeling Procedure Two millicuries of fleshly generated carrier-flee J25I (100 mCi/ml) is added to 100/xl of an interferon solution in PBS adjusted to 100 ~g protein per ml. Then 20/zl of Affigel-10-1actoperoxidase gel suspension is added prior to initiation of the iodination which is done by addition of 10/zl of diluted H2Oz to give a final concentration of 10-4 M. After 20 rain incubation at room temperature under continuous agitation the reaction is stopped by addition of 20/zl of 0.25 M sodium azide. Separation of free iodine is carried out on a prepacked Sephadex G-25 column equilibrated with PBS containing 1% fetal calf serum. Labeled interferon is eluted in a pool of 2.5 m! and subsequently diluted 8-fold in RPMI-1640 containing 10% fetal calf serum. Aliquots of 1 ml are kept frozen at -70 °. Under these conditions the material is stable for approximately 2 months; the loss of specific radioactivity follows the decay of J25I. This procedure yields iodinated interferon with a specific radioactivity of approximately 50 /.tCi//zg of protein while 25 to 50% of the biological activity is recovered. Procedure for Studying Binding to Cells Growing in Suspension The expression of mouse interferon receptors depends on a rather rapid turnover rate 2 and is markedly increased on exponentially growing cells as compared to cells resting at saturation (unpublished observation). To yield reproducible results binding experiments should be carried out on cells which have been cultured for at least 8 hr. Changes in cell culture conditions such as temporary temperature shifts or centrifugation of the cells result in decreased receptor expression with a latency of 1-2 hr upon reincubation at 370.2 Therefore, monitoring J25I-labeled interferon binding at 37° requires careful addition of labeled interferon to precultivated samples of cell suspensions while avoiding any perturbation of culture condi-
324
INTERFERON RECEPTOR AND UPTAKE OF INTERFERON
[47]
tions. In contrast receptor expression on cells kept at 4° in tissue culture medium remains virtually unchanged over at least 12 hr. Cell density is a critical variable in experiments designed to calculate constants defined by mass action law. It is only below a critical limit that the amount of ligand bound per cell at a given ligand concentration becomes independent of cell density. L2 This critical density or receptor concentration corresponds to the binding constant Kd and has to be verified experimentally. In the case of mouse L 1210 cells which express about 103 receptors per cell this critical threshold value corresponds to about 107 cells per ml. 2 In experiments carried out under culture conditions at 37° cells are incubated with uSI-labeled interferon in tissue culture flasks at cell densities compatible with exponential growth (i.e., 0.5-1.5 × 106 cells per ml for most lymphoid cells in nonagitated culture). Cell associated radioactivity can be determined simply by washing three times at 200 g for 5 min with ice cold culture medium containing 1% fetal calf serum and subsequently transferring the cells into counting vials. Dissociation of specifically bound ligand during this procedure is negligible. Multiple samples can be washed efficiently by transferring the cells into U-bottomed microtiter plates (Greiner) with 1-2 x l 0 6 cells per well. The plates are centrifuged at 200 g for 5 min in the cold and the supernatants discarded. Prior to addition of fresh medium the cell pellets are disrupted by stirring on a vortex. Before the last centrifugation the cells are transferred by means o f a multichannel pipette into U-bottomed minivials placed into the wells of a fiat bottomed microtiter plate (Greiner) that serves as a tray. Interferon incubation at 4° can be carried out directly in U-bottomed microtiter plates at a cell density of 0.5-1.0 x 107 cells per ml with 200/zl of cell suspension per well. To determine nonspecific binding, specific binding sites have to be saturated with unlabeled interferon. Therefore, an excess of interferon in a final concentration of 5 × 104 units/ml is added simultaneously with labeled interferon. Washing cells by repeated centrifugation to remove unbound ligand is certainly more time consuming than other commonly applied single step procedures such as centrifugation on sucrose gradients or oil. However, it has the advantage of maintaining the cells viable in a physiological medium and therefore of allowing recultivation or consecutive incubations with different ligands (e.g., to reveal specifically bound interferon by means of anti-interferon antibodiesS).
5 H. Arnheiter, M. Ohno, M. Smith, B. Gutte, and K. C. Zoon, Proc. Natl. Acad. Sci. U.S.A. 80, 2539 (1983).
[47]
BINDING OF MOUSE INTERFERON
325
Procedure for Studying Binding to Cells Growing as Monolayer Cultures Binding of ~25I-labeled interferon to monolayer cells is best carried out in Costar 3524 24-well plates on exponentially growing cells that reach confluency. 6 To improve cell adherence the plates are pretreated with a 1% solution of gelatine in PBS which is applied for a few minutes at room temperature and subsequently removed prior to drying the plates under sterile conditions. For most monolayer cells beginning confluency corresponds to approximately 105 cells per well. To avoid perturbation of culture conditions interferon incubation at 37° is carried out in a waterbath with prewarmed media and interferon samples. After incubation cells are washed three times by addition of 1.0 ml per well of ice cold culture medium which should remain in contact with the cells for about 1 rain prior to being removed. Subsequently, cell-associated radioactivity is determined by solubilization of the cells with 0.5 ml 1 N NaOH for 1 hr at 37 ° and transferring the lysates into counting vials. Nondisplaceable binding is determined as described in the previous section. Interpretation of Binding D a t a Binding data are most commonly analyzed by mass action law to estimate binding affinity and number of binding sites involved.W The limitations have been already emphasized elsewhere, 8,9 yet two aspects should be briefly recalled. As stated above cell density is a critical parameter for interpreting binding data by mass action law. To define a ligandreceptor interaction, mass action law requires that the reaction be reversible, bimolecular, and in an equilibrium state. Obviously these criteria are more convincingly fulfilled at low temperature, whereas at 37° dynamic processes such as receptor turnover, ligand internalization and degradation, or ligand induced modulation of receptor expression represent additional variables difficult to define by mass action law. Therefore, it is important to distinguish between experiments designed to determine or compare physical binding properties and experiments conceived to investigate dynamic events involved or induced by receptor binding under physiological conditions. To our knowledge a generally valid mathematical model to describe these dynamic processes has not yet been proposed. 6 M. Aguet, 1. Gresser, A. G. Hovanessian, M. T. Bandu, B. Blanchard, and D. Blangy, Virology 114, 585 (1981). 7 G. Scatchard, Ann. N . Y . Acad. Sci. 51, 660 (1949). 8 I. M. Klotz, Science 217, 1247 (1982). 9 M. Aguet and K. E. Mogensen, in " I n t e r f e r o n " (I. Gresser, ed.), Vol. 5, p. 1. Academic Press, New York, 1984.