Immunol. Cell Bioi. (1990) 68. 231-234
In vivo effects of recombinant granulocyte-colony stimulating factor in athymic nude mice Zygmunt Pojda,* Yoshiro Aoki and Atsushi Tsuboi Division of Radiation Hazards and Division of Radiation Health. National Institute of Radiological Sciences. Chiha. Japan and * Department of Radiation Haematology. WIHiE Institute of Hygiene and Epidemiology. Warsaw. Poland (Submitted
3 April 1990. Accepted for publication
6 June 1990.)
Summary The possible role ofT lymphocytes in in vivo regulation of haemopoiesis by recombinant human graniilocyte-colony stimulating factor (rhG-CSF) was evaluated, Athymic nude (nu/nu) mice andtheirnormal( + /+)littermateswereinjectedsubcutaneousiy twice daily with lOO/ig/kgpcrdayof rhG-CSF for 5 days. Such parameters as number of neutrophils in blood, spleen weight and cellularily. bone marrow ccllularily, and number of stem and progenitor cells (colony forming units in spleen [CFU-S], mix colony forming cells [Mix-CFC]. granuloeyte-macrophage colony forming cells [GM-CFC]) in bone marrow and spleen were evaluated. Some effects ofthe rhG-CSF treatment were similar in nu/nu and 47+ mice. Others, however, were to some extent dilicrent in the two groups of animals. It is concluded that T lymphocytes may be partially responsible for some of the effects of rhG-CSF in vivo activity.
IP^RODUCTION Granulocyte-colony stimulating factor (G-CSF) is a natural glycopeptide. belonging to Ihe family of haemopoictic growth factors. Cloning of its cDNA (1) allowed us to obtain large quantities of its recombinant form and. as a consequence. to introduce recombinant human granulocytecolony stimulating factor (rhG-CSF) for clinical use as a stimulator of granulopoiesis (2). Both in vitro and in vivo rhG-CSF stimulate division and maturation ofthe neutrophil progenitors (3.4). However, some in vivo effects of rhG-CSF treatment cannot be explained on the basis ofthe in vitro observations, and probably are the result of interactions of other cells present in the living organism (5). Talmadge et al. (6) reported no effect from treatment of athymic nude {nu/nu) mice with G-CSF. suggesting that these cells could be T lymphocytes. Correspondence: Dr Atsushi Tsuboi. Division of Radiation Hazards, National Institute of Radiological Sciences, 9-1, Anagawa 4-chome, Chiba-shi 260. Japan. Abbreviations used in this paper: rhG-CSF. recombinant human granulocyte-colony stimulating factor: BSA, bovine serum albumin; CFU-S, colony forming units in spleen; Mix-CFC, mix colony forming cells; GM-CFC. granuloeyte-macrophage colony forming cells: IMDM. Iscove's modified Dulbecco's medium.
The objective ofthe present study was to evaluate a possible role ofT lymphocytes in the in v/v'o response to rhG-CSF treatment, by the comparison ofthe elfects of rhG-CSF injections inT lymphocyte deficient nu/nu miee and their normal ( + / + ) littermates. MATERIAL AND METHODS Animals Speeifie pathogen-free female athymic Balb/c nu/nu mice and their normal Balb/c + / + littermates, aged 8 weeks, were obtained from Shizuoka Laboratory Animal Center, Hamamatsu, Japan. Recombinant human G-CSF This was kindly supplied by Chugai Pharmaceutical Company Ltd. Us activity was 10*' units/mg, endotoxin contamination did not exceed 2 ng/;ig. Stock solution was diluted with buffered salt solution with 0-1% bovine serum albumin (BSA). Nucleated blood cells These were counted in a haemocytometer chamber. Differentia! counting was performed on smears stained with May-Grunwald-Giemsa. CPU'S a.ssay Bone marrow or spleen cells were injected to lethally irradiated mice. After 9 days the number of spleen colonies was scored.
232
Z.
MiX'CFC as.say Bone marrow or spleen cells were cultured for 9 days in a medium composed of Iscovc's modified Dulbccco's medium (IMDM). 20% fetal calf serum, 1% BSA, 2units/mLerythropoietin. 10% of 8313 celUine conditioned medium (7) and 0-8% w/v methylcellulose. at 37°C. The frequency of mixed colonies was evaluated under an inverted microscope. GM-CFC assay Bone marrow or spleen cells were cultured in a medium composed of Fischer's modified medium. 20% horse serum, 15% 83! 3 CM and 0-3% Noble agar, al 37°C. After 7 days the number of neutrophil-macrophage cell colonies was scored. Statistics Each group consisted of no less than five mice. The experiment was repeated twice. Means, varianciesand standard errors were calculated for each group of data. Results were re-calculated for graphic presentation as percent of control values ± s,c,m. Statistical significance was estimated by /-test {/'
In vivo effects of recombinant granulocyte-colony stimulating factor in athymic nude mice Zygmunt Pojda,* Yoshiro Aoki and Atsushi Tsuboi Division of Radiation Hazards and Division of Radiation Health. National Institute of Radiological Sciences. Chiha. Japan and * Department of Radiation Haematology. WIHiE Institute of Hygiene and Epidemiology. Warsaw. Poland (Submitted
3 April 1990. Accepted for publication
6 June 1990.)
Summary The possible role ofT lymphocytes in in vivo regulation of haemopoiesis by recombinant human graniilocyte-colony stimulating factor (rhG-CSF) was evaluated, Athymic nude (nu/nu) mice andtheirnormal( + /+)littermateswereinjectedsubcutaneousiy twice daily with lOO/ig/kgpcrdayof rhG-CSF for 5 days. Such parameters as number of neutrophils in blood, spleen weight and cellularily. bone marrow ccllularily, and number of stem and progenitor cells (colony forming units in spleen [CFU-S], mix colony forming cells [Mix-CFC]. granuloeyte-macrophage colony forming cells [GM-CFC]) in bone marrow and spleen were evaluated. Some effects ofthe rhG-CSF treatment were similar in nu/nu and 47+ mice. Others, however, were to some extent dilicrent in the two groups of animals. It is concluded that T lymphocytes may be partially responsible for some of the effects of rhG-CSF in vivo activity.
IP^RODUCTION Granulocyte-colony stimulating factor (G-CSF) is a natural glycopeptide. belonging to Ihe family of haemopoictic growth factors. Cloning of its cDNA (1) allowed us to obtain large quantities of its recombinant form and. as a consequence. to introduce recombinant human granulocytecolony stimulating factor (rhG-CSF) for clinical use as a stimulator of granulopoiesis (2). Both in vitro and in vivo rhG-CSF stimulate division and maturation ofthe neutrophil progenitors (3.4). However, some in vivo effects of rhG-CSF treatment cannot be explained on the basis ofthe in vitro observations, and probably are the result of interactions of other cells present in the living organism (5). Talmadge et al. (6) reported no effect from treatment of athymic nude {nu/nu) mice with G-CSF. suggesting that these cells could be T lymphocytes. Correspondence: Dr Atsushi Tsuboi. Division of Radiation Hazards, National Institute of Radiological Sciences, 9-1, Anagawa 4-chome, Chiba-shi 260. Japan. Abbreviations used in this paper: rhG-CSF. recombinant human granulocyte-colony stimulating factor: BSA, bovine serum albumin; CFU-S, colony forming units in spleen; Mix-CFC, mix colony forming cells; GM-CFC. granuloeyte-macrophage colony forming cells: IMDM. Iscove's modified Dulbecco's medium.
The objective ofthe present study was to evaluate a possible role ofT lymphocytes in the in v/v'o response to rhG-CSF treatment, by the comparison ofthe elfects of rhG-CSF injections inT lymphocyte deficient nu/nu miee and their normal ( + / + ) littermates. MATERIAL AND METHODS Animals Speeifie pathogen-free female athymic Balb/c nu/nu mice and their normal Balb/c + / + littermates, aged 8 weeks, were obtained from Shizuoka Laboratory Animal Center, Hamamatsu, Japan. Recombinant human G-CSF This was kindly supplied by Chugai Pharmaceutical Company Ltd. Us activity was 10*' units/mg, endotoxin contamination did not exceed 2 ng/;ig. Stock solution was diluted with buffered salt solution with 0-1% bovine serum albumin (BSA). Nucleated blood cells These were counted in a haemocytometer chamber. Differentia! counting was performed on smears stained with May-Grunwald-Giemsa. CPU'S a.ssay Bone marrow or spleen cells were injected to lethally irradiated mice. After 9 days the number of spleen colonies was scored.
232
Z.
MiX'CFC as.say Bone marrow or spleen cells were cultured for 9 days in a medium composed of Iscovc's modified Dulbccco's medium (IMDM). 20% fetal calf serum, 1% BSA, 2units/mLerythropoietin. 10% of 8313 celUine conditioned medium (7) and 0-8% w/v methylcellulose. at 37°C. The frequency of mixed colonies was evaluated under an inverted microscope. GM-CFC assay Bone marrow or spleen cells were cultured in a medium composed of Fischer's modified medium. 20% horse serum, 15% 83! 3 CM and 0-3% Noble agar, al 37°C. After 7 days the number of neutrophil-macrophage cell colonies was scored. Statistics Each group consisted of no less than five mice. The experiment was repeated twice. Means, varianciesand standard errors were calculated for each group of data. Results were re-calculated for graphic presentation as percent of control values ± s,c,m. Statistical significance was estimated by /-test {/'
