Ann Hematol (1991) 62:25-31
Annals of
Hematology 9 Springer-Verlag 1991
Original article Acute hematologic effects of interferon alpha, interferon gamma, tumor necrosis factor alpha and Interleukin 2 Walter E. Aulitzky ~, Herbert Tilg ~, Wolfgang Vogel 2, Wolfgang Aulitzky 3, Manuela Berger ~, Giinther GastP, Manfred Herold ~, and Christoph Huber 1 1Division of Clinical Immunobiology and z Division of Gastroenterology, Department of Internal Medicine, University Hospital Innsbruck, Austria 3 Department of Urology, General Hospital Salzburg, Austria Received August 1, 1990/Accepted October 12, 1990
Summary. This study was designed to investigate acute effects of various doses of the cytokines IFN-alpha, IFNgamma, Interleukin 2 and t u m o r necrosis factor alpha on white blood cell differential counts. Before initiation of phase II trials, a dose-determination phase was performed, where three different dose levels of each cytokine were applied as a single dose. White blood cell differential counts were assessed immediately before and 2, 12, 24, 48 and 168 h after injection. Patients enrolled suffered from metastatic cancer or chronic active hepatitis. In addition, IFN-alpha was administered to five healthy volunteers. Results indicate that cytokines cause rapid and transient changes in the numbers of leukocyte subsets. Hematologic changes were cell-type- and cytokine-specific: transient lymphopenia was observed after administration of all four cytokines, reaching a nadir 12 to 24 h after subcutaneous injection. Administration of TNF-alpha and IFNg a m m a also caused transient monocytopenia. Neutrophilia developed after administration of Interleukin 2, IFN-alpha and TNF-alpha. We conclude that cytokines play a key role in the regulation of peripheral blood cell traffic by their capacity to influence homing patterns of peripheral blood leukocytes.
Key words: T N F a - I F N a - IFN7 - IL-2 - Cell adhesion
Introduction Cytokines are peptide hormones that are produced by virtually every cell of an organism and play a key role in the regulation o f a variety of biological processes. They influence specific and unspecific immune responses, act on growth and differentiation o f normal and malignant cells Offprint requests to: W.E. Aulitzky, Division of Hematology, IIIrd Department of Internal Medicine, Johannes Gutenberg University Mainz, Langenbeckstrasse 1, W-6500 Mainz, Federal Republic of Germany
and modify various metabolic pathways [14, 26, 31]. In recent years several cytokines were purified, biochemically characterized and produced by recombinant D N A technology. Subsequently, recombinant cytokines were entered into clinical studies and their efficacy has been demonstrated in certain malignant, infectious and autoimmune disease states [2, 5, 15, 29, 30]. In addition to clinical response assessment, numerous studies also investigated a large variety o f biological response parameters. In particular, the influence of cytokine treatment on the effector functions of various immune cells has attracted much attention [21,23, 24]. The final goal of these studies was to define key parameters which would enable optimization of dose and schedule and prediction of clinical outcome. During interpretation of treatment-related effects on cellular functions, most investigators assumed that the relative composition and numbers of various leukocyte subsets would not change subsequent to cytokine treatment. Recently, results of several studies have raised serious doubts on the general validity of this assumption [4, 32]. The effect of single doses of cytokines on white blood cell counts, however, has yet not been studied in detail. Such data for recombinant interferon alpha (IFN-alpha), recombinant inferferon g a m m a (IFN-gamma), recombinant t u m o r necrosis factor alpha (TNF-alpha) and natural purified Interleukin 2 (IL-2) are presented in this study.
Material and methods Patients and treatment plan.
The four studies reported were performed according to the Declaration of Helsinki. The protocols were approved by the Ethics committee at the University of Innsbruck. All patients had given their written informed consent prior to start of treatment. Cytokines were administered subcutaneously as single doses at three different dose levels. The dose levels tested ranged from low doses without known toxicity up to a dose representing approximately 50% of the maximum tolerated dose. Cytokine doses were escalated after a therapy free interval of two weeks. Differential blood counts were performed before and 2, 12, 24 and 48 h.
26 Interleukin 2 Patients. Ten patients suffering from chronic active hepatitis B were entered into the study. All patients were positive for HBs and HBe antigen. All patients were males ranging from 18 to 66 (median 26) years of age. Trial substance. Purified natural human Interleukin 2 with a specific activity of 1x 10v U/mg protein was kindly provided by Biotest Incorporated, Dreieich, FRG. Dose levels of IL-2 tested were 30,000, 300,000 and 1,000,000 U. TNF-Alpha Patients. Seventeen patients with advanced malignant disease were treated in this Phase I/II study. Five patients were females, 12 were males ranging from 20 to 80 (median 62) years of age. Gastrointestinal tumors (hepatocellular carcinoma, colorectal carcinoma and apudoma of the pancreas) represented the diagnosis in eleven patients. The remaining patients suffered from malignant melanoma, soft tissue sarcoma and pharyngeal carcinoma. Patients were treated with either 1, 10 or 100 pg/m z subcutaneously. Trial substance. Recombinant tumor necrosis factor with a specific activity of 4x107 U/ml, originally expressed by Genentech was kindly provided by Boehringer Ingelheim Inc, Ingelheim, FRG.
Leukocytes IFN alpha (5x106 U)
Interleukin 2 (1 x l 0 6 U)
Cells/p.I (Thousands)
Thousands
20
15 /
10
o
.J J [3
5
0
' ' 1o ' ' '203 ' ' ' 0 '
L 20 ~ ~ 3'0 ~ 40 ~ 1 O~ 5O Hours after IFN alpha treatment
40 Hours after IL-2 treatment
TNF alpha (100p.g/m 2)
IFN gamma (0.5mg) 20
Ce]ls/gl (Thousands)
Thousands
j/"~\
J .vf
J
\
j
\
\
Interferon Alpha Patients. Five Patients suffering from chronic hepatitis C and five healthy volunteers were treated with 1, 3 and 5• 106 U Interferon alpha 2 b. Four patients were males, 1 female, ranging from 23 to 56 years of age (median 38). All healthy volunteers were males aged from 24 to 38 (median 32) years. Trial substance. Recombinant interferon alpha 2 b with a specific activity of 1.8 • 108 U/ml originally produced by Schering Inc. was kindly provided by Aesca Inc., Austria. Interferon Gamma Patients. Sixteen patients suffering from metastasizing renal cell carcinoma were treated with 10, 100 and 500 pg recombinant interferon gamma. Fourteen of the patients were male and 2 female. Their ages ranged from 41 to 75 years (median 59). Major manifestation of the disease were lung, bone and liver metastases. The data reported for these patients were in part published previously [4], but for comparison to the effects of other cytokines these data are also shown in the graphs. Trial substance. Recombinant IFN-gamma, originally produced by Genentech Inc., with a specific activity of 2x107 IU/mg protein was obtained from Boehringer Ingelheim International (Ingelheim, FRO). Differential blood counts Differential blood counts were performed using established standard methods. Briefly, cell counts were assessed with a coulter counter and differential blood counts were performed on Giemsa stained blood smears by counting a minimum of 200 cells. Statistical analysis Descriptive statistics are given for all variables before and after application of the cytokines. Statistical significance of the differences before and after application of the cytokines were analyzed by means of the confidence limits of the mean difference [13].
Results L e u k o c y t e counts
O n l y m i n o r effects o f cytokine t r e a t m e n t o n t o t a l white b l o o d cell counts were observed (Fig. 1). M o s t p r o m i n e n t a n d d o s e - d e p e n d e n t changes were seen after a d m i n i s t r a tion o f I b 2 . Twelve h o u r s after t r e a t m e n t with 5 x 106 U r e c o m b i n a n t IL-2, leukocyte counts increased to 42 +
50
\ \
\
10
\
i
0
'
1'o' 2'o . .30. . . 40 50 Hours after IFN gamma treatment
0
i
i
i
i
~
i
\
i
\
i
10 20 30 40 50 Hours after rTNF administration
Fig. 1. Kinetics of leukocyte counts after subcutaneous administration of 5 • 106 units IFN-alpha, 1• 106 U IL-2, 500 pg IFN-gamma and 100 pg TNF-alpha. The solid line represents the median, dotted lines minimum and maximum
12% ( m e a n + sem) above p r e t r e a t m e n t values. S m a l l e r increments were seen at the two lower dose levels ( d a t a n o t shown). Treatment with I F N - g a m m a led to a m i n o r decrease o f the n u m b e r o f p e r i p h e r a l white b l o o d cells reaching the n a d i r after 24 h (72 _+ 4 % o f p r e t r e a t m e n t values). A f t e r a p p l i c a t i o n o f I F N - a l p h a a n d T N F - a l p h a , leukocyte counts r e m a i n e d u n c h a n g e d .
Granulocyte counts
A n a l y s i s o f g r a n u l o c y t e counts a n d b a n d f o r m s before a n d after a d m i n i s t r a t i o n o f cytokines revealed a different p a t t e r n (Fig. 2): T r e a t m e n t with all cytokines except I F N g a m m a led to a transient increase o f granulocyte counts p e a k i n g within the first 24 h after the injection. T h e m o s t p r o m i n e n t changes were o b s e r v e d after t r e a t m e n t with IL-2 with a m e a n increase o f 115% after 1 x 106 U (Fig. 3). I n j e c t i o n o f I F N - a l p h a was also followed by a dosed e p e n d e n t increase o f n e u t r o p h i l counts. T h e n u m b e r o f n e u t r o p h i l s increased after 1• U by a p p r o x i m a t e l y 40%, while 5 x 106 units were followed by an increase o f m o r e t h a n 70%. I n c r e m e n t s after a d m i n i s t r a t i o n o f IL-2 a n d I F N - a l p h a were n o t a c c o m p a n i e d by a n increase o f the n u m b e r s o f b a n d f o r m s in the p e r i p h e r a l b l o o d iFig. 4). I n contrast, T N F - a l p h a caused o n l y a m i n o r in-
27 Interleukin 2
IFN alpha
Neutrophils
Percent ef pretreatment value (12 h)
Interleukin 2 (1 x l O 6 U)
IFN alpha ( 5 x 106 U)
250
Cells/gl (Thousands)
Thousands
Percent of pretreatment value (12h)
16 200
150
8 t I I1"\'\\ ,L;/o.
Annals of
Hematology 9 Springer-Verlag 1991
Original article Acute hematologic effects of interferon alpha, interferon gamma, tumor necrosis factor alpha and Interleukin 2 Walter E. Aulitzky ~, Herbert Tilg ~, Wolfgang Vogel 2, Wolfgang Aulitzky 3, Manuela Berger ~, Giinther GastP, Manfred Herold ~, and Christoph Huber 1 1Division of Clinical Immunobiology and z Division of Gastroenterology, Department of Internal Medicine, University Hospital Innsbruck, Austria 3 Department of Urology, General Hospital Salzburg, Austria Received August 1, 1990/Accepted October 12, 1990
Summary. This study was designed to investigate acute effects of various doses of the cytokines IFN-alpha, IFNgamma, Interleukin 2 and t u m o r necrosis factor alpha on white blood cell differential counts. Before initiation of phase II trials, a dose-determination phase was performed, where three different dose levels of each cytokine were applied as a single dose. White blood cell differential counts were assessed immediately before and 2, 12, 24, 48 and 168 h after injection. Patients enrolled suffered from metastatic cancer or chronic active hepatitis. In addition, IFN-alpha was administered to five healthy volunteers. Results indicate that cytokines cause rapid and transient changes in the numbers of leukocyte subsets. Hematologic changes were cell-type- and cytokine-specific: transient lymphopenia was observed after administration of all four cytokines, reaching a nadir 12 to 24 h after subcutaneous injection. Administration of TNF-alpha and IFNg a m m a also caused transient monocytopenia. Neutrophilia developed after administration of Interleukin 2, IFN-alpha and TNF-alpha. We conclude that cytokines play a key role in the regulation of peripheral blood cell traffic by their capacity to influence homing patterns of peripheral blood leukocytes.
Key words: T N F a - I F N a - IFN7 - IL-2 - Cell adhesion
Introduction Cytokines are peptide hormones that are produced by virtually every cell of an organism and play a key role in the regulation o f a variety of biological processes. They influence specific and unspecific immune responses, act on growth and differentiation o f normal and malignant cells Offprint requests to: W.E. Aulitzky, Division of Hematology, IIIrd Department of Internal Medicine, Johannes Gutenberg University Mainz, Langenbeckstrasse 1, W-6500 Mainz, Federal Republic of Germany
and modify various metabolic pathways [14, 26, 31]. In recent years several cytokines were purified, biochemically characterized and produced by recombinant D N A technology. Subsequently, recombinant cytokines were entered into clinical studies and their efficacy has been demonstrated in certain malignant, infectious and autoimmune disease states [2, 5, 15, 29, 30]. In addition to clinical response assessment, numerous studies also investigated a large variety o f biological response parameters. In particular, the influence of cytokine treatment on the effector functions of various immune cells has attracted much attention [21,23, 24]. The final goal of these studies was to define key parameters which would enable optimization of dose and schedule and prediction of clinical outcome. During interpretation of treatment-related effects on cellular functions, most investigators assumed that the relative composition and numbers of various leukocyte subsets would not change subsequent to cytokine treatment. Recently, results of several studies have raised serious doubts on the general validity of this assumption [4, 32]. The effect of single doses of cytokines on white blood cell counts, however, has yet not been studied in detail. Such data for recombinant interferon alpha (IFN-alpha), recombinant inferferon g a m m a (IFN-gamma), recombinant t u m o r necrosis factor alpha (TNF-alpha) and natural purified Interleukin 2 (IL-2) are presented in this study.
Material and methods Patients and treatment plan.
The four studies reported were performed according to the Declaration of Helsinki. The protocols were approved by the Ethics committee at the University of Innsbruck. All patients had given their written informed consent prior to start of treatment. Cytokines were administered subcutaneously as single doses at three different dose levels. The dose levels tested ranged from low doses without known toxicity up to a dose representing approximately 50% of the maximum tolerated dose. Cytokine doses were escalated after a therapy free interval of two weeks. Differential blood counts were performed before and 2, 12, 24 and 48 h.
26 Interleukin 2 Patients. Ten patients suffering from chronic active hepatitis B were entered into the study. All patients were positive for HBs and HBe antigen. All patients were males ranging from 18 to 66 (median 26) years of age. Trial substance. Purified natural human Interleukin 2 with a specific activity of 1x 10v U/mg protein was kindly provided by Biotest Incorporated, Dreieich, FRG. Dose levels of IL-2 tested were 30,000, 300,000 and 1,000,000 U. TNF-Alpha Patients. Seventeen patients with advanced malignant disease were treated in this Phase I/II study. Five patients were females, 12 were males ranging from 20 to 80 (median 62) years of age. Gastrointestinal tumors (hepatocellular carcinoma, colorectal carcinoma and apudoma of the pancreas) represented the diagnosis in eleven patients. The remaining patients suffered from malignant melanoma, soft tissue sarcoma and pharyngeal carcinoma. Patients were treated with either 1, 10 or 100 pg/m z subcutaneously. Trial substance. Recombinant tumor necrosis factor with a specific activity of 4x107 U/ml, originally expressed by Genentech was kindly provided by Boehringer Ingelheim Inc, Ingelheim, FRG.
Leukocytes IFN alpha (5x106 U)
Interleukin 2 (1 x l 0 6 U)
Cells/p.I (Thousands)
Thousands
20
15 /
10
o
.J J [3
5
0
' ' 1o ' ' '203 ' ' ' 0 '
L 20 ~ ~ 3'0 ~ 40 ~ 1 O~ 5O Hours after IFN alpha treatment
40 Hours after IL-2 treatment
TNF alpha (100p.g/m 2)
IFN gamma (0.5mg) 20
Ce]ls/gl (Thousands)
Thousands
j/"~\
J .vf
J
\
j
\
\
Interferon Alpha Patients. Five Patients suffering from chronic hepatitis C and five healthy volunteers were treated with 1, 3 and 5• 106 U Interferon alpha 2 b. Four patients were males, 1 female, ranging from 23 to 56 years of age (median 38). All healthy volunteers were males aged from 24 to 38 (median 32) years. Trial substance. Recombinant interferon alpha 2 b with a specific activity of 1.8 • 108 U/ml originally produced by Schering Inc. was kindly provided by Aesca Inc., Austria. Interferon Gamma Patients. Sixteen patients suffering from metastasizing renal cell carcinoma were treated with 10, 100 and 500 pg recombinant interferon gamma. Fourteen of the patients were male and 2 female. Their ages ranged from 41 to 75 years (median 59). Major manifestation of the disease were lung, bone and liver metastases. The data reported for these patients were in part published previously [4], but for comparison to the effects of other cytokines these data are also shown in the graphs. Trial substance. Recombinant IFN-gamma, originally produced by Genentech Inc., with a specific activity of 2x107 IU/mg protein was obtained from Boehringer Ingelheim International (Ingelheim, FRO). Differential blood counts Differential blood counts were performed using established standard methods. Briefly, cell counts were assessed with a coulter counter and differential blood counts were performed on Giemsa stained blood smears by counting a minimum of 200 cells. Statistical analysis Descriptive statistics are given for all variables before and after application of the cytokines. Statistical significance of the differences before and after application of the cytokines were analyzed by means of the confidence limits of the mean difference [13].
Results L e u k o c y t e counts
O n l y m i n o r effects o f cytokine t r e a t m e n t o n t o t a l white b l o o d cell counts were observed (Fig. 1). M o s t p r o m i n e n t a n d d o s e - d e p e n d e n t changes were seen after a d m i n i s t r a tion o f I b 2 . Twelve h o u r s after t r e a t m e n t with 5 x 106 U r e c o m b i n a n t IL-2, leukocyte counts increased to 42 +
50
\ \
\
10
\
i
0
'
1'o' 2'o . .30. . . 40 50 Hours after IFN gamma treatment
0
i
i
i
i
~
i
\
i
\
i
10 20 30 40 50 Hours after rTNF administration
Fig. 1. Kinetics of leukocyte counts after subcutaneous administration of 5 • 106 units IFN-alpha, 1• 106 U IL-2, 500 pg IFN-gamma and 100 pg TNF-alpha. The solid line represents the median, dotted lines minimum and maximum
12% ( m e a n + sem) above p r e t r e a t m e n t values. S m a l l e r increments were seen at the two lower dose levels ( d a t a n o t shown). Treatment with I F N - g a m m a led to a m i n o r decrease o f the n u m b e r o f p e r i p h e r a l white b l o o d cells reaching the n a d i r after 24 h (72 _+ 4 % o f p r e t r e a t m e n t values). A f t e r a p p l i c a t i o n o f I F N - a l p h a a n d T N F - a l p h a , leukocyte counts r e m a i n e d u n c h a n g e d .
Granulocyte counts
A n a l y s i s o f g r a n u l o c y t e counts a n d b a n d f o r m s before a n d after a d m i n i s t r a t i o n o f cytokines revealed a different p a t t e r n (Fig. 2): T r e a t m e n t with all cytokines except I F N g a m m a led to a transient increase o f granulocyte counts p e a k i n g within the first 24 h after the injection. T h e m o s t p r o m i n e n t changes were o b s e r v e d after t r e a t m e n t with IL-2 with a m e a n increase o f 115% after 1 x 106 U (Fig. 3). I n j e c t i o n o f I F N - a l p h a was also followed by a dosed e p e n d e n t increase o f n e u t r o p h i l counts. T h e n u m b e r o f n e u t r o p h i l s increased after 1• U by a p p r o x i m a t e l y 40%, while 5 x 106 units were followed by an increase o f m o r e t h a n 70%. I n c r e m e n t s after a d m i n i s t r a t i o n o f IL-2 a n d I F N - a l p h a were n o t a c c o m p a n i e d by a n increase o f the n u m b e r s o f b a n d f o r m s in the p e r i p h e r a l b l o o d iFig. 4). I n contrast, T N F - a l p h a caused o n l y a m i n o r in-
27 Interleukin 2
IFN alpha
Neutrophils
Percent ef pretreatment value (12 h)
Interleukin 2 (1 x l O 6 U)
IFN alpha ( 5 x 106 U)
250
Cells/gl (Thousands)
Thousands
Percent of pretreatment value (12h)
16 200
150
8 t I I1"\'\\ ,L;/o.
