Immunology 1979 37 715
Cellular co-operation in the production of human leucocyte inhibitory factor by lymphocyte subpopulations stimulated with antigens and allogeneic cells LIISA RASANEN Institute of Biomedical Sciences, University of Tampere, Finland
Acceptedfor publication 17 January 1979
Summary. The ability of antigens and allogeneic cells to induce lymphokine synthesis and cellular co-operation in lymphokine production was investigated. Human peripheral blood lymphocytes were separated into T- and B-cell populations by sheep red blood cell rosette formation and centrifugation on Ficoll-Isopaque. The cells were then stimulated with PPD, SK-SD, candida and with allogeneic cells. The presence of leucocyte inhibitory factor (LIF) in the culture supernatants was tested by the agarose migration method. The results indicated that T lymphocytes produced LIF after stimulation with antigens and allogeneic cells. In addition, B cells responded to PPD. Except for PPD the stimulants did not induce significant T-cell LIF production in the absence of monocytes. Only autologous monocytes enhanced LIF synthesis after antigenic stimulation, whereas in mixed lymphocyte cultures allogeneic monocytes were as effective as autologous ones. The monocyte helper effect was mediated by soluble factors in mixed lymphocyte cultures and by soluble factors and direct cell-cell contact in antigen-stimulated cultures. No co-operation between T and B lymphocytes was found. B cells did not enhance LIF production by T cells, nor could T cells induce a B-cell response to antigens or allogeneic cells. Correspondence: Dr Liisa Rasanen, Institute of Biomedical Sciences, University of Tampere, SF-33 101 Tampere 10, Finland. 0019-2805/79/0800-0715 $02.00 © 1979 Blackwell Scientific Publications
INTRODUCTION
Lymphokines, the soluble mediators elaborated by lymphocytes, have been connected with cell-mediated immunity and T cells (Lawrence & Landy, 1969). This concept has become the subject of re-evaluation after several workers have found that B lymphocytes are able to produce lymphokines as well (Yoshida, Sonozaki & Cohen, 1973; Mackler, Altman, Rosenstreich & Oppenheim, 1974; Rocklin, MacDermott, Chess, Schlossman & David, 1974). The requirement for phagocytic cells in lymphokine synthesis (Nelson & Leu, 1974; Ohishi & Onoue, 1975; Wahl, Wilton, Rosenstreich & Oppenheim, 1975; Twomey, Lazar & Rocklin, 1977) has also diminished T-cell dominauce in the concept. At present, lymphokine synthesis in vivo is not believed to represent the function of a single cell population but to be the result of interactions between various cells. While it is generally accepted that phagocytic cells play a role in antigen-induced T-cell lymphokine synthesis, it is not settled whether this role is facilitative but not obligatory, or obligatory. Controversy also exists as to whether antigens activate B lymphocytes directly to elaborate lymphokines (Rocklin et al., 1974) or whether B-cell lymphokine synthesis is dependent on T cells (Wahl & Rosenstreich, 1976). The other side of T cell-B cell interactions, viz. the effect of B lymphocytes on T-cell lymphokine synthesis has been studied very little (Twomey et al., 1977; Weisbart, Yu, Billing, Fan, Clements & Paulus, 1978). 715
716
Liisa Rdsdnen
In this investigation, the ability of antigens and allogeneic cells to induce leucocyte inhibitory factor (LIF) synthesis and the interactions between T cells and monocytes and between T and B cells in lymphokine production were studied.
MATERIALS AND METHODS Separation and identification of cells Mononuclear cells from healthy human individuals were obtained by Ficoll-Isopaque centrifugation of heparinized or citrated peripheral blood (Boyum, 1968). These cells were then incubated at 370 for 45 min with 0 2 g of carbonyl iron in Hanks's balanced salt solution (HBSS) supplemented with 10% foetal bovine serum (Flow Laboratories, Irvine, Scotland), after which the phagocytic cells were removed with a magnet. In order to obtain purified T and B cells the lymphocyte suspension was rosetted with sheep red blood cells (SRBC) and centrifuged on Ficoll-Isopaque as described before (Rasanen, Karhumaki & Krohn, 1978). Monocytes were purified as follows. 40 x 106 mononuclear cells were incubated for 30 min in a tissue culture flask (growth area 25 cm2, Falcon, Oxnard, Ca., U.S.A.) containing 10 ml of HBSS supplemented with 10% foetal bovine serum. In order to remove the non-adherent cells the flasks were shaken vigorously with HBSS, after which phosphate-buffered saline (PBS) containing 0-02% EDTA was added to the flasks. After a 2 h incubation at room temperature the adherent cells were removed by pipetting vigorously with a Pasteur pipette. The viability of the recovered monocytes was > 85%. The purity of the cell populations was studied as previously described (Rasanen et al., 1978) using SRBC rosette formation to demonstrate T cells, staining surface membrane immunoglobulin to demonstrate B cells and staining non-specific esterase to demonstrate monocytes.
Cell cultures In the first series of experiments, the ability of antigens and allogeneic cells to induce LIF synthesis was investigated. The cells were suspended at a concentration of 106 cells/ml in RPMI 1640 (Flow Laboratories) supplemented with 10% horse serum (Microbiological Associates, Bethesda, Maryland, U.S.A.). Duplicate cultures containing 105 cells/well were set up in Ubottomed microplates (Sterilin Ltd, Middlesex). The
cells were incubated in the absence (controls) or in the presence of various concentrations of the following antigens: PPD (Statens Seruminstitut, Copenhagen, Denmark), SK-SD (dialysed for 48 h against PBS, Lederle, Pearl River, N.Y., U.S.A.) and candida (dialysed, Hollister-Stier, Spokane, Wash., U.S.A.) at 37' in a 5% CO2 atmosphere for 3 days. After incubation the controls were reconstituted with corresponding amounts of these antigens. In mixed lymphocyte cultures the cells were stimulated with allogeneic mononuclear, T or B cells. The controls were cultured in the presence of corresponding amounts of autologous cells. In subsequent experiments, cellular co-operation in LIF synthesis was investigated. When the effect of monocytes on LIF production was studied, increasing amounts of monocytes were added to the T cells while keeping the lymphocyte number constant, i.e. 105 cells/well. When the effect of B cells on T-cell LIF synthesis was studied the sum of T and B lymphocytes was kept constant. Unless otherwise stated, the cells were stimulated with 12 5 pg/ml of PPD, 400 u/ml of SK-SD, a 1:20 dilution of candida or 105 allogeneic T or B lymphocytes. These concentrations were found to be optimum in previous experiments. Puromycin treatment of cells The protein synthesis of cells was blocked by puromycin as described by Gorski, Dupont, Hansen & Good (1976). In brief, the cells were pulse-treated with 500 pg/ml of puromycin for 90 min, after which the cells were washed five times. The stimulator cells in oneway mixed lymphocyte cultures and monocytes and T lymphocytes in co-operation experiments were treated in this way. Monocytes were treated by puromycin in order to prevent the elaboration of soluble factors affecting LIF production by T cells. In oder to study whether the presence of T lymphocytes enables B cells to respond to antigens and allogeneic cells, B lymphocytes were cultured together with puromycin-treated T lymphocytes.
Cell cultures in Marbrook-type culture tessels In order to study whether the monocyte helper effect was mediated by soluble factors, T lymphocytes and monocytes were cultured in double-chambered Marbrook-type culture vessels. The vessels were constructed by glueing a 0 22 pore-size filter with non-toxic cement (both from Millipore Co., Bedford, Mass., U.S.A.) onto a bottomless tissue culture tube and sterilizing this inner part with UV light before
717
Cellular co-operation and human leucocyte inhibitoryfactor placing it in a bigger tube. Monocytes were cultured in the upper compartment and T lymphocytes in the lower one. The total volume of the culture was 0 7 ml. B-cell cultures in T-cell supernatants In order to study whether T lymphocytes are able to produce soluble factors which enable B cells to respond to antigens and allogeneic cells, B lymphocytes were incubated in the culture supernatants of T cells. To produce T-cell culture supernatants, T cells supplemented with 5% monocytes were pulse-treated with 800 u/ml of SK-SD for 30 min after which the cells were washed three times, or they were cultured in the presence of allogeneic B cells (responder:stimulator ratio 1:1). The culture supernatants were collected after 12, 24 or 48 h of incubation. B lymphocytes stimulated with 400 u/ml SK-SD or 105 allogeneic B cells were then cultured in various dilutions of antigenor allogeneic cell-stimulated T-cell supernatants, respectively. Leucocyte migration inhibitory factor assay LIF activity was tested in the culture supernatants by the agarose migration method as described by Rasanen et al., (1978). The migration index (MI) was calculated as follows: Ml=
area of migration in the presence of test supernatant
1.2 PPD w
0 z
0
SK-SD
Candida
Z+NiI
1.0
0.8
o 0.6
0.4J
, i, 6.2 12.5 25 100 200 400 800 1:80 1:40 1:20 1:10 CONCENTRATION OF ANTIGENS
3.1
Figure 1. Elaboration of LIF by antigen-stimulated cells. The concentrations of PPD, SK-SD and candida are expressed as ug/ml, units of streptokinase/ml and dilution of the stock solution, respectively. (A) Mononuclear cells; (m) T lymphocytes; (-) B lymphocytes. Each point represents the mean £ SD of four to seven experiments.
not with SK-SD or candida. At optimum concentrations of antigens PPD induced greater LIF production than SK-SD or candida. In these experiments the blood donors were not skin-tested. In a separate investigation we skin-tested seventy-one healthy individuals. More than 90% were positive to PPD (highest dose 10 TU), SK-SD (highest dose 50 u streptokinase) or candida (highest dose 1:5). Thus healthy persons lacking cell-mediated immunity to these antigens are rare.
area of migration in the presence of control supernatant
1.2rMN Generally, MIs smaller than 0-85 represented significant inhibition of migration as calculated by Student's t test. The significance of differences between Mls was calculated by Mann-Whitney's U test.
x
Antigen- and allogeneic cell-induced LIF production The separation method used yielded T-cell populations contaminated with an average of 2-4% B lymphocytes and
Cellular co-operation in the production of human leucocyte inhibitory factor by lymphocyte subpopulations stimulated with antigens and allogeneic cells LIISA RASANEN Institute of Biomedical Sciences, University of Tampere, Finland
Acceptedfor publication 17 January 1979
Summary. The ability of antigens and allogeneic cells to induce lymphokine synthesis and cellular co-operation in lymphokine production was investigated. Human peripheral blood lymphocytes were separated into T- and B-cell populations by sheep red blood cell rosette formation and centrifugation on Ficoll-Isopaque. The cells were then stimulated with PPD, SK-SD, candida and with allogeneic cells. The presence of leucocyte inhibitory factor (LIF) in the culture supernatants was tested by the agarose migration method. The results indicated that T lymphocytes produced LIF after stimulation with antigens and allogeneic cells. In addition, B cells responded to PPD. Except for PPD the stimulants did not induce significant T-cell LIF production in the absence of monocytes. Only autologous monocytes enhanced LIF synthesis after antigenic stimulation, whereas in mixed lymphocyte cultures allogeneic monocytes were as effective as autologous ones. The monocyte helper effect was mediated by soluble factors in mixed lymphocyte cultures and by soluble factors and direct cell-cell contact in antigen-stimulated cultures. No co-operation between T and B lymphocytes was found. B cells did not enhance LIF production by T cells, nor could T cells induce a B-cell response to antigens or allogeneic cells. Correspondence: Dr Liisa Rasanen, Institute of Biomedical Sciences, University of Tampere, SF-33 101 Tampere 10, Finland. 0019-2805/79/0800-0715 $02.00 © 1979 Blackwell Scientific Publications
INTRODUCTION
Lymphokines, the soluble mediators elaborated by lymphocytes, have been connected with cell-mediated immunity and T cells (Lawrence & Landy, 1969). This concept has become the subject of re-evaluation after several workers have found that B lymphocytes are able to produce lymphokines as well (Yoshida, Sonozaki & Cohen, 1973; Mackler, Altman, Rosenstreich & Oppenheim, 1974; Rocklin, MacDermott, Chess, Schlossman & David, 1974). The requirement for phagocytic cells in lymphokine synthesis (Nelson & Leu, 1974; Ohishi & Onoue, 1975; Wahl, Wilton, Rosenstreich & Oppenheim, 1975; Twomey, Lazar & Rocklin, 1977) has also diminished T-cell dominauce in the concept. At present, lymphokine synthesis in vivo is not believed to represent the function of a single cell population but to be the result of interactions between various cells. While it is generally accepted that phagocytic cells play a role in antigen-induced T-cell lymphokine synthesis, it is not settled whether this role is facilitative but not obligatory, or obligatory. Controversy also exists as to whether antigens activate B lymphocytes directly to elaborate lymphokines (Rocklin et al., 1974) or whether B-cell lymphokine synthesis is dependent on T cells (Wahl & Rosenstreich, 1976). The other side of T cell-B cell interactions, viz. the effect of B lymphocytes on T-cell lymphokine synthesis has been studied very little (Twomey et al., 1977; Weisbart, Yu, Billing, Fan, Clements & Paulus, 1978). 715
716
Liisa Rdsdnen
In this investigation, the ability of antigens and allogeneic cells to induce leucocyte inhibitory factor (LIF) synthesis and the interactions between T cells and monocytes and between T and B cells in lymphokine production were studied.
MATERIALS AND METHODS Separation and identification of cells Mononuclear cells from healthy human individuals were obtained by Ficoll-Isopaque centrifugation of heparinized or citrated peripheral blood (Boyum, 1968). These cells were then incubated at 370 for 45 min with 0 2 g of carbonyl iron in Hanks's balanced salt solution (HBSS) supplemented with 10% foetal bovine serum (Flow Laboratories, Irvine, Scotland), after which the phagocytic cells were removed with a magnet. In order to obtain purified T and B cells the lymphocyte suspension was rosetted with sheep red blood cells (SRBC) and centrifuged on Ficoll-Isopaque as described before (Rasanen, Karhumaki & Krohn, 1978). Monocytes were purified as follows. 40 x 106 mononuclear cells were incubated for 30 min in a tissue culture flask (growth area 25 cm2, Falcon, Oxnard, Ca., U.S.A.) containing 10 ml of HBSS supplemented with 10% foetal bovine serum. In order to remove the non-adherent cells the flasks were shaken vigorously with HBSS, after which phosphate-buffered saline (PBS) containing 0-02% EDTA was added to the flasks. After a 2 h incubation at room temperature the adherent cells were removed by pipetting vigorously with a Pasteur pipette. The viability of the recovered monocytes was > 85%. The purity of the cell populations was studied as previously described (Rasanen et al., 1978) using SRBC rosette formation to demonstrate T cells, staining surface membrane immunoglobulin to demonstrate B cells and staining non-specific esterase to demonstrate monocytes.
Cell cultures In the first series of experiments, the ability of antigens and allogeneic cells to induce LIF synthesis was investigated. The cells were suspended at a concentration of 106 cells/ml in RPMI 1640 (Flow Laboratories) supplemented with 10% horse serum (Microbiological Associates, Bethesda, Maryland, U.S.A.). Duplicate cultures containing 105 cells/well were set up in Ubottomed microplates (Sterilin Ltd, Middlesex). The
cells were incubated in the absence (controls) or in the presence of various concentrations of the following antigens: PPD (Statens Seruminstitut, Copenhagen, Denmark), SK-SD (dialysed for 48 h against PBS, Lederle, Pearl River, N.Y., U.S.A.) and candida (dialysed, Hollister-Stier, Spokane, Wash., U.S.A.) at 37' in a 5% CO2 atmosphere for 3 days. After incubation the controls were reconstituted with corresponding amounts of these antigens. In mixed lymphocyte cultures the cells were stimulated with allogeneic mononuclear, T or B cells. The controls were cultured in the presence of corresponding amounts of autologous cells. In subsequent experiments, cellular co-operation in LIF synthesis was investigated. When the effect of monocytes on LIF production was studied, increasing amounts of monocytes were added to the T cells while keeping the lymphocyte number constant, i.e. 105 cells/well. When the effect of B cells on T-cell LIF synthesis was studied the sum of T and B lymphocytes was kept constant. Unless otherwise stated, the cells were stimulated with 12 5 pg/ml of PPD, 400 u/ml of SK-SD, a 1:20 dilution of candida or 105 allogeneic T or B lymphocytes. These concentrations were found to be optimum in previous experiments. Puromycin treatment of cells The protein synthesis of cells was blocked by puromycin as described by Gorski, Dupont, Hansen & Good (1976). In brief, the cells were pulse-treated with 500 pg/ml of puromycin for 90 min, after which the cells were washed five times. The stimulator cells in oneway mixed lymphocyte cultures and monocytes and T lymphocytes in co-operation experiments were treated in this way. Monocytes were treated by puromycin in order to prevent the elaboration of soluble factors affecting LIF production by T cells. In oder to study whether the presence of T lymphocytes enables B cells to respond to antigens and allogeneic cells, B lymphocytes were cultured together with puromycin-treated T lymphocytes.
Cell cultures in Marbrook-type culture tessels In order to study whether the monocyte helper effect was mediated by soluble factors, T lymphocytes and monocytes were cultured in double-chambered Marbrook-type culture vessels. The vessels were constructed by glueing a 0 22 pore-size filter with non-toxic cement (both from Millipore Co., Bedford, Mass., U.S.A.) onto a bottomless tissue culture tube and sterilizing this inner part with UV light before
717
Cellular co-operation and human leucocyte inhibitoryfactor placing it in a bigger tube. Monocytes were cultured in the upper compartment and T lymphocytes in the lower one. The total volume of the culture was 0 7 ml. B-cell cultures in T-cell supernatants In order to study whether T lymphocytes are able to produce soluble factors which enable B cells to respond to antigens and allogeneic cells, B lymphocytes were incubated in the culture supernatants of T cells. To produce T-cell culture supernatants, T cells supplemented with 5% monocytes were pulse-treated with 800 u/ml of SK-SD for 30 min after which the cells were washed three times, or they were cultured in the presence of allogeneic B cells (responder:stimulator ratio 1:1). The culture supernatants were collected after 12, 24 or 48 h of incubation. B lymphocytes stimulated with 400 u/ml SK-SD or 105 allogeneic B cells were then cultured in various dilutions of antigenor allogeneic cell-stimulated T-cell supernatants, respectively. Leucocyte migration inhibitory factor assay LIF activity was tested in the culture supernatants by the agarose migration method as described by Rasanen et al., (1978). The migration index (MI) was calculated as follows: Ml=
area of migration in the presence of test supernatant
1.2 PPD w
0 z
0
SK-SD
Candida
Z+NiI
1.0
0.8
o 0.6
0.4J
, i, 6.2 12.5 25 100 200 400 800 1:80 1:40 1:20 1:10 CONCENTRATION OF ANTIGENS
3.1
Figure 1. Elaboration of LIF by antigen-stimulated cells. The concentrations of PPD, SK-SD and candida are expressed as ug/ml, units of streptokinase/ml and dilution of the stock solution, respectively. (A) Mononuclear cells; (m) T lymphocytes; (-) B lymphocytes. Each point represents the mean £ SD of four to seven experiments.
not with SK-SD or candida. At optimum concentrations of antigens PPD induced greater LIF production than SK-SD or candida. In these experiments the blood donors were not skin-tested. In a separate investigation we skin-tested seventy-one healthy individuals. More than 90% were positive to PPD (highest dose 10 TU), SK-SD (highest dose 50 u streptokinase) or candida (highest dose 1:5). Thus healthy persons lacking cell-mediated immunity to these antigens are rare.
area of migration in the presence of control supernatant
1.2rMN Generally, MIs smaller than 0-85 represented significant inhibition of migration as calculated by Student's t test. The significance of differences between Mls was calculated by Mann-Whitney's U test.
x
Antigen- and allogeneic cell-induced LIF production The separation method used yielded T-cell populations contaminated with an average of 2-4% B lymphocytes and
