Acta Med Scand 206: 345-350, 1979
Effect of Prolonged In Vivo Administration of Leukocyte Interferon on the Mitogen Responsiveness of Human Lymphocytes Stefan Einhorn, Henric Blomgren, Kari Cantell and Hans Strander From Rtrdirrmlrrmmrt, Kurolinska Hospital, Stockholm. Sweden, and the Department of Virology, Central Public Health Luboratoq, Helsinki, Finland
ABSTRACT. Peripheral lymphocytes from patients with osteosarcoma receiving interferon (IF) as adjuvant therapy were tested in vitro for response to various mitogens. Prolonged parenteral administration of IF preparations caused no major change of the mitogen responses. The ability of IF, when added in vitro, to inhibit lymphocyte response to mitogens was not altered to any major extent by in vivo administration of IF. K e y words: interferon, lymphocytes, mitogens. Acta Med Scand 206: 345, 1979.
Since 1969 more than 100 patients with various neoplastic diseases have received interferon (IF) at the Karolinska Hospital. In late 1971 a clinical trial was initiated with the object of examining the effect of adjuvant therapy in patients with osteosarcoma (1, 14).
Among other effects on the immune system (7), IF preparations have been shown to suppress lymphocytes’ response to mitogens and allogeneic cells in vitro (4,12). The evidence that IF may interfere with lymphocyte functions prompted a study in a group of these osteosarcoma patients of I ) whether in vivo administration of IF affects the reactivity of the peripheral lymphocytes to mitogens, and 2 ) whether this therapy modifies the capacity of IF to inhibit mitogen responses in vitro.
STUDY POPULATION The study comprises 20 patients (5 females, 1.5 males) with osteosarcoma of the long bones without evidence of metastases on admission who received IF as adjuvant therapy. Their ages ranged from 7 to 30 years (mean 19). In one experiment a control group was used; it comprised 14 healthy members of the laboratory staff (9 men, 5 women) with an age range of 26-61 years (mean 34).
METHODS Treatment Human leukocyte IF (see below) was given for a period of 18 months after primary surgery (amputation, exarticulation or resection). To 9 of the 20 patients local irradiation was given (40004800 rdd) prior to surgery. The I F was administered by intramuscular injection 3 times a week except during the first months and if the patient returned to the hospital, when it was given once a day. Each injection consisted of 3X lo8 IF units (14). The IF therapy was discontinued in some patients when metastases developed. Blood sampling When the patients were tested before IF therapy, the blood sample was collected after biopsy but before major surgery. From patients receiving radiotherapy blood was drawn when this treatment had been completed. During IF therapy, blood was collected 24-72 hours after the last injection. Separa iion of lymphocytes Lymphocytes were separated from heparinized venous blood by centrifugation on a layer of Ficoll-Isopaque (11) and washed twice by centrifugation in Eagle’s minimal essential medium (MEM), supplemented with 10% of heat-inactivated human serum (HS) from AB+ donors. Approximately 90% of the cells were classified as lymphocytes after crystal violet staining, the rest as monocytes and granulocytes. Mitogens The contents of vials containing phytohaemagglutinin (PHA, Bacto Phytohaemagglutinin M, Difco Lab., Detroit, USA) and poke-weed mitogen (PWM, Grand Island Biological Company, NY, USA) were dissolved in 5 ml of MEM. These solutions will be referred to a s 100% ofPHA and PWM respectively. Concanavalin A (Con A, Sigma Chemical Co., St. Louis, USA) was dissolved in MEM.
Abbreviations: IF=interferon, MEM=Eagle’s minimal essential medium, HS=human serum, PHA=phytohaemagglutinin, PWM=poke-weed mitogen, Con A= concanavalin A. Acrli
Med S c m d 206
346
S. Einhorn et (11.
Table I . Mitogen rc'Jponse of periphrrrrl lymphocytes t o P H A , PWM und Coil A byfore mujor surgrry und IF therrrpy Incorporated IT-thymidine (cpm) Controls (n = 14)
Patients (n= 1 I ) Mitogen PHA PWM
Con A
Concentration
Mean
S.D.
Mean
S.D.
I'
1.5% 0.75% 1.0% 0.1 % 14.0 pg/ml 3.5 pg/ml
100600 85 200 26 600 27 900 65 200 33 400
10 100 9 500
1 1 700 51 200
Effect of Prolonged In Vivo Administration of Leukocyte Interferon on the Mitogen Responsiveness of Human Lymphocytes Stefan Einhorn, Henric Blomgren, Kari Cantell and Hans Strander From Rtrdirrmlrrmmrt, Kurolinska Hospital, Stockholm. Sweden, and the Department of Virology, Central Public Health Luboratoq, Helsinki, Finland
ABSTRACT. Peripheral lymphocytes from patients with osteosarcoma receiving interferon (IF) as adjuvant therapy were tested in vitro for response to various mitogens. Prolonged parenteral administration of IF preparations caused no major change of the mitogen responses. The ability of IF, when added in vitro, to inhibit lymphocyte response to mitogens was not altered to any major extent by in vivo administration of IF. K e y words: interferon, lymphocytes, mitogens. Acta Med Scand 206: 345, 1979.
Since 1969 more than 100 patients with various neoplastic diseases have received interferon (IF) at the Karolinska Hospital. In late 1971 a clinical trial was initiated with the object of examining the effect of adjuvant therapy in patients with osteosarcoma (1, 14).
Among other effects on the immune system (7), IF preparations have been shown to suppress lymphocytes’ response to mitogens and allogeneic cells in vitro (4,12). The evidence that IF may interfere with lymphocyte functions prompted a study in a group of these osteosarcoma patients of I ) whether in vivo administration of IF affects the reactivity of the peripheral lymphocytes to mitogens, and 2 ) whether this therapy modifies the capacity of IF to inhibit mitogen responses in vitro.
STUDY POPULATION The study comprises 20 patients (5 females, 1.5 males) with osteosarcoma of the long bones without evidence of metastases on admission who received IF as adjuvant therapy. Their ages ranged from 7 to 30 years (mean 19). In one experiment a control group was used; it comprised 14 healthy members of the laboratory staff (9 men, 5 women) with an age range of 26-61 years (mean 34).
METHODS Treatment Human leukocyte IF (see below) was given for a period of 18 months after primary surgery (amputation, exarticulation or resection). To 9 of the 20 patients local irradiation was given (40004800 rdd) prior to surgery. The I F was administered by intramuscular injection 3 times a week except during the first months and if the patient returned to the hospital, when it was given once a day. Each injection consisted of 3X lo8 IF units (14). The IF therapy was discontinued in some patients when metastases developed. Blood sampling When the patients were tested before IF therapy, the blood sample was collected after biopsy but before major surgery. From patients receiving radiotherapy blood was drawn when this treatment had been completed. During IF therapy, blood was collected 24-72 hours after the last injection. Separa iion of lymphocytes Lymphocytes were separated from heparinized venous blood by centrifugation on a layer of Ficoll-Isopaque (11) and washed twice by centrifugation in Eagle’s minimal essential medium (MEM), supplemented with 10% of heat-inactivated human serum (HS) from AB+ donors. Approximately 90% of the cells were classified as lymphocytes after crystal violet staining, the rest as monocytes and granulocytes. Mitogens The contents of vials containing phytohaemagglutinin (PHA, Bacto Phytohaemagglutinin M, Difco Lab., Detroit, USA) and poke-weed mitogen (PWM, Grand Island Biological Company, NY, USA) were dissolved in 5 ml of MEM. These solutions will be referred to a s 100% ofPHA and PWM respectively. Concanavalin A (Con A, Sigma Chemical Co., St. Louis, USA) was dissolved in MEM.
Abbreviations: IF=interferon, MEM=Eagle’s minimal essential medium, HS=human serum, PHA=phytohaemagglutinin, PWM=poke-weed mitogen, Con A= concanavalin A. Acrli
Med S c m d 206
346
S. Einhorn et (11.
Table I . Mitogen rc'Jponse of periphrrrrl lymphocytes t o P H A , PWM und Coil A byfore mujor surgrry und IF therrrpy Incorporated IT-thymidine (cpm) Controls (n = 14)
Patients (n= 1 I ) Mitogen PHA PWM
Con A
Concentration
Mean
S.D.
Mean
S.D.
I'
1.5% 0.75% 1.0% 0.1 % 14.0 pg/ml 3.5 pg/ml
100600 85 200 26 600 27 900 65 200 33 400
10 100 9 500
1 1 700 51 200
