Journal of Neuroimmunology, 32 (1991) 269-278
269
© 1991 Elsevier Science Publishers B.V. 0165-5728/91/$03.50 JNI 02000
Modulation by tumor necrosis factor-a of human astroglial cell production of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) D a v i d J. T w e a r d y , E d w a r d W. G l a z e r , P a m e l a L. M o t t a n d K f i s t i n A n d e r s o n Departments of Medicine and Molecular Genetics and Biochemistry, Unioersity of Pittsburgh School of Medicine and the Pittsburgh Cancer Institute, Pittsburgh, PA 15213, U.S.A.
(Received 22 August 1990) (Revised, received 21 November 1990 and 8 January 1991) (Accepted 10 January 1991)
Key words: Astroglial cell; Colony-stimulatingfactor; Tumor necrosis factor
Summary Phagocyte survival and function are enhanced by G M - C S F and G-CSF. The production of both CSFs can be induced in mesenchymal cells by tumor necrosis factor-a ( T N F - a ) and interleukin-1 (IL-1). We have recently demonstrated that I L - l a and fl induced the production of G M - C S F and G - C S F by two h u m a n astroglial cell lines. In the present study, we examined the effects of T N F - a on the production of G M - C S F and G - C S F by U87MG, a human astroglial cell line that constitutively expresses G M - C S F and G-CSF, and U373MG, a second ]iuman astroglial cell line that does not produce CSF. We demonstrate that U 8 7 M G can be induced to ir~crease its production of G M - C S F and G - C S F by exposure to T N F - a while U 3 7 3 M G is induced to produce G M - C S F but not G-CSF. These responses, measured by accumulation of elevated levels of CSF protein and m R N A , are rapid and sensitive. The implications of these findings to the immunopathogenesis of'central nervous system infections are discussed.
Address for correspondence: David J. Tweardy, M.D., The Pittsburgh Cancer Institute, 3343 Forbes Avenue, Pittsburgh, PA 15213, U.S.A. This work was presented in part at the National Meeting of the American Federation for Clinical Research, April 29-May 2, 1988. This work was supported by grants HL10763 from the National Institutes of Health and CH-384 from the American Cancer Society. D.J.T. is a recipient of a Physician Scientist Award from the National Heart, Lung and Blood Institute, U.S.A.
InWoduction G r a n u l o c y t e - m a c r o p h a g e colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) have profound effects upon survival and activation of mature neutrophils, eosinophils and macrophages, in addition to their potent effects on the growth and development of myeloid cells (reviewed by Clark and Kamen,
270
1987; Whetton and Dexter, 1989). In light of these wide-ranging effects on phagocyte activation, it is not surprising that GM-CSF and G-CSF have been shown to be produced by several cell types distributed throughout the body including T lymphocytes, macrophages, fibroblasts, and endothelial cells (Clarke and Kamen, 1987; Whetton and Dexter, 1989). These cells do not constitutively produce GM-CSF and G-CSF; rather, they produce CSF in response to an activation stimulus. Normal T cells activated by an antigen-specific or non-specific stimulus produce GM-CSF, as well as interleukin-3 (IL-3), but not G-CSF (Clark and Kamen, 1987). Stimulation of human macrophages with lipopolysaccharide (LPS) and phorbol esters results in G-CSF production (Ernst et al., 1989). Fibroblasts and endothelial cells can be induced to produce both GM-CSF and G-CSF by monokines such as IL-1 (Bagby et al., 1986; Zucoli et al., 1986) and tumor necrosis factor-a (TNF-c0 (Broudy et al., 1986a; Munker et al., 1986). In addition, primary human stromal cell lines can be induced to produce GM-CSF and G-CSF m R N A in response to IL-1 and T N F - a (Broudy et al., 1986b; Yang et al., 1988). Our knowledge of the mechanisms controlling inflammation in the central nervous system (CNS) lags behind our understanding of inflammatory mechanisms in extraneural tissues. Rat and mouse astrocytes and microglial cells have been shown to produce IL-1 (Fontana et al., 1982; Giulian et al., 1986), T N F (Robbins et al., 1987), and IL-6 (Lieberman et al., 1989; Frei et al., 1989). However, it is not known whether factors that activate phagocytes and prolong their survival, such as GM-CSF or G-CSF, are produced by human neural-derived cells and, if so, what stimulus induces their production. We have recently demonstrated that the astrocytoma cell line, U87MG, constitutively produces G-CSF and GM-CSF (Tweardy et al., 1987a) and a full-length G-CSF cDNA was molecularly cloned from a U87MG cDNA library (Tweardy et al., 1987b). Since U87MG was the only neural tumor cell line of several lines screened that constitutively produced either G-CSF or GMCSF and we could not demonstrate an effect of G-CSF or GM-CSF upon neuron growth and maturation, we hypothesized that astrocytes perform a local CSF-producing function similar to
fibroblasts and endothelial cells. To test this hypothesis, we examined whether the monokine, TNF-c~, modulated levels of GM-CSF and G-CSF production by U87MG and whether TNF-c~ could induce the production of these CSFs by another astrocytoma cell line, U373MG, that does not constitutively produce CSF. We chose to examine the effect of TNF-a, in part, because elevated levels of T N F - a have been detected in the cerebrospinal fluid of adults and children with bacterial meningitis (Mustafa et al., 1989; Nadal et al., 1989; Waage, 1989). Our results demonstrate that T N F - a increases the levels of G M - C S F and G-CSF protein and m R N A production by U87MG and induces production of GM-CSF mRNA by U373MG. The m R N A accumulation in these cells in response to T N F is rapid and sensitive.
Material and methods
Cell lines and cytokines The human astrocytoma cell lines, U 8 7 M G and U373MG, were obtained from ATCC and grown in Iscove's modified Dulbecco's medium (IMDM, Gibco, Grand Island, NY, U.S.A.) supplemented with penicillin (50 U / m l ) , streptomycin (50 /xg/ml), and 10% heat-inactivated fetal bovine serum (FBS, Gibco). Each cell line was originally derived from explants of grade III human astrocytomas and tested negative for Mycoplasma contamination. Cells were transferred to I M D M / I % FBS 24 h before addition of monokines. Recombinant human TNF-a, expressed in Escherichia coli, purified to homogeneity, and free of contaminating LPS as determined by limulus lysate assay was kindly provided by Cetus. GM-CSF and G-CSF bioassays Supernatants were assayed for GM-CSF activity using the human GM-CSF-dependent cell line TALL-101 (Valtieri et al., 1987a). Supernatants were assayed f o r G-CSF by their ability to stimulate the proliferation of 32Dc13, a murine myeloid progenitor cell line that is exquisitely sensitive to human G-CSF but not to any other known human cytokines (Valtieri et al., 1987b). Dilutions of supernatants were tested in triplicate along with a
271
rhGM-CSF or rhG-CSF, kindly provided by Genetics Institute (Cambridge, MA, U.S.A.). The concentration of GM-CSF (ng/ml) and G-CSF (U/ml) in the test supernatants was calculated on the basis of comparison with dilution curves of these known standards as described (Valtieri et al., 1987a, b).
al., 1977), blotted onto Zetabind (Cuno, Meridan, CT, U.S.A.) according to the manufacturer's instructions, and covalently bound to the membrane by brief ultraviolet irradiation (Church and Gilbert, 1984). A full-length eDNA clone of G-CSF was obtained from U87MG (Tweardy et al., 1987b) and a full-length eDNA clone of GM-CSF was kindly provided by Dr. Gordon Wong of Genetics Institute (Wong et al., 1985). The eDNA probe for fl-tubulin was obtained from Dr. J. Hall et al. (1983). Nucleic acid probes were radiolabeled to high specific activity (2-5 × 108 cpm/#g eDNA) by nick translation with [32p]dCTP and used to hybridize membranes according to the manufacturer's instructions. In those cases where a single blot was hybridized with more than one probe, the
RNA isolation and hybridization studies Cells were washed in phosphate-buffered saline (PBS) and lysed in guanidine isothiocyanate (BRL, Bethesda, MD, U.S.A.) solution; total cellular RNA was isolated by centrifugation through a cesium chloride (BRL) cushion as described (Chirgwin et al., 1979). 10 #g of RNA were separated on agarose-formaldehyde gels (Lehrach et 30
30
O
b 20 ~0
u
{J
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lO
1o
O
0
0
l
!
I 0
20
0 3'0
4'0
5'0
1
60
5OO
~ 400-
i 10,000
d
4oo
300.
u
o rO
i 1.000
600
¢
500 '
~
100
[TNFI U / m l
TIME (hr) 6OO
| 10
200
0
100 •
0
20O 100
i
i 10
i 20
i 30
40
TIME (hr)
i 50
60
i 0
i 1
i
10
i
100
i i 1,00010,000
{TNFI U / m l
Fig. 1. Effects of T N F - a on G M - C S F and G-CSF protein production by U87MG. Confluent cultures of U 8 7 M G were incubated in T N F - a (1000 U / m l ) for the times indicated. Supernatants were harvested and tested for G M - C S F (panel a) and G - C S F (panel c) as described in Material and Methods. In other experiments, confluent cultures of U 8 7 M G were incubated in T N F - a at the concentrations indicated. At 32 h, supernatants were harvested and tested for G M - C S F (panel b) and G - C S F (panel d ) as above. The results shown are representative of two experiments.
272 sequence of p r o b i n g was G M - C S F , G - C S F , then fl-tubulin. Blots were s t r i p p e d b e f o r e p r o b i n g with /3-tubulin only. Blots were e x p o s e d to K o d a k X A R - 5 film at - 8 0 ° C with two intensifying screens for the length of time indicated. A u t o r a d i o g r a m s were a n a l y z e d b y d e n s i t o m e t e r in one d i m e n s i o n ( m o d e l 620, B i o R a d , R i c h m o n d , C A , U.S.A.).
Results
Induction by TNF-a of increased production of GMCSF and G-CSF protein and mRNA in U87MG cells I n e a r l i e r studies, we d e m o n s t r a t e d t h a t U87MG constitutively produced GM-CSF and G - C S F ; the G M - C S F activity in the s u p e r n a t a n t s of u n s t i m u l a t e d U 8 7 M G was n e u t r a l i z e d b y antiG M - C S F a n t i s e r a ( T w e a r d y et al., 1987a). T o d e t e r m i n e the effect of m o n o k i n e s on the p r o d u c tion of C S F b y these cells, U 8 7 M G were inc u b a t e d with TNF-c~. C o n t i n u o u s e x p o s u r e of U 8 7 M G cells to T N F - a (1000 U / m l ) i n d u c e d the p r o d u c t i o n of G M - C S F a n d G - C S F b i o a c t i v i t y as
A.
0 0.5
TIME ['HR-~ 1 1.5 2
4
8
early as 4 h f r o m the start o f c u l t u r e (Fig. 1). P r o d u c t i o n of G M - C S F b e g a n to p l a t e a u at 32 h while p r o d u c t i o n of G - C S F p e a k s at 32 h a n d b i o a c t i v i t y decreases t h e r e a f t e r p o s s i b l y d u e to d e g r a d a t i o n . C h o o s i n g 32 h as the o p t i m u m time to e x a m i n e the effect of v a r y i n g c o n c e n t r a t i o n s of TNF-a on CSF protein production by U87MG cells, we f o u n d that G M - C S F p r o d u c t i o n was i n d u c e d b y as little as 10 U / m l TNF-c~ while G - C S F p r o d u c t i o n was i n c r e a s e d at 1 U / m l . Peak p r o d u c t i o n of b o t h G M - C S F a n d G - C S F was i n d u c e d b y T N F - a at 1000 U / m l . T h e i n c r e a s e in C S F b i o a c t i v i t y i n d u c e d b y T N F was a c c o m p a n i e d b y a d e t e c t a b l e increase in G M - C S F a n d G - C S F m R N A levels (Fig. 2). A t 1000 U / m l , the i n d u c t i o n of G M - C S F a n d G - C S F m R N A occ u r r e d within 30 m i n of e x p o s u r e a n d p e a k e d at 6 0 - 9 0 m i n (Fig. 2). C h o o s i n g 1 h as the o p t i m u m time for e x a m i n i n g the sensitivity of C S F m R N A p r o d u c t i o n b y U 8 7 M G cells in r e s p o n s e to TNF-a, we f o u n d that i n d u c t i o n of G M - C S F m R N A occ u r r e d at a c o n c e n t r a t i o n of T N F - a as low as 1 U / m l a n d r e a c h e d a p l a t e a u at 1000 U / m l , while i n d u c t i o n of G - C S F m R N A o c c u r r e d at a conc e n t r a t i o n of T N F - a as low as 1 U / m l a n d d i d
B.
0.800 T
GM-CSF
o.,oo
0.600 t
0,500 t G-CSF
i
B-TUBULIN
0,200 0.100 0.000
0
0.5
1
1,5
2
4
8
HUE Chd
Fig. 2. Kinetics of G-CSF and GM-CSF mRNA accumulation by U87MG in response to TNF-a. U87MG cells were harvested for extraction of total RNA after incubation in TNF-a, 1000 U/nil, at the indicated time points. A Northern blot of total RNA (10 #g, panel A) was probed with GM-CSF (top row; band of 0.9 kb), G-CSF (middle row; band of 1.6 kb) and fl-tubulin (bottom row; bands of 2.8 and 1.8 kb) and exposed to film for 5 days, 5 days, and 18 h, respectively, before developing. The autoradiograms were analyzed by densitometry and the scanning areas for the bands detected by GM-CSF were corrected by dividing them by the area of the corresponding bottom band of/~-tubulin and the results plotted in panel B using the open bars. The corrected scanning ratio for G-CSF is plotted in panel B using the closed bars. The results shown are representative of two experiments.
273
Ao 0
1
B.
ITNFI U/ml 10 102 103 104
©
GM-CSF
L~ z Z
269
© 1991 Elsevier Science Publishers B.V. 0165-5728/91/$03.50 JNI 02000
Modulation by tumor necrosis factor-a of human astroglial cell production of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) D a v i d J. T w e a r d y , E d w a r d W. G l a z e r , P a m e l a L. M o t t a n d K f i s t i n A n d e r s o n Departments of Medicine and Molecular Genetics and Biochemistry, Unioersity of Pittsburgh School of Medicine and the Pittsburgh Cancer Institute, Pittsburgh, PA 15213, U.S.A.
(Received 22 August 1990) (Revised, received 21 November 1990 and 8 January 1991) (Accepted 10 January 1991)
Key words: Astroglial cell; Colony-stimulatingfactor; Tumor necrosis factor
Summary Phagocyte survival and function are enhanced by G M - C S F and G-CSF. The production of both CSFs can be induced in mesenchymal cells by tumor necrosis factor-a ( T N F - a ) and interleukin-1 (IL-1). We have recently demonstrated that I L - l a and fl induced the production of G M - C S F and G - C S F by two h u m a n astroglial cell lines. In the present study, we examined the effects of T N F - a on the production of G M - C S F and G - C S F by U87MG, a human astroglial cell line that constitutively expresses G M - C S F and G-CSF, and U373MG, a second ]iuman astroglial cell line that does not produce CSF. We demonstrate that U 8 7 M G can be induced to ir~crease its production of G M - C S F and G - C S F by exposure to T N F - a while U 3 7 3 M G is induced to produce G M - C S F but not G-CSF. These responses, measured by accumulation of elevated levels of CSF protein and m R N A , are rapid and sensitive. The implications of these findings to the immunopathogenesis of'central nervous system infections are discussed.
Address for correspondence: David J. Tweardy, M.D., The Pittsburgh Cancer Institute, 3343 Forbes Avenue, Pittsburgh, PA 15213, U.S.A. This work was presented in part at the National Meeting of the American Federation for Clinical Research, April 29-May 2, 1988. This work was supported by grants HL10763 from the National Institutes of Health and CH-384 from the American Cancer Society. D.J.T. is a recipient of a Physician Scientist Award from the National Heart, Lung and Blood Institute, U.S.A.
InWoduction G r a n u l o c y t e - m a c r o p h a g e colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) have profound effects upon survival and activation of mature neutrophils, eosinophils and macrophages, in addition to their potent effects on the growth and development of myeloid cells (reviewed by Clark and Kamen,
270
1987; Whetton and Dexter, 1989). In light of these wide-ranging effects on phagocyte activation, it is not surprising that GM-CSF and G-CSF have been shown to be produced by several cell types distributed throughout the body including T lymphocytes, macrophages, fibroblasts, and endothelial cells (Clarke and Kamen, 1987; Whetton and Dexter, 1989). These cells do not constitutively produce GM-CSF and G-CSF; rather, they produce CSF in response to an activation stimulus. Normal T cells activated by an antigen-specific or non-specific stimulus produce GM-CSF, as well as interleukin-3 (IL-3), but not G-CSF (Clark and Kamen, 1987). Stimulation of human macrophages with lipopolysaccharide (LPS) and phorbol esters results in G-CSF production (Ernst et al., 1989). Fibroblasts and endothelial cells can be induced to produce both GM-CSF and G-CSF by monokines such as IL-1 (Bagby et al., 1986; Zucoli et al., 1986) and tumor necrosis factor-a (TNF-c0 (Broudy et al., 1986a; Munker et al., 1986). In addition, primary human stromal cell lines can be induced to produce GM-CSF and G-CSF m R N A in response to IL-1 and T N F - a (Broudy et al., 1986b; Yang et al., 1988). Our knowledge of the mechanisms controlling inflammation in the central nervous system (CNS) lags behind our understanding of inflammatory mechanisms in extraneural tissues. Rat and mouse astrocytes and microglial cells have been shown to produce IL-1 (Fontana et al., 1982; Giulian et al., 1986), T N F (Robbins et al., 1987), and IL-6 (Lieberman et al., 1989; Frei et al., 1989). However, it is not known whether factors that activate phagocytes and prolong their survival, such as GM-CSF or G-CSF, are produced by human neural-derived cells and, if so, what stimulus induces their production. We have recently demonstrated that the astrocytoma cell line, U87MG, constitutively produces G-CSF and GM-CSF (Tweardy et al., 1987a) and a full-length G-CSF cDNA was molecularly cloned from a U87MG cDNA library (Tweardy et al., 1987b). Since U87MG was the only neural tumor cell line of several lines screened that constitutively produced either G-CSF or GMCSF and we could not demonstrate an effect of G-CSF or GM-CSF upon neuron growth and maturation, we hypothesized that astrocytes perform a local CSF-producing function similar to
fibroblasts and endothelial cells. To test this hypothesis, we examined whether the monokine, TNF-c~, modulated levels of GM-CSF and G-CSF production by U87MG and whether TNF-c~ could induce the production of these CSFs by another astrocytoma cell line, U373MG, that does not constitutively produce CSF. We chose to examine the effect of TNF-a, in part, because elevated levels of T N F - a have been detected in the cerebrospinal fluid of adults and children with bacterial meningitis (Mustafa et al., 1989; Nadal et al., 1989; Waage, 1989). Our results demonstrate that T N F - a increases the levels of G M - C S F and G-CSF protein and m R N A production by U87MG and induces production of GM-CSF mRNA by U373MG. The m R N A accumulation in these cells in response to T N F is rapid and sensitive.
Material and methods
Cell lines and cytokines The human astrocytoma cell lines, U 8 7 M G and U373MG, were obtained from ATCC and grown in Iscove's modified Dulbecco's medium (IMDM, Gibco, Grand Island, NY, U.S.A.) supplemented with penicillin (50 U / m l ) , streptomycin (50 /xg/ml), and 10% heat-inactivated fetal bovine serum (FBS, Gibco). Each cell line was originally derived from explants of grade III human astrocytomas and tested negative for Mycoplasma contamination. Cells were transferred to I M D M / I % FBS 24 h before addition of monokines. Recombinant human TNF-a, expressed in Escherichia coli, purified to homogeneity, and free of contaminating LPS as determined by limulus lysate assay was kindly provided by Cetus. GM-CSF and G-CSF bioassays Supernatants were assayed for GM-CSF activity using the human GM-CSF-dependent cell line TALL-101 (Valtieri et al., 1987a). Supernatants were assayed f o r G-CSF by their ability to stimulate the proliferation of 32Dc13, a murine myeloid progenitor cell line that is exquisitely sensitive to human G-CSF but not to any other known human cytokines (Valtieri et al., 1987b). Dilutions of supernatants were tested in triplicate along with a
271
rhGM-CSF or rhG-CSF, kindly provided by Genetics Institute (Cambridge, MA, U.S.A.). The concentration of GM-CSF (ng/ml) and G-CSF (U/ml) in the test supernatants was calculated on the basis of comparison with dilution curves of these known standards as described (Valtieri et al., 1987a, b).
al., 1977), blotted onto Zetabind (Cuno, Meridan, CT, U.S.A.) according to the manufacturer's instructions, and covalently bound to the membrane by brief ultraviolet irradiation (Church and Gilbert, 1984). A full-length eDNA clone of G-CSF was obtained from U87MG (Tweardy et al., 1987b) and a full-length eDNA clone of GM-CSF was kindly provided by Dr. Gordon Wong of Genetics Institute (Wong et al., 1985). The eDNA probe for fl-tubulin was obtained from Dr. J. Hall et al. (1983). Nucleic acid probes were radiolabeled to high specific activity (2-5 × 108 cpm/#g eDNA) by nick translation with [32p]dCTP and used to hybridize membranes according to the manufacturer's instructions. In those cases where a single blot was hybridized with more than one probe, the
RNA isolation and hybridization studies Cells were washed in phosphate-buffered saline (PBS) and lysed in guanidine isothiocyanate (BRL, Bethesda, MD, U.S.A.) solution; total cellular RNA was isolated by centrifugation through a cesium chloride (BRL) cushion as described (Chirgwin et al., 1979). 10 #g of RNA were separated on agarose-formaldehyde gels (Lehrach et 30
30
O
b 20 ~0
u
{J
©
lO
1o
O
0
0
l
!
I 0
20
0 3'0
4'0
5'0
1
60
5OO
~ 400-
i 10,000
d
4oo
300.
u
o rO
i 1.000
600
¢
500 '
~
100
[TNFI U / m l
TIME (hr) 6OO
| 10
200
0
100 •
0
20O 100
i
i 10
i 20
i 30
40
TIME (hr)
i 50
60
i 0
i 1
i
10
i
100
i i 1,00010,000
{TNFI U / m l
Fig. 1. Effects of T N F - a on G M - C S F and G-CSF protein production by U87MG. Confluent cultures of U 8 7 M G were incubated in T N F - a (1000 U / m l ) for the times indicated. Supernatants were harvested and tested for G M - C S F (panel a) and G - C S F (panel c) as described in Material and Methods. In other experiments, confluent cultures of U 8 7 M G were incubated in T N F - a at the concentrations indicated. At 32 h, supernatants were harvested and tested for G M - C S F (panel b) and G - C S F (panel d ) as above. The results shown are representative of two experiments.
272 sequence of p r o b i n g was G M - C S F , G - C S F , then fl-tubulin. Blots were s t r i p p e d b e f o r e p r o b i n g with /3-tubulin only. Blots were e x p o s e d to K o d a k X A R - 5 film at - 8 0 ° C with two intensifying screens for the length of time indicated. A u t o r a d i o g r a m s were a n a l y z e d b y d e n s i t o m e t e r in one d i m e n s i o n ( m o d e l 620, B i o R a d , R i c h m o n d , C A , U.S.A.).
Results
Induction by TNF-a of increased production of GMCSF and G-CSF protein and mRNA in U87MG cells I n e a r l i e r studies, we d e m o n s t r a t e d t h a t U87MG constitutively produced GM-CSF and G - C S F ; the G M - C S F activity in the s u p e r n a t a n t s of u n s t i m u l a t e d U 8 7 M G was n e u t r a l i z e d b y antiG M - C S F a n t i s e r a ( T w e a r d y et al., 1987a). T o d e t e r m i n e the effect of m o n o k i n e s on the p r o d u c tion of C S F b y these cells, U 8 7 M G were inc u b a t e d with TNF-c~. C o n t i n u o u s e x p o s u r e of U 8 7 M G cells to T N F - a (1000 U / m l ) i n d u c e d the p r o d u c t i o n of G M - C S F a n d G - C S F b i o a c t i v i t y as
A.
0 0.5
TIME ['HR-~ 1 1.5 2
4
8
early as 4 h f r o m the start o f c u l t u r e (Fig. 1). P r o d u c t i o n of G M - C S F b e g a n to p l a t e a u at 32 h while p r o d u c t i o n of G - C S F p e a k s at 32 h a n d b i o a c t i v i t y decreases t h e r e a f t e r p o s s i b l y d u e to d e g r a d a t i o n . C h o o s i n g 32 h as the o p t i m u m time to e x a m i n e the effect of v a r y i n g c o n c e n t r a t i o n s of TNF-a on CSF protein production by U87MG cells, we f o u n d that G M - C S F p r o d u c t i o n was i n d u c e d b y as little as 10 U / m l TNF-c~ while G - C S F p r o d u c t i o n was i n c r e a s e d at 1 U / m l . Peak p r o d u c t i o n of b o t h G M - C S F a n d G - C S F was i n d u c e d b y T N F - a at 1000 U / m l . T h e i n c r e a s e in C S F b i o a c t i v i t y i n d u c e d b y T N F was a c c o m p a n i e d b y a d e t e c t a b l e increase in G M - C S F a n d G - C S F m R N A levels (Fig. 2). A t 1000 U / m l , the i n d u c t i o n of G M - C S F a n d G - C S F m R N A occ u r r e d within 30 m i n of e x p o s u r e a n d p e a k e d at 6 0 - 9 0 m i n (Fig. 2). C h o o s i n g 1 h as the o p t i m u m time for e x a m i n i n g the sensitivity of C S F m R N A p r o d u c t i o n b y U 8 7 M G cells in r e s p o n s e to TNF-a, we f o u n d that i n d u c t i o n of G M - C S F m R N A occ u r r e d at a c o n c e n t r a t i o n of T N F - a as low as 1 U / m l a n d r e a c h e d a p l a t e a u at 1000 U / m l , while i n d u c t i o n of G - C S F m R N A o c c u r r e d at a conc e n t r a t i o n of T N F - a as low as 1 U / m l a n d d i d
B.
0.800 T
GM-CSF
o.,oo
0.600 t
0,500 t G-CSF
i
B-TUBULIN
0,200 0.100 0.000
0
0.5
1
1,5
2
4
8
HUE Chd
Fig. 2. Kinetics of G-CSF and GM-CSF mRNA accumulation by U87MG in response to TNF-a. U87MG cells were harvested for extraction of total RNA after incubation in TNF-a, 1000 U/nil, at the indicated time points. A Northern blot of total RNA (10 #g, panel A) was probed with GM-CSF (top row; band of 0.9 kb), G-CSF (middle row; band of 1.6 kb) and fl-tubulin (bottom row; bands of 2.8 and 1.8 kb) and exposed to film for 5 days, 5 days, and 18 h, respectively, before developing. The autoradiograms were analyzed by densitometry and the scanning areas for the bands detected by GM-CSF were corrected by dividing them by the area of the corresponding bottom band of/~-tubulin and the results plotted in panel B using the open bars. The corrected scanning ratio for G-CSF is plotted in panel B using the closed bars. The results shown are representative of two experiments.
273
Ao 0
1
B.
ITNFI U/ml 10 102 103 104
©
GM-CSF
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