Journal of Immunological Methods, 11 (1976)147--151
147
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
A F F I N I T Y CHROMATOGRAPHY. A NEW TECHNIQUE FOR PARTIAL PURIFICATION OF HUMAN LEUCOCYTE MIGRATION INHIBITORY FACTOR
KLAUS BENDTZEN The Laboratory of Clinical Immunology, Medical Department TA, R igshospitalet University Hospital, Tagensvej 18, DK-2200 Copenhagen N, Denmark
(Received 5 November 1975, accepted 16 December 1975)
Human leucocyte migration inhibitory factor (LIF) was subjected to affinity column chromatography on Sepharose-bound a-L-fucose. Following the addition of a-L-fucose to the running buffer adsorbed lymphokine was eluted almost quantitatively. A minimum of 70-fold purification of LIF was achieved.
Activated l y m p h o c y t e s elaborate a number of chemical substances which are t h o u g h t to play a role in cell-mediated immunity. The biochemical identification of these lymphokines is still in its preliminary stages. Although m a n y of the methods known to protein chemists have been used, no specific lymphokine is available in a highly purified form. Thus, gel filtration, ion-exchange chromatography, fractional precipitation with a m m o n i u m sulphate and preparatory gel electrophoresis have been used either alone or in combination, all with rather disappointing results. Even macrophage migration inhibitory factor (MIF) (Rocklin et al., 1972} and leucocyte migration inhibitory factor (LIF) (Rocklin, 1975), some of the best studied lymphokines, have been characterized only in terms of general physicochemical properties. Since the monosaccharide a-L-fucose seems to be a part of a receptor on the macrophage for guinea pig MIF (Remold, 1973) and on the polymorphonuclear leucocyte for h u m a n LIF (Bendtzen, 1975a), a-L-fucose affinity chromatography might be used for the purification of these mediators. This paper describes a simple and lenient procedure based on affinity chromatography on Sepharose-bound a-L-fucose, which allows a 70-fold purification of serum- and antigen-free LIF material. MATERIALS AND METHODS Lymphokine production
Peripheral blood mononuclear cells were obtained from normal adults as previously detailed (Bendtzen et al., 1975). 2.5 X 106 cells/ml were incub-
148 ated in medium TC-199 (Difco Labor., Michigan, U.S.A.) in the presence (active supernatant) or in the absence (control supernatant) of concanavalin A 80 pg/ml (Pharmacia, Uppsala, Sweden) (Con A). After 22 h at 37°C in a 2% CO2 98% air atmosphere the supernatants were harvested and the control supernatant was reconstituted with Con A 80 pg/ml. The supernatants were desalted and depleted of Con A by passage through calibrated columns of Sephadex G-100 (Pharmacia) (Bendtzen et al., 1975). The mitogen-free supernatants were then passed through Millipore filters (0.45 p m pore size), lyophilized and stored at --20°C.
Assay for LIF The indirect leucocyte migration agarose technique described by Clausen (1972) was employed using unrelated peripheral blood leucocytes as migratory cells. 22 X 106 cells/90 pl culture supernatant were tested in 7 pl aliquots for migration under agarose and a migration index (MI) was determined: MI =
mean area of migration in the presence of LIF supernatant _ mean area of migration in the presence of control supernatant
Preparation of affinity columns All procedures were carried out at 20°C unless otherwise indicated. Three grams Epoxy-activated Sepharose 6B (Pharmacia) was swollen and washed with distilled water (300 ml) for 1 h in a glass column. After w~shing with coupling solution (NaOH, pH 11,5) (15 ml) the gel was mixed with 0.2 M a-L-fucose in coupling solution (15 ml) and incubated for 20 h at 37°C. As control ligands a-L-rhamnose and a-D-glucose were employed. All sugars were obtained in analytical grade form from Sigma, St. Louis, Mo., U.S.A. After incubation the gel was washed free of sugar and remaining free active groups were then blocked by incubating the product in 1 M ethanolamine, pH 9.0 for 20 h. Finally, the gel was washed with distilled water (15 ml), 0.1 M borate buffer, pH 8.0 (15 ml), 0.1 M acetate buffer, pH 4.0 (15 ml) and 0.1 M phosphate buffer, pH 7.4, each containing 0.5 M NaC1. Four ml gel suspension was then poured into each of two glass columns and stored at 4°C until use.
Affinity chromatography of supernatants Lyophilized active and control supernatant material were dissolved in 1 ml 0.1 M phosphate buffer, pH 7.4 containing 0.5 M NaC1 and drained at 6 ml/ h in t w o a-L-fucose--Sepharose columns and as controls two a-L-rhamnose or a-D-glucose columns. After washing of the columns with the buffer specific elution of adsorbed lymphokine was carried out by the addition of the
149
corresponding ligand (0.3 M solution) to the buffer. Six ml eluates from each column were dialyzed for 48 h at 4°C against three changes of water, lyophilized and reconstituted in 1 ml TC-199 to original supernatant concentration before LIF assay. Protein m e a s u r e m e n t s
The m e t h o d of Lowry et al. (1951) was employed. The sensitivity level of the m e t h o d as it is used in our laboratory is 20 pg/ml. RESULTS
All lymphokine activity was retained in the columns containing ~-L-fucose--Sepharose 6B even when five times concentrated supernatants were chromatographed {table 1). As shown in table 2, it was possible to elute adherent lymphokine with 0.3 M ~-L-fucose with a recovery of almost the same mediator activity as that found in untreated LIF-rich supernatants. Semi-quantitative experiments presently in progress (Bendtzen, 1975b) have shown that a recovery of 60-80% of lymphokine activity can be expected. When control columns were eluted with s-D-glucose and ~-L-rhamnose respectively, no lymphokine activity was eluted (table 2). When subjected to Sephadex G-100 gel filtration the migration inhibitory activity eluted from ~-L-fucose--Sepharose appeared in the same fractions as untreated human LIF (Bendtzen, 1975a) (table 3). These three experiments offered partial p r o o f that the lymphokine was in fact unaffected by the affinity chromatography procedure. An approximate 70-fold purification of LIF was achieved. The average amount of protein applied on each column was 1.2 mg (standard deviation: 0.5 mg, 4 experiments), and the protein content of eluted material was 10
TABLE 1 LIF - - activity o f s u p e r n a t a n t s b e f o r e and a f t e r passage t h r o u g h c o l u m n s o f ~ - L - f u c o s e - S e p h a r o s c 6B. Matrix Ligand Exp. no.
: ........
Before passage t h r o u g h c o l u m n s A f t e r passage t h r o u g h c o l u m n s **
E p o x y - a c t i v a t e d S e p h a r o s e 6B ~-L-fucose 1 2 3 *
4 *
0.7.8 0.98
0.48 0.93
0.67 1.02
0.44 1.04
* Five t i m e s c o n c e n t r a t e d s u p e r n a t a n t s . ** Eiuates were c o n c e n t r a t e d t o original v o l u m e o f o n e ml b e f o r e assay. T h e activity o f a s u p e r n a t a n t is e x p r e s s e d as a m i g r a t i o n i n d e x (see t e x t ) .
150 TABLE 2 LIF activity of supernatants before and after affinity chromatography on columns of (~-L-fucose--Sepharose 6B and control columns. -
-
Matrix Ligand No. of experiments
Epoxy-activated Sepharose 6B ~-L-fucose a-D-glucose (7) (4)
~-L-rhamnose (3)
Before affinity chromatography After affinity chromatography *
0.70 _+0.05 0.76 -+ 0.06
0.73 -+ 0.08 0.95 -+ 0.04
0.66 ± 0.02 0.98 -+ 0.02
* Eluates were concentrated to original volume of one ml before assay. The activity of a supernatant is expressed as a migration index _+standard deviation (see text).
TABLE 3 Sephadex G-100 elution pattern of lymphokine material after affinity chromatography on ~-L-fucose--Sepharose 6B. Fraction no. *
I II III IV V
Molecular weight range (daltons) > 130,000 130,000--70,000 70,000--40,000 40,000--10,000 < 10,000
Migration index Exp. no. 1
2
1.00 0.72 0.74 0.91 0.98
0.93 0.70 0.70 1.02 0.98
0.97 0.82 0.80 0.96 1.04
* Each fraction was concentrated to original volume of one ml before assay.
pg or less, w h i c h was b e l o w t h e sensitivity level o f the L o w r y m e t h o d . Thus, w i t h a r e c o v e r y o f 6 0 - - 8 0 % the degree of p u r i f i c a t i o n was at least 70-fold. DISCUSSION T h e a t t a i n m e n t o f 1 3 0 0 - t o 1 6 0 0 - f o l d p u r i f i c a t i o n o f guinea pig M I F has been claimed b y using c o m b i n a t i o n s o f several laborious s e p a r a t i o n techniques ( R e m o l d et al., 1 9 7 0 ; D u m o n d e et al., 1 9 7 2 ) including fractional salt p r e c i p i t a t i o n , gel filtration, i o n - e x c h a n g e c h r o m a t o g r a p h y , p r e p a r a t o r y a c r y l a m i d e gel e l e c t r o p h o r e s i s and i m m u n e p r e c i p i t a t i o n o f i n d u c i n g antigen and serum. H o w e v e r , these a p p a r e n t high degrees o f p u r i f i c a t i o n were calc u l a t e d o n the basis o f initial s u p e r n a t a n t s ' c o n t a m i n a t e d ' with antigen at c o n c e n t r a t i o n s o f 1 0 0 p g / m l and 1 0 0 0 p g / m l o f u n c o n c e n t r a t e d s u p e r n a t a n t . A l t h o u g h s o m e o f the antigen m i g h t have p r e c i p i t a t e d d u r i n g dialysis and
151
lyophilization, even slight amounts of antigen left in the supernatants before protein determinations would have contributed heavily to the estimated degree of purification. Furthermore, it must be realized that guinea pig MIF, unlike human LIF, is easy to separate from albumin, the major contaminating cell metabolite, because of different molecular weight and electrophoretic mobility. In relation to these considerations, the present technique seems to offer a powerful supplementary step in the efforts to obtain high purity preparations of human LIF. Experiments designed to disclose the LIF-binding capacity of a-L-fucose-Sepharose and to provide more detailed information on the degree purification and recovery are now in preparation (Bendtzen, 1975b). ACKNOWLEDGEMENTS
This study was supported by grants from the Danish Hospital Foundation for Medical Research, Region of Copenhagen, Greenland and the Faroe Islands, and the Danish Medical Research Council.
REFERENCES Bendtzen, K., 1975a, Acta Allergol. 31, in press. Bendtzen, K., 1975b, Acta Allergol. (In preparation). Bendtzen, K., V. Andersen and G. Bendixen, 1975, Acta AUergol. 30, 133. Clausen, J.E., 1972, J. Immunol. 108,453. Dumonde, D.C., D.A. Page, M. Matthew and R.A. Wolstencroft, 1972, Clin. Exp. Immunol. 10, 25. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, J. Biol. Chem. 193, 265. Remold, H.G., 1973, J. Exp. Med. 138, 1065. Remold, H.G., A.B. Katz, E. Haber and J.R. David, 1970, Cell. Immunol. 1,133. Rocklin, R.E., 1975, J. Immunol. 114, 1161. Rocklin, R.E., H.G. Remold and J.R. David, 1972, Cell. Immunol. 5,436.
147
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
A F F I N I T Y CHROMATOGRAPHY. A NEW TECHNIQUE FOR PARTIAL PURIFICATION OF HUMAN LEUCOCYTE MIGRATION INHIBITORY FACTOR
KLAUS BENDTZEN The Laboratory of Clinical Immunology, Medical Department TA, R igshospitalet University Hospital, Tagensvej 18, DK-2200 Copenhagen N, Denmark
(Received 5 November 1975, accepted 16 December 1975)
Human leucocyte migration inhibitory factor (LIF) was subjected to affinity column chromatography on Sepharose-bound a-L-fucose. Following the addition of a-L-fucose to the running buffer adsorbed lymphokine was eluted almost quantitatively. A minimum of 70-fold purification of LIF was achieved.
Activated l y m p h o c y t e s elaborate a number of chemical substances which are t h o u g h t to play a role in cell-mediated immunity. The biochemical identification of these lymphokines is still in its preliminary stages. Although m a n y of the methods known to protein chemists have been used, no specific lymphokine is available in a highly purified form. Thus, gel filtration, ion-exchange chromatography, fractional precipitation with a m m o n i u m sulphate and preparatory gel electrophoresis have been used either alone or in combination, all with rather disappointing results. Even macrophage migration inhibitory factor (MIF) (Rocklin et al., 1972} and leucocyte migration inhibitory factor (LIF) (Rocklin, 1975), some of the best studied lymphokines, have been characterized only in terms of general physicochemical properties. Since the monosaccharide a-L-fucose seems to be a part of a receptor on the macrophage for guinea pig MIF (Remold, 1973) and on the polymorphonuclear leucocyte for h u m a n LIF (Bendtzen, 1975a), a-L-fucose affinity chromatography might be used for the purification of these mediators. This paper describes a simple and lenient procedure based on affinity chromatography on Sepharose-bound a-L-fucose, which allows a 70-fold purification of serum- and antigen-free LIF material. MATERIALS AND METHODS Lymphokine production
Peripheral blood mononuclear cells were obtained from normal adults as previously detailed (Bendtzen et al., 1975). 2.5 X 106 cells/ml were incub-
148 ated in medium TC-199 (Difco Labor., Michigan, U.S.A.) in the presence (active supernatant) or in the absence (control supernatant) of concanavalin A 80 pg/ml (Pharmacia, Uppsala, Sweden) (Con A). After 22 h at 37°C in a 2% CO2 98% air atmosphere the supernatants were harvested and the control supernatant was reconstituted with Con A 80 pg/ml. The supernatants were desalted and depleted of Con A by passage through calibrated columns of Sephadex G-100 (Pharmacia) (Bendtzen et al., 1975). The mitogen-free supernatants were then passed through Millipore filters (0.45 p m pore size), lyophilized and stored at --20°C.
Assay for LIF The indirect leucocyte migration agarose technique described by Clausen (1972) was employed using unrelated peripheral blood leucocytes as migratory cells. 22 X 106 cells/90 pl culture supernatant were tested in 7 pl aliquots for migration under agarose and a migration index (MI) was determined: MI =
mean area of migration in the presence of LIF supernatant _ mean area of migration in the presence of control supernatant
Preparation of affinity columns All procedures were carried out at 20°C unless otherwise indicated. Three grams Epoxy-activated Sepharose 6B (Pharmacia) was swollen and washed with distilled water (300 ml) for 1 h in a glass column. After w~shing with coupling solution (NaOH, pH 11,5) (15 ml) the gel was mixed with 0.2 M a-L-fucose in coupling solution (15 ml) and incubated for 20 h at 37°C. As control ligands a-L-rhamnose and a-D-glucose were employed. All sugars were obtained in analytical grade form from Sigma, St. Louis, Mo., U.S.A. After incubation the gel was washed free of sugar and remaining free active groups were then blocked by incubating the product in 1 M ethanolamine, pH 9.0 for 20 h. Finally, the gel was washed with distilled water (15 ml), 0.1 M borate buffer, pH 8.0 (15 ml), 0.1 M acetate buffer, pH 4.0 (15 ml) and 0.1 M phosphate buffer, pH 7.4, each containing 0.5 M NaC1. Four ml gel suspension was then poured into each of two glass columns and stored at 4°C until use.
Affinity chromatography of supernatants Lyophilized active and control supernatant material were dissolved in 1 ml 0.1 M phosphate buffer, pH 7.4 containing 0.5 M NaC1 and drained at 6 ml/ h in t w o a-L-fucose--Sepharose columns and as controls two a-L-rhamnose or a-D-glucose columns. After washing of the columns with the buffer specific elution of adsorbed lymphokine was carried out by the addition of the
149
corresponding ligand (0.3 M solution) to the buffer. Six ml eluates from each column were dialyzed for 48 h at 4°C against three changes of water, lyophilized and reconstituted in 1 ml TC-199 to original supernatant concentration before LIF assay. Protein m e a s u r e m e n t s
The m e t h o d of Lowry et al. (1951) was employed. The sensitivity level of the m e t h o d as it is used in our laboratory is 20 pg/ml. RESULTS
All lymphokine activity was retained in the columns containing ~-L-fucose--Sepharose 6B even when five times concentrated supernatants were chromatographed {table 1). As shown in table 2, it was possible to elute adherent lymphokine with 0.3 M ~-L-fucose with a recovery of almost the same mediator activity as that found in untreated LIF-rich supernatants. Semi-quantitative experiments presently in progress (Bendtzen, 1975b) have shown that a recovery of 60-80% of lymphokine activity can be expected. When control columns were eluted with s-D-glucose and ~-L-rhamnose respectively, no lymphokine activity was eluted (table 2). When subjected to Sephadex G-100 gel filtration the migration inhibitory activity eluted from ~-L-fucose--Sepharose appeared in the same fractions as untreated human LIF (Bendtzen, 1975a) (table 3). These three experiments offered partial p r o o f that the lymphokine was in fact unaffected by the affinity chromatography procedure. An approximate 70-fold purification of LIF was achieved. The average amount of protein applied on each column was 1.2 mg (standard deviation: 0.5 mg, 4 experiments), and the protein content of eluted material was 10
TABLE 1 LIF - - activity o f s u p e r n a t a n t s b e f o r e and a f t e r passage t h r o u g h c o l u m n s o f ~ - L - f u c o s e - S e p h a r o s c 6B. Matrix Ligand Exp. no.
: ........
Before passage t h r o u g h c o l u m n s A f t e r passage t h r o u g h c o l u m n s **
E p o x y - a c t i v a t e d S e p h a r o s e 6B ~-L-fucose 1 2 3 *
4 *
0.7.8 0.98
0.48 0.93
0.67 1.02
0.44 1.04
* Five t i m e s c o n c e n t r a t e d s u p e r n a t a n t s . ** Eiuates were c o n c e n t r a t e d t o original v o l u m e o f o n e ml b e f o r e assay. T h e activity o f a s u p e r n a t a n t is e x p r e s s e d as a m i g r a t i o n i n d e x (see t e x t ) .
150 TABLE 2 LIF activity of supernatants before and after affinity chromatography on columns of (~-L-fucose--Sepharose 6B and control columns. -
-
Matrix Ligand No. of experiments
Epoxy-activated Sepharose 6B ~-L-fucose a-D-glucose (7) (4)
~-L-rhamnose (3)
Before affinity chromatography After affinity chromatography *
0.70 _+0.05 0.76 -+ 0.06
0.73 -+ 0.08 0.95 -+ 0.04
0.66 ± 0.02 0.98 -+ 0.02
* Eluates were concentrated to original volume of one ml before assay. The activity of a supernatant is expressed as a migration index _+standard deviation (see text).
TABLE 3 Sephadex G-100 elution pattern of lymphokine material after affinity chromatography on ~-L-fucose--Sepharose 6B. Fraction no. *
I II III IV V
Molecular weight range (daltons) > 130,000 130,000--70,000 70,000--40,000 40,000--10,000 < 10,000
Migration index Exp. no. 1
2
1.00 0.72 0.74 0.91 0.98
0.93 0.70 0.70 1.02 0.98
0.97 0.82 0.80 0.96 1.04
* Each fraction was concentrated to original volume of one ml before assay.
pg or less, w h i c h was b e l o w t h e sensitivity level o f the L o w r y m e t h o d . Thus, w i t h a r e c o v e r y o f 6 0 - - 8 0 % the degree of p u r i f i c a t i o n was at least 70-fold. DISCUSSION T h e a t t a i n m e n t o f 1 3 0 0 - t o 1 6 0 0 - f o l d p u r i f i c a t i o n o f guinea pig M I F has been claimed b y using c o m b i n a t i o n s o f several laborious s e p a r a t i o n techniques ( R e m o l d et al., 1 9 7 0 ; D u m o n d e et al., 1 9 7 2 ) including fractional salt p r e c i p i t a t i o n , gel filtration, i o n - e x c h a n g e c h r o m a t o g r a p h y , p r e p a r a t o r y a c r y l a m i d e gel e l e c t r o p h o r e s i s and i m m u n e p r e c i p i t a t i o n o f i n d u c i n g antigen and serum. H o w e v e r , these a p p a r e n t high degrees o f p u r i f i c a t i o n were calc u l a t e d o n the basis o f initial s u p e r n a t a n t s ' c o n t a m i n a t e d ' with antigen at c o n c e n t r a t i o n s o f 1 0 0 p g / m l and 1 0 0 0 p g / m l o f u n c o n c e n t r a t e d s u p e r n a t a n t . A l t h o u g h s o m e o f the antigen m i g h t have p r e c i p i t a t e d d u r i n g dialysis and
151
lyophilization, even slight amounts of antigen left in the supernatants before protein determinations would have contributed heavily to the estimated degree of purification. Furthermore, it must be realized that guinea pig MIF, unlike human LIF, is easy to separate from albumin, the major contaminating cell metabolite, because of different molecular weight and electrophoretic mobility. In relation to these considerations, the present technique seems to offer a powerful supplementary step in the efforts to obtain high purity preparations of human LIF. Experiments designed to disclose the LIF-binding capacity of a-L-fucose-Sepharose and to provide more detailed information on the degree purification and recovery are now in preparation (Bendtzen, 1975b). ACKNOWLEDGEMENTS
This study was supported by grants from the Danish Hospital Foundation for Medical Research, Region of Copenhagen, Greenland and the Faroe Islands, and the Danish Medical Research Council.
REFERENCES Bendtzen, K., 1975a, Acta Allergol. 31, in press. Bendtzen, K., 1975b, Acta Allergol. (In preparation). Bendtzen, K., V. Andersen and G. Bendixen, 1975, Acta AUergol. 30, 133. Clausen, J.E., 1972, J. Immunol. 108,453. Dumonde, D.C., D.A. Page, M. Matthew and R.A. Wolstencroft, 1972, Clin. Exp. Immunol. 10, 25. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, J. Biol. Chem. 193, 265. Remold, H.G., 1973, J. Exp. Med. 138, 1065. Remold, H.G., A.B. Katz, E. Haber and J.R. David, 1970, Cell. Immunol. 1,133. Rocklin, R.E., 1975, J. Immunol. 114, 1161. Rocklin, R.E., H.G. Remold and J.R. David, 1972, Cell. Immunol. 5,436.
