Journal o f Immunological Methods, 14 ( 1977 ) 141--146
141
© Elsevier/North-Holland Biomedical Press
A SIMPLE METHOD FOR THE PRODUCTION OF MIGRATION INHIBITORY FACTOR BY CONCANAVALIN A - STIMULATED LYMPHOCYTES
EDGAR PICK and PHILIP KOTKES Department o f Human Microbiology, Tel-Aviv University, Sackler School o f Medicine, Tel-Arty, Israel
(Received 25 July 1976, accepted 1 September 1976) A simple method for preparing culture supernatants containing macrophage migration inhibitory factor (MIF) by pulse exposure of guinea pig lymphocytes to concanavalin A (Con A), is described. The method is based on the property of originally nonadherent lymphocytes to attach to the flask surface in the presence of Con A. The Con A-induced lymphocyte 'monolayer' can be easily rinsed, allowing the removal of free Con A without repeated, cell damaging centrifugation. The Con A-pulsed lymphocytes are cultured for 24 h in medium and supernatants containing MIF but free of undesired Con A are regularly obtained. INTRODUCTION Production of macrophage migration inhibitory factor (MIF) by lymphoc y t e s can be i n d u c e d b y specific a n t i g e n , w h e n l y m p h o c y t e s are derived f r o m sensitized a n i m a l s , a n d b y a n u m b e r o f n o n - s p e c i f i c agents i n t e r a c t i n g w i t h r e c e p t o r s o n t h e cell m e m b r a n e ( r e v i e w e d b y Pick a n d T u r k , 1 9 7 2 ) . T h e use of non-antigenic stimulants offers the following advantages: no need for p r e v i o u s s e n s i t i z a t i o n , b e t t e r d e f i n i t i o n o f cell r e c e p t o r s a n d larger a m o u n t s o f l y m p h o k i n e b e i n g p r o d u c e d ( R e m o l d et al., 1972). A m o n g n o n - s p e c i f i c s t i m u l a n t s , c o n c a n a v a l i n A ( C o n A) o c c u p i e s a c e n t r a l place b e c a u s e o f t h e reliability w i t h w h i c h it i n d u c e s M I F s y n t h e s i s (Pick et al., 1 9 7 0 ) , its availability in highly p u r i f i e d f o r m a n d its ability to b i n d to a - D - g l u c o s y l a n d r e l a t e d sugar residues ( G o l d s t e i n et al., 1 9 6 5 ) . T h e use o f C o n A as a M I F i n d u c e r is, h o w e v e r , c o m p l i c a t e d b y t h e f a c t t h a t t h e m i t o g e n left in t h e s u p e r n a t a n t s a f t e r c u l t u r e has a d i r e c t i n h i b i t o r y e f f e c t o n m a c r o p h a g e m i g r a t i o n , at suba g g l u t i n a t i n g c o n c e n t r a t i o n s ( K u m a g a i a n d Arai, 1 9 7 3 ; T a y l o r et al., 1975). In a d d i t i o n to t h a t , since it is c u s t o m a r y to use u n f r a c t i o n a t e d p e r i t o n e a l e x u d a t e cells, c o n t a i n i n g a s u b s t a n t i a l a m o u n t o f l y m p h o c y t e s , as t a r g e t cells f o r t h e p e r f o r m a n c e o f m o s t M I F assays, C o n A r e m a i n i n g in t h e s u p e r n a t a n t can s t i m u l a t e l y m p h o c y t e s p r e s e n t in t h e e x u d a t e to p r o d u c e M I F , thus f u r t h e r c o m p l i c a t i n g t h e a s s e s s m e n t o f t h e a m o u n t o f M I F p r e s e n t in t h e s u p e r n a t a n t . I t t h e r e f o r e b e c a m e n e c e s s a r y t o find m e t h o d s to e l i m i n a t e or n e u t r a l i z e C o n A, b e f o r e t e s t i n g s u p e r n a t a n t s f o r t h e p r e s e n c e o f MIF. T h e
142 following methods have been used: 1) Absorption of Con A left in supernatants by Sephadex G-75, using a batch procedure (Pick et al., 1970) or passage through Sephadex G-100 columns (Remold et al., 1972). 2) Use of Con A, insolubilized by binding to Sepharose beads, for induction of MIF synthesis (Pick, 1972; Friedrich et al., 1975). 3) Addition of a-methyl-Dmannoside to the supernatants (Friedrich et al., 1975). 4) Use of a multistep procedure including passage through Sephadex G-100 columns, filtration through 0.22 pm porosity cellulose nitrate or esters of cellulose membranes and addition of a-methyl-D-mannoside to the supernatants (Taylor et al., 1975). The main problems encountered with these procedures were: incomplete removal of Con A by Sephadex, dilution of the supematant during batch or column absorption which requires subsequent reconcentration, leakage of Con A from cyanogen bromide-activated Sepharose beads used to induce MIF production, and nonspecific effects on macrophage migration by the relatively high concentration of a-methyl-D-mannoside used to block Con A. We describe a simple procedure for the preparation of Con A-induced MIF which uses stimulation by regular soluble Con A but does n o t require elimination of Con A from the supernatant. MATERIALS AND METHODS
Animals and immunization Male, Hartley strain guinea pigs, weighing 300--500 g, were used as donors for both lymph node cells (LNC) and peritoneal exudate cells. The animals were injected with 1 ml Freund's complete adjuvant (FCA, Difco Detroit, Michigan) containing 0 . 5 m g / m l of killed Mycobacterium tuberculosis H37Ra, divided among the four f o o t pads and the nuchal region. The reason for immunizing with FCA was to cause an increase in the size of draining lymph nodes so as to yield larger amounts of lymphocytes for culture. It has also been reported that immunization with FCA increases the ability of lymphocytes to produce MIF in response to a number of non-antigenic stimulants (Pekarek et al., 1973).
Lymphocyte preparation LNC were obtained from the draining nodes and made into a suspension in serum-free Eagle's minimum essential medium (referred to as MEM), supplemented with nonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/ml penicillin and 100 pg/ml streptomycin, as described earlier (Pick and Manheimer, 1974). The LNC were suspended in MEM containing 20% heat-inactivated calf serum and filtered at 37°C through columns of 60 mesh glass beads, using the technique previously described (Manheimer and Pick, 1973) The filtered cells were washed twice in ice-cold
143 MEM and brought to a concentration of 107 trypan blue,excluding cells per ml.
Culture of lymphocytes and Con A pulse Glass bead-filtered LNC, suspended in serum-free MEM, were added to disposable Falcon polystyrene culture flasks of 30 ml capacity (surface area 25 cm 2, catalogue number 3012) or 250 ml capacity (surface area 75 cm 2, catalogue number 3024) (Falcon Plastics, Oxnard, California). Similar flasks manufactured by Nunc Products, Roskilde, Denmark, were f o u n d to be equally satisfactory. Five ml of cell suspension were added to 30-ml flasks, and 15 ml, to 250-ml flasks. To the desired number of flasks Con A (A grade, lyophilized in NaCl,. Calbiochem, San Diego, California, catalogue number 234567) was added to a final concentration of 10 pg/ml. For this purpose a stock solution of 500 pg/ml Con A in distilled water was prepared, filtered through 0.22 pm pore size Millipore membrane (Millipore Corporation, Bedford, Massachusetts), divided into small aliquots and kept frozen at --70°C. To control flasks an identical volume of distilled water was added. The culture flasks were placed in a horizontal position and carefully shaken in order to insure an even distribution of the cells over the b o t t o m surface of the flask. Both Con A supplemented and coltrol cultures were incubated for 2 h at 37°C, with partially open caps, in a humidified incubator, gassed with 90% air--10% CO2. After the end of the 2 h interval the flasks to which Con A was added were removed from the incubator and transferred in their original horizontal position to a laminar flow sterile hood. We observed that guinea pig LNC, exposed to 10 pg/ml Con A for 2 h at 37°C, firmly adhere to the flask surface. This was in sharp contrast to the complete lack of adherence of glass bead-filtered control LNC, since normally adherent or damaged, sticky cells were retained on the columns (Manheimer and Pick, 1973). This observation was at the basis of the development of the present m e t h o d . Flasks containing Con A--pulsed LNC were tilted several times around their long axis and the medium poured off. The adherent l y m p h o c y t e 'monolayer' was washed five times with 20 ml (per 250 ml flask) or 5 ml (per 30 ml flask) of MEM warmed to 37°C, taking care n o t to direct the stream of medium emerging from the pipette tip onto the cell layer. After the last wash the original a m o u n t of fresh medium was added and the flask returned to the incubator. Control fla'sks were left undisturbed. Both Con A--pulsed and control LNC were cultured for 18--24 at 37°C. At the completion of culture the cell suspensions were centrifuged at 1000 g for 30 min and the supernatants stored at --70 ° C. A substantial part of the Con A--pulsed LNC were still adherent to the flask surface after 24 h of incubation, but variable numbers of cells detached spontaneously. In some experiments a number of flasks with Con A--pulsed cells were treated as described above b u t instead of being incubated at 37°C they were kept for 18--24 h at 4°C, a temperature at which no MIF is produced. Super-
144 natants of Con A--pulsed cells, incubated in the cold, were used to test for leakage of Con A from the cells or flask surface. Any macrophage migration inhibitory activity detected in such material could only be due to leaked Con A.
Detection of free Con A Supernatants of Con A--pulsed LNC were examined for the presence of free Con A by their ability to agglutinate guinea pig red cells (GPRC) (Pick et al., 1970; Taylor et al., 1975). 0.1 ml washed GPttC, suspended to a concentration of 1% v/v in 0.15 m NaC1, were added to 0.1 ml of culture supern a t a n t in 12 X 75 m m glass tubes and shaken for 4 h in a water bath at 37°C. Agglutination was assessed macroscopically and microscopically using as controls GPRC added to MEM, to supernatant of unstimulated cultures, and to Con A dissolved at various concentrations in supernatant of unstimulated cultures. In agreement with Taylor et al. (1975), this m e t h o d detected the presence of Con A down to a concentration of 0.5 pg/ml. Additional methods of checking for the presence of free Con A, were to test for the ability of supernatants of Con A--pulsed LNC, cultured at 4 ° C, to inhibit macrophage migration, and to determine the effect of 0.1 M amethyl-D-mannoside (Sigma Chemical Co., St. Louis, Missouri) on the migration inhibitory capacity of Con A--pulsed LNC supernatants, obtained by culturing the cells at 37 ° C.
Assessment of MIF in supernatants MIF was quantitated by the classical capillary tube assay using oil-induced guinea pig peritoneal exudate cells and disposable plastic migration chambers (Sterilin, Teddington, Middlesex, England), as previously described (Manheimer and Pick, 1973). Supernatants were supplemented with 15% foetal calf serum (Gibco, Grand Island, N.Y.) just prior to the performance of the assay. Areas of migration were measured after 8--18 h of incubation and results expressed as per cent inhibition of migration, which was calculated as follows:
Percent inhibition = 100--
Area of migration in supernatant of Con A--pulsed lymphocytes Area of migration in supernatant of unstimulated lymphocytes
X 100
Each supernatant was tested in at least three chambers. RESULTS In a series of 9 successive cultures performed by the technique described under Materials and Methods and using LNC pooled from 10 guinea pigs per
145 culture, we regularly obtained supernatants with strong migration inhibitory activity. Per cent inhibition values were regularly higher than 50. On no occasion was migration inhibitory activity detected in supernatants of Con A--pulsed l y m p h o c y t e s , cultured at 4 ° C. Addition of 0.1 M ~-methyl-D-mannoside to supernatants of Con A-pulsed cells, exPressing MIF activity, did n o t reduce the extent of migration inhibition caused by these supernatants. • f4. Migration inhibitory activity was undxmmlshed after extensive dialysis against MEM, proving that it was not due to depletion of nutritional factors from the medium. All supernatants were tested for residual free Con A by GPRC agglutination and were found to be negative. DISCUSSION Pulse exposure of guinea pig l y m p h o c y t e s to Con A for 2 h at 37°C regularly results in the production of MIF. Such a methodological approach has been used in the past by Lamelin and Vassalli (1971), who described the production of MIF by guinea pig l y m p h o c y t e s pulsed in suspension with phytohaemagglutinin and a n t i l y m p h o c y t e serum. The main disadvantage of their technique was the need for thorough washing of the cells, employing repeated centrifugation. Removal of mitogens by centrifugation is further complicated by the fact that the cells are heavily clumped, which makes resuspension technically difficult and unavoidably damaging. The alternative m e t h o d , based on the use of insolubilized Con A, suffers from c o m m o n l y encountered leakage of lectin from the Sepharose matrix and from the difficulty with which such preparations are standardized, stored for longer periods and handled. It is also probable that a certain proportion of l y m p h o c y t e s will not come in contact with the Con A beads, even though rocking of cultures was recommended (Friedrich et al., 1975). On the other hand, the extreme difficulties met with when soluble Con A has to be removed from supernatants, have been amply listed by Taylor et al. (1975). If such removal is to be complete, it involves a sequence of technically cumbersome steps with the inherent danger of loss of part of the MIF. The m e t h o d we describe is simple, uses regular soluble Con A, avoids repeated handling of the cells and does n o t require any t r e a t m e n t of the supernatant after culture. It is based on the observation that Con A causes strong a t t a c h m e n t of an originally nonadherent l y m p h o c y t e population to the surface of Falcon or equivalent flasks. The mechanism of this a t t a c h m e n t is not clear but it probably involves the specific binding of Con A to lymphocyte membrane glycoproteins and its nonspecific absorption to the flask surface, therefore acting as a bridge between the cell and the flask. Con A was recently found to increase the strength of a t t a c h m e n t of baby hamster kidney cells to Falcon flasks (Grinnell, 1973).
146
ACKNOWLEDGEMENTS This research was s u p p o r t e d b y g r a n t 5 R 0 1 I n s t i t u t e s o f Health, Bethesda, Md., by a g r a n t B i n a t i o n a l Science F o u n d a t i o n a n d b y a g r a n t & Co. L t d . , Basle, S w i t z e r l a n d . T h e c o m p e t e n t S c h w a r t z is greatfully a c k n o w l e d g e d .
A I 1 1 1 9 4 f r o m the N a t i o n a l f r o m the U n i t e d States-Israel f r o m F. H o f f m a n n - L a R o c h e secretarial help o f Mrs. Julia
REFERENCES Friedrich, W., S. Lazary, C. Geczy and A.L. De Weck, 1975, Int. Arch. Allergy 49,504. Goldstein, I.J., C.E. Hollerman and E.E. Smith, 1965, Biochemistry 4,876. Grinnell, F., 1973, J. Cell. Biol. 58,602. Kumagai, K. and S. Arai, 1973, J. of Reticuloendothel. Soc. 13,507. Lamelin, J.P. and P. Vassali, 1971, Nature 229,426. Manheimer, S. and E. Pick, 1973, Immunology 24, 1027. Pekarek, J., J. Svejcar, V. Hribalova and J. Johanovsky, 1973, Folia Microbiol. 18,393. Pick, E., 1972, Israel J. Med. Sci. 8,654. Pick, E., J. Brostoff, J. Krejci and J.L. Turk, 1970, Cell. Immunol. 1, 92. Pick, E. and J.L. Turk, 1972, Clin. Exp. Immunol. 10, 1. Pick, E. and S. Manheimer, 1974, Cell. Immunol. 11, 30. Remold, H.G., R.A. David and J.R. David, 1972. J. Immunol. 109, 578. Taylor, M., C.J. Burman and K.H. Fantes, 1975, Cell. Immunol. 19, 41.
141
© Elsevier/North-Holland Biomedical Press
A SIMPLE METHOD FOR THE PRODUCTION OF MIGRATION INHIBITORY FACTOR BY CONCANAVALIN A - STIMULATED LYMPHOCYTES
EDGAR PICK and PHILIP KOTKES Department o f Human Microbiology, Tel-Aviv University, Sackler School o f Medicine, Tel-Arty, Israel
(Received 25 July 1976, accepted 1 September 1976) A simple method for preparing culture supernatants containing macrophage migration inhibitory factor (MIF) by pulse exposure of guinea pig lymphocytes to concanavalin A (Con A), is described. The method is based on the property of originally nonadherent lymphocytes to attach to the flask surface in the presence of Con A. The Con A-induced lymphocyte 'monolayer' can be easily rinsed, allowing the removal of free Con A without repeated, cell damaging centrifugation. The Con A-pulsed lymphocytes are cultured for 24 h in medium and supernatants containing MIF but free of undesired Con A are regularly obtained. INTRODUCTION Production of macrophage migration inhibitory factor (MIF) by lymphoc y t e s can be i n d u c e d b y specific a n t i g e n , w h e n l y m p h o c y t e s are derived f r o m sensitized a n i m a l s , a n d b y a n u m b e r o f n o n - s p e c i f i c agents i n t e r a c t i n g w i t h r e c e p t o r s o n t h e cell m e m b r a n e ( r e v i e w e d b y Pick a n d T u r k , 1 9 7 2 ) . T h e use of non-antigenic stimulants offers the following advantages: no need for p r e v i o u s s e n s i t i z a t i o n , b e t t e r d e f i n i t i o n o f cell r e c e p t o r s a n d larger a m o u n t s o f l y m p h o k i n e b e i n g p r o d u c e d ( R e m o l d et al., 1972). A m o n g n o n - s p e c i f i c s t i m u l a n t s , c o n c a n a v a l i n A ( C o n A) o c c u p i e s a c e n t r a l place b e c a u s e o f t h e reliability w i t h w h i c h it i n d u c e s M I F s y n t h e s i s (Pick et al., 1 9 7 0 ) , its availability in highly p u r i f i e d f o r m a n d its ability to b i n d to a - D - g l u c o s y l a n d r e l a t e d sugar residues ( G o l d s t e i n et al., 1 9 6 5 ) . T h e use o f C o n A as a M I F i n d u c e r is, h o w e v e r , c o m p l i c a t e d b y t h e f a c t t h a t t h e m i t o g e n left in t h e s u p e r n a t a n t s a f t e r c u l t u r e has a d i r e c t i n h i b i t o r y e f f e c t o n m a c r o p h a g e m i g r a t i o n , at suba g g l u t i n a t i n g c o n c e n t r a t i o n s ( K u m a g a i a n d Arai, 1 9 7 3 ; T a y l o r et al., 1975). In a d d i t i o n to t h a t , since it is c u s t o m a r y to use u n f r a c t i o n a t e d p e r i t o n e a l e x u d a t e cells, c o n t a i n i n g a s u b s t a n t i a l a m o u n t o f l y m p h o c y t e s , as t a r g e t cells f o r t h e p e r f o r m a n c e o f m o s t M I F assays, C o n A r e m a i n i n g in t h e s u p e r n a t a n t can s t i m u l a t e l y m p h o c y t e s p r e s e n t in t h e e x u d a t e to p r o d u c e M I F , thus f u r t h e r c o m p l i c a t i n g t h e a s s e s s m e n t o f t h e a m o u n t o f M I F p r e s e n t in t h e s u p e r n a t a n t . I t t h e r e f o r e b e c a m e n e c e s s a r y t o find m e t h o d s to e l i m i n a t e or n e u t r a l i z e C o n A, b e f o r e t e s t i n g s u p e r n a t a n t s f o r t h e p r e s e n c e o f MIF. T h e
142 following methods have been used: 1) Absorption of Con A left in supernatants by Sephadex G-75, using a batch procedure (Pick et al., 1970) or passage through Sephadex G-100 columns (Remold et al., 1972). 2) Use of Con A, insolubilized by binding to Sepharose beads, for induction of MIF synthesis (Pick, 1972; Friedrich et al., 1975). 3) Addition of a-methyl-Dmannoside to the supernatants (Friedrich et al., 1975). 4) Use of a multistep procedure including passage through Sephadex G-100 columns, filtration through 0.22 pm porosity cellulose nitrate or esters of cellulose membranes and addition of a-methyl-D-mannoside to the supernatants (Taylor et al., 1975). The main problems encountered with these procedures were: incomplete removal of Con A by Sephadex, dilution of the supematant during batch or column absorption which requires subsequent reconcentration, leakage of Con A from cyanogen bromide-activated Sepharose beads used to induce MIF production, and nonspecific effects on macrophage migration by the relatively high concentration of a-methyl-D-mannoside used to block Con A. We describe a simple procedure for the preparation of Con A-induced MIF which uses stimulation by regular soluble Con A but does n o t require elimination of Con A from the supernatant. MATERIALS AND METHODS
Animals and immunization Male, Hartley strain guinea pigs, weighing 300--500 g, were used as donors for both lymph node cells (LNC) and peritoneal exudate cells. The animals were injected with 1 ml Freund's complete adjuvant (FCA, Difco Detroit, Michigan) containing 0 . 5 m g / m l of killed Mycobacterium tuberculosis H37Ra, divided among the four f o o t pads and the nuchal region. The reason for immunizing with FCA was to cause an increase in the size of draining lymph nodes so as to yield larger amounts of lymphocytes for culture. It has also been reported that immunization with FCA increases the ability of lymphocytes to produce MIF in response to a number of non-antigenic stimulants (Pekarek et al., 1973).
Lymphocyte preparation LNC were obtained from the draining nodes and made into a suspension in serum-free Eagle's minimum essential medium (referred to as MEM), supplemented with nonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/ml penicillin and 100 pg/ml streptomycin, as described earlier (Pick and Manheimer, 1974). The LNC were suspended in MEM containing 20% heat-inactivated calf serum and filtered at 37°C through columns of 60 mesh glass beads, using the technique previously described (Manheimer and Pick, 1973) The filtered cells were washed twice in ice-cold
143 MEM and brought to a concentration of 107 trypan blue,excluding cells per ml.
Culture of lymphocytes and Con A pulse Glass bead-filtered LNC, suspended in serum-free MEM, were added to disposable Falcon polystyrene culture flasks of 30 ml capacity (surface area 25 cm 2, catalogue number 3012) or 250 ml capacity (surface area 75 cm 2, catalogue number 3024) (Falcon Plastics, Oxnard, California). Similar flasks manufactured by Nunc Products, Roskilde, Denmark, were f o u n d to be equally satisfactory. Five ml of cell suspension were added to 30-ml flasks, and 15 ml, to 250-ml flasks. To the desired number of flasks Con A (A grade, lyophilized in NaCl,. Calbiochem, San Diego, California, catalogue number 234567) was added to a final concentration of 10 pg/ml. For this purpose a stock solution of 500 pg/ml Con A in distilled water was prepared, filtered through 0.22 pm pore size Millipore membrane (Millipore Corporation, Bedford, Massachusetts), divided into small aliquots and kept frozen at --70°C. To control flasks an identical volume of distilled water was added. The culture flasks were placed in a horizontal position and carefully shaken in order to insure an even distribution of the cells over the b o t t o m surface of the flask. Both Con A supplemented and coltrol cultures were incubated for 2 h at 37°C, with partially open caps, in a humidified incubator, gassed with 90% air--10% CO2. After the end of the 2 h interval the flasks to which Con A was added were removed from the incubator and transferred in their original horizontal position to a laminar flow sterile hood. We observed that guinea pig LNC, exposed to 10 pg/ml Con A for 2 h at 37°C, firmly adhere to the flask surface. This was in sharp contrast to the complete lack of adherence of glass bead-filtered control LNC, since normally adherent or damaged, sticky cells were retained on the columns (Manheimer and Pick, 1973). This observation was at the basis of the development of the present m e t h o d . Flasks containing Con A--pulsed LNC were tilted several times around their long axis and the medium poured off. The adherent l y m p h o c y t e 'monolayer' was washed five times with 20 ml (per 250 ml flask) or 5 ml (per 30 ml flask) of MEM warmed to 37°C, taking care n o t to direct the stream of medium emerging from the pipette tip onto the cell layer. After the last wash the original a m o u n t of fresh medium was added and the flask returned to the incubator. Control fla'sks were left undisturbed. Both Con A--pulsed and control LNC were cultured for 18--24 at 37°C. At the completion of culture the cell suspensions were centrifuged at 1000 g for 30 min and the supernatants stored at --70 ° C. A substantial part of the Con A--pulsed LNC were still adherent to the flask surface after 24 h of incubation, but variable numbers of cells detached spontaneously. In some experiments a number of flasks with Con A--pulsed cells were treated as described above b u t instead of being incubated at 37°C they were kept for 18--24 h at 4°C, a temperature at which no MIF is produced. Super-
144 natants of Con A--pulsed cells, incubated in the cold, were used to test for leakage of Con A from the cells or flask surface. Any macrophage migration inhibitory activity detected in such material could only be due to leaked Con A.
Detection of free Con A Supernatants of Con A--pulsed LNC were examined for the presence of free Con A by their ability to agglutinate guinea pig red cells (GPRC) (Pick et al., 1970; Taylor et al., 1975). 0.1 ml washed GPttC, suspended to a concentration of 1% v/v in 0.15 m NaC1, were added to 0.1 ml of culture supern a t a n t in 12 X 75 m m glass tubes and shaken for 4 h in a water bath at 37°C. Agglutination was assessed macroscopically and microscopically using as controls GPRC added to MEM, to supernatant of unstimulated cultures, and to Con A dissolved at various concentrations in supernatant of unstimulated cultures. In agreement with Taylor et al. (1975), this m e t h o d detected the presence of Con A down to a concentration of 0.5 pg/ml. Additional methods of checking for the presence of free Con A, were to test for the ability of supernatants of Con A--pulsed LNC, cultured at 4 ° C, to inhibit macrophage migration, and to determine the effect of 0.1 M amethyl-D-mannoside (Sigma Chemical Co., St. Louis, Missouri) on the migration inhibitory capacity of Con A--pulsed LNC supernatants, obtained by culturing the cells at 37 ° C.
Assessment of MIF in supernatants MIF was quantitated by the classical capillary tube assay using oil-induced guinea pig peritoneal exudate cells and disposable plastic migration chambers (Sterilin, Teddington, Middlesex, England), as previously described (Manheimer and Pick, 1973). Supernatants were supplemented with 15% foetal calf serum (Gibco, Grand Island, N.Y.) just prior to the performance of the assay. Areas of migration were measured after 8--18 h of incubation and results expressed as per cent inhibition of migration, which was calculated as follows:
Percent inhibition = 100--
Area of migration in supernatant of Con A--pulsed lymphocytes Area of migration in supernatant of unstimulated lymphocytes
X 100
Each supernatant was tested in at least three chambers. RESULTS In a series of 9 successive cultures performed by the technique described under Materials and Methods and using LNC pooled from 10 guinea pigs per
145 culture, we regularly obtained supernatants with strong migration inhibitory activity. Per cent inhibition values were regularly higher than 50. On no occasion was migration inhibitory activity detected in supernatants of Con A--pulsed l y m p h o c y t e s , cultured at 4 ° C. Addition of 0.1 M ~-methyl-D-mannoside to supernatants of Con A-pulsed cells, exPressing MIF activity, did n o t reduce the extent of migration inhibition caused by these supernatants. • f4. Migration inhibitory activity was undxmmlshed after extensive dialysis against MEM, proving that it was not due to depletion of nutritional factors from the medium. All supernatants were tested for residual free Con A by GPRC agglutination and were found to be negative. DISCUSSION Pulse exposure of guinea pig l y m p h o c y t e s to Con A for 2 h at 37°C regularly results in the production of MIF. Such a methodological approach has been used in the past by Lamelin and Vassalli (1971), who described the production of MIF by guinea pig l y m p h o c y t e s pulsed in suspension with phytohaemagglutinin and a n t i l y m p h o c y t e serum. The main disadvantage of their technique was the need for thorough washing of the cells, employing repeated centrifugation. Removal of mitogens by centrifugation is further complicated by the fact that the cells are heavily clumped, which makes resuspension technically difficult and unavoidably damaging. The alternative m e t h o d , based on the use of insolubilized Con A, suffers from c o m m o n l y encountered leakage of lectin from the Sepharose matrix and from the difficulty with which such preparations are standardized, stored for longer periods and handled. It is also probable that a certain proportion of l y m p h o c y t e s will not come in contact with the Con A beads, even though rocking of cultures was recommended (Friedrich et al., 1975). On the other hand, the extreme difficulties met with when soluble Con A has to be removed from supernatants, have been amply listed by Taylor et al. (1975). If such removal is to be complete, it involves a sequence of technically cumbersome steps with the inherent danger of loss of part of the MIF. The m e t h o d we describe is simple, uses regular soluble Con A, avoids repeated handling of the cells and does n o t require any t r e a t m e n t of the supernatant after culture. It is based on the observation that Con A causes strong a t t a c h m e n t of an originally nonadherent l y m p h o c y t e population to the surface of Falcon or equivalent flasks. The mechanism of this a t t a c h m e n t is not clear but it probably involves the specific binding of Con A to lymphocyte membrane glycoproteins and its nonspecific absorption to the flask surface, therefore acting as a bridge between the cell and the flask. Con A was recently found to increase the strength of a t t a c h m e n t of baby hamster kidney cells to Falcon flasks (Grinnell, 1973).
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ACKNOWLEDGEMENTS This research was s u p p o r t e d b y g r a n t 5 R 0 1 I n s t i t u t e s o f Health, Bethesda, Md., by a g r a n t B i n a t i o n a l Science F o u n d a t i o n a n d b y a g r a n t & Co. L t d . , Basle, S w i t z e r l a n d . T h e c o m p e t e n t S c h w a r t z is greatfully a c k n o w l e d g e d .
A I 1 1 1 9 4 f r o m the N a t i o n a l f r o m the U n i t e d States-Israel f r o m F. H o f f m a n n - L a R o c h e secretarial help o f Mrs. Julia
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