JOURNAL OF INTERFERON RESEARCH 11:319-325 (1991) Mary Ann Liebert, Inc., Publishers
Natural Human Interferon-ct Augments Interleukin-2 Production by a Direct Action on the Activated IL-2-Producing T Cells VLADIMIR
HOLÁN,
KEIZO KOHNO, and JUN MINOWADA
ABSTRACT
production of interleukin-2 (IL-2) by Concanavalin A (ConA)-stimulated peripheral blood leukocytes (PBL) from normal human donors was enhanced by natural human interferon-a (IFN-a). The mechanism of the action of IFN-a on IL-2 production was studied further using cloned human leukemic T-cell lines, which produce IL-2 spontaneously and/or after mitogen stimulation. It was found that IFN-a alone did not stimulate The
production, but increased it in the activated cells. The ability of IFN-a to increase IL-2 production was abrogated by the treatment of IFN-a preparations with specific anti-IFN-a antibody, and the induced IL-2 activity was completely inhibited by monoclonal antibody against human IL-2. We suggest that the augmentation by IFN-a of IL-2 production in the activated T cells may be one of the mechanisms attributable to the immunotherapeutic action of IFN-a. IL-2
INTRODUCTION
MATERIALS AND METHODS
(IFN) represents a family of molecules that modulate immune response in both a positive and negative manIt has been shown that IFN inhibits many facets of the immune response, but some manifestations of the immune system can be rather enhanced by IFN. Among the immunostimulatory effects of IFN, enhancement of cytokine production was found in some models,'3 8I but in the other experimental systems no effects of IFN on lymphokine production have been
Cells: Fresh heparinized blood was obtained by venipuncfrom healthy volunteers. The mononuclear cell fraction was isolated by Ficoll-Paque (Pharmacia Chemicals, Uppsala, Sweden) density gradient centrifugation. The cells were washed three times in serum-free RPMI-1640 medium and resuspended in a medium containing 10% heat-inactivated fetal bovine serum (FBS, GIBCO, Grand Island, NY), 100 U/ml penicillin and 50 (xg/ml streptomycin to make a final cell concentration of 4 x 106 cells/ml. Human leukemic T-cell lines MOLT 4,"" MOLT 16,(12) HUT-102,"1' and Jurkat"4' were maintained in our laboratory in RPMI-1640 medium containing 5% heat-inactivated FBS.
I ner."'2'
NTERFERON
observed/9'101
We evaluated the question of the action of IFN on lymphokine production, and we found that natural human IFN-a, when added to the cultures of peripheral blood leukocytes (PBL), increased interleukin-2 (IL-2) production. Using monoclonal leukemic T-cell lines that produce IL-2 constitutively or inducibly, we demonstrated that IFN-ct alone does not directly stimulate IL-2 production, but increases IL-2 production by the activated cells. By means of specific antibodies, we showed that the stimulated lymphokine activity was antigenically and functionally identical to IL-2, and we suggest that the augmentation by IFN-a of lymphokine production in the activated cells may be one of the mechanisms attributable to the immunotherapeutic action of IFN-a.
ture
IFN-a: Highly purified natural human IFN-a prepared by Hayashibara Biochemical Laboratories Inc., (Okayama, Japan) was prepared from BALL-1 cells stimulated with the HVJ strain of Sendai virus.'IS) The IFN was a mixture of subspecies purified by an affinity column using monoclonal anti-human IFN-a antibody technique to a specific activity of 2 x 10H IU/mg of
protein."5'
Fujisaki Cell Center. Hayashibara Biochemical Laboratories. Inc., 675-1. Fujisaki, Okayama 702, Japan. 319
HOLÁN ET AL.
320 IL-2 Production: PBL were incubated at a concentration of 10" cells/ml in 96-well flat-bottomed tissue culture plates 4 (Corning Glass Works, Corning, NY) in a volume of 300 u.1 of medium alone, with 5 p,g/ml of Concanavalin A (ConA, Sigma Chemical Co., St. Louis, MO) and/or with varying concentrations of IFN-a. In the case of the leukemic cell lines, 2 x 106 cells/well were incubated with IFN-a in the presence or absence of 20 u-g/ml of phytohemagglutinin P (PHA, Difco Laboratories, Detroit, MI) in 24-well tissue culture plates (Corning) in 1 ml of RPMI-1640 medium containing 5% FBS. After 24 h of incubation at 37°C, the supernatants were collected and tested for their ability to support the growth of IL-2-dependent murine CTLL-2 cells."6' x
IL-2 Assay: One hundred microliters (5 X 104 cells/ml) per well of the IL-2-dependent CTLL-2 cells"6' were cultured in 96-well tissue culture plates with the same volume of serially diluted test supernatants. To determine cell proliferation, |3H]thymidine (0.5 u-Ci/well, sp. act. 6.7 Ci/mmole, DuPont, New England Nuclear, Boston, MA) was added into the cultures for the last 6 h of the 24-h incubation period. The standard preparation of recombinant human IL-2 (Genzyme Laboratories, Boston, MA) was used in all experiments as a positive control to determine the maximum of |3H]thymidine incorporation. The results were calculated as units of IL-2. One unit of IL-2 is defined as the activity in the sample yielding a proliferation equal to 50% of the maximum [3H]thymidine uptake obtained with the standard IL-2 preparation. Since IL-2 production was low in some experiments, the results are expressed in cpm (the mean of corresponding cultures) of [3H]thymidine
incorporated into CTLL-2 cells rather than units of IL-2. The standard deviations derived from multiple assays for each experiment were regularly less than 10% of the mean. The statistical significance of differences between the mean values was calculated using the Student's /-test. Neutralization Assays: Specific rabbit antiserum against huIFN-a (1.6 x 106 neutralization U/ml) and control antiserum against human IFN-7 (2.4 x 105 neutralization U/ml) were obtained from Hayashibara Biochemical Labs. Inc., (Okayama, Japan). To inhibit IFN-a activity, equal volumes of IFN-a (2 x 103 IU/ml) and the antiserum (diluted 1:100) were mixed and preincubated for 30 min at 37°C before being added to the cultures of IL-2-producing cells. The supernatants containing IL-2 activity were mixed with the control mouse immunoglobulin (IgG,, Cappel Laboratories, West Chester, PA) or with the mouse monoclonal antibody against human IL-2 (Genzyme) at a concentration of 250 u,g of antibody per 0.1 unit of IL-2 (as recommended by the manufacturer, on the basis of the results of Smith et a/."7'). After 60 min of incubation at 37°C, the supernatants were tested for IL-2 activity. man
RESULTS
Effects of IFN-a
on
IL-2
production by PBL
PBL from normal healthy blood donors produced spontanea low (5 U/ml) of IL-2. Again, this ConA-induced IL-2 production was further increased by IFN-a (Fig. IB). The augmentation of IL-2 production depended on the concentration of IFN-a and was reproducibly significant (p < 0.05), even in a range of low concentrations (10 and 1 IU/ml) of IFN-a.
adding
Effects of IFN-a
on
IL-2
T-cell lines
production by
leukemic
The leukemic T-cell line MOLT 4 does not produce constituany detectable IL-2, nor was any IL-2 production observed after adding 103 IU/ml of IFN-a into the cultures of MOLT4 cells (Fig. 2A). However, in the presence of 20 (xg/ml PHA, MOLT 4 cells produced a measurable quantity (2-4 U/ ml) of IL-2 and this mitogen-induced production of IL-2 was significantly (p < 0.001) increased by adding IFN-a to the cultures (Fig. 2A). Contrary to MOLT 4 cells, another leukemic T-cell line, MOLT 16, produces a small quantity (
Natural Human Interferon-ct Augments Interleukin-2 Production by a Direct Action on the Activated IL-2-Producing T Cells VLADIMIR
HOLÁN,
KEIZO KOHNO, and JUN MINOWADA
ABSTRACT
production of interleukin-2 (IL-2) by Concanavalin A (ConA)-stimulated peripheral blood leukocytes (PBL) from normal human donors was enhanced by natural human interferon-a (IFN-a). The mechanism of the action of IFN-a on IL-2 production was studied further using cloned human leukemic T-cell lines, which produce IL-2 spontaneously and/or after mitogen stimulation. It was found that IFN-a alone did not stimulate The
production, but increased it in the activated cells. The ability of IFN-a to increase IL-2 production was abrogated by the treatment of IFN-a preparations with specific anti-IFN-a antibody, and the induced IL-2 activity was completely inhibited by monoclonal antibody against human IL-2. We suggest that the augmentation by IFN-a of IL-2 production in the activated T cells may be one of the mechanisms attributable to the immunotherapeutic action of IFN-a. IL-2
INTRODUCTION
MATERIALS AND METHODS
(IFN) represents a family of molecules that modulate immune response in both a positive and negative manIt has been shown that IFN inhibits many facets of the immune response, but some manifestations of the immune system can be rather enhanced by IFN. Among the immunostimulatory effects of IFN, enhancement of cytokine production was found in some models,'3 8I but in the other experimental systems no effects of IFN on lymphokine production have been
Cells: Fresh heparinized blood was obtained by venipuncfrom healthy volunteers. The mononuclear cell fraction was isolated by Ficoll-Paque (Pharmacia Chemicals, Uppsala, Sweden) density gradient centrifugation. The cells were washed three times in serum-free RPMI-1640 medium and resuspended in a medium containing 10% heat-inactivated fetal bovine serum (FBS, GIBCO, Grand Island, NY), 100 U/ml penicillin and 50 (xg/ml streptomycin to make a final cell concentration of 4 x 106 cells/ml. Human leukemic T-cell lines MOLT 4,"" MOLT 16,(12) HUT-102,"1' and Jurkat"4' were maintained in our laboratory in RPMI-1640 medium containing 5% heat-inactivated FBS.
I ner."'2'
NTERFERON
observed/9'101
We evaluated the question of the action of IFN on lymphokine production, and we found that natural human IFN-a, when added to the cultures of peripheral blood leukocytes (PBL), increased interleukin-2 (IL-2) production. Using monoclonal leukemic T-cell lines that produce IL-2 constitutively or inducibly, we demonstrated that IFN-ct alone does not directly stimulate IL-2 production, but increases IL-2 production by the activated cells. By means of specific antibodies, we showed that the stimulated lymphokine activity was antigenically and functionally identical to IL-2, and we suggest that the augmentation by IFN-a of lymphokine production in the activated cells may be one of the mechanisms attributable to the immunotherapeutic action of IFN-a.
ture
IFN-a: Highly purified natural human IFN-a prepared by Hayashibara Biochemical Laboratories Inc., (Okayama, Japan) was prepared from BALL-1 cells stimulated with the HVJ strain of Sendai virus.'IS) The IFN was a mixture of subspecies purified by an affinity column using monoclonal anti-human IFN-a antibody technique to a specific activity of 2 x 10H IU/mg of
protein."5'
Fujisaki Cell Center. Hayashibara Biochemical Laboratories. Inc., 675-1. Fujisaki, Okayama 702, Japan. 319
HOLÁN ET AL.
320 IL-2 Production: PBL were incubated at a concentration of 10" cells/ml in 96-well flat-bottomed tissue culture plates 4 (Corning Glass Works, Corning, NY) in a volume of 300 u.1 of medium alone, with 5 p,g/ml of Concanavalin A (ConA, Sigma Chemical Co., St. Louis, MO) and/or with varying concentrations of IFN-a. In the case of the leukemic cell lines, 2 x 106 cells/well were incubated with IFN-a in the presence or absence of 20 u-g/ml of phytohemagglutinin P (PHA, Difco Laboratories, Detroit, MI) in 24-well tissue culture plates (Corning) in 1 ml of RPMI-1640 medium containing 5% FBS. After 24 h of incubation at 37°C, the supernatants were collected and tested for their ability to support the growth of IL-2-dependent murine CTLL-2 cells."6' x
IL-2 Assay: One hundred microliters (5 X 104 cells/ml) per well of the IL-2-dependent CTLL-2 cells"6' were cultured in 96-well tissue culture plates with the same volume of serially diluted test supernatants. To determine cell proliferation, |3H]thymidine (0.5 u-Ci/well, sp. act. 6.7 Ci/mmole, DuPont, New England Nuclear, Boston, MA) was added into the cultures for the last 6 h of the 24-h incubation period. The standard preparation of recombinant human IL-2 (Genzyme Laboratories, Boston, MA) was used in all experiments as a positive control to determine the maximum of |3H]thymidine incorporation. The results were calculated as units of IL-2. One unit of IL-2 is defined as the activity in the sample yielding a proliferation equal to 50% of the maximum [3H]thymidine uptake obtained with the standard IL-2 preparation. Since IL-2 production was low in some experiments, the results are expressed in cpm (the mean of corresponding cultures) of [3H]thymidine
incorporated into CTLL-2 cells rather than units of IL-2. The standard deviations derived from multiple assays for each experiment were regularly less than 10% of the mean. The statistical significance of differences between the mean values was calculated using the Student's /-test. Neutralization Assays: Specific rabbit antiserum against huIFN-a (1.6 x 106 neutralization U/ml) and control antiserum against human IFN-7 (2.4 x 105 neutralization U/ml) were obtained from Hayashibara Biochemical Labs. Inc., (Okayama, Japan). To inhibit IFN-a activity, equal volumes of IFN-a (2 x 103 IU/ml) and the antiserum (diluted 1:100) were mixed and preincubated for 30 min at 37°C before being added to the cultures of IL-2-producing cells. The supernatants containing IL-2 activity were mixed with the control mouse immunoglobulin (IgG,, Cappel Laboratories, West Chester, PA) or with the mouse monoclonal antibody against human IL-2 (Genzyme) at a concentration of 250 u,g of antibody per 0.1 unit of IL-2 (as recommended by the manufacturer, on the basis of the results of Smith et a/."7'). After 60 min of incubation at 37°C, the supernatants were tested for IL-2 activity. man
RESULTS
Effects of IFN-a
on
IL-2
production by PBL
PBL from normal healthy blood donors produced spontanea low (5 U/ml) of IL-2. Again, this ConA-induced IL-2 production was further increased by IFN-a (Fig. IB). The augmentation of IL-2 production depended on the concentration of IFN-a and was reproducibly significant (p < 0.05), even in a range of low concentrations (10 and 1 IU/ml) of IFN-a.
adding
Effects of IFN-a
on
IL-2
T-cell lines
production by
leukemic
The leukemic T-cell line MOLT 4 does not produce constituany detectable IL-2, nor was any IL-2 production observed after adding 103 IU/ml of IFN-a into the cultures of MOLT4 cells (Fig. 2A). However, in the presence of 20 (xg/ml PHA, MOLT 4 cells produced a measurable quantity (2-4 U/ ml) of IL-2 and this mitogen-induced production of IL-2 was significantly (p < 0.001) increased by adding IFN-a to the cultures (Fig. 2A). Contrary to MOLT 4 cells, another leukemic T-cell line, MOLT 16, produces a small quantity (
