Brain Research, 564 (1991) 79-85 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91l$03.50 ADONIS 0006899391171411
79
BRES 17141
Tumor necrosis factor and interleukin-1 down-regulate receptors for substance P in human astrocytoma cells C.L. Johnson and C.G. Johnson Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0575 (U.S.A.) (Accepted 18 June 1991) Key words: Astrocytoma cell; Substance P; Tachykinin; Tumor necrosis factor; Interleukin-1; Cytokine; Receptor binding; Inositolphosphate formation
This study examined the influence of cytokines on substance P (SP) receptors (NK1 subtype) in the human astrocytoma cell line UCI1. Following trypsinization and passage, the density of SP receptors in these cells was rather low but gradually increased several fold over the course of a few days in culture. Frequent replacement of the growth medium enhanced the density of receptors even more, suggesting that growth factors in the culture medium may determine the levels of receptor. Exposure of the cells to sub-nanomolar concentrations of tumor necrosis factor (TNFa) or interleukin-lfl (ILlfl), but not interleukin-2 or interleukin-6, decreased the density of SP receptors. This was accompanied by a decrease in the ability of SP to stimulate inositolphosphate formation. The ability of histamine to activate inositolphosphate formation was not influenced by the cytokines. The decrease in SP receptor density was readily reversible on washout of the cytokines. The ECso for TNFa was approximately 0.5 ng/ml, the ECs0 for ILlfl was approximately 0.1 ng/ml. Radioligand binding studies with [ 125I]TNFa indicated the presence of a low density of high affinity binding sites for this ligand: K d = 2.5 + 0.6 ng/ml, B m a x = 14.8 _+ 2.7 fmol bound/mg protein (assuming trimeric form of ligand bound). The most likely explanation for the cytokine effect is an inhibition of the synthesis of new receptors.
INTRODUCTION Although long viewed as simple support cells, glial cells are now thought to perform critical homeostatic functions within the central nervous system. These include fairly well established roles for astrocytes in the accumulation of certain neurotransmitters such as glutamate, the buffering of extracellular K +, and the regulation of interstitial fluid volume. In addition, recent studies have suggested that astrocytes may play a central role in immune responses in the CNS. These cells may express class I and II M H C antigens 1"1s. In addition, astrocytes have been r e p o r t e d 3"8 to synthesize and release a variety of immunoregulatory molecules, including tum o r necrosis factor ( T N F a ) and interleukin-1 (ILl/3). Substance P (SP) is an undecapeptide widely distributed in the central and peripheral nervous Systems. In the periphery, in addition to causing increased production and release of proinflammatory mediators such as histamine, prostaglandins and thromboxanes, SP has recently been reported to induce the production by monocytes of several cytokines, including I L l f l , interleukin-6 (IL6), and T N F a 4A7'21. Thus some of the inflammatory
effects of SP in the periphery may be m e d i a t e d by these or related cytokines. A l t h o u g h a role for SP in pain responses is clearly established, other actions of SP in the CNS are poorly understood. We have summarized elsewhere 13, the evidence for the presence of SP receptors on astrocytes. The physiological significance of these receptors remains unclear, but we have found that SP does induce significant alterations in glutamate and K + transport by the human astrocytoma cell line U C l l . It is also of considerable interest that SP has very recently been r e p o r t e d to induce the formation of IL6 by the human astrocytoma cell line U37315'3°. In view of the p r o b a b l e influence of SP on cytokine production by astrocytes and the fact that astrocytes may be target cells for cytokines in the CNS, we w o n d e r e d whether these compounds would influence SP receptors and the biochemical response to activation of these receptors, namely inositolphosphate lIP) formation. In the present p a p e r we demonstrate that prolonged exposure to ILl/3 or T N F a causes a m a r k e d down regulation of SP receptors and an accompanying decrease in IP formation.
Correspondence: C.L. Johnson, Department Pharmacology, University Cincinnati, Cincinnati, OH 45267-0575, U.S.A. Fax: (1) (513) 5581169.
80 MATERIALS AND METHODS
[~251]TN Fa binding All binding assays were conducted on intact cells in 24-well plastic culture dishes at room temperature for 2 h. Cells were rinsed and BSA buffer was added to each well for 30 min to temperature equilibrate. The buffer was aspirated and fresh buffer (300 #1) containing various concentrations of [lZSI]TNFa was added. At the end of the incubation period, the plate was inverted in a sink and then immersed for 20 s under 500 ml of ice-cold 0.9% NaCI-10 mM HEPES (pH 7.2) wash buffer. 500 #1 5% SDS-0,01 N HCI was added to each well to solubilize the cells, and the plate was incubated at 37 °C for 1 h. The solubilized material was removed and counted in a scintillation counter. The binding data were fit to the following equation by a non-linear least-squares procedure:
Materials [3H][Sar9,Met(O2)ll]-sP (SarMetSe) (spec. act. 32.5 Ci/mmol), [125I]TNFa (spec. act. 35.2/~Ci/#g) and [3H]myo-inositol (spec. act. 15.6 Ci/mmol) were from New England Nuclear (Boston MA). Sterile solutions of RPMI-1640, glutamine, HEPES, antibiotic-antimycotic were from Sigma (St. Louis MO). Bovine serum albumin (BSA) and SP were from Sigma. Calf serum was from Hyclone (Logan UT). Inositol-free RPMI-1640 was from ICN Biomedicals (Costa Mesa, CA). Unlabeled SarMetSP was from Peninsula Labs (Belmont CA). All cytokines were human recombinant products and were purchased from Boehringer Mannheim (Indianapolis, IN): TNFa (10 #g/ml; 1088 939); ILlfl (1000 U/ml; 1059 394); IL2 (10000 U/ml; 1011 456); IL6 (200000 U/ml; 1138 600).
N'[L]
Cell culture The U C l l MG human astrocytoma cell line was originally isolated at the University of Cincinnati 2° and was kindly provided by Dr. Eric Gruenstein. The cells were grown in RPMI-1640 (supplemented with glutamine, HEPES, antibiotic-antimycotic) plus 10% calf serum.
where [L] is the free concentration of TNFa (expressed in ng/mi). The first term represents a saturable binding site with maximum binding Bmax (expressed as ng bound/rag cell protein) and binding constant K d (in ng/ml). The second term represents non-specific binding. Using the fitted value for N. the calculated non-specific binding was subtracted from total binding to yield specific binding. The specific binding data were then converted to Scatchard format for graphical presentation.
[3H]SarMetSP binding All binding assays were conducted on intact cells in 24-well plastic culture dishes on ice (4 °C). Cells were rinsed and ice-cold BSA buffer (same ionic composition as RPMI-1640 plus 0.2% glucose plus 1% BSA) was added to each well for 30 min to temperature equilibrate. The buffer was aspirated and fresh cold buffer (300/A) containing [3H]SarMetSP was added followed by 75 gl of buffer (total binding) or buffer containing 500 nM unlabeled SP (non-specific binding). Binding studies were conducted for 3 h. At the end of the incubation period, the plate was inverted in a sink and then immersed for 20 s under 500 ml of ice-cold 0.9% NaCI-10 mM HEPES (pH 7.2) wash buffer. 500/tl 5% SDS-0.01 N HCI was added to each well to solubilize the cells, and the plate was incubated at 37 °C for 1 h. The solubilized material was removed and counted in a scintillation counter. Protein content of the wells was determined by a modified Lowry procedure 29 and usually ranged from 50 to 200/~g.
Inositolphosphate formation Cells in 24-well plates were labeled for approximately 24 h in 300 #1 inositoi-free RPMI-1640 medium (serum was also reduced to 0.1%) containing 1/~Ci/ml[aH]myo-inositol. The cells were rinsed and BSA buffer was added for 30 min at room temperature. Twenty mM LiC1 was added for 15 min prior to addition of agonist Cells were incubated at room temperature for 60 min. The reaction was terminated by aspiration of the buffer and rapid addition of l ml of cold MeOH-0.1 N HCI (50:50). The extraction of the water soluble inositoiphosphates was allowed to continue for 1
TABLE I
Effect of pretreatment of UC11 cells with TNFa or 1Llfl on inositolphosphate formation stimulated by substance P or histamine The table shows 5 independent experiments. The period of exposure to 10 ng/ml TNFa or 10 U/ml IL lfl was 1 to 6 days, To correct for differences in the incorporation of inositol, IP 1 was expressed as dpm IP 1 formed x I00/dpm inositol incorporated into: the lipids. ResUlts shown represent mean ± S.E.M. for triplicate determinations. The concentration of SP was 100 nM and the concentration of histamine was t mM. The incubation time was 60 min. The increase in inositol incorporation into the IL 1 t-treated: cells was not due to an increase in cell number as estimated by cell protein per well. The protein content in the fourth experiment was 263 ± 10 ~g (control) and 261 ± 6 #g (IL lfl), and in the fifth experiment was 218 ± 11 #g (control) and 203 ± 6 ~g (IL lfl).
Treatment
dpm in phosphoinositides
100 × dpm 1P1/dpm in phosphoinositides Basal
Substance P
Histamine
Control TNFa, 1 day
83247 ± 1241 57078 ± 1228
0.20 -+ 0.03 0.17 _+ 0.01
22.9 ± 1.23 16.9 -+ 0.66
1.08 ± 0.03 1.23 -+ 0.07
Control TNFa, 2 days
57921 ± 2290 84432 ± 1492
0.36 ± 0.00 0.35 ± 0.02
18.9 ± 0.9 8.2 ± 0.3
0.91 + 0.04 1.18 ± 0.05
Control TNFa, 3 days
63048 ± 3716 51194 ± 370
0.38 ± 0.03 0.38 ± 0.02
13.4 ± 1.9 4.5 ± 0.5
1.27 -+ 0,05 1.56 + 0.07
Control ILlfl, 6 days
63021 +-- 1628 131882 ± 1663
0.54 ± 0.01 0.43 _+ 0.01
10.3 -+ 1.0 1.49 ± 0.04
0.93 - 0.04 1.16 ± 0.03
Control ILlfl, 6 days
84095 _+ 3086 142416 ± 1504
0.43 ± 0.02 0.47 ± 0.01
4.84 -+ 0.48 1.41 ± 0.07
0.78 -- 0;03 1.01 ± 0.05
81 h. The extract was then removed, diluted with 6 ml H20, and applied to a Dowex anion exchange column to isolate the inositolphosphate fractions as previously described 14. The water insoluble cell membranes remaining on the tissue culture plates after extraction with MeOH-HCI were solubilized with SDS and directly counted as a rough measure of total inositol incorporation into the phosphoinositide pools. Dose-response curve data were analyzed with the ALLFIT program 7.
RESULTS
Effect of TNFa or ILlfl treatment on inositolphosphate formation by UCll cells Cells w e r e t r e a t e d for 1 - 3 days with 10 ng/ml T N F a t h e n tested for i n o s i t o l p h o s p h a t e r e s p o n s e s (Table I). F o r this study, the cells for the 3 e x p e r i m e n t s with T N F a
Cytokine treatment Cells were plated on 24-well plastic culture dishes, usually at a density of 2-4 x 104 cells/well. The cells were treated by adding small volumes of concentrated solutions of cytokines directly to the culture medium on the plates. No changes in culture medium were made during the course of the experiments except in the case of the inositolphosphate studies. In this latter case, efficient labeling of the phosphoinositide pools with [3H]inositol required that the medium be replaced with medium containing no unlabeled inositol and low serum concentrations. Fresh cytokine was added back to the cells during this 24 h labeling period.
s h o w n in the table w e r e p l a t e d at d i f f e r e n t densities in o r d e r to o b t a i n a p p r o x i m a t e l y the s a m e cell density at the time of the assays. E v e n with this p r e c a u t i o n , s o m e differences w e r e s e e n in the total a m o u n t of [3H]inositol i n c o r p o r a t e d in the control vs the T N F a - t r e a t e d cells. B e c a u s e of this variation, the data w e r e n o r m a l i z e d by dividing the d p m of IP~ f o r m e d by the total D P M incorp o r a t e d into the p h o s p h o i n o s i t i d e
pools. This should
also correct for any differences in cell n u m b e r b e t w e e n wells. A s s h o w n in the table, T N F a t r e a t m e n t did not influence basal levels of IP[. H o w e v e r , the ability of SP to stimulate IP 1 f o r m a t i o n was m a r k e d l y r e d u c e d . O n a 600
i
i
i
p e r c e n t a g e basis, the T N F a
i
effect at t h r e e days (68%
d e c r e a s e ) was g r e a t e r than that at two days (58% dep, m
o ~.
crease) which in turn was g r e a t e r than at o n e day (26%
500
79
BRES 17141
Tumor necrosis factor and interleukin-1 down-regulate receptors for substance P in human astrocytoma cells C.L. Johnson and C.G. Johnson Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0575 (U.S.A.) (Accepted 18 June 1991) Key words: Astrocytoma cell; Substance P; Tachykinin; Tumor necrosis factor; Interleukin-1; Cytokine; Receptor binding; Inositolphosphate formation
This study examined the influence of cytokines on substance P (SP) receptors (NK1 subtype) in the human astrocytoma cell line UCI1. Following trypsinization and passage, the density of SP receptors in these cells was rather low but gradually increased several fold over the course of a few days in culture. Frequent replacement of the growth medium enhanced the density of receptors even more, suggesting that growth factors in the culture medium may determine the levels of receptor. Exposure of the cells to sub-nanomolar concentrations of tumor necrosis factor (TNFa) or interleukin-lfl (ILlfl), but not interleukin-2 or interleukin-6, decreased the density of SP receptors. This was accompanied by a decrease in the ability of SP to stimulate inositolphosphate formation. The ability of histamine to activate inositolphosphate formation was not influenced by the cytokines. The decrease in SP receptor density was readily reversible on washout of the cytokines. The ECso for TNFa was approximately 0.5 ng/ml, the ECs0 for ILlfl was approximately 0.1 ng/ml. Radioligand binding studies with [ 125I]TNFa indicated the presence of a low density of high affinity binding sites for this ligand: K d = 2.5 + 0.6 ng/ml, B m a x = 14.8 _+ 2.7 fmol bound/mg protein (assuming trimeric form of ligand bound). The most likely explanation for the cytokine effect is an inhibition of the synthesis of new receptors.
INTRODUCTION Although long viewed as simple support cells, glial cells are now thought to perform critical homeostatic functions within the central nervous system. These include fairly well established roles for astrocytes in the accumulation of certain neurotransmitters such as glutamate, the buffering of extracellular K +, and the regulation of interstitial fluid volume. In addition, recent studies have suggested that astrocytes may play a central role in immune responses in the CNS. These cells may express class I and II M H C antigens 1"1s. In addition, astrocytes have been r e p o r t e d 3"8 to synthesize and release a variety of immunoregulatory molecules, including tum o r necrosis factor ( T N F a ) and interleukin-1 (ILl/3). Substance P (SP) is an undecapeptide widely distributed in the central and peripheral nervous Systems. In the periphery, in addition to causing increased production and release of proinflammatory mediators such as histamine, prostaglandins and thromboxanes, SP has recently been reported to induce the production by monocytes of several cytokines, including I L l f l , interleukin-6 (IL6), and T N F a 4A7'21. Thus some of the inflammatory
effects of SP in the periphery may be m e d i a t e d by these or related cytokines. A l t h o u g h a role for SP in pain responses is clearly established, other actions of SP in the CNS are poorly understood. We have summarized elsewhere 13, the evidence for the presence of SP receptors on astrocytes. The physiological significance of these receptors remains unclear, but we have found that SP does induce significant alterations in glutamate and K + transport by the human astrocytoma cell line U C l l . It is also of considerable interest that SP has very recently been r e p o r t e d to induce the formation of IL6 by the human astrocytoma cell line U37315'3°. In view of the p r o b a b l e influence of SP on cytokine production by astrocytes and the fact that astrocytes may be target cells for cytokines in the CNS, we w o n d e r e d whether these compounds would influence SP receptors and the biochemical response to activation of these receptors, namely inositolphosphate lIP) formation. In the present p a p e r we demonstrate that prolonged exposure to ILl/3 or T N F a causes a m a r k e d down regulation of SP receptors and an accompanying decrease in IP formation.
Correspondence: C.L. Johnson, Department Pharmacology, University Cincinnati, Cincinnati, OH 45267-0575, U.S.A. Fax: (1) (513) 5581169.
80 MATERIALS AND METHODS
[~251]TN Fa binding All binding assays were conducted on intact cells in 24-well plastic culture dishes at room temperature for 2 h. Cells were rinsed and BSA buffer was added to each well for 30 min to temperature equilibrate. The buffer was aspirated and fresh buffer (300 #1) containing various concentrations of [lZSI]TNFa was added. At the end of the incubation period, the plate was inverted in a sink and then immersed for 20 s under 500 ml of ice-cold 0.9% NaCI-10 mM HEPES (pH 7.2) wash buffer. 500 #1 5% SDS-0,01 N HCI was added to each well to solubilize the cells, and the plate was incubated at 37 °C for 1 h. The solubilized material was removed and counted in a scintillation counter. The binding data were fit to the following equation by a non-linear least-squares procedure:
Materials [3H][Sar9,Met(O2)ll]-sP (SarMetSe) (spec. act. 32.5 Ci/mmol), [125I]TNFa (spec. act. 35.2/~Ci/#g) and [3H]myo-inositol (spec. act. 15.6 Ci/mmol) were from New England Nuclear (Boston MA). Sterile solutions of RPMI-1640, glutamine, HEPES, antibiotic-antimycotic were from Sigma (St. Louis MO). Bovine serum albumin (BSA) and SP were from Sigma. Calf serum was from Hyclone (Logan UT). Inositol-free RPMI-1640 was from ICN Biomedicals (Costa Mesa, CA). Unlabeled SarMetSP was from Peninsula Labs (Belmont CA). All cytokines were human recombinant products and were purchased from Boehringer Mannheim (Indianapolis, IN): TNFa (10 #g/ml; 1088 939); ILlfl (1000 U/ml; 1059 394); IL2 (10000 U/ml; 1011 456); IL6 (200000 U/ml; 1138 600).
N'[L]
Cell culture The U C l l MG human astrocytoma cell line was originally isolated at the University of Cincinnati 2° and was kindly provided by Dr. Eric Gruenstein. The cells were grown in RPMI-1640 (supplemented with glutamine, HEPES, antibiotic-antimycotic) plus 10% calf serum.
where [L] is the free concentration of TNFa (expressed in ng/mi). The first term represents a saturable binding site with maximum binding Bmax (expressed as ng bound/rag cell protein) and binding constant K d (in ng/ml). The second term represents non-specific binding. Using the fitted value for N. the calculated non-specific binding was subtracted from total binding to yield specific binding. The specific binding data were then converted to Scatchard format for graphical presentation.
[3H]SarMetSP binding All binding assays were conducted on intact cells in 24-well plastic culture dishes on ice (4 °C). Cells were rinsed and ice-cold BSA buffer (same ionic composition as RPMI-1640 plus 0.2% glucose plus 1% BSA) was added to each well for 30 min to temperature equilibrate. The buffer was aspirated and fresh cold buffer (300/A) containing [3H]SarMetSP was added followed by 75 gl of buffer (total binding) or buffer containing 500 nM unlabeled SP (non-specific binding). Binding studies were conducted for 3 h. At the end of the incubation period, the plate was inverted in a sink and then immersed for 20 s under 500 ml of ice-cold 0.9% NaCI-10 mM HEPES (pH 7.2) wash buffer. 500/tl 5% SDS-0.01 N HCI was added to each well to solubilize the cells, and the plate was incubated at 37 °C for 1 h. The solubilized material was removed and counted in a scintillation counter. Protein content of the wells was determined by a modified Lowry procedure 29 and usually ranged from 50 to 200/~g.
Inositolphosphate formation Cells in 24-well plates were labeled for approximately 24 h in 300 #1 inositoi-free RPMI-1640 medium (serum was also reduced to 0.1%) containing 1/~Ci/ml[aH]myo-inositol. The cells were rinsed and BSA buffer was added for 30 min at room temperature. Twenty mM LiC1 was added for 15 min prior to addition of agonist Cells were incubated at room temperature for 60 min. The reaction was terminated by aspiration of the buffer and rapid addition of l ml of cold MeOH-0.1 N HCI (50:50). The extraction of the water soluble inositoiphosphates was allowed to continue for 1
TABLE I
Effect of pretreatment of UC11 cells with TNFa or 1Llfl on inositolphosphate formation stimulated by substance P or histamine The table shows 5 independent experiments. The period of exposure to 10 ng/ml TNFa or 10 U/ml IL lfl was 1 to 6 days, To correct for differences in the incorporation of inositol, IP 1 was expressed as dpm IP 1 formed x I00/dpm inositol incorporated into: the lipids. ResUlts shown represent mean ± S.E.M. for triplicate determinations. The concentration of SP was 100 nM and the concentration of histamine was t mM. The incubation time was 60 min. The increase in inositol incorporation into the IL 1 t-treated: cells was not due to an increase in cell number as estimated by cell protein per well. The protein content in the fourth experiment was 263 ± 10 ~g (control) and 261 ± 6 #g (IL lfl), and in the fifth experiment was 218 ± 11 #g (control) and 203 ± 6 ~g (IL lfl).
Treatment
dpm in phosphoinositides
100 × dpm 1P1/dpm in phosphoinositides Basal
Substance P
Histamine
Control TNFa, 1 day
83247 ± 1241 57078 ± 1228
0.20 -+ 0.03 0.17 _+ 0.01
22.9 ± 1.23 16.9 -+ 0.66
1.08 ± 0.03 1.23 -+ 0.07
Control TNFa, 2 days
57921 ± 2290 84432 ± 1492
0.36 ± 0.00 0.35 ± 0.02
18.9 ± 0.9 8.2 ± 0.3
0.91 + 0.04 1.18 ± 0.05
Control TNFa, 3 days
63048 ± 3716 51194 ± 370
0.38 ± 0.03 0.38 ± 0.02
13.4 ± 1.9 4.5 ± 0.5
1.27 -+ 0,05 1.56 + 0.07
Control ILlfl, 6 days
63021 +-- 1628 131882 ± 1663
0.54 ± 0.01 0.43 _+ 0.01
10.3 -+ 1.0 1.49 ± 0.04
0.93 - 0.04 1.16 ± 0.03
Control ILlfl, 6 days
84095 _+ 3086 142416 ± 1504
0.43 ± 0.02 0.47 ± 0.01
4.84 -+ 0.48 1.41 ± 0.07
0.78 -- 0;03 1.01 ± 0.05
81 h. The extract was then removed, diluted with 6 ml H20, and applied to a Dowex anion exchange column to isolate the inositolphosphate fractions as previously described 14. The water insoluble cell membranes remaining on the tissue culture plates after extraction with MeOH-HCI were solubilized with SDS and directly counted as a rough measure of total inositol incorporation into the phosphoinositide pools. Dose-response curve data were analyzed with the ALLFIT program 7.
RESULTS
Effect of TNFa or ILlfl treatment on inositolphosphate formation by UCll cells Cells w e r e t r e a t e d for 1 - 3 days with 10 ng/ml T N F a t h e n tested for i n o s i t o l p h o s p h a t e r e s p o n s e s (Table I). F o r this study, the cells for the 3 e x p e r i m e n t s with T N F a
Cytokine treatment Cells were plated on 24-well plastic culture dishes, usually at a density of 2-4 x 104 cells/well. The cells were treated by adding small volumes of concentrated solutions of cytokines directly to the culture medium on the plates. No changes in culture medium were made during the course of the experiments except in the case of the inositolphosphate studies. In this latter case, efficient labeling of the phosphoinositide pools with [3H]inositol required that the medium be replaced with medium containing no unlabeled inositol and low serum concentrations. Fresh cytokine was added back to the cells during this 24 h labeling period.
s h o w n in the table w e r e p l a t e d at d i f f e r e n t densities in o r d e r to o b t a i n a p p r o x i m a t e l y the s a m e cell density at the time of the assays. E v e n with this p r e c a u t i o n , s o m e differences w e r e s e e n in the total a m o u n t of [3H]inositol i n c o r p o r a t e d in the control vs the T N F a - t r e a t e d cells. B e c a u s e of this variation, the data w e r e n o r m a l i z e d by dividing the d p m of IP~ f o r m e d by the total D P M incorp o r a t e d into the p h o s p h o i n o s i t i d e
pools. This should
also correct for any differences in cell n u m b e r b e t w e e n wells. A s s h o w n in the table, T N F a t r e a t m e n t did not influence basal levels of IP[. H o w e v e r , the ability of SP to stimulate IP 1 f o r m a t i o n was m a r k e d l y r e d u c e d . O n a 600
i
i
i
p e r c e n t a g e basis, the T N F a
i
effect at t h r e e days (68%
d e c r e a s e ) was g r e a t e r than that at two days (58% dep, m
o ~.
crease) which in turn was g r e a t e r than at o n e day (26%
500
