Clin. exp. Immunol. (1975) 22, 522-527.
Production of leucocyte inhibitory factor (LIF) and macrophage inhibitory factor (MIF) by PHA-stimulated lymphocytes RUTH LOMNITZER, A. R. RABSON & H. J. KOORNHOF Department of Microbiology, School ofPathology, University of Witwatersrand, and the South African Institute for Medical Research, Johannesburg, South Africa
(Received 2 January 1975) SUM MARY
Supernatants from human mononuclear cells cultured with PHA inhibited the migration of both human polymorphonuclear leucocytes and guinea-pig peritoneal exudate cells, but not human mononuclear cells. Using ultrafiltration it was shown that these supernatants contained two inhibiting factors, the one with a molecular weight of 15,000-50,000 inhibited only guinea-pig peritoneal exudate cells (MIF), whereas the fraction containing molecules of a size between 50,000 and 75,000 specifically inhibited the migration of polymorphonuclear leucocytes (LIF). The polymorphonuclear leucocyte inhibiting activity was heat labile. It is suggested that the leucocyte migration inhibition test is dependent upon the production of a lymphokine (LIF) which acts specifically on polymorphonuclear leucocytes causing their inhibition of migration. INTRODUCTION The leucocyte migration inhibition test has been shown to be a specific measure of cellmediated immunity in man (S0borg & Bendixen, 1967). It has been employed to determine sensitization to a variety of antigens such as tuberculin (Federlin et al., 1971), Candida (Astor et al., 1973), tumour-specific (Segal et al., 1972) and transplantation antigens (Falk et al., 1972). Various workers have suggested that the test relies upon different indicator cells, and Clausen (1970) pointed out that both polymorphonuclear leucocytes (PMN) and mononuclear cells (MN) are essential for inhibition to occur in the presence of specific antigens. Different authors, however, have shown more reliable results using a PMN-free leucocyte preparation (Bull et al., 1973), whereas other observations suggest that direct interaction of antigen with PMN cells is important in this test. The technique as originally performed relied on the direct inhibition of leucocytes by antigen. Later studies have shown that human leucocytes both in capillary tubes (Rocklin, 1974) or in agarose (Clausen, 1973) are inhibited from migrating by products of activated lymphocytes. To investigate the nature of this lymphokine and its effector cells, a two-stage test was used whereby PHA-induced supernatants from human lymphocyte cultures were tested for their inhibiting effect on the migration of different indicator cells. In this system it was found that a soluble mediator different from migratory inhibitory factor (MIF) inhibited PMN specifically. MATERIALS AND METHODS Production of migration inhibiting supernatants. Normal venous blood was collected and mixed with preservative-free heparin (Panheparin Abbott) (10 i.u./ml). The mononuclear cells (MN) were separated by Correspondence: Mrs Ruth Lomnitzer, School of Pathology, P.O. Box 1038, Johannesburg, South Africa.
522
Production of leucocyte inhibitory factor (LIF)
523
centrifugation in a Ficoll-Hypaque gradient (Boyum, 1968) and the MN cell fraction at the medium-Ficoll interphase was found to consist of approximately 60-70% lymphocytes, 15-20% monocytes and 10% PMN. Viability was greater than 950 as assessed by trypan blue exclusion. The cells were washed twice in minimal essential medium (MEM) and the lymphocyte count was adjusted to 2 x 106/ml in MEM containing 10% FCS (Burroughs-Wellcome) 2 mM L-glutamine (Flow Laboratories), 100 u/mI of penicillin and 100 jug/ml of streptomycin. Lymphocytes were incubated in the presence (active supernatant) and in the absence (control supernatant) of 10 jug/ml of PHA (reagent grade Burroughs Wellcome) in plastic tissue culture flasks (Falcon). Cultures were incubated at 370C in 5% CO2 in air for 72 hr. The cell-free supernatants were obtained by centrifugation at 250 g for 10 min. The supernatants from the control cultures were divided into two and to one part 10 ,ug/ml of PHA was added. This supernatant will be referred to as PHA-reconstituted control supernatant. Fractionation by ultrafiltration. Active supernataits and PHA-reconstituted control supernatants were crudely fractionated using the Amicon Centriflo membrane which retains molecules above 50,000 molecular weight and the Minicon-B 15 and A 75 with a molecular weight cut-off of 15,000 and 75,000 respectively. (Amicon Corporation, Lexington, Massachusetts). The supernatants were centrifuged in the Centriflo membrane at 250 g for 30 min until they were concentrated approximately ten-fold. The fraction which passed through the membrane was collected and recentrifuged and this process was repeated three times. The resultant concentrate retained by the membrane was diluted with MEM containing 10% FCS to threequarters of the original volume (upper fraction). The fraction which passed through the membrane was concentrated in the Minicon B 15 approximately ten-fold and diluted with MEM + 10% FCS to about three-quarters of the original volume (bottom fraction). Supernatants were also concentrated ten-fold in the Minicon A 75 and were then reconstituted with MEM to the original volume. This process was repeated three times and the resultant concentrate was diluted with MEM+ 10% FCS to three-quarters of the original volume. All samples were sterilized by millipore filtration (045 pum Millipore Corporation, Bedford, Massachusetts). Migration inhibition assay. Oil-induced guinea-pig peritoneal exudate cells (PEC) were obtained from normal laboratory guinea-pigs according to the method of Thor (1970). Human PMN and MN leucocytes were obtained by separating whole blood diluted 1:2 with MEM on Ficoll-Hypaque gradients. The MN cell fraction was collected from the medium-Ficoll interphase and washed three times in MEM. The pellet containing erythrocytes and PMN was treated with 0-830 ammonium chloride at 4°C for 10 min to lyse the erythrocytes. The remaining PMN were centrifuged at 250 g for 5 min and washed three times in MEM. The resultant suspension contained 98-99% PMN. Siliconized capillary tubes (50 A Yankee disposable micropet, Clay Adams) were used in all the experiments, and were filled with cell suspensions containing 3 x 107 cells/ml. The capillary tubes were then sealed with seal-ease (Clay Adams) and centrifuged for 5 min at 300 g. The tubes were cut at the cell-fluid interface, and the capillary stumps were then placed on the floor of small tissue culture chambers (Steriline, Middlesex) held in place by a drop of silicon grease. In testing the effect of supernatants on the migration pattern of different indicator cells, active, control, and PHA reconstituted control supernatants were used to fill the chamber. The chambers were incubated at 37°C in 500 CO2 in air. When migration of human MN and PMN leucocytes was being assessed the chambers were incubated for 16-18 hr, and in the case of G.P. PEC for 40-42 h. The migration pattern was projected and traced and the area of migration was measured by planimetry. Two indices of migration were calculated. (a) For active supernatants: migration index (MI) = (area of migration in the presence of active supernatant)/(area of migration in the presence of PHA-reconstituted supernatant). (b) for PHA-reconstituted control supernatants: MI = (area of migration in the presence of PHAreconstituted control supernatant)/(area of migration in the presence of control supernatant). When the ultrafiltration fractions were assessed for their effect on the migration of the different indicator cells, the MI was calculated as follows: MI = (area of migration in the presence of active supernatant fraction)/(area of migration in the presence of PHA-reconstituted control supernatant fraction). The mean + s.e.m. migrations were determined by averaging the migration from four to eight capillary tubes, and statistical significance was determined by the Student's t-test for paired means. A P value higher than 0-05 was considered not significant. Heat stability. Samples of active, control and PHA-reconstituted control supernatants were heated in a water bath at 56°C for 30 min. Their effect on PMN migration was assessed before and after the heating.
RESULTS The effect of active supernatants on the migration of different indicator cells Active supernatants from normal human lymphocyte cultures were tested for their effect
524
Ruth Lomnitzer, A. R. Rabson and H. J. Koornhof 0
l
0 0 0
-LI
1.0 0
0
0
x
I
.c
-
0
L 0 0
.2
0-4~
0-2k Human MN
Human PMN
Guinea-pig PEC
FIG. 1. The effects of active supernatants (0) and PHA-reconstituted supernatants (o) on the migration of different indicator cells.
on normal human MN and PMN leucocyte migration and normal guinea-pig PEC
migration. As can be seen from Fig. 1 the active supernatants caused inhibition of human PMN and guinea-pig PEC but not of human MN cells. To rule out the possibility that the inhibition of migration was caused non-specifically by the PHA present in the active supernatants the effect of reconstituted PHA control supernatants on the migration of the various indicator cells was tested. It was shown that human PMN were markedly inhibited by the active supernatants with a mean migration index of 0-71 (P
Production of leucocyte inhibitory factor (LIF) and macrophage inhibitory factor (MIF) by PHA-stimulated lymphocytes RUTH LOMNITZER, A. R. RABSON & H. J. KOORNHOF Department of Microbiology, School ofPathology, University of Witwatersrand, and the South African Institute for Medical Research, Johannesburg, South Africa
(Received 2 January 1975) SUM MARY
Supernatants from human mononuclear cells cultured with PHA inhibited the migration of both human polymorphonuclear leucocytes and guinea-pig peritoneal exudate cells, but not human mononuclear cells. Using ultrafiltration it was shown that these supernatants contained two inhibiting factors, the one with a molecular weight of 15,000-50,000 inhibited only guinea-pig peritoneal exudate cells (MIF), whereas the fraction containing molecules of a size between 50,000 and 75,000 specifically inhibited the migration of polymorphonuclear leucocytes (LIF). The polymorphonuclear leucocyte inhibiting activity was heat labile. It is suggested that the leucocyte migration inhibition test is dependent upon the production of a lymphokine (LIF) which acts specifically on polymorphonuclear leucocytes causing their inhibition of migration. INTRODUCTION The leucocyte migration inhibition test has been shown to be a specific measure of cellmediated immunity in man (S0borg & Bendixen, 1967). It has been employed to determine sensitization to a variety of antigens such as tuberculin (Federlin et al., 1971), Candida (Astor et al., 1973), tumour-specific (Segal et al., 1972) and transplantation antigens (Falk et al., 1972). Various workers have suggested that the test relies upon different indicator cells, and Clausen (1970) pointed out that both polymorphonuclear leucocytes (PMN) and mononuclear cells (MN) are essential for inhibition to occur in the presence of specific antigens. Different authors, however, have shown more reliable results using a PMN-free leucocyte preparation (Bull et al., 1973), whereas other observations suggest that direct interaction of antigen with PMN cells is important in this test. The technique as originally performed relied on the direct inhibition of leucocytes by antigen. Later studies have shown that human leucocytes both in capillary tubes (Rocklin, 1974) or in agarose (Clausen, 1973) are inhibited from migrating by products of activated lymphocytes. To investigate the nature of this lymphokine and its effector cells, a two-stage test was used whereby PHA-induced supernatants from human lymphocyte cultures were tested for their inhibiting effect on the migration of different indicator cells. In this system it was found that a soluble mediator different from migratory inhibitory factor (MIF) inhibited PMN specifically. MATERIALS AND METHODS Production of migration inhibiting supernatants. Normal venous blood was collected and mixed with preservative-free heparin (Panheparin Abbott) (10 i.u./ml). The mononuclear cells (MN) were separated by Correspondence: Mrs Ruth Lomnitzer, School of Pathology, P.O. Box 1038, Johannesburg, South Africa.
522
Production of leucocyte inhibitory factor (LIF)
523
centrifugation in a Ficoll-Hypaque gradient (Boyum, 1968) and the MN cell fraction at the medium-Ficoll interphase was found to consist of approximately 60-70% lymphocytes, 15-20% monocytes and 10% PMN. Viability was greater than 950 as assessed by trypan blue exclusion. The cells were washed twice in minimal essential medium (MEM) and the lymphocyte count was adjusted to 2 x 106/ml in MEM containing 10% FCS (Burroughs-Wellcome) 2 mM L-glutamine (Flow Laboratories), 100 u/mI of penicillin and 100 jug/ml of streptomycin. Lymphocytes were incubated in the presence (active supernatant) and in the absence (control supernatant) of 10 jug/ml of PHA (reagent grade Burroughs Wellcome) in plastic tissue culture flasks (Falcon). Cultures were incubated at 370C in 5% CO2 in air for 72 hr. The cell-free supernatants were obtained by centrifugation at 250 g for 10 min. The supernatants from the control cultures were divided into two and to one part 10 ,ug/ml of PHA was added. This supernatant will be referred to as PHA-reconstituted control supernatant. Fractionation by ultrafiltration. Active supernataits and PHA-reconstituted control supernatants were crudely fractionated using the Amicon Centriflo membrane which retains molecules above 50,000 molecular weight and the Minicon-B 15 and A 75 with a molecular weight cut-off of 15,000 and 75,000 respectively. (Amicon Corporation, Lexington, Massachusetts). The supernatants were centrifuged in the Centriflo membrane at 250 g for 30 min until they were concentrated approximately ten-fold. The fraction which passed through the membrane was collected and recentrifuged and this process was repeated three times. The resultant concentrate retained by the membrane was diluted with MEM containing 10% FCS to threequarters of the original volume (upper fraction). The fraction which passed through the membrane was concentrated in the Minicon B 15 approximately ten-fold and diluted with MEM + 10% FCS to about three-quarters of the original volume (bottom fraction). Supernatants were also concentrated ten-fold in the Minicon A 75 and were then reconstituted with MEM to the original volume. This process was repeated three times and the resultant concentrate was diluted with MEM+ 10% FCS to three-quarters of the original volume. All samples were sterilized by millipore filtration (045 pum Millipore Corporation, Bedford, Massachusetts). Migration inhibition assay. Oil-induced guinea-pig peritoneal exudate cells (PEC) were obtained from normal laboratory guinea-pigs according to the method of Thor (1970). Human PMN and MN leucocytes were obtained by separating whole blood diluted 1:2 with MEM on Ficoll-Hypaque gradients. The MN cell fraction was collected from the medium-Ficoll interphase and washed three times in MEM. The pellet containing erythrocytes and PMN was treated with 0-830 ammonium chloride at 4°C for 10 min to lyse the erythrocytes. The remaining PMN were centrifuged at 250 g for 5 min and washed three times in MEM. The resultant suspension contained 98-99% PMN. Siliconized capillary tubes (50 A Yankee disposable micropet, Clay Adams) were used in all the experiments, and were filled with cell suspensions containing 3 x 107 cells/ml. The capillary tubes were then sealed with seal-ease (Clay Adams) and centrifuged for 5 min at 300 g. The tubes were cut at the cell-fluid interface, and the capillary stumps were then placed on the floor of small tissue culture chambers (Steriline, Middlesex) held in place by a drop of silicon grease. In testing the effect of supernatants on the migration pattern of different indicator cells, active, control, and PHA reconstituted control supernatants were used to fill the chamber. The chambers were incubated at 37°C in 500 CO2 in air. When migration of human MN and PMN leucocytes was being assessed the chambers were incubated for 16-18 hr, and in the case of G.P. PEC for 40-42 h. The migration pattern was projected and traced and the area of migration was measured by planimetry. Two indices of migration were calculated. (a) For active supernatants: migration index (MI) = (area of migration in the presence of active supernatant)/(area of migration in the presence of PHA-reconstituted supernatant). (b) for PHA-reconstituted control supernatants: MI = (area of migration in the presence of PHAreconstituted control supernatant)/(area of migration in the presence of control supernatant). When the ultrafiltration fractions were assessed for their effect on the migration of the different indicator cells, the MI was calculated as follows: MI = (area of migration in the presence of active supernatant fraction)/(area of migration in the presence of PHA-reconstituted control supernatant fraction). The mean + s.e.m. migrations were determined by averaging the migration from four to eight capillary tubes, and statistical significance was determined by the Student's t-test for paired means. A P value higher than 0-05 was considered not significant. Heat stability. Samples of active, control and PHA-reconstituted control supernatants were heated in a water bath at 56°C for 30 min. Their effect on PMN migration was assessed before and after the heating.
RESULTS The effect of active supernatants on the migration of different indicator cells Active supernatants from normal human lymphocyte cultures were tested for their effect
524
Ruth Lomnitzer, A. R. Rabson and H. J. Koornhof 0
l
0 0 0
-LI
1.0 0
0
0
x
I
.c
-
0
L 0 0
.2
0-4~
0-2k Human MN
Human PMN
Guinea-pig PEC
FIG. 1. The effects of active supernatants (0) and PHA-reconstituted supernatants (o) on the migration of different indicator cells.
on normal human MN and PMN leucocyte migration and normal guinea-pig PEC
migration. As can be seen from Fig. 1 the active supernatants caused inhibition of human PMN and guinea-pig PEC but not of human MN cells. To rule out the possibility that the inhibition of migration was caused non-specifically by the PHA present in the active supernatants the effect of reconstituted PHA control supernatants on the migration of the various indicator cells was tested. It was shown that human PMN were markedly inhibited by the active supernatants with a mean migration index of 0-71 (P
