Modulation of macrophage TNF production by temperature
Eur. J. Immunol. 1992. 22: 2983-2987
2983
Bruno Fouqueray, Carole Philippe, Abdelaziz Amrani, Joelle Perez and Laurent Baud
Heat shock prevents lipopolysaccharide-induced tumor necrosis factor-a synthesis by rat mononuclear phagocytes"
INSERM U. 64, H6pital Tenon, Paris
Tumor necrosis factor-a (TNF-a), a mononuclear phagocyte-derived peptide is known to participate in the pathogenesis of fever. To determine whether a feedback mechanism exists by which elevated temperatures influence TNF-a generation,we have examined the effects of heat shock on the in vitro synthesis of TNF-a by rat glomeruli, inflammatory peritoneal macrophages and blood monocytes. Preexposure of peritoneal macrophages to elevated temperatures for 20 min decreased the subsequent lipopolysaccharide-induced release of TNF-a bioactivity. The mean reductions were 11.9 5.0 YO, 86.3 k 12.0 YO, and 95.2 k 3.5 % after pretreatment at 39, 41 and 43 "C, respectively. Reductions, that were transient, were maximum when lipopolysaccharide was added 0-2 h after heat shock. They correlated with the decreased release of immunoreactive TNF-a and the decreased expression of both cell-associated TNF-a molecule and TNF-a mRNA. Heat shock-induced inhibition of TNF-a release was independent of variations of prostaglandin synthesis, but was possibly related to the induction of heat-shock proteins since (a) macrophages exposed to heat shock synthesized the major 70- and 90-kDa heat-shock proteins, and (b) chemical inducers of the heat-shock response were also effective inhibitors of TNF-a release. The mean reduction of TNF-a release after pretreatment at 41 "C was found to be identical in glomerular tissue (82.0 IfI 7.5 YO),but significantly less in blood monocytes (43.9 k 10.9 %).This supports the hypothesis that a negative-feedback mechanism exists between elevated temperature and lipopolysaccharide-induced TNF-a synthesis, and suggests that this regulation is less active in blood monocytes than in tissue macrophages.
*
1 Introduction A number of bacterial products including Gram-negative endotoxin (LPS) [1] cause blood monocytes and tissue macrophages to synthesize and release tumor necrosis factor-a (TNF-a). Negative regulatory mechanisms exist to limit this response. For example, LPS has been shown in vitro to induce the simultaneous production of TNF-a and prostaglandin E2 (PGE2) 121. Indomethacin, a potent inhibitor of PGE? production, enhances LPS-induced TNF-a production by monocytes/macrophages [3]. Conversely, the addition of exogenous PGE2 promotes a dose-dependent reduction in LPS-induced TNF-a production via regulation at a transcriptional and post-transcriptional level [2]. There is strong evidence that TNF-a production is limited, in vivo, by systemic host responses to LPS as well. LPS injection promotes the release of corticosteroids into the plasma by stimulating the formation of adrenocorticotrophic hormone [4].In turn, corticosteroids limit the synthesis of TNF-a [5]. LPS also initiates fever. However, little is
[I 102411
* This work was supported by grants from the Institut National de la Sant@et de la Recherche MCdicale and from the FacultC de MCdccine Saint-Antoine. Correspondence: Laurent Baud, INSERM U. 64, HBpital Tenon, 4 rue dc la Chine, F-75020 Paris, France
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992
known about the regulatory role of elevated temperatures on TNF-a synthesis. Although inhibitory effects of heat stress on TNF-a synthesis by astroglial cells have been reported [6], different findings have been presented which show no effect of elevated temperature on TNF-a synthesis by blood mononuclear cells [7]. The purpose of the present study was to determine the effect of heat stress on TNF-a biosynthesis by rat glomeruli, inflammatory peritoneal macrophages and blood monocytes.
2 Materials and methods 2.1 Isolation and incubation of cells Peritoneal exudate cells were obtained from 6-week-old male Sprague Dawley rats. Animals received an i.p. injection of 5 ml 3 YOthioglycollate medium (Difco Laboratories, Detroit, MI) 5 days before harvesting peritoneal cells using HBSS (Flow Laboratories, Irvine, Scotland) containing 20 IU/ml heparin. Peritoneal exudate cells were washed by centrifugation, then suspended in RPMI 1640 (Flow Laboratories) containing 30 YO fetal bovine serum (FBS), 2 mM glutamine, and 100 U/ml penicillin/100 pg/ml streptomycin (medium 1). They were plated at 2 x lo6 cells/ml in 0.5 ml medium in 24-well culture plates (Nunc, Roskilde, Denmark) and were allowed to adhere for 2 h at 37 "C in a 5 % CO2-95 YOair atmosphere. The nonadherent cells were removed by three washings and the macrophage monolayers were then overlaid with 0.3 ml RPMI 1640 medium containing 1YO FBS (medium 2). Rat peripheral blood mononuclear cells were obtained by density centri0014-2980/92/1111-2983$3.50+ .25/0
2984
B. Fouqueray, C. Philippe, A . Amrani et a1
fugation as previously described [S]. Monocytes were isolated by a 4-h adherence in 24-well culture plates (1.5 x lo6 mononuclear cells/well) and incubated with 0.3 ml medium 2. Glomeruli, isolated from rat kidneys as previously described [9], were resuspended in culture medium and added to 24-well culture plates (10 000 isolated glomeruWwel1).
Eur. J. Immunol. 1992. 22: 2983-2987
4°C for 10 min and lysed in sample buffer (50 mM Tris/HCl, p H 6.8, 1% SDS, 10 % glycerol and bromopheno1 blue). Proteins were resolved by SDS-PAGE on 10 % polyacrylamide gels. The gels were fixed, dried and developed by autoradiography. 2.5 Statistical analysis
After 20 min of exposure at 37"C, 39"C, 41 "C or 43 "C, peritoneal macrophages, blood monocytes and isolated glomeruli were stimulated with 1 to 1000ng/ml LPS (Escherichia coli 026: B6, Sigma Chemical Co, St. Louis, MO). At indicated times cell-free culture supernatants were harvested and macrophages were fixed with 2 YO paraformaldehyde [ 101, before both components were assayed for TNF-a activity.
Results are given as the mean k standard error of the mean (SEM). The statistical significance of differences between groups was analyzed by the Student's t test for unpaired samples. A p value < 0.05 was considered significant.
3 Results
2.2 TNF-a assays and lymphocyte-activating factor (LAF) assay
3.1 Effect of heat shock on the release of TNF-a from LPS-stimulated macrophages
TNF-a biological activity was determined by the assay for cytotoxicity using L-929 cells as previously described [11]. Specificity for TNF-a was demonstrated by neutralization of TNF-a-induced cytotoxicity with anti-mouseTNF-a (lo6 neutralizing units/ml, Genzyme, Cambridge, MA) at a titer of 1: 100. Extracellular levels of TNF-a were also measured using specific ELISA (mouse TNF-a ELISA kit, Genzyme, Cambridge, MA) according to the supplier's instructions.
In response to LPS concentrations of 1to 100 ng/ml,TNF-a production was enhanced in a dose-dependent way (Fig. 1). Cell pretreatment at 41 "C significantly reduced the amount of TNF-a produced, especially at the lower LPS concentration. Cell pretreatment at 43 "C completely blocked the release of TNF-a at all the LPS concentrations used. These experimental conditions have been shown to preserve the cell viability as determined by trypan blue exclusion and ability to adhere to the tissue culture plate. A time-course study showed that TNF-a release from stimulated macrophages was detectable within 60 min following LPS challenge, increased with time and reached a plateau within 6 h (Fig. 2A). Heat shock reduced this release at all periods of time tested. In parallel experiments, the presence of IL-1 was analyzed by the LAFassay. IL-1 was detected in the cell extracts but not in the cell-free supernatants. Significantly less IL-1 bioactivity was synthesized by cells after exposure at 41 "C (Fig. 2B). Reversibility of the heat shock-induced reduction of TNF-a release was also examined. This reduction was maximum when LPS was added immediately
In order to detect intracellular IL-1 activity, macrophages were sonicated, centrifuged and IL-1 activity was determined in the supernatant by the standard thymocyte proliferation assay [12]. 2.3 Northern blot analysis Following a 20-min preincubation period at 37 "C or 41 "C, macrophages were treated at 37 "C with 1 ng/ml LPS. After 120 min, total RNA was extracted by the phenol-chloroform method [13], submitted to agarose gel (1.2%) electrophoresis (10 yg/lane), and Northern blot transfer on Gene Screen Plus membrane (New England Nuclear, Boston, MA). After prehybridization, the filters were hybridized with a "P-labeled mouse TNF-a probe [13] donated by Dr. W. Fiers, Biogen, Ghent, Belgium, and rehybridized with a "P-labeled oligonucleotide for p-actin. Messanger RNA for TNF-a was compared with mRNA for 13-actin by analyzing the autoradiographs by laser densitometry.
LPS (ng/ml)
2.4 Analysis of heat-induced proteins in macrophages Macrophages in 50-mm diameter culture dishes (lo7 cells/dish) were heat shocked in culture medium at 39 "C, 41 "C and 43 "C for 20 min. Immediately after heat shock, the medium was replaced by 3 ml of cysteine- and methionine-free RPMI 1640 medium (Institut Jacques Boy, Compikgne, France) supplemented with 1 ng/ml LPS and 70 yCi ['%I methionine (14 mCi/ml, Amersham Int., Amersham, GB). After a 2-h incubation at 37 "C, cell-free supernatants were collected and cells were washed twice with cold culture medium. Both fractions were precipitated in 10 % TCA at
37
39
41
43
TEMPF.RATLRE(00
Figure 1. Effect of heat shock on the release of TNF-a bioactivity from macrophages. Macrophages were incubated at 37 "C, 39 "C, 41 "C, or 43°C for 20 min and then at 37°C with various concentrations of LPS. After 2 h of incubation, the cell-free culture medium was harvested and TNF-a concentration was determined by bioassay. Means and SEM of values obtained in 4 experiments are given. * p < 0.05 vs. 37°C; ** p < 0.005 vs. 37°C.
Modulation of macrophage TNF production by temperature
Eur. J. Immunol. 1992. 3:2083-2987
2985
after the end of heat shock, and then declined progressively to be undctectable after 6--8 h (Fig. 3).
mKNA for TNF-a. Cell pretreatment at 41°C reduced markedly this expression.
3.2 Regulatory mechanisms involved in heat shock-induced reduction of TNF-a release from LPS-stimulated macrophages
3.3 Relationship between heat shock-induced reduction of TNF-a release and heat-shock protein synthesis
The amount of immunoreactive TNF-a in the medium of unstimulated macrophages was near the limit of detectability. Its mean concentration reached 1348 k 295 pg/ml after 2 h of incubation with 100 nglml LPS and was significantly reduced when macrophages were first exposed for 20 min to 41°C (555 f 186pg/ml; p = 0.05) or 43°C (168 k 57 pg/ml; p < 0.05). The diminished accumulation of bioactive and immunoreactiveTNF-a in the medium was paralleled by a similar decrease in its expression as a cell-associated molecule. Indeed, fixed LPS-activated macrophages were capable of promoting the lysi~of the L-929 cells. The mean cytotoxic activity induced by 20000 fixed macrophages reached 44.1 k 0.2% after 2 h of incubation with 1 ng/ml LPS and was significantly reduced when macrophages were first exposed for 20 min to 41 "C (30.6 t- 2 . 9 % ; ~= 0.01 vs. 37°C) or43"C (17.8 1. 4.0%; p = 0.003 vs. 37°C). To determine whether the heat shock-induced reduction of cell expression and release of TNF-a from LPS-activated macrophages was associated with changes in TNF-a gene transcription, total KNA was isolated from macrophages and TNF-a-specific mRNA was detected by Northern blot analysis using a TNF-a-specific cDNA probe. Fig. 4 demonstrates that, after LPS treatment, macrophages contained substantial amounts of
To determine whether PGE2 synthesis could account for the heat shock-induced reduction of TNF-a release, macrophages were preincubated at 37 "C or 41 "C in the presence of 1 pM indomethacin. Indomethacin did not significantly affect TNF-a release from macrophages (Table 1). The possibility that reduction of TNF-a release by heat shock was rather related to the synthesis of heat shock proteins was then investigated. After exposure to a range of temperatures, macrophages were pulse-labeled with [35S]methionine. Labeled proteins from identical amounts of cells or cell-free supernatants were then analyzed by SDS-PAGE (Fig. 5 ) . Cell pretreatment at 41"C, that did not reduce total protein synthesis, induced the synthesis of
0
2
4
6
8
10
12
14
16
18
20
TIME0
Figure 3. Reversibility of the heat shock-induced reduction of TNF-u release from macrophages. Macrophages were incubated at 41 "C for 20 min. LPS (1 ng/ml) was added at indicated time points before or after the heat shock. After 2 h of incubation at 37"C, the cell-free culture medium was harvested and TNF-a concentration was determined by bioassay. Means and SEM of values obtained in three experiments are given. * p < 0.05 vs control value; ** p < 0.005 vs. control value.
B
n
300
37'C
41'C
I
.61
T
28 S 185
*
* 37
n 0
5
10
15
41
zn
TI\.fF(H)
Fzgure 2. Time-course of LPS-induced expression of TNF-u (A) and IL-1 (B). Macrophages were incubated at 37°C (0) or 41°C (W) for 20 min and then at 37°C with 1 ng/ml LPS. At indicated times, cell-free culture medium and cell extracts were harvested to determine the concentrations of TNF-u and IL-1, respectively. Means and SEM of values obtained in 3-4 experiments are given. -* p < 0.05 V F 37 "C. ** p < 0 005 vs. 37 "C.
Figure 4. Effect of heat shock on the expression of TNF-u mFWA in macrophages. Macrophages were incubated at 37°C or 41 "C for 20 min and then at 37°C with 1ng/ml LPS. After2 h of incubation. RNAwas extracted, blotted and probed for TNF-u and p-actin. A) Northern blot probed for TNF-a mRNA; B) ethidium bromidestained gel used in panel A depicting 28s and 18s RNA bands; C) amounts of TNFu mRNA determined in four different blots and adjusted for amount of p-actin in each lane. * p < 0.05 vs. control value.
Eur. J. Immunol. 1992. 22: 2983-2987
B. Fouqueray, C. Philippe, A. Amrani et al.
2986
Table 1. Effect of indomethacin on TNF-a release from LPSactivatcd macrophaged Addition
Release of cytotoxic activity (%) 37 "C 41 "C
LPS (1 ng/ml) LPS (1 ng/ml)
11.6 k 4.3**
47.5 k 2.8
+ Indomethacin (1 FM)
44.5 k 3.6
8.1
* 4.2**
a) Elicited rat peritoneal macrophages were incubated at 37" or 41 "C for 20 min with or without indomethacin and then at 37°C with 1 ngiml LPS. After 2 h of incubation, the cell-free culture medium was harvested and TNF-a concentration was determined by bioassay. ** p < 0.005 vs. 37°C (n = 3).
37
A
39
41
43
37
6
39
41
43
200, 200,
116, 97,
116,
66b
97b 45,
two cell-associated proteins of 70 and 90 kDa. These were the predominant proteins synthesized by macrophages at 43 "C. To confirm a possible relationship between the synthesis of such heat-shock proteins and the reduction of TNF-a release, experiments have been performed with chemical inducers of the heat-shock response [14]. Macrophages were preincubated with sodium arsenite (20 PM) or iodoacetamide (5 pM) before exposure to 1 ng/ml LPS. These compounds reduced the concentration of immunoreactive TNF-a in the cell-free culture medium of macrophages from 1560 208 to 460 zk 347 (p = 0.05) and 400 -t 345 (p < 0.05) pg/ml, respectively.
*
3.4 Comparison of the heat-shock effects on the release of TNF-a from isolated glomeruli and blood monocytes
To determine whether the results obtained were unique to elicited peritoneal macrophages, glomerular cells and blood monocytes were similarly screened for heat-shock effects on the release of TNF-a. Pretreatment at 41°C significantly and transiently reduced the amount of TNF-a produced (Fig. 6). The inhibition was found to be almost complete in glomerular cells (82.0 f 7.5 YO), but significantly less in blood monocytes (43.8 k 10.9%; p < 0.05).
66r 31b 34,
4 Discussion 4.1 Effects of heat shock on the release of TNF-a from LPS-stimulated macrophages
21, 14*
14'
Figure.5. Effect of heat shock on protein synthesis by maerophages. Macrophages were incubated at 37"C, 39 "C, 41 "C or 43 "C for 20 min and then at 37 "C with 1 ng/ml LPS and [3sS]methionine. After 2 h of labcling, aliquots of cell lysates (A) and cell-free supernatants (B) werc subjected to SDS-PAGE.
80
60
-
40
-
20
**
0
I
I
I
Wnr)
Figure 6. Revcrsibility of the heat shock-induced reduction of TNF-a release from isolated glomeruli and blood monocytes. Thcse cells were incubated at 41 "C for 20 min. One yg LPS was added at indicated time points before or after the heat shock. After 2 h of incubation at 37"C, the cell-free culture medium was harvested and TNF-a concentration was determined by bioassay. Means and SEM of values obtained in five experiments are given. * p < 0.05 vs. control value; ** p < 0.005 vs. control value.
The data presented here give further evidence for the modulating role of elevated temperatures on TNF-a synthesis.We show that heat stress can decrease the expression of TNF-a mRNA and hence limit both cell expression and release of TNF-a bioactivity in LPS-stimulated peritoneal macrophages. Stress responses occur following exposure of macrophages to 41 "C for 20 min (Fig. 1). Because this temperature is reached during acute fever, our in vitro observations may have potential implications in vivo. Heat shock at 41°C for 20 min is not toxic to macrophages as suggested by the ability of cells (a) to still adhere to the tissue culture plates, and (b) to incorporate [35S]methionine into proteins to a similar extent than control cells (Fig. 5). In this regard, it is also important to note that the inhibitory effect of heat shock is rapidly reversible: a total recovery is observed when macrophages are incubated at 37°C for 6-8 h following the end of heat shock (Fig. 3). These data are comparable to those reported for the recovery of NADPH-oxidase activity in human neutrophils exposed to 43 "C for 20 min [15]. Our data on the time course study show that heat shockinduced reduction of TNF-a release is a rapid process which begins after less than 2 hours (Fig. 2).This finding suggests that heat shock reducesTNF-a bioactivity by modifying its release from macrophages rather than its stability in the medium. Previous studies demonstrated that the released form of TNF-a (17 kDa) is derived from the integral transmembrane TNF-a precursor molecule (26 kDa) possibly by proteolytic cleavage [ 161. Reduction of this cleavage
Eur. J. Immunol. 1992. 22: 2983-2987
Modulation of macrophage TNF production by temperature
could contribute to the reduced levels of TNF-a bioactivity and immunoreactivity in the medium of macrophages exposed to elevated temperatures. However, such a mechanism is unlikely since heat shock reduces cell expression and release of TNF-a bioactivity to a similar extent. Because of the known shedding of TNF-a receptors from stimulated cells [16], and the evidence that soluble TNF-a receptors reduce the bioactivity of TNF-a [17], the inhibitory effect of heat shock on the bioactivity of TNF-a could also be explained by a more extensive cleavage of TNF-a receptor. However, preliminary studies indicate that the concentration of immunoreactive TNF-a binding protein (TNF-BP75 and TNF-BP55) in the cell-free supernatant of mononuclear phagocytes is not affected by heat stress (data not shown). Exposure of macrophages to elevated temperatures similarly affected the expression of IL-1, suggesting that these observations were not unique to the particular cytokine tested (Fig. 2B). 4.2 Mechanisms by which heat shock reduces the release of TNF-a from LPS-stimulated macrophages
Our studies suggest that the effects of heat shock are independent of variations of prostaglandin synthesis. They are rather related to the synthesis of heat-shock proteins, since (a) after treatment at 41 "C,at least two proteins of 70 and 90 kDa are induced in macrophages, [they are the predominant proteins synthesized at 43 "C (Fig. 5)] and (b) chemical inducers of the heat shock response are also effective inhibitors of the release of immunoreactive TNFa. This finding is consistent with a previous report showing that elevated temperatures down-regulate IL-1 biosynthesis through a mechanism related to the induction of heat-shock proteins [14]. Because the synthesis of stress proteins (70 and 90 kDa) is induced by LPS as well [18], these proteins might down-regulate macrophage activation in an autoregulatory circuit, even in the absence of fever. 4.3 Comparison of the heat-shock effects on tissue macrophages and blood monocytes
In response to LPS,TNF-a is produced by tissues, including glomeruli [19], as well as by circulating monocytes [20].We show in this report that the priming effect of LPS (1 pg/ml) on TNF-a release is identical in isolated glomeruli and in blood monocytes (Fig. 6). However, the effect of heat shock on TNF-a release is clearly greater in isolated glomeruli than in blood monocytes. The difference in
2987
heat-stress response could be explained by the weaker stability of TNF-a mRNA in tissue macrophages compared to that observed in blood monocytes [20]. Such a different modulation by temperature could contribute, in vivo, to reduce the local generation of TNF-a more than its systemic availability. We thank Mrs. Morin, Miranda and Knobloch for secretarial assistance. Received January 2, 1992; in final revised form July 6, 1992.
5 References 1 Fong,Y and Lowry, S. E , Clin.Immunol. Immunopathol. 1990. 55: 157. 2 Kunkel, S. L., Spengler, M.. May, M. A., Spengler, R., Larrick, J. and Remick, D., J. Biol. Chem. 1988. 263: 5380. 3 Kunkel, S. L., Wiggins, R. C., Chensue, S. W. and Larrick, J., Biochem. Biophys. Res. Commun. 1986. 137: 404. 4 Michie, H. R., Manogue, K. R., Spriggs, D. R., Revhaug, A . , O'Dwyer, S., Dinarello, C. A., Cerami, A., Wolff, S. M. and Wilmore, D. W., N. Engl. J. Med. 1988. 318: 1481. 5 Waage, A., Clin.Immunol. Immunopathol. 1987. 45: 348. 6 Velasco, S., Tarlow, M . , Olsen, K., Shay, J. W., McCracken, G. H. and Nisen, F! D., J. Clin. Invest. 1991. 87: 1674. 7 Kappel, M., Diamant, M., Hansen, M. B., Klokker, M. and Pedersen, B. K., Immunology 1991. 73: 304. 8 Cadranel, J., Philippe, C., Perez, J., Milleron, B., Akoun, G., Ardaillou, R. and Baud, L., Clin. Exp. Immunol. 1990. 81: 319. 9 Schambelan, M., Blake, S., Sraer, J., Bens, M., Nivez, M.-P. and Wahbe, F., J. Clin. Invest. 1985. 75: 404. 10 Ichinose, Y., Bakouche, O., Tsao, J. Y. and Fidler, I. J., J. Irnmunol. 1988. 141: 512. 11 Affres, H., Perez, J., Hagege, J., Fouqueray, B., Kornprobst, M., Ardaillou, R. and Baud, L., Kidney Int. 1991. 39: 822. 12 Herbelin, A., Nguyen, A. T., Zingraff, J., Urena, P., Descamps-Latscha, B., Kidney Int. 1990. 37: 116. 13 Baud, L., Oudinet, J.-I?, Bens, M., Noe, L., Peraldi, M.-N., Rondeau, E . , Etienne, J. and Ardaillou, R . , Kidney Int. 1989. 35: 1111. 14 Schmidt, J. A. and Abdulla, E . , J. Immunol. 1988. 141: 2027. 15 Maridonneau-Parini, I., Clerc, J. and Polla, B. S., Biochem. Biophys. Res. Commun. 1988. 154: 179. 16 Vilcek, J. and Le, T. H . , J. Biol. Chem. 1991. 266: 7313. 17 Loetscher, H . , Gentz, R., Zulauf, M., Lustig, A . ,Tabuchi, H., Schlaeger, E.-J., Brockhaus. M., Gallati. H . , Manneberg, M. and Lesslauer, W., J. Biol. Chem. 1991. 266: 18324. 18 J o s h , G . , Hafeez,W. and Perlmutter, D. H . , J. Immunol. 1991. 147: 1614. 19 Fouqueray, B., Philippe, C., Perez, J., Ardaillou, R. and Baud, L., J. Am. Soc. Nephrol. 1991. 2: 427. 20 Burchett, S. K., Weaver, W. M., Westall, J. A., Larsen, A., Kronhein, S. and Wilson, C. B., J. Imrnunol. 1988. 140: 3473.
Eur. J. Immunol. 1992. 22: 2983-2987
2983
Bruno Fouqueray, Carole Philippe, Abdelaziz Amrani, Joelle Perez and Laurent Baud
Heat shock prevents lipopolysaccharide-induced tumor necrosis factor-a synthesis by rat mononuclear phagocytes"
INSERM U. 64, H6pital Tenon, Paris
Tumor necrosis factor-a (TNF-a), a mononuclear phagocyte-derived peptide is known to participate in the pathogenesis of fever. To determine whether a feedback mechanism exists by which elevated temperatures influence TNF-a generation,we have examined the effects of heat shock on the in vitro synthesis of TNF-a by rat glomeruli, inflammatory peritoneal macrophages and blood monocytes. Preexposure of peritoneal macrophages to elevated temperatures for 20 min decreased the subsequent lipopolysaccharide-induced release of TNF-a bioactivity. The mean reductions were 11.9 5.0 YO, 86.3 k 12.0 YO, and 95.2 k 3.5 % after pretreatment at 39, 41 and 43 "C, respectively. Reductions, that were transient, were maximum when lipopolysaccharide was added 0-2 h after heat shock. They correlated with the decreased release of immunoreactive TNF-a and the decreased expression of both cell-associated TNF-a molecule and TNF-a mRNA. Heat shock-induced inhibition of TNF-a release was independent of variations of prostaglandin synthesis, but was possibly related to the induction of heat-shock proteins since (a) macrophages exposed to heat shock synthesized the major 70- and 90-kDa heat-shock proteins, and (b) chemical inducers of the heat-shock response were also effective inhibitors of TNF-a release. The mean reduction of TNF-a release after pretreatment at 41 "C was found to be identical in glomerular tissue (82.0 IfI 7.5 YO),but significantly less in blood monocytes (43.9 k 10.9 %).This supports the hypothesis that a negative-feedback mechanism exists between elevated temperature and lipopolysaccharide-induced TNF-a synthesis, and suggests that this regulation is less active in blood monocytes than in tissue macrophages.
*
1 Introduction A number of bacterial products including Gram-negative endotoxin (LPS) [1] cause blood monocytes and tissue macrophages to synthesize and release tumor necrosis factor-a (TNF-a). Negative regulatory mechanisms exist to limit this response. For example, LPS has been shown in vitro to induce the simultaneous production of TNF-a and prostaglandin E2 (PGE2) 121. Indomethacin, a potent inhibitor of PGE? production, enhances LPS-induced TNF-a production by monocytes/macrophages [3]. Conversely, the addition of exogenous PGE2 promotes a dose-dependent reduction in LPS-induced TNF-a production via regulation at a transcriptional and post-transcriptional level [2]. There is strong evidence that TNF-a production is limited, in vivo, by systemic host responses to LPS as well. LPS injection promotes the release of corticosteroids into the plasma by stimulating the formation of adrenocorticotrophic hormone [4].In turn, corticosteroids limit the synthesis of TNF-a [5]. LPS also initiates fever. However, little is
[I 102411
* This work was supported by grants from the Institut National de la Sant@et de la Recherche MCdicale and from the FacultC de MCdccine Saint-Antoine. Correspondence: Laurent Baud, INSERM U. 64, HBpital Tenon, 4 rue dc la Chine, F-75020 Paris, France
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992
known about the regulatory role of elevated temperatures on TNF-a synthesis. Although inhibitory effects of heat stress on TNF-a synthesis by astroglial cells have been reported [6], different findings have been presented which show no effect of elevated temperature on TNF-a synthesis by blood mononuclear cells [7]. The purpose of the present study was to determine the effect of heat stress on TNF-a biosynthesis by rat glomeruli, inflammatory peritoneal macrophages and blood monocytes.
2 Materials and methods 2.1 Isolation and incubation of cells Peritoneal exudate cells were obtained from 6-week-old male Sprague Dawley rats. Animals received an i.p. injection of 5 ml 3 YOthioglycollate medium (Difco Laboratories, Detroit, MI) 5 days before harvesting peritoneal cells using HBSS (Flow Laboratories, Irvine, Scotland) containing 20 IU/ml heparin. Peritoneal exudate cells were washed by centrifugation, then suspended in RPMI 1640 (Flow Laboratories) containing 30 YO fetal bovine serum (FBS), 2 mM glutamine, and 100 U/ml penicillin/100 pg/ml streptomycin (medium 1). They were plated at 2 x lo6 cells/ml in 0.5 ml medium in 24-well culture plates (Nunc, Roskilde, Denmark) and were allowed to adhere for 2 h at 37 "C in a 5 % CO2-95 YOair atmosphere. The nonadherent cells were removed by three washings and the macrophage monolayers were then overlaid with 0.3 ml RPMI 1640 medium containing 1YO FBS (medium 2). Rat peripheral blood mononuclear cells were obtained by density centri0014-2980/92/1111-2983$3.50+ .25/0
2984
B. Fouqueray, C. Philippe, A . Amrani et a1
fugation as previously described [S]. Monocytes were isolated by a 4-h adherence in 24-well culture plates (1.5 x lo6 mononuclear cells/well) and incubated with 0.3 ml medium 2. Glomeruli, isolated from rat kidneys as previously described [9], were resuspended in culture medium and added to 24-well culture plates (10 000 isolated glomeruWwel1).
Eur. J. Immunol. 1992. 22: 2983-2987
4°C for 10 min and lysed in sample buffer (50 mM Tris/HCl, p H 6.8, 1% SDS, 10 % glycerol and bromopheno1 blue). Proteins were resolved by SDS-PAGE on 10 % polyacrylamide gels. The gels were fixed, dried and developed by autoradiography. 2.5 Statistical analysis
After 20 min of exposure at 37"C, 39"C, 41 "C or 43 "C, peritoneal macrophages, blood monocytes and isolated glomeruli were stimulated with 1 to 1000ng/ml LPS (Escherichia coli 026: B6, Sigma Chemical Co, St. Louis, MO). At indicated times cell-free culture supernatants were harvested and macrophages were fixed with 2 YO paraformaldehyde [ 101, before both components were assayed for TNF-a activity.
Results are given as the mean k standard error of the mean (SEM). The statistical significance of differences between groups was analyzed by the Student's t test for unpaired samples. A p value < 0.05 was considered significant.
3 Results
2.2 TNF-a assays and lymphocyte-activating factor (LAF) assay
3.1 Effect of heat shock on the release of TNF-a from LPS-stimulated macrophages
TNF-a biological activity was determined by the assay for cytotoxicity using L-929 cells as previously described [11]. Specificity for TNF-a was demonstrated by neutralization of TNF-a-induced cytotoxicity with anti-mouseTNF-a (lo6 neutralizing units/ml, Genzyme, Cambridge, MA) at a titer of 1: 100. Extracellular levels of TNF-a were also measured using specific ELISA (mouse TNF-a ELISA kit, Genzyme, Cambridge, MA) according to the supplier's instructions.
In response to LPS concentrations of 1to 100 ng/ml,TNF-a production was enhanced in a dose-dependent way (Fig. 1). Cell pretreatment at 41 "C significantly reduced the amount of TNF-a produced, especially at the lower LPS concentration. Cell pretreatment at 43 "C completely blocked the release of TNF-a at all the LPS concentrations used. These experimental conditions have been shown to preserve the cell viability as determined by trypan blue exclusion and ability to adhere to the tissue culture plate. A time-course study showed that TNF-a release from stimulated macrophages was detectable within 60 min following LPS challenge, increased with time and reached a plateau within 6 h (Fig. 2A). Heat shock reduced this release at all periods of time tested. In parallel experiments, the presence of IL-1 was analyzed by the LAFassay. IL-1 was detected in the cell extracts but not in the cell-free supernatants. Significantly less IL-1 bioactivity was synthesized by cells after exposure at 41 "C (Fig. 2B). Reversibility of the heat shock-induced reduction of TNF-a release was also examined. This reduction was maximum when LPS was added immediately
In order to detect intracellular IL-1 activity, macrophages were sonicated, centrifuged and IL-1 activity was determined in the supernatant by the standard thymocyte proliferation assay [12]. 2.3 Northern blot analysis Following a 20-min preincubation period at 37 "C or 41 "C, macrophages were treated at 37 "C with 1 ng/ml LPS. After 120 min, total RNA was extracted by the phenol-chloroform method [13], submitted to agarose gel (1.2%) electrophoresis (10 yg/lane), and Northern blot transfer on Gene Screen Plus membrane (New England Nuclear, Boston, MA). After prehybridization, the filters were hybridized with a "P-labeled mouse TNF-a probe [13] donated by Dr. W. Fiers, Biogen, Ghent, Belgium, and rehybridized with a "P-labeled oligonucleotide for p-actin. Messanger RNA for TNF-a was compared with mRNA for 13-actin by analyzing the autoradiographs by laser densitometry.
LPS (ng/ml)
2.4 Analysis of heat-induced proteins in macrophages Macrophages in 50-mm diameter culture dishes (lo7 cells/dish) were heat shocked in culture medium at 39 "C, 41 "C and 43 "C for 20 min. Immediately after heat shock, the medium was replaced by 3 ml of cysteine- and methionine-free RPMI 1640 medium (Institut Jacques Boy, Compikgne, France) supplemented with 1 ng/ml LPS and 70 yCi ['%I methionine (14 mCi/ml, Amersham Int., Amersham, GB). After a 2-h incubation at 37 "C, cell-free supernatants were collected and cells were washed twice with cold culture medium. Both fractions were precipitated in 10 % TCA at
37
39
41
43
TEMPF.RATLRE(00
Figure 1. Effect of heat shock on the release of TNF-a bioactivity from macrophages. Macrophages were incubated at 37 "C, 39 "C, 41 "C, or 43°C for 20 min and then at 37°C with various concentrations of LPS. After 2 h of incubation, the cell-free culture medium was harvested and TNF-a concentration was determined by bioassay. Means and SEM of values obtained in 4 experiments are given. * p < 0.05 vs. 37°C; ** p < 0.005 vs. 37°C.
Modulation of macrophage TNF production by temperature
Eur. J. Immunol. 1992. 3:2083-2987
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after the end of heat shock, and then declined progressively to be undctectable after 6--8 h (Fig. 3).
mKNA for TNF-a. Cell pretreatment at 41°C reduced markedly this expression.
3.2 Regulatory mechanisms involved in heat shock-induced reduction of TNF-a release from LPS-stimulated macrophages
3.3 Relationship between heat shock-induced reduction of TNF-a release and heat-shock protein synthesis
The amount of immunoreactive TNF-a in the medium of unstimulated macrophages was near the limit of detectability. Its mean concentration reached 1348 k 295 pg/ml after 2 h of incubation with 100 nglml LPS and was significantly reduced when macrophages were first exposed for 20 min to 41°C (555 f 186pg/ml; p = 0.05) or 43°C (168 k 57 pg/ml; p < 0.05). The diminished accumulation of bioactive and immunoreactiveTNF-a in the medium was paralleled by a similar decrease in its expression as a cell-associated molecule. Indeed, fixed LPS-activated macrophages were capable of promoting the lysi~of the L-929 cells. The mean cytotoxic activity induced by 20000 fixed macrophages reached 44.1 k 0.2% after 2 h of incubation with 1 ng/ml LPS and was significantly reduced when macrophages were first exposed for 20 min to 41 "C (30.6 t- 2 . 9 % ; ~= 0.01 vs. 37°C) or43"C (17.8 1. 4.0%; p = 0.003 vs. 37°C). To determine whether the heat shock-induced reduction of cell expression and release of TNF-a from LPS-activated macrophages was associated with changes in TNF-a gene transcription, total KNA was isolated from macrophages and TNF-a-specific mRNA was detected by Northern blot analysis using a TNF-a-specific cDNA probe. Fig. 4 demonstrates that, after LPS treatment, macrophages contained substantial amounts of
To determine whether PGE2 synthesis could account for the heat shock-induced reduction of TNF-a release, macrophages were preincubated at 37 "C or 41 "C in the presence of 1 pM indomethacin. Indomethacin did not significantly affect TNF-a release from macrophages (Table 1). The possibility that reduction of TNF-a release by heat shock was rather related to the synthesis of heat shock proteins was then investigated. After exposure to a range of temperatures, macrophages were pulse-labeled with [35S]methionine. Labeled proteins from identical amounts of cells or cell-free supernatants were then analyzed by SDS-PAGE (Fig. 5 ) . Cell pretreatment at 41"C, that did not reduce total protein synthesis, induced the synthesis of
0
2
4
6
8
10
12
14
16
18
20
TIME0
Figure 3. Reversibility of the heat shock-induced reduction of TNF-u release from macrophages. Macrophages were incubated at 41 "C for 20 min. LPS (1 ng/ml) was added at indicated time points before or after the heat shock. After 2 h of incubation at 37"C, the cell-free culture medium was harvested and TNF-a concentration was determined by bioassay. Means and SEM of values obtained in three experiments are given. * p < 0.05 vs control value; ** p < 0.005 vs. control value.
B
n
300
37'C
41'C
I
.61
T
28 S 185
*
* 37
n 0
5
10
15
41
zn
TI\.fF(H)
Fzgure 2. Time-course of LPS-induced expression of TNF-u (A) and IL-1 (B). Macrophages were incubated at 37°C (0) or 41°C (W) for 20 min and then at 37°C with 1 ng/ml LPS. At indicated times, cell-free culture medium and cell extracts were harvested to determine the concentrations of TNF-u and IL-1, respectively. Means and SEM of values obtained in 3-4 experiments are given. -* p < 0.05 V F 37 "C. ** p < 0 005 vs. 37 "C.
Figure 4. Effect of heat shock on the expression of TNF-u mFWA in macrophages. Macrophages were incubated at 37°C or 41 "C for 20 min and then at 37°C with 1ng/ml LPS. After2 h of incubation. RNAwas extracted, blotted and probed for TNF-u and p-actin. A) Northern blot probed for TNF-a mRNA; B) ethidium bromidestained gel used in panel A depicting 28s and 18s RNA bands; C) amounts of TNFu mRNA determined in four different blots and adjusted for amount of p-actin in each lane. * p < 0.05 vs. control value.
Eur. J. Immunol. 1992. 22: 2983-2987
B. Fouqueray, C. Philippe, A. Amrani et al.
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Table 1. Effect of indomethacin on TNF-a release from LPSactivatcd macrophaged Addition
Release of cytotoxic activity (%) 37 "C 41 "C
LPS (1 ng/ml) LPS (1 ng/ml)
11.6 k 4.3**
47.5 k 2.8
+ Indomethacin (1 FM)
44.5 k 3.6
8.1
* 4.2**
a) Elicited rat peritoneal macrophages were incubated at 37" or 41 "C for 20 min with or without indomethacin and then at 37°C with 1 ngiml LPS. After 2 h of incubation, the cell-free culture medium was harvested and TNF-a concentration was determined by bioassay. ** p < 0.005 vs. 37°C (n = 3).
37
A
39
41
43
37
6
39
41
43
200, 200,
116, 97,
116,
66b
97b 45,
two cell-associated proteins of 70 and 90 kDa. These were the predominant proteins synthesized by macrophages at 43 "C. To confirm a possible relationship between the synthesis of such heat-shock proteins and the reduction of TNF-a release, experiments have been performed with chemical inducers of the heat-shock response [14]. Macrophages were preincubated with sodium arsenite (20 PM) or iodoacetamide (5 pM) before exposure to 1 ng/ml LPS. These compounds reduced the concentration of immunoreactive TNF-a in the cell-free culture medium of macrophages from 1560 208 to 460 zk 347 (p = 0.05) and 400 -t 345 (p < 0.05) pg/ml, respectively.
*
3.4 Comparison of the heat-shock effects on the release of TNF-a from isolated glomeruli and blood monocytes
To determine whether the results obtained were unique to elicited peritoneal macrophages, glomerular cells and blood monocytes were similarly screened for heat-shock effects on the release of TNF-a. Pretreatment at 41°C significantly and transiently reduced the amount of TNF-a produced (Fig. 6). The inhibition was found to be almost complete in glomerular cells (82.0 f 7.5 YO), but significantly less in blood monocytes (43.8 k 10.9%; p < 0.05).
66r 31b 34,
4 Discussion 4.1 Effects of heat shock on the release of TNF-a from LPS-stimulated macrophages
21, 14*
14'
Figure.5. Effect of heat shock on protein synthesis by maerophages. Macrophages were incubated at 37"C, 39 "C, 41 "C or 43 "C for 20 min and then at 37 "C with 1 ng/ml LPS and [3sS]methionine. After 2 h of labcling, aliquots of cell lysates (A) and cell-free supernatants (B) werc subjected to SDS-PAGE.
80
60
-
40
-
20
**
0
I
I
I
Wnr)
Figure 6. Revcrsibility of the heat shock-induced reduction of TNF-a release from isolated glomeruli and blood monocytes. Thcse cells were incubated at 41 "C for 20 min. One yg LPS was added at indicated time points before or after the heat shock. After 2 h of incubation at 37"C, the cell-free culture medium was harvested and TNF-a concentration was determined by bioassay. Means and SEM of values obtained in five experiments are given. * p < 0.05 vs. control value; ** p < 0.005 vs. control value.
The data presented here give further evidence for the modulating role of elevated temperatures on TNF-a synthesis.We show that heat stress can decrease the expression of TNF-a mRNA and hence limit both cell expression and release of TNF-a bioactivity in LPS-stimulated peritoneal macrophages. Stress responses occur following exposure of macrophages to 41 "C for 20 min (Fig. 1). Because this temperature is reached during acute fever, our in vitro observations may have potential implications in vivo. Heat shock at 41°C for 20 min is not toxic to macrophages as suggested by the ability of cells (a) to still adhere to the tissue culture plates, and (b) to incorporate [35S]methionine into proteins to a similar extent than control cells (Fig. 5). In this regard, it is also important to note that the inhibitory effect of heat shock is rapidly reversible: a total recovery is observed when macrophages are incubated at 37°C for 6-8 h following the end of heat shock (Fig. 3). These data are comparable to those reported for the recovery of NADPH-oxidase activity in human neutrophils exposed to 43 "C for 20 min [15]. Our data on the time course study show that heat shockinduced reduction of TNF-a release is a rapid process which begins after less than 2 hours (Fig. 2).This finding suggests that heat shock reducesTNF-a bioactivity by modifying its release from macrophages rather than its stability in the medium. Previous studies demonstrated that the released form of TNF-a (17 kDa) is derived from the integral transmembrane TNF-a precursor molecule (26 kDa) possibly by proteolytic cleavage [ 161. Reduction of this cleavage
Eur. J. Immunol. 1992. 22: 2983-2987
Modulation of macrophage TNF production by temperature
could contribute to the reduced levels of TNF-a bioactivity and immunoreactivity in the medium of macrophages exposed to elevated temperatures. However, such a mechanism is unlikely since heat shock reduces cell expression and release of TNF-a bioactivity to a similar extent. Because of the known shedding of TNF-a receptors from stimulated cells [16], and the evidence that soluble TNF-a receptors reduce the bioactivity of TNF-a [17], the inhibitory effect of heat shock on the bioactivity of TNF-a could also be explained by a more extensive cleavage of TNF-a receptor. However, preliminary studies indicate that the concentration of immunoreactive TNF-a binding protein (TNF-BP75 and TNF-BP55) in the cell-free supernatant of mononuclear phagocytes is not affected by heat stress (data not shown). Exposure of macrophages to elevated temperatures similarly affected the expression of IL-1, suggesting that these observations were not unique to the particular cytokine tested (Fig. 2B). 4.2 Mechanisms by which heat shock reduces the release of TNF-a from LPS-stimulated macrophages
Our studies suggest that the effects of heat shock are independent of variations of prostaglandin synthesis. They are rather related to the synthesis of heat-shock proteins, since (a) after treatment at 41 "C,at least two proteins of 70 and 90 kDa are induced in macrophages, [they are the predominant proteins synthesized at 43 "C (Fig. 5)] and (b) chemical inducers of the heat shock response are also effective inhibitors of the release of immunoreactive TNFa. This finding is consistent with a previous report showing that elevated temperatures down-regulate IL-1 biosynthesis through a mechanism related to the induction of heat-shock proteins [14]. Because the synthesis of stress proteins (70 and 90 kDa) is induced by LPS as well [18], these proteins might down-regulate macrophage activation in an autoregulatory circuit, even in the absence of fever. 4.3 Comparison of the heat-shock effects on tissue macrophages and blood monocytes
In response to LPS,TNF-a is produced by tissues, including glomeruli [19], as well as by circulating monocytes [20].We show in this report that the priming effect of LPS (1 pg/ml) on TNF-a release is identical in isolated glomeruli and in blood monocytes (Fig. 6). However, the effect of heat shock on TNF-a release is clearly greater in isolated glomeruli than in blood monocytes. The difference in
2987
heat-stress response could be explained by the weaker stability of TNF-a mRNA in tissue macrophages compared to that observed in blood monocytes [20]. Such a different modulation by temperature could contribute, in vivo, to reduce the local generation of TNF-a more than its systemic availability. We thank Mrs. Morin, Miranda and Knobloch for secretarial assistance. Received January 2, 1992; in final revised form July 6, 1992.
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